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Mechanisms of altered cell-mediated immune responsiveness in mice infected with Trichinella spiralis

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Mechanisms of altered cell-mediated immune responsiveness in mice infected with Trichinella spiralis
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Hall, Bruce T
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English
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x, 84 leaves : ill. ; 29 cm.

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Antibodies ( jstor )
Antigens ( jstor )
Graft rejection ( jstor )
In vitro fertilization ( jstor )
Infections ( jstor )
Larvae ( jstor )
Lymphocytes ( jstor )
Spleen ( jstor )
Spleen cells ( jstor )
Transponders ( jstor )
Concanavalin A ( mesh )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( mesh )
Immunology and Medical Microbiology thesis Ph.D ( mesh )
Phytohemagglutinins ( mesh )
Trichinella ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 75-83.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Bruce T. Hall.

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MECHANISMS OF ALTERED CELL-MEDIATED IMMUNE RESPONSIVENESS
IN MICE INFECTED WITH Trichinella spiralis










By

BRUCE T. HALL


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY








UNIVERSITY OF FLORIDA


1981

















ACKNOWLEDGEMENTS

I would sincerely like to thank my mentors, Dr. Richard Crandall and Dr. Catherine Crandall, for all their time, help, advise, friendship and especially for their patience. I thank each of the members of my committee, Dr. Paul Klein, Dr. Ammon Peck and Dr. Kenneth Berns, for encouragement and assistance in planning my dissertation research.

I would like to thank all my friends in the department who helped make the past five years a most memorable experience. Rick Kris, Erv Faulmann, Cindi Donnelly and Tom Doyle, in particular, helped to make graduate school bearable.

I want to thank my wonderful parents, Mr. and Mrs. Edgar Hall, for their constant support and love. Without them this work would never have begun. Lastly I want to thank the best friend I have ever had, Ms. Judy Emiko Yoshioka. Without her love this work would never have been completed. It is to my wonderful parents and my best friend that I dedicate this dissertation.

















TABLE OF CONTENTS


ACKNOWLEDGEMENTS ...... ..............

LIST OF TABLES ...... ................

LIST OF FIGURES ...... ................

KEY TO ABBREVIATIONS ..... .............

ABSTRACT ........ ..................

INTRODUCTION ....... ................

BACKGROUND REVIEW ..... ...............

The Biology of Trichinella spiralis..

Immunity to Trichinosis ...........

Trichinella-Induced Alterations of Host Mechanisms of Altered Immune Responses Statement of the Problem ... ...... MATERIALS AND METHODS ............

Mice ....... ..................

Trichinella infection ... ..........

Leukocyte Preparation ... ..........

Nylon Wool Filtration of Leukocytes. .

Differential Cell Counts ..........

Cytotoxicity Assays .... ...... .....

Mitogen Stimulation ... ...........

Mixed Leukocyte Reaction .. ........

LR Supernatant Fluid .............


Immune


PAGE

. . . . . ii



.. vi


S. . . . . vii S. . . . . . ix



* 2 * 2 * 4

Responsiveness 7 . . . . . . . 13

. . . . . . . 18

. . . . . . . 19

. . . . . . . 19

. . . . . . . 19

. . . . . . . 19

. . . . . . . 20

. . . . . . . 20

. . . . . . . 20

. . . . . . . 21

. . . . . . . 22

. . . . . . . 23













Cell-Mediated Lympholysis Assay ....

Delayed-Type Hypersensitivity Assays .

Skin Grafts ..... ...............

Statistical Analysis .... .........

RESULTS ........ ....................

Alteration of Blastogenic Responses..

Mixed Leukocyte Responses. ...

Spleen cells ..........

Lymph node cells .......

Mitogen Responses ...........

Kinetics of Altered Blastogenic R

Mechanism of Altered Blastogenic Respon


PAGE

. . . . . . . . .. . 23

. . . . . . . . .. . 24

. . . . . . . . .. . 24

. . . . . . . . .. . 25

. . . . . . . . .. . 26

. . . . . . . . .. . 26

. . . . . . . . .. . 26

. . . . . . . . .. . 26

. . . . . . . . .. . 31

. . . . . . . . . . 36

esponses ......... ...36

ses ... ......... ..36


Alterations in Cell-Mediated Lympholysis (CML) Activity. . 52 Changes in Splenic T-Cell Subsets .... .............. ...52

Delayed-Type Hypersensitivity (DTH) to Allogeneic Cells. . 56 Effect of Adoptively Transferred Cells of Allograft Rejection 60 DISCUSSION ........... ............................ 62

CONCLUSION ........... ............................ 74

LIST OF REFERENCES .......... ........................ 75

BIOGRAPHICAL SKETCH .......... ........................ ..85

















LIST OF TABLES


TABLE PAGE

1 THE MIXED LEUKOCYTE RESPONSE OF SPLEEN CELLS ........... ...27

2 THE INFLUENCE OF INCUBATION TIME ON THE MLR RESPONSE OF
SPLEEN CELLS .......... ......................... ..29

3 THE MLR RESPONSE OF NYLON-WOOL ENRICHED SPLENIC T-CELLS . . . 33

4 SPLEEN CELL POPULATIONS DURING INFECTION WITH
TRICHINELLA SPIRALIS ........ ..................... . 44

5 SPLEEN CELLS FROM C57BL/6 MICE AS STIMULATORS IN
THE MLR ASSAY ........... ...................... 45

6 RELEASE OF 51Cr FROM 51Cr-LABELED CBA/Ca STIMULATOR CELLS . 46

7 ACTIVE SUPPRESSION OF THE MLR RESPONSE IS MEDIATED BY
SPLEEN CELLS FROM INFECTED MICE . ..... .............. ..47

8 SUPPRESSION OF THE MLR RESPONSE MEDIATED BY NYLON WOOLADHERANT AND NONADHERANT SPLENOCYTES .... ............. ...50

9 LACK OF SUPPRESSION BY SUPERNATANT FROM "REDUCED" MLR
CULTURES ........... ........................... .54

10 CHANGES IN SPLENIC T-CELL SUBSETS ..... .............. 57

11 DTH RESPONSE TO ALLOGENEIC CELLS ..... ............... ..58

12 EFFECT OF ADOPTIVELY TRANSFERRED CELLS ON ALLOGRAFT REJECTION ........... .......................... 61
















LIST OF FIGURES


FIGURE PAGE

1 Life cycle of Trichinella spiralis ...... ............. 3

2 A graphic representation of the data in Table 1 (the MLR
response of spleen cells from uninfected and Trichinellainfected C57BL/6 mice) ....... ................... . 28

3 The influence of varying the ratios of responder cells
to stimulator cells on the MLR ..... ............... . 30
5
4 Comparison of the MLR response using 2.5 x 10 and 5.0
x 105 responder cells ........ ................... .32

5 The MLR response of uninfected and Trichinella-infected
B10.AQR mice ......... ........................ . 34

6 The NLR response of lymph node cells .... ............ . 35

7 The MLR response of nylon wool-enriched lymph node T-cells . 37 8 The Con A response of spleen cells .... ............. . 38

9 The PHA response of spleen cells ..... .............. . 39

10 The MLR response of spleen cells during infection with
T. spiralis . ......... ...................... 40

11 The T-cell mitogen responses of spleen cells during infection with T. spiralis ........ .................... . 41

12 A comparison of the reduced MLR and Con A responses during
infection with T. spiralis ...... ................. .42

13 Spleen cell-mediated active suppression of the MLR
response during infection ....... .................. .49

14 Identification of the MLR suppressor cell by negative
selection using specific antisera plus complement ........ ...51

15 Identification of the MLR suppressor cell by negative
selection using anti-Ly antisera plus complement ....... . 53










FIGURE


PAGE


16 Cell-mediated lympholysis (CML) mediated by spleen cells
from Trichinella-infected C57BL/6 mice ... ........... ...55

17 Adoptive transfer of DTH responsiveness ... .......... . 59
















KEY TO ABBREVIATIONS

BCG bacillus Calmette-Guerin CML cell-mediated lympholysis Con A concanavalin A cpm count per minute CTL cytotoxic T-lymphocyte DNP dinitrophenol DTH delayed-type hypersensitivity DxS dextran sulfate FcR Fc receptor H-2 histocompatibility-2 complex Ig immunoglobulin LPS lipopolysaccharide MIF macrophage inhibition factor MLR mixed leukocyte reaction PHA phytohemagglutinin PPD purified protein derivative SRBC sheep red blood cell


viii

















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


MECHANISMS OF ALTERED CELL-MEDIATED IMMUNE RESPONSIVENESS
IN MICE INFECTED WITH Trichinella spiralis



By

Bruce T. Hall

December 1981

Chairman: Richard B. Crandall
Major Department: Immunology and Medical Microbiology

Cell-mediated immune functions were evaluated during infection

with Trichinella spiralis in C57BL/6 mice. The response of spleen cells to concanavalin A (Con A) was reduced by 70%, while the response to phytohemagglutinin was unaltered. There was a 60% reduction in the response of spleen cells to allogeneic stimulation in the mixed leukocyte reaction (1,R), with no detectable effect on the ability of spleen cells to mediate lysis of allogeneic cells in cell-mediated lympholysis assays. The MLR response to H-2K plus H-2D region stimulation was significantly more reduced than the response to H-21 plus H-2S region stimulation. The reduced MLR and Con A responses showed similar patterns of suppression; they were both maximally reduced at 2-3 weeks of infection and returned to normal after 3-4 months. Spleen cells from Trichinella-infected mice actively suppressed the MLR response, but not the Con A response, of spleen cells from uninfected mice.










This in vitro suppression was not mediated by soluble factors, was enriched by treatment with anti-Ig and was completely abrogated by treatment with anti-Ly-2, but not anti-Ly-l, antibody plus complement. The reduced MLR response observed during infection was not due to a change in the timing of optimal responsiveness or to lysis of the allogeneic stimulator cells. There was no change in the percentage of T-cells during infection, but there was a decrease in the percentage of cells sensitive to complement-mediated lysis with anti-Ly-l, but not anti-Ly-2, antibody. These data are consistent with the hypothesis that Ly-l +,2/3+ T-cells convert to Ly-l -,2/3+ T-cells during infection. There was no alteration of the delayed-type hypersensitivity (DTH) response, as measured by the injection of allogeneic cells into footpads of Trichinella-infected mice. Although not statistically significant (P = 0.17), there was some reduction in the DTH responsiveness of spleen cells from infected mice when transferred to footpads of uninfected mice. The results indicate that the impaired rejection of skin allografts during trichinosis results from a Ly-l-,2/3+ T-cellmediated suppression of Ly-l +,2/3- T-cells, which are the presumed effector cells of allograft rejection.
















INTRODUCTION

Parasites continue to be a major health problem throughout the world, particularly in the developing countries of Africa, Asia and South America. It is estimated that over half of the world's population is infected with medically important parasites. In addition, parasitic infections of domestic animals result in serious economic loss and contribute to a vicious cycle of poverty and malnutrition. Unfortunately it is presently impractical or impossible to control these diseases through improved sanitation and living conditions or chemotherapy, and vaccines do not yet exist.

It is well known that parasites elicit strong immune responses in uncompromised hosts, and one of the major goals of research today is the development of vaccines for parasitic diseases. However, efforts to develop vaccines have been frustrated by the complexity of the life cycles and the fact that parasites have evolved elaborate means of resisting or evading the host's immune defense mechanisms. In addition, parasites frequently induce a state of altered immune responsiveness to nonparasite antigens, thus rendering the victim more susceptible to infection by other diseases and less able to respond to immunization. Knowledge of parasite-induced alterations of the immune system will not only help us to understand how parasites survive in an immunologically competent host, but may also provide the key for developing effective vaccines.
















BACKGROUND REVIEW

The Biology of Trichinella spiralis Trichinella spiralis was discovered in 1835 by James Paget, a first year medical student, while dissecting the cadaver of a patient who died from pulmonary tuberculosis (1). In the same year Richard Owen presented the first description of the parasite and named it Trichina spiralis (2). The details of the life cycle were established during the next 25 years (3) and in 1896 Railliet changed the name to Trichinella spiralis (4).

Trichinosis, an infection with the tissue-inhabiting nematode T. spiralis, has a worldwide distribution and infects a large variety of carnivorous animals. The disease is essentially a zoonosis, with man being an incidental host. Most human infections are light and asymptomatic, but heavy infections may result in clinical disease characterized by fever, myositis, periorbital edema, eosinophilia and sometimes death. Human infections result primarily from the ingestion of improperly cooked meat from pigs, bears and sea mammals.

Infection is initiated by the ingestion of meat which contains viable, encysted larvae (Figure 1). The cyst wall is digested in the stomach of the host and the larvae pass to the proximal small intestine. The larvae then embed themselves within the lamina propia, where they molt four times within thirty-six hours and transform into immature adults (5). After 5-6 days the worms have reached full length, mated

















larvae encyst in muscle fibers


larvae penetrate the sarcolemma oc the muscle fibers


ingestion of
viable, encysted larvae


larvae excyst in stomach


larvae pass to small intestine


newborn larvae migrate through the circulatory and lymphatic systems


larvae mature, mate and females deposit second stage larvae in lamina propia


Life cycle of Trichinella spiralis.


FIGURE 1.









and the gravid females deposit hundreds of second stage larvae into the lamina propia (6). The newborn larvae migrate through the lymphatic and circulatory systems and become distributed throughout the body (7). They leave the circulatory system in the striated muscle, penetrate the sarcolemma of the muscle fibers and become encapsulated (8). By 30 days postinfection the muscle larvae are fully mature and infective (9). The encapsulated larvae may survive many months, or even years, but will eventually die and calcify. The intestinal adults are expelled from the gut by a local inflammatory response, which in a primary rodent infection, begins about 14 days postinfection and is complete by about Day 30 (10).

Immunity to Trichinosis

A degree of protective immunity is developed with trichinosis, but as in many other parasitic infections, it is not complete (5). Resistance to reinfection with T. spiralis appears to be induced primarily by antigens secreted by both larval and adult stages. Most, if not all, of these secreted antigens originate from stichocyte granules (5, 11).

Stichocytes are large, discoid cells lying in a single row

dorsal to the esophagus and comprise the stichosome, which is an organ characteristic of roundworms of the superfamily Trichuroidea. Stichocytes contain two types of secretory granules, a and 0 granules (11). It is noteworthy that complete cross-reactivity between the antigens secreted in vitro and the antigens of the a and 0 granules has been shown in double diffusion assays (12).

Acquired resistance to trichinosis may be expressed in a number of ways; stunting of adult worms (13, 14), impairment in their ability







5

to reproduce (15), a more rapid expulsion of adults from the gut (16, 17) and fewer larvae encysting in the host muscle cells (17). Once inside the muscle cells, the larvae appear to be protected from the effects of the host's immune system (5).

The precise mechanisms of acquired resistance are not completely understood, but experiments have indicated roles for both humoral and cellular immune mechanisms, as well as nonspecific mechanisms (16, 18, 19). T. spiralis stimulates production of circulating antibodies, predominantly IgG but also IgM and IgA (20, 21). The IgG antibody exhibits specificity for stichosome cells and cuticle, while IgM and IgA show specificity for membranes of the larvae. While the role of these antibodies remains unclear, the fact that purified immune B-cells are capable of transferring resistance (22) indicates that they do play an active role in immunity.

One possible role for antibody may be in assisting in the attachment of eosinophils to newborn larvae. It has been shown that newborn larvae, but not adults or muscle larvae, are destroyed by eosinophils in the presence of immune, but not nonimmune serum in vitro (23). Evidence for a role of eosinophils in mediating resistance to the systemic phase of trichinosis has also been demonstrated in vivo. Mice infected with T. spiralis and depleted of circulating eosinophils by treatment with anti-eosinophil serum had increased numbers of larvae in the muscles relative to controls (24). Treatment with anti-eosinophil serum had no effect on the expulsion of adults from the small intestine, supporting the supposition that different immune mechanisms are effective against the intestinal and parenteral stages of T. spiralis.









Perrudet-Badous et al. (25) demonstrated that mice genetically selected for high and low antibody production had the same number of larvae encysting in the muscles. These results probably indicate that antibody alone is not sufficient protection against infection.

A T-cell involvement in host resistance to T. spiralis has been clearly demonstrated in studies using T-cell deficient mice and in adoptive transfer experiments using immune T-cells. Ruitenberg and Steerenberg (26,27) found that athymic (nude) mice continue to accumulate parasites in the muscles and do not expel adults worms from the intestine. Implantation of thymus tissue from heterozygous (+/nu) mice restores the immune capacities and the infection is terminated (5). Using thymectomized, X-irradiated, bone marrow-reconstituted mice, Walls et al. (28) found that adult parasites persisted longer in the gut and more larvae encysted in the muscles of T-cell deficient mice. The persistence of intestinal adults corresponded to a defective local inflammatory response in the gut.

Another principal arm of host defense against reinfection is the rapid expulsion of 90% or more of a challenge infection from the intestine. In elegant experiments utilizing parabiosed rats, Bell and McGregor (29,30) showed that rapid expulsion results from the synergistic actions of two distinct host responses. One component of rapid expulsion is generated in response to a priming effect on the intestine by adult worms. This effect is a local, nonspecific phenomenon which is not transferred to parabionts and lacks an immunological bisis. The other component is a circulating factor that is specific for Trichinella and is presumably either antibody or specifically sensitized lymphocytes.









Trichinella-Induced Alterations of Host Immune Responses

Infection with T. spiralis induces complex immunological responses which not only result in specific resistance to the parasite, but also modify the response of the host to heterologous antigens. Alterations of host immune responses in trichinosis include examples of both immunopotentiation and immunosuppression and are most pronounced during a limited time period, approximately 2-6 weeks following infection. This time period corresponds roughly with the late intestinal, larval migration, muscle invasion and intracellular development stages of infection, and a time when the host is undergoing intense immune responses to parasite antigens (5, 18, 19).

Parasite-Induced Immunopotentiation

Effect on macrophage activity. Although the role of macrophages during Trichinella-infection has not been fully established, there are several quantitative and functional changes in peritoneal macrophages that warrant mention.

Wing et al. (31) observed that the number of mononuclear cells in the peritoneal cavaties of Trichinella-infected mice increased as much as eightfold over uninfected control mice. This increase in peritoneal exudate cells reached a maximum 18 days after infection and coincided with the infiltration of macrophages and lymphocytes into the intestinal wall and elimination of adult worms from the gut. While the absolute number of macrophages and lymphocytes increased in the peritoneum following infection, the relative proportions remained the same.

In studies of macrophage function, it was found that infection

with T. spiralis results in a nonspecific activation of the host reticuloendothelial system. The increased activity of macrophages during










trichinosis is similar, but not identical, to the immunopotentiating activity of BCG and C. parvum. Wing et al. (31) found that macrophages from Trichinella-infected mice and C. parvum-injected mice both inhibited DNA synthesis of EL-4 tumor cells in vitro and had comparable cytocidal activity as determined by 51Cr release assays. However, whereas macrophages from C. parvum-injected mice, or mice infected with Toxoplasma $ondii, inhibited the intracellular replication of T. gondii, macrophages from Trichinella-infected mice did not. It is possible that different subsets of macrophages are responsible for activity against tumor cells and cells infected with an intracellular parasite such as T. gondii, and while both subsets are activated by C. parvum or infection with T. gondii, only the former subset is activated in trichinosis.

Infection with T. spiralis also appears to enhance the activity of the fixed reticuloendothelial system. Trichinella-infected mice exhibit an increased rate of clearance of intravenously injected colloidal carbon 14 to 28 days after infection (32).

Effect on tumor growth. Infection with T. spiralis is reported to lead to an increased resistance to transplantable tumors and a reduction in the incidence of spontaneous tumors. Lubiniecki and Cypess

(33) studied the effect of T. spiralis infection on the incubation time and survival time of mice given an ascitic sarcoma. They found that the tumor developed more slowly in mice infected for 28 days with T. spiralis and the mice survived for longer periods of time. Mice infected for 56 days showed no differences in tumor development relative to control mice. The authors suggested that the T. spiralis infection activated macrophages to be more efficient killers of tumor cells.









Molinari and Ebersole (34) found an even more dramatic effect on the development of a melanoma following infection with T. spiralis. Control mice developed tumors by Day 28 following tumor challenge and all died within 60 days, while none of the Trichinella-infected mice exhibited any signs of neoplasia.

Effect on other infections. Mice infected with T. spiralis for

7 to 21 days are less susceptible to intravenous challenge with Listeria monocytogenes. Trichinella-infected mice had approximately an eightfold higher LD50 and fewer viable bacteria could be isolated from their livers (35). T. spiralis has also been reported to exert effects against infections with Trypanosoma lewisi (36) and Leishmania tropica

(37).

Effect on delayed-type hypersensitivity. Molinari et al. (38, 39 40) studied the effect of trichinosis on delayed-type hypersensitivity (DTH) responses to BCG in mice, as measured by footpad swelling. Mice were sensitized with live or heat-killed BCG and subsequently challenged with Old Tuberculin (OT). The response to OT was found to be dependent on i) the route of BCG injection, ii) the timing of BCG sensitization relative to the initiation of Trichinella infection and iii) whether the BCG was live or heat-killed.

When mice were sensitized with BCG at varying times after infection with T. spiralis there was an initial suppression of the DTH response which, by 21 days after helminth-infection, converted to a stimulation of DTH responsiveness. The route of sensitization had no effect when using live BCG. When using heat-killed BCG, however, there was no initial suppression of the DTH response and the subsequent stimulation of DTH responsiveness occurred only when the sensitizing BCG was injected intravenously or intraperitoneally, not when injected subcutaneously.









When mice were sensitized to BCG 14 days before infection with T. spiralisthe DTH response was suppressed 14 days after helminthinfection but, again, it converted to an enhanced response 20 to 85 days after Trichinella-infection.

Jones (41) examined the DTH response to sheep erythrocytes and oxazalone in mice 20 days postinfection with T. spiralis. In contrast to the enhanced response observed to BCG, there was little or no effect on the DTH response to those antigens. Interestingly, the response of parasitized, but unsensitized, mice sometimes had significantly greater responses to challenge with the sheep erythrocytes and oxazalone.

Effect on responses to B-cell mitogens. Ljungstrom (42) used

three polyclonal B-cell activators to examine the effect which Trichinella-infection has on B-cells which are at different stages of maturation. Purified protein derivative (PPD), lipopolysaccharide (LPS) and dextran sulfate (DxS) were used to stimulate mature, intermediate and immature B-cells respectively. No significant alteration in the response of intermediate or mature B-cells from parasitized mice was observed. Immature B-cells, however, were more reactive in parasitized mice than uninfected controls during the late muscular stage of infection.

Effect on antibody responses to T-independent antigens. The

ability of Trichinella-infected mice to produce antibody in response to T-independent antigens was assessed using polyvinyl pyrrolidone (PVP) and DNP 52-Ficoll. Serum antibody specific for PVP was only slightly, but consistently, higher 1, 2 and 4 weeks postinfection in A/Sn mice, which are "high responders" to PVP. CBA mice, which are "low responders" to PVP, showed a more pronounced, and significant, elevation of the antibody response to PVP (43), Jones (41) observed that the antibody









response to DNP 52-Ficoll, as measured by the number of antibody forming cells in Jerne plaque assays, was significantly enhanced 20 days after Trichinella-infection following either in vivo or in vitro immunization. Parasite-Induced Immunosuppression

In contrast to the reports of enhanced reactivity where the major effector cells are probably macrophages or B-lymphocytes, there is a pronounced suppression of several functions which are dependent on Tlymphocytes.

Effect on virus infections. Suitably timed challenge with Japanese B encephalitis (JBE) virus results in a higher mortality rate and decreased survival times of Trichinella-infected mice (44). Although no increase in the duration or magnitude of JBE viremia was observed, mice infected for 7 to 28 days with T. spiralis had reduced serum antibody titers to JBE. Also, Kilham and Oliver (45) observed that rats infected with encephalomyocarditis virus 10 days after infection with T. spiralis had higher death rates than unparasitized rats challenged with the same virus.

Effect on antibody responses to T-dependent antigens. Suppression of the antibody response to sheep red blood cells (SRBC) is perhaps the most throughly investigated, and best understood, alteration of host immune responsiveness during trichinosis. Mice immunized with SRBC during the intestinal stage (Day 7) of trichinosis exhibit no change in the antibody response either at the humoral or the single cell level (43,46,47,48). When immunized during the second week of infection they have either normal or reduced antibody responses, depending on the strain of mouse, route of immunization and dose of SRBC used. During the early muscular stage (Day 20), both the humoral antibody level and the number









of spleen cells producing anti-SRBC antibodies are reduced (43,44,46,47 48). Spleen cells from 20-day infected mice are also less responsive to immunization with SRBC in vitro (47). By the late muscular stage (Day 30) the response to SRBC is essentially normal. Jones et al.(47) determined that the transient suppression of humoral immunity to SRBC is mediated by suppressor T-cells (T S) which can be found in the spleens of infected mice. Interestingly, while the humoral response to SRBC was reduced in the spleen, the response of lymph node lymphocytes was enhanced. The antibody response to SRBC can also be diminished, even eliminated, by the injection of solubilized extract of T. spiralis, without affecting the antibody response to the T-independent antigen PVP (48).

Trichinosis was also found to depress both the systemic and local antibody responses to orally administered cholera toxin (49). Local antibody production was evaluated by determining the amount of antibody synthesized in vitro by tissue-cultured intestine. The intestines from uninfected mice produced significant levels of both IgA and IgG in vitro, while no antibody synthesis could be detected in the cultured intestine from parasitized mice. The suppressive effect was more pronounced during the intestinal stage of infection.

Effect on passive cutaneous anaphylaxis (PCA). Munoz and Cole

(50) demonstrated that mice infected with T. spiralis were relatively unresponsive to passive cutaneous anaphylaxis following injection of egg albumin and anti-egg albumin (IgG1 and IgE) antibodies. Presumably the Trichinella infection induces the formation of IgE antibodies which bind to mast cells and prevent the binding of passively transferred antigen-specfic IgE.








13

Effect on responses to T-cell mitogens. Conflicting results exist in the literature relating to the responsiveness of lymphocytes from Trichinella-infected mice to the polyclonal T-cell activators concanavalin A (Con A) and phytohemagglutinin (PHA). A majority of studies indicate that the response of splenic lymphocytes to Con A is significantly reduced 14 to 20 days after infection (42,47,51,52), while the response to PHA is reported to be only slightly depressed (42,47) or not affected at all (51).

Effect on graft versus host and allograft rejection responses.

Svet-Moldavsky et al. (53) and Chimyshkyan et al. (54) showed that spleen cells from mice infected with T. spiralis for 22 to 72 days were impaired in their ability to induce a graft versus host reaction, and that mice infected for 20 to 40 days were suppressed in their ability to reject skin allografts. Using split heart allografts, Ljungstrom and Huldt

(43) confirmed the observation that transplant rejection is impaired in mice infected with T. spiralis.

In summary, infection with T. spiralis alters the ability of the host to respond immunologically to a wide range of antigens. These alterations are complex, and the underlying basis of most of them has not been determined.

Mechanisms of Altered Immune Responses

Numerous mechanisms have been proposed which may explain the alterations of immune responsiveness induced by infections with T. spiralis, but in most cases these mechanisms are hypothetical only. In any case, each altered immune response which is identified most likely reflects the summation of numerous synergistic and antagonistic factors.









Parasite-Produced Immunosuppressive Factors

Faubert and Tanner (55) provide evidence that serum from Trichinella-infected mice may contain parasite-derived immunosuppressive factors. Sera from infected mice agglutinates and kills homologous lymphocytes in vitro beginning 7 days after infection. These leukoagglutinating and cytotoxic activities reached a maximum 30 days after infection and declined thereafter. Transfer of sera from infected mice also prolongs the survival of allografts in normal recipients. Based on the kinetics of appearance of these activities and the fact that saline extracts of T. spiralis possess both the leukoagglutinating and leukotoxic activities, the authors have concluded that these activities are due to parasite produced substances and not antibody.

Others have also shown that saline extracts of T. spiralis are immunosuppressive. Barriga (56) has shown that normal mice injected with a saline extract exhibited depressed antibody responses to the Tdependent antigen SRBC, depressed responses to the mitogens LPS and Con A and a delayed allograft rejection response. Saline extracts of T. spiralis are also reported to suppress the formation of sheep red cell rosette forming cells (57).

Altered Structure of Lymphoid Organs

Changes in the structure, or architecture, of lymphoid organs (e.g., splenomegaly, lymphadenopathy) may affect the circulation of antigens, or the trafficking of lymphoid cells through these organs and affect the cell-cell interactions requisite for mounting an immune response.

Faubert and Tanner (58) showed that the increase in size of lymph nodes in mice following infection was a T-cell dependent phenomenon









since the increase in size did not occur in thymectomized animals. Molinari et al.(40) described changes in the microscopic anatomy of the thymus during trichinosis in mice, including an increase in the total cell number and distinct anatomical changes in the thymic cortex and medulla. In contrast to the increase in cell numbers in the thymus observed by Molinari et al.(40), Ljungstrom and Huldt (43) reported that the thymic cortex is depleted of lymphocytes, with some infected mice being almost completely devoid of lymphocytes in the cortex. Ljungstrom and Sundqvist (59) and Jones (41) reported that although trichinosis in mice results in splenomegaly, there is no change in the proportion of B-cells and T-cells.

Immunosuppressive Serum Factors

A number of factors have been identified that are found normally in the serum, or are induced following infection, which are capable of suppressing in vitro tests of cellular immunity. Although some of these factors act specifically and depress reactivity only to antigens of the infecting agent, most act nonspecifically and depress the response of sensitized lymphocytes to unrelated antigens.

C-reactive protein. C-reactive protein (CRP) is a trace component of serum that is not readily demonstrable in normal serum but which increases in concentration during the acute phase of febrile illnesses

(60). The detection of CRP has long served as an indicator of the presence and degree of inflammatory activity. CRP is known to bind to some, but not all, T-lymphocytes and to suppress their activity. CRP can suppress the proliferative response of T-cells in the mixed lymphocyte response (61,62,63), the generation of cytotoxic T-cells in vitro









(63), antigen-induced blastogenesis (64) and mitogen-induced MIF production (64).

Histamine. It has been shown that some, but not all, murine Tcells have receptors for histamine. These receptors are of the H-2 type and are found on T-cells which inhibit antigen-induced MIF production (65,66,67), antigen-induced blastogenesis (65,66,67), cytotoxic activity against allogeneic cells (68) and the antibody response to SRBC (69).

Bekish (70) demonstrated that there is a considerable increase in the free histamine level in the liver, muscle and blood in rodent trichinosis. The author suggests that the rise in histamine level in the tissues and circulation may be an important factor in promoting the migration of Trichinella larvae through the capillary beds since histamine causes the dilatation of capillary vessels and increases their permeability. It is possible that the free histamine also binds to the H-2 receptor-bearing lymphocytes and modulates those functions mentioned above.

Antibody-Mediated Alterations

Antibody, either alone or complexed with antigen, is capable of both enhancing and suppressing immune responses. Perhaps the simplest explanation of the suppressive effect of antibody is that antibody which is bound to antigen may block any subsequent recognition of that antigen by lymphocytes. Such "blocking antibodies" have been shown to play an important role in the survival of tumor cells in immunocompetent hosts (71,72). It has been demonstrated in some systems that antibodymediated suppression requires intact Fc domains in addition to the antigen binding domains (73). The mechanism(s) of this type of suppression is not understood, although speculation exists that it is mediated









by soluble factors released by Fc-bearing lymphocytes following the binding of antigen-antibody complexes. Antigen-antibody complexes may also enhance immune responses. Dennert (74) found that complexes formed between IgM and SRBC enhanced the antibody response, relative to SRBC alone, both in vivo and in vitro. Dennert suggested that the IgM-SRBC complexes resulted in a more efficient antigen localization in vivo. Anti-idiotypic antibodies may also play an important role in the regulation of immune responses, both humoral and cellular (75,76). Antigenic Competition

Antigenic competition is the phenomenon whereby the injection of one antigen may inhibit the immune response to another, unrelated antigen. The mechanism of antigenic competition is not clear, but it is evidently dependent on T-cells since it involves only T-dependent antigens (77). Antigenic competition has been suggested as a possible explanation of reduced immune responsiveness during trichinosis (46,47). Suppressor Cells

One of the major areas of research in immunology today is the regulation of immune responses. It is well documented that distinct subpopulations of T-cells function not only as effector cells, but also as regulator cells. Some T-cells function as helper cells and others function as suppressor cells. Suppressor T-cells may be activated nonspecifically by mitogens such as Con A (78) to produce soluble factors which can mediate suppression in the absence of suppressor cells. Whether Con A induces suppressor cells that are truly nonspecific, or whether it stimulates a great number of antigen-specific clones is not clear. Antigen-specific suppression (reviewed in 79) consists of suppression of B-cells, or T-cells, by T-cells. Although suppression may







18

be exerted directly on B-cells, it appears that the effect is more likely in reducing the activity of helper T-cells, which in turn reduces the response of the B-cells. In only one case has the underlying basis of a Trichinella-induced alteration been determined. In that singular case, it was found that suppressor T-cells were responsible for the reduced antibody response to SRBC during infection (47).

Statement of the Problem

It has been shown by others that the alloresponsiveness of the

host is impaired during infection with T. spiralis. There is a reduced ability of spleen cells from Trichinella-infected mice to induce a graft versus host reaction (54) and Trichinella-infected mice do not reject allografts as rapidly as uninfected mice (43,53,54). However, the underlying basis of these impaired cell-mediated responses was not determined.

The purpose of this study is to determine the cellular basis of the impaired allograft rejection response during infection with T. spiralis.















MATERIALS AND METHODS

Mice

Female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) 8-14 weeks old were the principal experimental animals used in this study. Female CBA/Ca mice of varying ages were used as the source of allogeneic stimulator cells in the mixed leukocyte reactions. The congeneic strains of mice C57BL/10, BI0.AQR, BlO.A(2R) and BlO.T(6R) were obtained from the Department of Pathology animal colony.

Trichinella Infection

Mice were infected with 200 T. spiralis larvae via stomach intubation by the method of Larsh and Kent (80). The parasite was originally obtained from Dr. John E. Larsh (Department of Parasitology, University of North Carolina, Chapel Hill, NC) and maintained in this laboratory by serial passage in rats and mice.

Leukocyte Preparation

Spleens or lymph nodes were removed aseptically and gently pressed through wire screens into cold Dulbecco's phosphate buffered saline (D-PBS) contained in plastic petri dishes. The cells were transferred to sterile plastic centrifuge tubes and allowed to settle for 5 minutes on ice to permit the larger clumps to settle out. The remaining single cell suspension was transferred to a new tube and the cells washed 3 times with cold D-PBS. The red blood cells were lysed using warm (370) NH4 Cl and the remaining white cells were washed 3 times with cold D-PBS.









On the final wash the white cells were suspended in complete media (RPMI-1640 supplemented with L-glutamine, 5% heat-inactivated fetal calf serum and antibiotics). Viability was determined by trypan blue exclusion and the cells diluted to the desired concentration with complete media.

Nylon Wool Filtration of Leukocytes

Spleen cells were separated into B-cell and T-cell enriched fractions by passage of the cells over nylon wool columns, as first described by Julius et al. (81). Briefly, 100 x 106 spleen cells in complete media were filtered over 0.6 grams of nylon wool (LP-I Leuko-Pak Leukocyte Filters, Fenwall Laboratories, Deerfield, IL) packed into 6 ml in a 12 ml syringe. Details of the nylon wool preparation and cell elution technique were as described by Henry (82). The adherant and nonadherant populations were tested for purity by testing their susceptibility to lysis with anti-mouse Ig and anti-Thy-l.2 plus complement and their ability to respond to the mitogens Con A, PHA and LPS. The nonadherant fraction was routinely at least 95% pure T-cells while the adherant fraction was only 75-80% B-cells.

Differential Cell Counts

Leukocytes were suspended in complete media and pelleted on microscope slides with a cytocentrifuge (Shandon Scientific Co., Inc., Sewickley, PA). The cytocentrifuge preparations were stained with either May-Gruenwald's stain or Wright's stain. Differential cell counts were performed by counting at least 200 cells per sample.

Cytotoxicity Assays

Leukocytes were suspended to 5 x 106 cells per ml in complete media and incubated for 30 minutes with antisera at 4*C. Folloiwng incubation










with the antisera, the cells were washed once with cold media, complement added and the cells were reincubated for 45 minutes at 37*C. The amounts of antisera and complement giving optimal results were predetermined for each lot of reagents. Following cell lysis, killed cells were removed using a 40% Percoll solution (Pharmacia Fine Chemicals, Piscataway, NJ) according to the manufacturers' instructions.

Rabbit anti-mouse Ig (donated by Dr. Catherine Crandall, Department of Patholgy) was prepared by injection of rabbits with an antigenantibody precipitate of sera from mice infected with Ascaris suum (83). The rabbit anti-mouse Ig was shown to recognize all classes of mouse Ig by immunoelectrophoresis. Anti-Thy-l.2 alloantisera (donated by Dr. Michael Norcross, Department of Pathology) was prepared by repeated immunizations of AKR mice with thymocytes from young C3H mice. Monoclonal anti-Thy-l.2 antibody was purchased from New England Nuclear, Inc. (Boston, MA). Monoclonal anti-Lyt-I and anti-Lyt-2 were obtained by culturing hybridoma cell lines 53-7.313 (anti-Lyt-l) and 53-6.72 (anti-Lyt-2) obtained from the Cell Distribution Center, The Salk Institute for Biological Research (San Diego, CA). The spent tissue culture media from the hybridomas was centrifuged and filtered to remove cellular debris and used directly. Guinea pig complement was used with alloantisera and was purchased from GIBCO (Grand Island, NY). Rabbit complement (Low-Tox-M rabbit complement) was used with monoclonal antibodies and was purchased from Cedarlane Laboratories, Ltd. (Accurate Chemical and Scientific Corp., Hicksville, NY).

Mitogen Stimulation

The proliferative response of lymphocytes to mitogens was assayed in a modified micromethod of Hartzman et al. (84). Lymphocytes were










cultured with varying concentrations of concanavalin A (Con A; Miles Laboratories, Inc., Kankakee, IL), phytohemagglutinin (PHA; PHA-P, Difco Laboratories, Detroit, MI) and lipopolysaccharide (LPS; LPS-W, S. typhimurium, Difco Labs.). Five hundred thousand cells in complete media were cultured in quadruplicate cultures for 72 hours in 96-well U-bottom microtiter plates (Linbro Chemical Co., Inc., New Haven, CO) at 37-C, 5% CO2 with 0.5 VCi of 3H-TdR (thymidine methyl 3-H, sterile aqueous, 1.9 CMM, Schwarz/Mann, Orangeburg, NY) added per well for the final 24 hours of culture. The cells were collected mechanically (Hiller Harvester, Otto Hiller Co., Madison, WI) using distilled water and the radioactivity measured in a liquid scintillation counter. The LPS was boiled for one hour in PBS (pH 8.0) before use.

Mixed Leukocyte Reaction

Unidirectional mixed leukocyte reactions were performed essentially by the procedure described by Rich and Rich (85). Routinely, 5 x 105 or 2.5 x 105 C57BL/6 (H-2 b) responder spleen cells were incubated with

5 x 105 mitomycin C-treated CBA/Ca (H-2 k) stimulator cells in 96-well U-bottom microtiter plates. Quadruplicate cultures were incubated for 96 hours at 37*C, 5% CO2 with 0.5 pCi 3H-TdR added per well for the final 24 hours of culture. The cells were collected and the radioactivity measured as above (see Mitogen Stimulation).

In studies of suppressor activity, 2.5 x 105 responder cells and 2.5 x 105 mitomycin C-treated "regulator" cells from mice infected with T. spiralis, or uninfected controls, were cultured with 5 x 105 mitomycin C-treated stimulator cells.

The MLR was quantitated by comparing the incorporation of 3H-TdR by responder cells following stimulation with allogeneic cells to that










of identical cultures stimulated with syngeneic cells. Complete media for the MLR was the same as for the mitogen stimulation with the addition
-5
of 5 x 10 M 2-mercaptoethanol.

MLR Supernatant Fluid

Culture media from mixed leukocyte reactions of spleen cells from Trichinella-infected mice responding to allogeneic stimulation for 24 hours was rendered cell free by centrifugation (400 x g, 10 minutes) and filtration through 0.22 p Millipore filters. This supernatant fluid was tested for suppressor activity at a 1:1 dilution in MLR cultures of leukocytes from normal, uninfected mice.

Cell-Mediated Lympholysis Assay

The ability of lymphocytes from Trichinella-infected mice to develop cytolytic effector cells (T c) was evaluated in vitro as described by Grabstein (86). Briefly, mixed leukocyte cultures were established in 30 ml volumes (75 x 106 C57BL/6 responder cells plus 75 x 106 mitomycin
3
C-treated stimulator cells) in 75 mm plastic tissue culture flasks (Falcon #3024; Becton, Dickinson and Co., Cockeysville, MD). Aliquots 51
were removed at various times and tested for cytotoxic activity in Cr release assays. Two million L-929 (H-2 k) target cells were incubated with 0.1 ml (Na)2 51Cr04 (sodium chromate in sterile saline, 1 mCi/ml, New England Nuclear, Boston, MA) for 60 minutes and gently washed 3 times with cold media. Varying numbers of cells were incubated with 104 51Cr-labeled L-929 target cells for 4 hours at 37*C, 5% CO2 in 96well U-bottom microtiter plates. The contents of each well were then resuspended by the forceful addition of 0.05 ml media, and the cells pelleted by gently centrifuging the microtiter plates. Equal volumes of supernatant fluid were removed from each well and quantitated in a gamma counter. Background release was determined by incubating









5Cr-labeled target cells alone. Maximum release was determined by adding 0.1 ml of 1% Nonidet P40 (NP40; Sigma Chemical Co., St. Louis, MO) to 51Cr-labeled target cells. The percentage of specific release was calculated using the following formula: experimental cpm - background cpm
% specific cytotoxicity = x 100% total release cpm - background cpm

Delayed-Type Hypersensitivity Assays

Delayed-type hypersensitivity to allogeneic cells was assessed in two ways: 1) footpad swelling in normal and Trichinella-infected mice, and 2) by adoptive transfer of spleen cells from allosensitized control and infected mice into the footpads of uninfected mice. The method used was essentially that of Loveland et al. (87). Control and Trichinellainfected mice were sensitized by subcutaneous injection of 3 x 107 allogeneic (CBA/Ca) spleen cells, followed 6 days later by footpad challenge with 5 x 106 allogeneic spleen cells in a volume of 0.05 ml. In the adoptive transfer experiment 4 x 106 spleen cells from allosensitized control and Trichinella-infected mice were transferred with 4 x 106 allogeneic or syngeneic spleen cells into the footpads of normal, uninfected C57BL/6 mice. The 24-hour footpad swelling was measured to the nearest 0.01 mm using a dial gauge micrometer (No. 25-441; L.S. Starrett Co., Athol, MA).

Skin Grafts

Graft beds were prepared aseptically in recipient mice maintained under Nembutal anesthesia. Back skin (approximately 1.0 cm 2) was removed aseptically from donor mice and put into place in the graft bed. The graft was sutured into place using a 6-0 silk fitted with an FS-3 needle








25

(Ethicon, Inc., Somerville, NJ). Skin graft recipients were caged individually. Graft rejection was read as total graft necrosis.

Statistical Analysis

Data were calculated to give mean and standard deviations, and statistical significance determined by Students' t-test. Analysis of delayed-type hypersensitivity experiments was performed using the Fisher Exact test with a footpad swelling of 0.3 mm or greater considered to be significant.















RESULTS

Alteration of Blastogenic Responses Mixed Leukocyte Responses

The ability of spleen cells and lymph node cells from C57BL/6

mice infected with Trichinella spiralis to respond in the one-way mixed leukocyte response (MLR) was assessed. The optimal conditions for the MLR were determined in preliminary experiments. Spleen cells

As seen in Table 1, spleen cells from C57BL/6 (H-2 b) mice infected 21(�l) days previously with 200 T. spiralis larvae did not respond as well to allogeneic stimulation with CBA/Ca (H-2k) spleen cells as did spleen cells from uninfected mice. Because the magnitude of the MLR response varies greatly between experiments, the results for each experiment were expressed as the percentage of the control MLR (calculated as described in Materials and Methods). The results of Table 1, presented in graphic form in Figure 2, show that the response of spleen cells from infected mice was approximately 40% of the control response. As shown in Table 2, the results were qualitatively unaffected by the incubation time of the cultures.

Experiments were performed using different concentrations of splenocytes to rule out the possibility that the reduction in MLR responsiveness of the Trichinella-infected mice was due to suboptimal concentrations of responder cells being used. Figure 3 shows that the reduced MLR response of spleen cells from infected mice was observed over a wide









TABLE 1

THE MIXED LEUKOCYTE RESPONSE OF SPLEEN CELLS


EXP. RESPONDER a STIMULATOR CELL (CPM � SD) b % CONTROLC P VALUE NO. CELL TYPE CBA/Ca C57BL/6 RESPONSE


1 Control Infected

2 Control Infected

3 Control Infected

4 Control Infected

5 Control Infected

6 Control Infected

7 Control Infected

8 Control Infected

9 Control Infected

10 Control
Infected


22,915 14,590

41,933 26,422

41,666 29,732

64,392 45,788

33,816 16,892

11,117 4,710

10,692 9,380

13,884 12,479

39,360 21,571

47,655 24,821


1,931 1,201

5,501 1,324

2,839 1,117

4,570 4,468

3,275 2,206

3,906
753

2,286 1,113

3,307 1,293

4,878 2,866

3,460 3,583


3,473 4,149

9,750 12,174

9,256 10,868

12,793 9,653

10,406 10,288

2,798 4,075

1,142 7,803

4,206 2,858

11,347 7,210

8,983 6,704


466 623

554 1,840

499 1,376

1,562 1,548

1,211 1,574

989 558

658 1,459

641 884

1,658 1,210

1,562 1,604


53.7 P< 0.005 44.3 P< 0.005 58.5 P< 0.005 70.0 P< 0.005 28.2 P< 0.005 7.6 P< 0.005 16.5 P< 0.005


100.0


51.3 P< 0.005 46.8 P< 0.005


a. Responder cell were 5 x 105 splenocytes
Trichinella-infected C57BL/6 mice at 21 b. Stimulator cells were 5 x 105 mitomycin


from uninfected (control) or days of infection. C-treated splenocytes from


allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice. c. The % control response was calculated as:

cpm, infected mice (allogeneic response - syngeneic response)
cpm, control mice (allogeneic response - syngeneic response) X 100%

Spleen cells from 3 to 7 mice were pooled for group in each
experiment.












100









z





0 50
0
50
z
















CONTROL INFECTED MICE MICE SOURCE OF RESPONDER CELLS


FIGURE 2. A graphic representation of the data in Table 1 (The MLR response of spleen cells from uninfected and Trichinella-infected C57BL/6 mice). See Table 1 for calculation of percentage of control MLR response.













TABLE 2

THE INFLUENCE OF INCUBATION TIME ON THE MLR RESPONSE OF SPLEEN CELLS



DAYS RESPONDERa STIMULATOR CELL (CPM � SD) b % CONTROL c IN VITRO CELL TYPE CBA/Ca C57BL/6 RESPONSE


3 Control 20,695 � 1,925 10,540 � 1,707
Infected 13,156 � 1,906 6,132 � 1,278 69.1

4 Control 65,004 � 7,723 14,358 � 1,141
Infected 25,824 � 2,204 7,389 � 294 36.1

5 Control 35,396 � 4,034 12,490 � 3,168
Infected 19,976 � 1,672 7,740 � 2,215 56.5



a. Responder cells were 5 x 105 splenocytes from uninfected
(control) or Trichinella-infected C57BL/6 mice at 21 days
of infection.

b. Stimulator cells were 5 x 105 mitomycin C-treated splenocytes
from allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice.

c. The percentage of control response was calculated as described
in Table 1.

Data are from one of three experiments giving identical
results; spleen cells from 3-7 mice were pooled for each group
in each of the experiments.













10 0


CONTROL
0

8


z �
0A
H



o 0
z


H - INFECTED
0 0
6



0
U



0






| I I I I I
0.5 1 2 3 4 5
2 -5




RESPONDER CELLS x l0

FIGURE 3. The influence of varying ratios of responder cells to stimulator cells on the MLR. Responder cells were splenocytes from uninfected C57BL/6 mice (control) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (infected). The cpm [3H] thymidine incorporation equals the cpm following stimulation with 5 x 10 allogeneic (CBA/Ca) spleen cells minus the cpm following stimulation with
5 x 105 syngeneic spleen cells. The results are from one of two experiments giving identical results; three mice were used in each group for each experiment.









range of responder cell to stimulator cell ratios. Based on these results, all remaining experiments were performed using either 0.25 x 106 or 0.5 x 106 responder cells and 0.5 x 106 stimulator cells. As seen in Figure 4, the MLR was qualitatively similar when using either

0.25 x 106 or 0.5 x 106 responder cells.

To rule out the possibility that the reduced IMR response of the infected mice was due to a decrease in the relative proportion of T-cells in the spleens, MLR assays were performed using nylon wool-enriched splenic T-cells from normal and infected mice. As seen in Table 3, purified T-cells from infected mice were less reactive in the MLR than T-cells from uninfected mice.

To evaluate the MLR response of defined subsets of T-cells,

assays were performed using spleen cells from normal and Trichinellainfected BI0.AQR mice responding in the MLR to spleen cells from congenic strains of mice. As seen in Figure 5, the T-cells from infected mice which respond in the MLR to H-2K plus H-2D region differences were even more suppressed, relative to cells from uninfected mice, than those cells which respond to entire H-2 differences or H-21 plus H-2S region differences.

Lymph node cells

To ascertain whether the reduced MLR response of splenocytes

was part of a general suppression, or restricted to the spleen, mixed leukocyte reactions were performed using lymph node cells. As seen in Figure 6, the MLR response of leukocytes from the axillary and brachial lymph nodes of Trichinella-infected mice was reduced approximately to the same degree as splenic leukocytes. Nylon wool-enriched










100 -


z
0




50 50


z
0









CONTROL INFECTED CONTROL INFECTED
2.5 x 10 RESPONDER CELLS 5.0 x 105 RESPONDER GELLS

FIGURE 4. Comparison of the MLR response using 2.5 x 105 and 5.0 x 105 responder cells. Responder cells were splenocytes from uninfected C57BL/6 mice (control) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (infected). Stimulator cells were 5.0 x 105 allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 1 for calculation of percentage of control MLR response. The results of ten experiments, with 3-7 mice per experiment, are averaged for each graph.


100











TABLE 3

THE MLR RESPONSE OF NYLON WOOL-ENRICHED SPLENIC T-CELLS


EXP. RESPONDERa STIMULATOR CELL (CPM � SD)b % CONTROLc NO. CELL TYPE CBA/Ca C57BL/6 RESPONSE


1 Control 12,501 � 1,748 2,840 � 208 Infected 9,031 � 1,330 363 � 128 89.7

2 Control 32,664 � 4,603 1,579 � 274 Infected 15,452 � 2,066 1,604 � 371 44.6

3 Control 18,645 � 1,486 1,041 � 842 Infected 13,167 � 2,129 1,543 � 478 66.0

4 Control 46,833 � 5,494 4,431 � 233 Infected 27,563 � 2,840 3,913 � 711 55.8

5 Control 22,391 � 2,149 3,609 � 670 Infected 17,330 � 1,533 4,497 � 1,208 68.3


a. Responder cells were 5 x 105 nylon wool-nonadherant spleen cells from uninfected (control) and Trichinella-infected C57BL/6 mice
at 21 days of infection.

b. Stimulator cells were 5 x 105 mitomycin C-treated allogeneic (CBA/Ca) or syngeneic (C57BL/6) splenocytes.

c. The percentage of control response was calculated as described in Table 1.

Spleen cells from 3-7 mice were pooled for each group in each
experiment.













100






80



z 0

M 60




z

40






20







H-2 K+D 1+5 MLR STIMULUS
FIGURE 5. The MLR response of uninfected (control) and Trichinellainfected B10.AQR mice. Stimulator cells were splenocytes from G57BL/l0 (entire 1H-2 difference; H-2), BlO.A(2R) (H-2K plus H-2D differences; K+D) and BlO.T(6R) (H1-21 plus H-25 differences; I+S) mice. Responder cells from infected mice were obtained 21 days after infection. The response of spleen cells from infected mice was compared to the response of spleen cells from uninfected mice following stimulation with the same congeneic strain of mouse. See Table 1 for calculation of percentage of control MLR response. The data is the average of two experiments; three mice were used in each group for each experiment.
















100 -


CONTROL INFECTED INFECTED
(POOLED) (AXILLARY) (BRACHIAL)
SOURCE OF RESPONDING LYMPH NODE CELLS

FIGURE 6. The MLR response of lymph node cells. Responder cells were
2.5 x 105 pooled lymph node cells from uninfected C57BL/6 mice (control) or from the axillary and brachial lymph nodes of C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (infected). Stimulator cells were 5.0 x 105 mitomycin C-treated allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 1 for calculation of percentage of control MLR response. The results of three experiements are averaged;
3 to 7 mice were used in each group for each experiment.







36

lymph node T-cell populations from infected mice were also less reactive in the MLR than enriched T-cells from normal, uninfected mice (Figure 7). Mitogen Responses

The ability of spleen cells from C57BL/6 mice infected with T. spiralis for 21 days to respond to the T-cell mitogens Con A and PHA was assessed. For each experiment a dose response was performed using at least three concentrations of mitogen. The response of spleen cells from infected mice to Con A was reduced relative to controls (Figure 8), while the response to PHA was unaltered (Figure 9). Kinetics of Altered Blastogenic Responses

To determine when during infection the MLR and mitogen responses of spleen cells from Trichinella-infected mice was reduced, a timecourse study was performed. As seen in Figure 10, the MLR response of splenocytes from infected mice was maximally reduced at 21 days of infection and the response to Con A (Figure 11) was reduced maximally at 14 days of infection; both responses returned to normal by 3 to 4 months. The response to PHA (Figure 11) was not significantly altered at any time during infection. A comparison of the altered MLR and Con A responses (Figure 12) shows that the kinetics of the reduced MLR and Con A responses were nearly identical.

Mechansim of Altered Blastogenic Responses

To determine whether the suppressed NLR response of spleen cells from infected mice was due to i) depletion of T-cells in the spleens of infected animals, ii) lysis of the allogeneic stimulator cells in the MLR assays or iii) an active suppression of the MLR responder cells, the experiments described below were done.















100











0 P.4




~ 50
0 H
z 0
U 0













CONTROL INFECTED MICE MICE

SOURCE OF RESPONDING LYMPH-NODE CELLS FIGURE 7. The MLR response of nylon wool-enriched lymph node T-cells. Responder cells were 2.5 x 105 pooled lymph node T-cells from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (INFECTED). Stimulator cells are 5.0 x 105 allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 1 for calculation of percentage of control MLR response. The results presented are from a single experiment.













CONTROL 80








z 60

E-4
0 0







z W
H

z


INFECTED


- 20 0


00



0

0 0.25 0.5 1 2 U'g Con A WELL FIGURE 8. The Con A response of spleen cells. Responder cells were
5.0 x 10 spleen cells from uninfected C57BL/6 mice (CONTROL) or C57BL16 mice infected 21 days previously with 200 T. spiralis larvae (INFECTED). This graph is a representative of 5 experiments, all of which gave the same qualitative result.













CONTROL


~aINFECTED


40 V


T0
0





z
o 0
rI




0

20
0





0 z





CY 100







ii I I

0 0.25 0.5 1 2 pg PHA / WELL

FIGURE 9. The PHA response of spleen cells. Responder cells were
5.0 x 105 spleen cells from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (INFECTED). This graph is a respresentative of 5 experiments; all of which gave the same qualitative result.










100 -


80 60




40 2


20 -


CONTROL 0
(2)

(6) 2



0
(3)
0 FCE
(8)

(8)

(1)~ INFECTED


7 14 21


I
131


DAYS OF TRICHINELLA SPIRALIS INFECTION

FIGURE 10. The MLR response of spleen cells during infection with T. spiralis. Responder cells were 5 x 105 splenocytes from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected for various times with 200 T. spiralis larvae (INFECTED). Stimulator cells were 5 x 10D allogeneic (CBA/Ca) or syngeneic spleen cells. Numbers in parenthesis indicate the number of experiments performed at eact time point. See Table 1 for calculation of percentage of control MLR response.












100 0
80 -0




060
80


z 40






PZ 0 Con A
0
60PR





20






7 14 21 40 63 84 131 DAYS OF TRICHINELLA SPIRALIS INFECTION FIGURE 11. The T-cell mitogen responses of spleen cells during infection with T. spiralis. Responder cells were 5 x 105 spleoncytes from uninfected C57BL/6 mice (control) or C57BL/6 mice infected for various times with 200 T. spiralis larvae. See Table 1 for calculation of % of control response.










100 -0 60
0 0













P4 602 0 38
800

00
z
O 60


9-10




20 0 0 MLR






7 14 21 40 63 84
DAYS OF TRICHINELLA SPIRALIS INFECTION FIGURE 12. A comparison of the reduced MLR and Con A responses during infection with T. spiralis. Data are the same as that presented in Figures 10 and 11.


~~0
*i�~


131









To determine if there was a decrease in the proportion of T-cells in the spleens of infected animals, the percentage of T-cells, and Bcells, in the spleens of normal and infected mice was determined in cytotoxic assays using anti-Thy-l.2 and anti-Ig antisera. As seen in Table 4, the relative proportion of B-cells and T-cells was the same following infection, even though there was an increase in the absolute number of leukocytes in the spleens of infected mice.

To determine whether the decreased MLR response was due to an increased lysis of the allogeneic stimulator cells, two experiments were performed. In the first, mitomycin C-treated spleen cells from normal and Trichinella-infected C57BL/6 mice were used as stimulators in the MLR, with CBA/Ca spleen cells as the responders. As seen in Table 5, the NLR responses of normal CBA/Ca spleen cells following stimulation with spleen cells from normal and infected C57BL/6 mice were equal. In the second experiment, spleen cells from normal and infected C57BL/6 mice were incubated with 51Cr-labeled CBA/Ca stimulator cells and the amount of lysis of stimulator cells determined after 4 hours. As seen in Table 6, there was no difference in the release of 51Cr from the stimulator cells following incubation with spleen cells from normal and Trichinella-infected mice. These results indicated that the reduced MLR response was not due to a more rapid lysis of stimulator cells in cultures containing splenocytes from infected mice.

Experiments were then done to determine whether the reduced MLR

response of spleen cells from infected mice was due to active suppression. Mitomycin C-treated spleen cells from normal and Trichinella-infected mice were added as third party regulator cells to MLR assays of normal spleen cells. As seen in Table 7, mitomycin C-treated regulator cells











TABLE 4

SPLEEN CELL POPULATION DURING INFECTION WITH TRICHINELLA SPIRALIS



TRICHINELLA- a
UNINFECTED MICE INFECTED MICE TOTAL CELLS PER SPLEEN 55.5 x 106 67.8 x 106 b

CELL TYPE % %

Mononuclear cells 100c 95c Eosinophils 0c 5c

B-lymphocytes 52 � 3d 48 � 3d T-lymphocytes 29 � 7d 23 � 5d




a. Spleen cells from Trichinella-infected C57BL/6 mice at 21
days of infection.

b. Total spleen cell counts are the average of 5 experiments. The
increase in total cell count during infection is significant to
P< 0.05.

c. Differential cell counts are the average of two experiments,
using spleen cells pooled from 4 mice in each group.

d. No significant difference between control and infected groups.











TABLE 5

SPLEEN CELLS FROM C57BL/6 MICE AS STIMULATORS IN THE MLR ASSAY



EXP. STIMULATOR CELL (CPM � SD)a NO. CBA/Ca UNINFECTED C57BL/6 INFECTED C57BL/6



1 1,370 � 286 12,678 � 1,620 13,483 � 286 2 4,559 � 2,350 23,667 � 4,621 24,872 � 3,309 3 2,788 � 4,339 9,135 � 1,438 10,198 � 1,268


a. Stimulator cells were 5 x 105


mitomycin C-treated spleen cells


from uninfected CBA/Ca and C57BL/6 mice, and Trichinella-infected C57BL/6 mice at 21 days of infection.

Responder cells were 5 x 105 spleen cells from uninfected CBA/Ca mice. Pooled spleen cells from 3 responder mice were used in each experiment.










TABLE 6

RELEASE OF 51Cr FROM 51Cr-LABELED CBA/Ca STIMULATOR CELLS


EXP. 1 EXP. 2 EXP. 3



Maximum Releasea 2,784 � 83 8,496 � 218 1,878 � 94 Background Releaseb 507 � 29 1,220 � 61 224 � 15 Uninfected Respondersc 436 � 31 980 � 39 303 � 18 Infected Respondersd 456 � 23 1,140 � 46 275 � 11


a. Amount b. Amount c. Amount
spleen d. Amount
spleen
mice.

Spleen


of 51Cr released following addition of 0.5% NP40. of 51Cr released spontaneously. of 51Cr released following incubation of 51Cr-labeled CBA/Ca cells with spleen cells from uninfected C57BL/6 mice. of 51Cr released following incubation of 51Cr-labeled CBA/Ca cells with spleen cells from Trichinella-infected C57BL/6


cells from 4-5 mice were pooled for each experiment.










TABLE 7

ACTIVE SUPPRESSION OF THE MLR RESPONSE IS MEDIATED BY SPLEEN CELLS FROM INFECTED MICE


EXP. REGULATORa STIMULATOR CELL (CPM � SD) b % CONTROLc NO. CELL TYPE CBA/Ca C57BL/6 RESPONSE


1 None 41,666 � 2,839 9,256 � 499 Control 47,710 � 2,829 6,508 � 1,703
Infected 27,845 � 1,762 6,282 � 609 52.3

2 None 64,392 � 4,570 12,793 � 1,526 Control 64,991 � 1,926 6,296 � 699
Infected 46,282 � 1,708 7,141 � 2,356 66.7

3 None 33,816 � 3,275 10,405 � 1,211 Control 30,574 � 1,628 7,898 � 887
Infected 22,296 � 3,305 7,735 � 1,195 64.2

4 None 11,564 � 1,369 4,323 � 906 Control 7,380 � 416 2,561 � 354
Infected 3,782 � 534 2,284 � 119 31.1


a. Regulator cells
from uninfected


were 2.5 x 105 mitomycin C-treated spleen cells (control) or Trichinella-infected C57BL/6 mice


at 21 days of infection.


b. Stimulator cells were 5 x 105 mitomycin C-treated spleen cells
from allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice.

c. The percentage of control response was calculated as described
in Table 1.

Responder cells were 2.5 x 105 spleen cells from uninfected C57BL/6
mice. Spleen cells from 3-7 mice were pooled for each group in
each experiment.









from normal mice showed little or no effect on the MLR response of normal spleen cells. Regulator cells from Trichinella-infected mice, however, caused a significant suppression of the MLR response of normal spleen cells. Figure 13 shows that the active suppression had similar kinetics to the reduced MLR response following infection.

Experiments similar to those performed to demonstrate active suppression in the MLR were performed to determine whether active suppression was also responsible for the reduced Con A response during infection. Interestingly, in 3 experiments, no evidence for an active suppression mechanism could be demonstrated.

The above experiments indicate the presence of a cell in the

spleens of infected mice that is capable of suppressing the MLR response, but not the Con A response. To identify the active suppressor cell, spleen cells from infected mice were separated into B-cell and T-cell enriched fractions and each fraction tested for its ability to mediate suppression of the MLR. Nylon wool adherant and nonadherant splenocytes were obtained from normal and infected mice and used as regulators in the MLR. As seen in Table 8, both the B-cell and T-cell enriched fractions contained suppressor activity. Negative selection utilizing specific antisera plus complement was then used to enrich for B-cells and T-cells. Some cells were treated sequentially with both anti-Thy1.2 and rabbit anti-mouse Ig plus complement to enrich for non-B, non-T cells ("null" cells). As shown in Figure 14, both B-cell and T-cell enriched fractions again contained suppressor activity, although the T-cell fraction was more suppressive than the B-cell fraction. The "null" cell fraction had only a slight, statistically insignificant, effect on the responder cells.













0 (2)0
(2) (2)


(4)


100




80


I I I I I 1 1
7 14 21 40 63 84 131


DAYS OF TRICHINELLA SPIRALIS INFECTION
FIGURE 13. Spleen cell-mediated active suppression of the MLR during infection. The influence of mitomycin C-treated third party regulator spleen cells from C57BL/6 mice infected with 200 T. spiralis larvae for various times in a control MLR is compared to the influence of third party regulator cells from uninfected mice. An equal number of responder cells and regulator cells (2.5 x 105) was added to 5 x 105 mitomycin C-treated stimulator cells. Numbers in parenthesis indicates the number of experiments performed at each time point. See Table 7 for calculation of percentage of control response.


(5)


(5)


60 1


20 -











TABLE 8

SUPPRESSION OF THE MLR RESPONSE MEDIATED BY NYLON WOOL-ADHERANT AND NONADHERANT SPLENOCYTES



RESPONDER' REGULATORb STIMULATOR CELL (CPM � SD)c % CONTROLd CELL TYPE CELL TYPE CBA/Ca C57BL/6 RESPONSE


Control None 17,854 � 1,646 2,849 � 243 Infected None 10,882 � 1,503 5,152 � 563 38.2 Control Control 19,807 � 1,089 3,423 � 432 109.2 Control Infected 7,506 � 779 2,654 � 570 33.0 Control Infectednonadherant 7,378 � 518 2,818 � 1,575 30.4 Control Infectedadherant 7,262 � 686 1,841 � 331 36.2



a. Responder cells were 2.5 x 105 spleen cells from uninfected (control)
or Trichinella-infected C57BL/6 mice at 21 days of infection.

b. Regulator cells were 2.5 x 105 mitomycin C-treated spleen cells that
were unfractionated from uninfected or Trichinella-infected C57BL/6
mice, or nylon wool-adherant and nonadherant cells from infected
mice.

c. Stimulator cells were 5 x 105 mitomycin C-treated spleen cells from
allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice.

d. The percentage of control response was calculated as described in
Table 1.

Spleen cells were pooled from 4 mice for this experiment. This
experiment is on of three which gave qualitatively identical
results.














100






80



z 0

M 60

0

0

40






20'







Con-Spl Tsp-Spl Tsp-T Tsp-B Tsp-Null SUPPRESSOR CELL TYPE FIGURE 14. Identification of the MLR suppressor cell by negative selection using specific antisera plus complement. Responder cells were 2.5 x 105 splenocytes from uninfected C57BL/6 mice. Regulator cells were
2.5 x 105 unfractionated spleen cells (Spl), or spleen cells treated with anti-Ig plus complement (T), anti-Thy-l.2 plus complement (B) or both anti-Ig and anti-Thy-l.2 plus complement (Null) from uninfected C57BL/6 mice (Con) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (Tsp). Stimulator cells were 5 x 105 mitomycin C-treated allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 7 for calculation of percentage of control response. Data are the average of three experiments.









A more precise identification of the suppressor T-cell was

accomplished through the use of monoclonal anti-Lyt antibody. As seen if Figure 15, treatment of third party regulator cells with anti-Lyt-2 plus complement removed essentially all suppressor activity, while treatment with anti-Lyt-l plus complement had essentially no effect. These experiments indicated that the suppressor T-cell found in the spleens of infected mice had the Lyt 1-,2/3+ surface phenotype.

To ascertain whether suppression could be mediated by soluble factors secreted by the suppressor cells, supernatant fluid was removed from the MLR cultures of Trichinella-infected mouse spleen cells responding to allogeneic cells and added to the MLR assays of spleen cells form normal, uninfected mice. As seen in Table 9, supernatant fluids from the MLR cultures of splenocytes from infected mice had no effect on the NLR response of normal spleen cells.

Alterations of Cell-Mediated Lympholysis (CML) Activity

To determine whether alterations in the effector arm of the allograft rejection response occur during infection, spleen cells from normal and Trichinella-infected mice were sensitized in vitro to allogeneic (CBA/Ca) spleen cells and then tested for their ability to mediate target cell lysis in chromium release assays using 51Cr-labeled L-929 cells as target cells. Maximum cytolytic activity was developed in the MLR after 7 days. As seen in Figure 16, there was no difference in the cytolytic ability of spleen cells from Trichinella-infected mice compared to spleen cells from normal, uninfected mice.

Changes in Splenic T-Cell Subsets

The relative proportions of T-cell subsets, as defined by the Ly-l and Ly-2 surface antigens, was determined for uninfected C57BL/6 mice














100





80


60 40


20 -


Con-Spl Tsp-Spl Tsp-Ly-I+ Tsp-Ly-23+
SUPPRESSOR CELL TYPE
FIGURE 15. Identification of the MLR suppressor cell by negative selection using anti-Ly antisera plus complement. Responder cells were 2.5 x 105 spleen cells from uninfected C57BL/6 mice. Regulator cells were 2.5 x 105 spleen cells treated with anti-Ly-2 plus complement (Ly-l+), anti-Ly-i plus complement (Ly-23+),or unfractionated spleen cells (Spl) from uninfected C57BL/6 mice (Con) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (Tsp). Stimulator cells were 5 x 105 mitomycin C-treated allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 7 for calculation of percentage of control response. Data are the average of three experiments.











TABLE 9

LACK OF SUPPRESSION BY SUPERNATANT FROM "REDUCED" MLR CULTURES




RESPONDER CELLa STIMULATOR CELL (CPM � SD)b % CONTROLc CBA/Ca C57BL/6 RESPONSE


Control 15,095 � 2,042 6,717 � 560

Infected 7,981 � 857 5,119 � 695 34.2

Control +
Supernatant from d
Infected Culture 15,549 � 1,408 6,210 � 360 111.5



a. Responder cells were 5 x 105 spleen cells from uninfected (control)
or Trichinella-infected C57BL/6 mice at 21 days of infection.

b. Stimulator cells were 5 x 105 mitomycin C-treated spleen cells from
allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice.

c. The percentage of control response was calculated as described in
Table 1.

d. Supernatant from infected cultures is tissue culture media from an
MLR assay of spleen cells from Trichinella-infected C57BL/6 mice in response to allogeneic (CBA/Ca) stimulation, removed after 24
hours of incubation and added at a 1:1 dilution (50% final volume).

These data are from one of three experiments giving the same qualitative results. Spleens from 3-7 mice were pooled for each group
in each experiment.













0

500



CONTROL

40 0
INFECTED




30


ale


20






10 0





0 -0O -0O
I I I I I I I I
1 2 4 8 10 25 50 100 EFFECTOR TO TARGET CELL RATIO FIGURE 16. Cell-mediated lympholysis (CNL) mediated by spleen cells from Trichinella-infected C57BL/6 mice. Effector cells were sensitized in MLR cultures against CBA/Ca (H-2k) spleen cells and tested for CML ability on 51Cr-labeled L-929 (H-2k) target cells. Effector cells were from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (INFECTED). The graph is data from one of three experiments giving nearly identical results. Percent of specific lysis was calculated as described in the Materials and Methods.


|







56

and for C57BL/6 mice at 21 days of infection with T. spiralis. As seen in Table 10, during infection there was a consistent decrease in the percentage of T-cells that were sensitive to monoclonal anti-Ly-i antibody plus complement, without change in the percentage of cells sensitive to anti-Ly-2 antibody plus complement. In a single exepriment (experiment 3, Table 10) there were fewer cells from infected mice that were susceptible to lysis by simultaneous treatment with both antibodies. Together these data indicate that there is a decrease in the relative proportion of Ly-l+ T-cells, but whether this reflects a conversion of Ly-l ,2/3 T-cells to blast cells (which cannot be lysed with these antibodies plus complement) or a conversion or Ly-l+,2/3+ T-cells to Ly-l-,2/3+ T-cells is not clear.

Delayed-Type Hypersensitivity (DTH) to Allogeneic Cells

To evaluate the DTH responsiveness of Trichinella-infected mice, uninfected C57BL/6 mice and C57BL/6 mice infected for 21 days with T. spiralis were sensitized with allogeneic (CBA/Ca) spleen cells and challenged 6 days later. The results (Table 11) show that there was no difference in the DTH response, as measured by footpad swelling, of control and infected mice. The DTH responsiveness of spleen cells from infected mice was also tested by injecting challenge cell together with sensitized spleen cells from control and infected mice into the footpads of uninfected mice. The results (Figure 17) were suggestive of a depressed DTH response of spleen cells from infected mice, but were not statistically significant (P = 0.17). Two of four mice receiving sensitized spleen cells from control mice showed significant DTH responses, while none of five animals receiving sensitized spleen cells from infected mice showed significant footpad swelling.










TABLE 10

CHANGES IN SPLENIC T-CELL SUBSETS


EXP. TREATMENT % OF CELLS KILLEDa NO. CONTROL INFECTED



1 C'b 6.3c 7.2 anti-Ly-1 + C' 76.1 62.3 anti-Ly-2 + C' 28.8 28.2 anti-Thy-l.2 + C' 84.1 82.7

2 C' 3.8 6.1 anti-Ly-1 + C' 63.8 55.1 anti-Ly-2 + C' 34.7 35.2 anti-Thy-1.2 + C' 81.9 80.8

3 C' 4.5 5.9 anti-Ly-l + C' 71.9 59.1 anti-Ly-2 + C' 29.7 31.9 anti-Thy-1.2 + C' 83.4 82.7
anti-Ly-i plus
anti-Ly-2 + C' 79.6 68.2


a. Target cells were nylon wool-enriched splenic
uninfected (control) and Trichinella-infected
at 21 days of infection.


T-cells from C57BL/6 mice


b. C' is rabbit complement at a final dilution of 1:40. c. At least 300 cells were counted for each determination.










TABLE 11

DTH RESPONSE TO ALLOGENEIC CELLS


FOOTPAD SWELLING (mm � SD)a
GROUP STATUS OF RESPONDING MICE 6-HOUR SWELLING 24-HOUR SWELLING



1 Unsensitized, control
C57BL/6 mice 0.07 � 0.04 0.05 � 0.06

2 Allosensitized, controlb
C57BL/6 mice 0.09 � 0.06 0.32 � 0.11

3 Allosensitized, Trichinella-c
infected C57B1/6 mice 0.06 � 0.04 0.34 � 0.10



a. Footpad swelling was measured after the injection of 5 x 106 allogeneic (CBA/Ca) spleen cells contained in 0.05 ml.

b. Allosensitized C57BL/6 mice were injected with 30 x 106 allogeneic (CBA/Ca) spleen cells subcutaneously 6 days prior to
footpad challenge.

c. Infected C57BL/6 mice were at 21 days of infection at the time
of allosensitization.

d. There was no significant difference between group 1 and group 2.

The measurements of 5 mice were averaged for each group.














0.5





0.4





S 0.3





S 0.2






0.1






(1) (2) (3) (4) (5) Control Infected Control Infected C57BL/6 C57BL/6 C57BL/6 C57BL/6 + + + + CBA/Ca CBA/Ca C57BL/6 C57BL/6 CBA/Ca

FIGURE 17. Adoptive transfer of DTH responsiveness. Control and Trichinella-infected C57BL/6 mice were sensitized by subcutaneous injection of 30 x 106 allogeneic (CBA/Ca) spleen cells. Six days after allosensitization, 4 x 106 spleen cells from allosensitized control and infected mice were transferred with 4 x 106 allogeneic (CBA/Ca) or syngeneic spleen cells into the footpads of uninfected C57BL/6 mice. The 24-hour footpad swelling, measured to the nearest 0.01 mm., of 5 mice was averaged for each group. There was no significant difference (P = 0.17) between group 1 and group 2.







60

Effect of Adoptively Transferred Cells on Allograft Rejection

To determine whether the Ly-l-,2/3+ T-cells from infected mice which are capable of actively suppressing the MLR response were also capable of impairing the allograft rejection response, adoptive transfer experiments were performed. As predicted (Table 12), there was no difference in the time required for complete graft rejection between mice receiving spleen cells from uninfected mice or Trichinellainfected mice, or splenocytes from infected mice enriched for Ly-l +,2/3or Ly-l ,2/3+ T-cells.
















TABLE 12


EFFECT OF ADOPTIVELY TRANSFERRED CELLS
ON ALLOGRAFT REJECTION


SOURCE OF a TIMES OF INDIVIDUAL AVERAGE TIME DONOR CELLS GRAFT REJECTION (DAYS) OF REJECTION



1. Control, Spleen b 16,17,17 16.7

2. Infected, Spleen 14,15,16 15.0

3. Infected, B-cells 14,17,18 16.3

4. Infected, T-cells 14,14,19 15.7 5. Infected, Ly-i+ T-cells 14,16,18 16.0 6. Infected, Ly-2/3+ T-cells 14,15,16 15.0




a. Donor cells (4 x 10 6) from uninfected (control) or Trichinellainfected C57BL/6 mice were injected I.V. into uninfected C57BL/6
mice immediately prior to placement of allograft from CBA/Ca
mice.

b. Transferred cells were spleen cells that were unfractionated
(spleen), treated with anti-Thy-1.2 plus complement (B-cells),
filtered over nylon w~ol (T-cells), treated with anti-Ly-2 plus complement (Ly-l) or treated with anti-Ly-l (Ly-2+).
















DISCUSSION

Infection with Trichinella spiralis results in a wide range of alterations in host immune responsiveness, including suppression and enhancement of both humoral and cellular immune functions. Little is known, however, concerning the underlying mechanisms of these altered immune responses. This research was to determine the mechanism(s) of an impaired cell-mediated immune function, the allograft rejection response, which has been reported in Trichinella infections by others (43,53,54).

At least two points relevant to the observation that mice infected with T. spiralis do not reject skin allografts as rapidly as uninfected mice, have been established by this research. First, the specific "defect" appears to be in the ability of the Ly-l +,2/3- Tcells to respond to allogeneic cells, and not in the ability of the Ly-l-,2/3+ cytotoxic T-lymphocytes (CTL's) to effect lysis of allogeneic target cells. Second, the reduced responsiveness of the Ly-i 2/3 T-cells is due, at least in part, to the suppressive effect of Ly-l-,2/3+ T-cells. Together, these results indicate that the impaired allograft rejection response of Trichinella-infected mice results from an active suppression of those T-cells which are responsible for the recognition of allogeneic cells.

The MLR and CML assays were used to study the recognition and killing pahses, respectively, of the allograft rejection response of mice after infection with Trichinella spiralis, with the response of










splenocytes to the T-cell mitogens Con A and PHA used as an additional indicator of altered T-cell function. The basic findings were that the response to allogeneic cells in the MLR (Table 1) and the response to Con A (Figure 8) were reduced following infection, while the response to PHA was unaltered (Figure 9) and there was no detectable change in the ability of splenocytes to effect lysis of allogeneic target cells in the CML assay (Figure 16). The reductions in MLR and Con A responsiveness were transient and showed strikingly similar patterns, in terms of duration and magnitude, of suppression (Figure 12).

The fact that the response to Con A was significantly reduced (to 30% of the control response at 14 days of infection), while the response to PHA was not affected, clearly indicates that the infection does not affect all T-cell subsets equally. This observation is in agreement with several other reports of parasite-induced reduction of mitogen responsiveness. Barriga (57) reported similar results after injecting a soluble extract of T. spiralis into mice. He found that spleen cells from mice injected with an extract of T. spiralis showed a "severe" depression in Con A responsiveness and only a "slight" depression in PHA responsiveness. Jones (41) also reported a greater depression of the response to Con A than to PHA following infection with T. spiralis in mice. A similar dichotomy in the reduction of T-cell mitogen responses has been observed in baboons infected with schistosomes. Cottrell et al. (88) found that serum from infected baboons dramatically suppressed both the MLR and Con A responses of lymphocytes from uninfected animals, while the inhibition of the PHA response was only marginal.










One possible explanation for the reduced Con A response in the

absence of any significant reduction in the PHA response is that immune complexes are formed during the course of infection. Suppression of the proliferative response of mouse spleen cells to Con A by immune complexes, in the absence of any effect on the response to PHA, has been reported by Ryan et al. (89) and confirmed by Stout and Herzenberg

(90). Immune complex-mediated suppression probably results from an interaction of the Fc portion or the immunoglobulin in the complex with an appropriate Fc receptor (FcR) on the T-cell. The differential effect of immune complexes on the Con A and PHA responses may be explained by the observation of Stout and Herzenberg (90), which was that the response to Con A was a characteristic of the FcR+ T-cells, while the removal of the FcR+ T-cells had no effect on the response to PHA. Since there is no direct correlation between mitogen responsiveness and the Ly phenotype of T-cells, it is not possible to relate the reduced Con A response to any particular subset of T-cells, as defined by the Ly surface antigens.

The fact that spleen cells, and lymph node cells, from Trichinellainfected mice do not respond as well to allogeneic stimulation in the MLR as do control cells is additional evidence that the cell-mediated immune system is impaired during infection. It is well known that the activity of lymphocytes in vitro is greatly influenced by the cellular concentration, and at least one report (91) suggests that most experiments showing helper and suppressor activities are simply artifacts of varying cell concentrations. It was found, however, that the response of spleen cells from Trichinella-infected mice was reduced over a wide range of responder cell to stimulator cell ratios (Figure 3), indicating









that the reduced MLR response observed in this study is not an artifact of cell density differences. Cells from infected mice also failed to achieve the magnitude of the response of control cells when incubated for shorter or longer periods of time (Table 2), ruling out the possibility that the reduced response was simply due to a change in the timing of optimal responsiveness.

In this study, three possible mechanisms which might explain the reduced MLR responses during T. spiralis infection were considered: 1) a decrease in the relative proportion of T-cells, or MLR-reactive cells, in the spleens following infection, 2) activation of suppressor cells which depress the MLR response and 3) preferential lysis of the allogeneic stimulator cells when incubated with spleen cells from the infected mice.

An overall decrease in the proportion of T-cells in the spleen

following infection was unlikely. Firstly, there was a marked decrease in the response to Con A with no reduction in the response to PHA. This would not be expected if there was an overall decrease in the frequency of T-cells. Secondly, nylon wool-purified T-cells from infected mice were also less responsive in the MLR than were purified T-cells from uninfected mice (Table 3). By using equal numbers of purified T-cells, it should have been possible to overcome any effect due to a simple decrease in the proportion of T-cells in the spleen during infection. Lastly, there was no change in the percentage of T-cells and B-cells in the spleen, following infection, when quantitated directly in cytotoxic assays using anti-mouse Ig and anti-Thy-l.2 (Table 4). These results are in agreement with Ljungstrom (59) and Jones (41), who










also reported no change in the relative proportions of T-cells and B-cells following infection with T. spiralis in mice.

These experiments have clearly ruled out the possibility that the reduced MLR response following infection is simply due to a decrease in the relative proportion of T-cells in the spleen. However, they do not reveal whether any shift in the relative proportions of the various T-cell subsets has occurred.

The possibility of a shift in the relative proportions of T-cell subsets occurring during infection was suggested by two observations. First, as already discussed, there was a significant depression of the response to Con A with no apparent effect on the response to PHA. Since no evidence for an active suppression of the Con A response could be demonstrated, the reduced response may reflect a decrease in the frequency of Con A, but not PHA, responsive cells during infection. Second, a similar dichotomy of T-cell responsiveness was observed when using Trichinella-infected B10.AQR mice as the source of responder cells in the MLR. By using select strains of congeneic mice, it was possible to induce a MLR response to defined regions of the H-2 complex. Once again, it was observed that not all T-cell subsets were affected equally by the infection. The H-2K/D responsive T-cells were significantly less responsive than were the H-21/S responsive T-cells (Figure 5).

It is fairly well established that in the MLR both the Ly-l+ and the Ly-2+ T-cells contribute to the proliferative response (92,93,94), however, when there is complete disparity at the MHC, as was the case in the experiments of this dissertation, the proliferating cell type is predominantly the Ly-l +,2/3- T-cell (94,95). The Ly-l +,2/3+ T-cells are the major responding cell type when responders and stimulators differ










only at the H-2K or H-2D region (94,95), while the Ly-l ,2/3 T-cells appear to contribute little, if at all, to proliferation in the MLR

(95). Thus, the impaired MLR response following infection with T. spiralis may be partially due to a relative depletion of the Ly-l ,2/3+ T-cells, and almost assuredly reflects a hyporesponsiveness of the Ly-l +,2/3- T-cell subset.

The most direct, yet still inconclusive, evidence for a shift in the relative proportions of T-cell subsets was the quantitation of the percentage of splenic T-cells expressing the Ly-l and Ly-2 surface antigens. There was a consistent decrease in the percentage of T-cells expressing the Ly-l antigen, with no change in the percentage of T-cells expressing the Ly-2 antigen (Table 10). This decrease in the frequency of Ly-l+ T-cells could result from a conversion of Ly-1+ T-cells to blast cells, which cannot be lysed using the monoclonal antibodies with complement; or it may reflect a conversion of the Ly-l +,2/3+ T-cells to Ly-I-,2/3+ T-cells, which is consistent with current dogma envisioning the Ly-l +,2/3+ T-cell as a precursor of Ly-l-,2/3+ T-cells (96,97).

In third party mixing experiments, mitomycin C-treated spleen

cells from Trichinella-infected mice were capable of actively suppressing the MLR response of control spleen cells (Table 7). This indicates that the reduced MLR response of splenocytes from infected mice is also due, at least in part, to an active suppression mechanism. Suppressor cells were enriched by treatment with anti-mouse Ig plus complement (Figure 14) and were completely abrogated by treatment with anti-Ly-2, but not anti-Ly-l, plus complement (Figure 15). Thus the suppressor cell was identified as a Ly-l-,2/3+ T-cell.










Another explanation for the reduced MLR response and the active

suppression, and which has precedence, is that spleen cells from infected mice may be lysing the allogeneic stimulator cells. Sugarbaker and Matthews (98) have demonstrated that cytotoxic cells can cause a suppression of mixed leukocyte cultures through lysing the stimulator cells. In their experiments, the cytotoxic cells were identified as T-cells. To test for the possibility that stimulator cell lysis may be responsible for the reduced MLR response following infection, mitomycin C-treated spleen cells from normal and Trichinella-infected C57BL/6 mice were used as stimulators in the MLR, with CBA/Ca spleen cells as the responders. No difference was noted in the MLR response when the stimulator cells were from normal and infected C57BL/6 mice (Table 5), indicating that spleen cells from infected mice did not lyse allogeneic cells more rapidly than spleen cells from control mice. In more direct experiments, mitomycin C-treated CBA/Ca stimulator cells were internally labelled with 51Cr, and the amount of stimulator cell lysis determined by measuring the release of 51Cr after incubation with responder cells from normal and infected mice. Again, there was no evidence of a more rapid lysis of stimulator cells when incubated with spleen cells from infected mice (Table 6).

Cell-mediated lympholysis (CML) assays comparing spleen cells from normal and Trichinella-infected C57BL/6 mice showed that there was no alteration in the cytolytic activity of splenic T-cells during infection (Figure 16). These results are similar to those of Hathcock (personal communication), who found that spleen cells from Trichinella-infected C57BL/lOSn (H-2 b) mice, sensitized either in vitro or in vivo, to BI0.BR (H-2 k) spleen cells, showed a slight, but statistically insignificant,










increase in the CML activity when tested in 51Cr release assays on RDM4 (H-2 k) target cells. These results indicate that there is no effect on the ability of the Ly-l-,2/3+ CTL's to lyse allogeneic target cells following infection.

After Bach and Hirschhorn (99) first described the MLR as an in vitro correlate of the allograft rejection response, it became apparent that distinct subsets of T-cells were required to interact in order to generate cytotoxic T-cells (CTL's) in vitro. Bach et al. (100) later showed that the cell population comprising the CTL's could be physically separated from the MLR responsive cells using monolayers of adherant allogeneic cells. Thus, it became established that distinct populations of T-lymphocytes were responsible for the recognition and killing phases of cell-mediated immunity. The MLR was accepted as the in vitro correlate of the recognition phase of allograft rejection, and was primarily a function of the Ly-1 +,2/3- T-cells, while the CML assay was accepted as the in vitro correlate of the killing phase, and was generally mediated by Ly-l-,2/3+ T-cells. One exception to this scheme is that CTL's a b b + + in mice possessing the Ly-i , Ly-1 and Ly-3 alleles are Ly-l ,2/3+ T-cells (101).

In spite of the numerous observations in vivo, and the many experiments in vitro, that supported the view that the generation of CTL's in vitro was an appropriate model for studying the cellular events that occur during allograft rejection, there was actually no direct proof that CTL's played any role in the rejection process. In a recent study Loveland et al. (87) have shown that the Ly-2/3+ T-cells, in fact, played no role in the rejection of skin allografts in CBA/H mice that










were thymectomized as adults, lethally irradiated, bone marrow-reconstituted and then repopulated with sensitized lymphocytes depleted of Ly-l +, Ly-2+ or Ly-l +,2/3+ T-cells. It was clearly demonstrated that the rejection of skin allografts was entirely dependent on the Ly-l 2/3 T-cells, and at the same time, entirely independent on the presence of Ly-2/3+ cytotoxic T-cells. This work is supported by that of Huber and Cantor (102), who demonstrated nearly identical results when transferring nonsensitized lymphocytes into "B mice" that had a surviving skin allograft still in place. The transfer of Ly-l-,2/3+ T-cells into the skin graft-bearing animals did not result in any evidence of graft +, -cel i edt rf
rejection, whereas the transfer of Ly-l ,2/3 T-cells did lead to graft rejection. In addition to identifying the Ly-l +,2/3- T-cell as the mediator of graft rejection, Loveland et al. (87) found that there was a perfect correlation between graft rejection and the DTH response, as measured by footpad swelling.

The studies of Loveland et al. (87) cast serious doubt on the

validity of the current dogma that graft rejection is effected by cytolytic T-cells, and that the generation of CTL's in vitro is an appropriate model to study the graft rejection response. These new data indicate, in fact, that graft rejection is actually another manifestation of delayed-type hypersensitivity. The observed suppression of Ly-l +,2/3T-cell function in the absence of any alteration of Ly-l-,2/3+ T-cell function in this dissertation research is consistent with the hypothesis that graft rejection is mediated by the Ly-l +,2/3- T-cells.

In light of the fact that skin allograft rejection appears to be mediated by Ly-l +,2/3- (DTH) T-cells, and not the "classic" Ly-l-,2/3+










CTL's, it is perhaps most important to consider what cells have been found capable of suppressing DTH responses. In fact, both Ly-l +,2/3and Ly-l ,2/3+ T-cells have been found capable of suppressing DTH responses. Thompson et al. (103) typed the T-cell which suppressed the DTH response of CBA/H mice to SRBC as Ly-l+ ,2/3-, while Huber et al. (104) typed the T-cell which suppressed the DTH response to the same antigen in C57BL/6 mice as Ly-l-,2/3 +. Not to be outdone, Benacerraf and colleagues (105,106,107) have been characterized the suppression of DTH to the azobenzenearsonate (ABA) determinant in A/J mice, and found a complex set of interactions involving three sets of T-cells; a Ly-i+, 2/3- (TsI) T-cell, a Ly-l +,2/3+ (Ts2) T-cell and a Ly-l -,2/3+ (TS3 ) T-cell. The development of suppressor activity is induced by intravenous injection of ABA-conjugated spleen cells, which induces TSI to secrete a soluble factor (TsF1), which bears both I-J determinants and ABA-antigen binding activity. The TsF1 activates Ts2 cells to secrete TsF2, which has anti-idiotypic binding activity and activates the final suppressor cell, the TS3 T-cell. The TS3 T-cell, once activated, acts in a nonspecific manner to suppress the DTH response irrespective of antigen specificity.

The identification of the MLR suppressor T-cell in this study as a Ly-l ,2/3+ T-cell is not unexpected, based on findings of suppressed DTH responses and MLR responses reported by others. The most extensively studied MLR suppressor T-cell system is that of S.S. Rich, R.R. Rich and their colleagues (108,109,110). They have shown that allosensitized T-cells, upon restimulation with allogeneic cells, secrete an antigen-specific molecule which actively suppresses proliferation of lymphocytes in the MLR assay. This molecule lacks conventional Ig










determinants and suppresses the proliferative response in a genetically restricted manner. Unfortunately, the Ly-l phenotype of the suppressor T-cell has not been determined, although it is known to by Ly-2/3+ (personal sommunication with S.S. Rich). In a similar situation, Bluestone et al. (111) characterized spleen cells from tumor-bearing mice which suppressed the NLR response as Ly-2/3+ T-cells, but again the Ly-l phenotype was not determined.

Interestingly, in the MR suppressor T-cell system of the Riches, the only strain of mouse examined and found not to produce a soluble MLR suppressor factor was the C57BL/6 strain (112). This may account for the inability to detect any evidence for a soluble mediator of MLR suppression in this study (Table 9).

If impaired allograft rejection during Trichinella infection does result from the suppression of the Ly-l +,2/3- (DTH) T-cells, then it might be expected that infected mice would not mount as strong a DTH response to allogeneic cells as would uninfected mice. A test of this prediction showed, in fact, that the DTH response, as measured by footpad swelling, to allogeneic cells appeared to be unaltered (Table 11). However, a suppressed DTH response might be masked by the parasiteinduced inflammatory response in the footpads of mice during infection. The DTH reactivity of lymphocytes was then tested by transferring allosensitized cells, from infected and uninfected mice, into the footpads of uninfected mice. The purpose of transferring the cells was to remove the DTH response from the environment of the parasite-induced inflammatory response. Although not statistically significant (P = 0.17), the results (Figure 17) suggested that the DTH response of spleen cells from infected mice was, in fact, suppressed relative to uninfected controls.







73

While this result seems contrary to the enhanced DTH response of Trichinella-infected mice to BCG reported by Molinari and colleagues (38,39, 40), or the unaltered DTH response to SRBC and oxazalone reported by Jones (41), it is consistent with the hypothesis that impaired allograft rejection results from suppression of the T-cells which mediate DTH responses to alloantigens.
















CONCLUSION

An impairment of the mixed leukocyte reactivity and Con A responsiveness, but not the CML activity and PHA responsiveness, was demonstrated during infection with T. spiralis, and the kinetics of the reduced responses characterized. An active, cell-mediated suppression of the MLR response, but not the Con A response, was demonstrated. It is hypothesized that the suppression of the Con A response, in the absense of any effect on the PHA response, reflects the known ability of immune-complexes to preferentially suppress the Con A response. An active suppression of the MLR response during infection was ascribed to the activity of Ly-l-,2/3+ T-cells. No evidence for a soluble mediator of MLR suppression was found, implying that cell-to-cell contact is required to effect suppression. The data suggested, but did not prove, that there is a shift in the relative proportions of T-cell subsets during infection, with a relative increase in the frequency of Ly-l-,2/3+ T-cells.

Together the results indicate that the impaired skin allograft rejection response following infection with Trichinella spiralis results from an active suppression of Ly-l +,2/3- (DTH) T-cell activity by Ly-l ,2/3+ suppressor T-cells. Whether this results from an increase in the proportion of Ly-l-,2/3+ T-cells during infection, or from an activation of preexisting Ly-l-,2/3+ T-cells is not established.
















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BIOGRAPHICAL SKETCH


Bruce "Ted" Hall was born on July 15, 1948, in Stroudsburg, Pennsylvania. His parents moved to Santa Maria, California, in 1958 and were kind enough to take him along. He graduated from U.C.L.A. sine laude in 1972 (or maybe it was 1973) and eventually came to the University of Florida. After completing his dissertation defense he will attempt to drive his hunk-a-junk car to Washington, D.C., where he will begin postdoctoral research at the Walter Reed Army Institute of Research.
















I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. "IN


Richard B. Crandall, P1. D., Chairman Professor of Immunology and Medical Microbiology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. //ZCatherine A. Crandall, Ph. D. Associate Professor of Pathology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and qualit a dissertation for the degree of Doctor of Philosophy. _ __Paul A. Klein, Ph. D.
Associate Professor of Pathology

















I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Assistant Professor of Pathology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. i . , D


Kenneth I. Berns, M.D., Ph. D. Professor of Immunology and Medical Microbiology


This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

December, 1981


D1ean olle of Medicine



Dean for Graduate Studies and Research




Full Text

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MECHANISMS OF ALTERED CELL-MEDIATED IMMUNE RESPONSIVENESS IN MICE INFECTED WITH Trichinella spira1is By BRUCE T. HALL A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1981

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ACKNOWLEDGEMENTS I would sincerely like to thank my mentors, Dr. Richard Crandall and Dr. Catherine Crandall, for all their time, help, advise, friendship and especially for their patience. I thank each of the members of my committee, Dr. Paul Klein, Dr. Ammon Peck and Dr. Kenneth Berns, for encouragement and assistance in planning my dissertation research. I would like to thank all my friends in the department who helped make the past five years a most memorable experience. Rick Kris, Erv Faulmann, Cindi Donnelly and Tom Doyle, in particular, helped to make graduate school bearable. I want to thank my wonderful parents, Mr. and Mrs. Edgar Hall, for their constant support and love. Without them this work would never have begun. Lastly I want to thank the best friend I have ever had, Ms. Judy Emiko Yo.shioka. Without her love this work would never have been completed. It is to my wonderful parents and my best friend that I dedicate this dissertation. 11

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TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES . LIST OF FIGURES. KEY TO ABBREVIATIONS ABSTRACT .. INTRODUCTION • BACKGROUND REVIEW. The Biology of Trichinella spiralis. Immunity to Trichinosis •• Trichinella-Induced Alterations of Host Immune Responsiveness Mechanisms of Altered Immune Responses Statement of the Problem MATERIALS AND METHODS; Mice • . • • • . Trichinella infection .• Leukocyte Preparation. Nylon Wool Filtration of Leukocytes. Differential Cell Counts • Cytotoxicity Assays •• Mitogen Stimulation. . Mixed Leukocyte Reaction • 11LR Supernatant Fluid •. iii PAGE ii v vi viii ix 1 2 2 4 7 13 18 19 19 19 19 20 20 20 21 22 23

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Cell-Mediated Lympholysis Assay ..•• Delayed-Type Hypersensitivity Assays Skin Grafts. Statistical Analysis RESULTS ...•. Alteration of Blastogenic Responses. Mixed Leukocyte Responses. Spleen cells . . Lymph node cells Mitogen Responses ••••• Kinetics of Altered Blastogenic Responses. Mechanism of Altered Blastogenic Responses • • PAGE 23 24 24 25 26 26 26 26 31 36 . . . . 36 36 Alterations in Cell-Mediated Lympholysis (CML) Activity. 52 Changes in Splenic T-Cell Subsets •.•• 52 Delayed-Type Hypersensitivity (DTH) to Allogeneic Cells.. 56 Effect of Transferred Cells of Allograft Rejection 60 DISCUSSION CONCLUSION • LIST OF REFERENCES • BIOGRAPHICAL SKETCH. iv 62 74 75 85

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LIST OF TABLES TABLE PAGE 1 THE MIXED LEUKOCYTE RESPONSE OF SPLEEN CELLS. . . . . . 27 2 THE INFLUENCE OF INCUBATION TIME ON THE MLR RESPONSE OF SPLEEN CELLS. 29 3 THE MLR RESPONSE OF NYLON-WOOL ENRICHED SPLENIC T-CELLS . 33 4 SPLEEN CELL POPULATIONS DURING INFECTION WITH TRICHINELLA SPIRALIS.. .....•.... 5 SPLEEN CELLS FROM C57BL/6 MICE AS STIMULATORS IN THE MLR ASSAY . . . . . . . . . • . . . . . . 44 45 6 RELEASE OF 51Cr FROM 51Cr-LABELED CBA/Ca STIMULATOR CELLS 46 7 ACTIVE SUPPRESSION OF THE MLR RESPONSE IS MEDIATED BY SPLEEN CELLS FROM INFECTED MICE . . . . 47 8 SUPPRESSION OF THE MLR RESPONSE MEDIATED BY NYLON WOOLADHERANT AND NONADHERANT SPLENOCYTES. . . . . . . 50 9 LACK OF SUPPRESSION BY SUPERNATANT FROM "REDUCED" MLR CULTURES. . . . . . . . . • . . . . 54 10 CHANGES IN SPLENIC T-CELL SUBSETS . 57 11 DTH RESPONSE TO ALLOGENEIC CELLS •. 58 12 EFFECT OF ADOPTIVELY TRANSFERRED CELLS ON ALLOGRAFT REJECTION . . . . . • . • . . • . . . . . . . . . . 61 v

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LIST OF FIGURES FIGURE 1 Life cycle of Trichinella spiralis . 2 A graphic representation of the data in Table 1 (the MLR response of spleen cells from uninfected and TrichinellaPAGE 3 infected C57BL/6 mice). .......... 28 3 The influence of varying the ratios of responder cells to stimulator cells on the MLR 30 4 Comparison of the MLR response using 2.5 x 105 and 5.0 x 105 responder cells. . . . . 32 5 The MLR response of uninfected and Trichinella-infected BIO .AQR mice . .. ..... 34 6 The MLR response of lymph node cells 35 / 7 The MLR response of nylon wool-enriched lymph node T-cells 37 8 The Con A response of spleen cells . 38 9 The PHA response of spleen cells . 39 10 The MLR response of spleen cells during infection with X. spiralis . : .... . . . . . 40 11 The T-cell mitogen responses of spleen cells during infec-tion with T. spiralis. . . . . . . • . . . . . . . • . 41 12 A comparison of the reduced MLR and Con A responses during infection with X. spiralis . . . . . . . . . . .. 42 13 Spleen cell-mediated active suppression of the MLR response during infection. . . . . . 49 14 Identification of the MLR suppressor cell by negative selection using specific antisera plus complement. . . 51 15 Identification of the MLR suppressor cell by negative selection using anti-Ly antisera plus complement . . . 53 vi

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FIGURE PAGE 16 Cell-mediated lympholysis (CML) mediated by spleen cells from Trichinella-infected C57BL/6 mice. 55 17 Adoptive transfer of DTH responsiveness 59 vii

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BCG CML Con A cpm CTL DNP DTH DxS FcR H-2 Ig LPS MIF MLR PHA PPD SRBC KEY TO ABBREVIATIONS bacillus Calmette-Guerin cell-mediated lympholysis concanavalin A count per minute cytotoxic T-Iymphocyte dinitrophenol delayed-type hypersensitivity dextran sulfate Fc receptor histocompatibility-2 complex immunoglobulin lipopolysaccharide macrophage inhibition factor mixed leukocyte reaction phytohemagglutinin purified protein derivative sheep red blood cell viii

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Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MECHANISMS OF ALTERED CELL-MEDIATED IMMUNE RESPONSIVENESS IN MICE INFECTED WITH Trichinella spiral is By Bruce T. Hall December 1981 Chairman: Richard B. Crandall Major Department: Immunology and Medical Microbiology Cell-mediated immune functions were evaluated during infection with Trichinella spiralis in C57BL/6 mice. The response of spleen cells to concanavalin A (Con A) was reduced by 70%, while the response to phytohemagglutinin was unaltered. There was a 60% reduction in the response of spleen cells to allogeneic stimulation in the mixed leuko-cyte reaction (taR), with no detectable effect on the ability of spleen cells to mediate lysis of allogeneic cells in cell-mediated lympholysis assays. The MLR response to H-2K plus H-2D region stimulation was significantly more reduced than the response to H-21 plus H-2S region stimulation. The reduced MLR and Con A responses showed similar patterns of suppression; they were both maximally reduced at 2-3 weeks of infection and returned to normal after 3-4 months. Spleen cells from Trichinella-infected mice actively suppressed the MLR response, but not the Con A response, of spleen cells from uninfected mice. ix

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This in vitro suppression was not mediated by soluble factors, was enriched by treatment with anti-Ig and was completely abrogated by treatment with anti-Ly-2, but not anti-Ly-l, antibody plus complement. The reduced MLR response observed during infection was not due to a change in the timing of optimal responsiveness or to lysis of the allogeneic stimulator cells. There was no change in the percentage of T-cells during infection, but there was a decrease in the percentage of cells sensitive to complement-mediated lysis with anti-Ly-l, but not anti-Ly-2, antibody. These data are consistent with the hypothesis + + -+ that Ly-l ,2/3 T-cells convert to Ly-l ,2/3 T-cells during infection. There was no alteration of the delayed-type hypersensitivity (DTH) response, as measured by the injection of allogeneic cells into foot-pads of Trichinella-infected mice. Although not statistically signi-ficant (P = 0.17), there was some reduction in the DTH responsiveness of spleen cells from infected mice when transferred to footpads of uninfected mice. The results indicate that the impaired rejection of -+ skin allografts during trichinosis results from a Ly-l ,2/3 T-cell-+ -mediated suppression of Ly-l ,2/3 T-cells, which are the presumed effector cells of allograft rejection. x

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INTRODUCTION Parasites continue to be a m ajor health problem throughout the world, particularly in the developing countries of Africa, Asia and South America. It is estimated that over half of the world's population is infected with medically important parasites. In addition, parasitic infections of domestic animals result in serious economic loss and contribute to a vicious cycle of poverty and malnutrition. Unfortunately it is presently impractical or impossible to control these diseases through improved sanitation and living conditions or chemotherapy, and vaccines do not yet exist. It is well known that parasites elicit strong immune responses in uncompromised hosts, and one of the major goals of research today is the development of vaccines for parasitic diseases. However, efforts to develop vaccines have been frustrated by the complexity of the life cycles and the fact that parasites have evolved elaborate means of resisting or evading the host's immune defense mechanisms. In addition, parasites frequently induce a state of altered immune responsiveness to nonparasite antigens, thus rendering the victim more susceptible to infection by other diseases and less able to respond to immunization. Knowledge of parasite-induced alterations of the immune system will not only help us to understand how parasites survive in an immunologically competent host, but may also provide the key for developing effective v accines. 1

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BACKGROUND REVIEW The Biology of Trichinella spira1is Trichinella spira1is was discovered in 1835 by James Paget, a first year medical student, while dissecting the cadaver of a patient who died from pulmonary tuberculosis (1). In the same year Richard Owen presented the first description of the parasite and named it Trichina spira1is (2). The details of the life cycle were established during the next 25 years (3) and in 1896 Rai11iet changed the name to Trichinella spira1is (4). Trichinosis, an infection with the tissue-inhabiting nematode T. spira1is, has a worldwide distribution and infects a large variety of carnivorous animals. The disease is essentially a zoonosis, with man being an incidental host. Most human infections are light and asymptomatic, but heavy infections may result in clinical disease characterized by fever, myositis, periorbital edema, eosinophilia and sometimes death. Human infections result primarily from the ingestion of improperly cooked meat from pigs, bears and sea mammals. Infection is initiated by the ingestion of meat which contains viable, encysted larvae (Figure 1). The cyst wall is digested in the stomach of the host and the larvae pass to the proximal small intestine. The larvae then embed themselves within the lamina propia, where they molt four times within thirty-six hours and transform into immature adults (5). After 5-6 days the worms have reached full length, mated 2

PAGE 13

larvae muscle ) ,. ) J larvae penetrate the sarcolemma oc the muscle fibers ingestion of viable, encysted larvae larvae excyst in stomach \ larvae pass to small intestine newborn larvae migrate through the circulatory and lymphatic systems \ / larvae mature, mate and females deposit second stage larvae in lamina propia / FIGURE 1. Life of Trichinella spiralis. 3

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and the gravid females deposit hundreds of second stage larvae into the lamina propia (6). The newborn larvae migrate through the lymphatic and circulatory systems and become distributed throughout the body (7). They leave the circulatory system in the striated muscle, penetrate the sarcolemma of the muscle fibers and become encapsulated (8). By 30 4 days postinfection the muscle larvae are fully mature and infective (9). The encapsulated larvae may survive many months, or even years, but will eventually die and calcify. The intestinal adults are expelled from the gut by a local inflammatory response, which in a primary rodent infection, begins about 14 days postinfection and is complete by about Day 30 (10). Immunity to Trichinosis A degree of protective immunity is developed with trichinosis, but as in many other parasitic infections, it is not complete (5). Resistance to reinfection with T. spira1is appears to be induced prima rily by antigens secreted by both larval and adult stages. Most, if not all, of these secreted antigens originate from stichocyte granules (5, 11). Stichocytes are large, discoid cells lying in a single row dorsal to the esophagus and comprise the stichosome, which is an organ characteristic of roundworms of the superfamily Trichuroidea. Stichocytes contain two types of secretory granules, a and B granules (11). It is noteworthy that complete cross-reactivity between the antigens secreted in vitro and the antigens of the a and B granules has been shown in double diffusion assays (12). Acquired resistance to trichinosis may be expressed in a number of ways; stunting of adult worms (13, 14), impairment in their ability

PAGE 15

5 to reproduce (15), a more rapid expulsion of adults from the gut (16, 17) and fewer larvae encysting in the host muscle cells (17). Once inside the muscle cells, the larvae appear to be protected from the effects of the host's immune system (5). The precise mechanisms of acquired resistance are not completely understood, but experiments have indicated roles for both humoral and cellular immune mechanisms, as well as nonspecific mechanisms (16, 18, 19). T. spiralis stimulates production of circulating antibodies, predominantly IgG but also IgM and IgA (20. 21). The IgG antibody e x -hibits specificity for stichosome cells and cuticle, while IgM and IgA show specificity for membranes of the larvae. While the role of these antibodies remains unclear, the fact that purified immune B-cells are capable of transferring resistance (22) indicates that they do play an active role in immunity. One possible role for antibody may be in assisting in the attach-ment of eosinophils to newborn larvae. It has been shown that newborn larvae, but not adults or muscle larvae, are destroyed by eosinophils in the presence of immune, but not non immune serum in vitro (23). Evidence for a role of eosinophils in mediating resistance to the sys-temic phase of trichinosis has also been demonstrated in vivo. Mice infected spiralis and depleted of circulating eosinophils by treatment with anti-eosinophil serum had increased numbers of larvae in the muscles relative to controls (24). Treatment with anti-eosinophil serum had no effect on the expulsion of adults from the small intestine, supporting the supposition that different immune mechanisms are effec-tive against the intestinal and p arenteral stages of T. spiralis.

PAGE 16

Perrudet-Badous et al. (25) demonstrated that mice genetically selected for high and low antibody production had the same number of larvae encysting in the muscles. These results probably indicate that antibody alone is not sufficient protection against infection. A T-cell involvement in host resistance to spiralis h a s been clearly demonstrated in studies using T-cell deficient mice and in adoptive transfer experiments using immune T-cells. Ruitenberg and Steerenberg (26,27) found that athymic (nude) mice continue to accumulate parasites in the muscles and do not expel adults worms from the intestine. Implantation of thymus tissue from heterozygous (+/nu) mice restores the immune capacities and the infection is terminated (5). Using thymectomized, X-irradiated, bone marrow-reconstituted mice, Walls et al. (28) found that adult parasites persisted longer in the gut and more larvae encysted in the muscles of T-cell deficient mice. The persistence of intestinal adults corresponded to a defective local inflammatory response in the gut. Another principal arm of host defense against reinfection is the rapid expulsion of 90 % or more of a challenge infection from the intestine. In elegant experiments utilizing parabiosed rats, Bell and McGregor (29,30) showed that rapid expulsion results from the synergistic actions of two distinct host responses. One component of rapid expulsion is generated in response to a priming effect on the intestine by adult worms. This effect is a local, nonspecific phenomenon which is not transferred to parabionts and lacks an immunological bisis. The other component is a circulating factor that is specific for Trichinella and is presumably either antibody or specifically sensitized lymphocytes. 6

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7 Trichinella-Induced Alterations of Host Immune Responses Infection with!. spiral is induces complex immunolo gical responses which not only result in specific resistance to the parasite, but also modify the response of the host to heterologous antigens. Alterations of host immune responses in trichinosis include examples of both immunopotentiation and immunosuppression and are most pronounced during a limited time period, approximately 2-6 weeks following infection. This time period corresponds roughly with the late intestinal, larval migration, muscle invasion and intracellular development stages of infection, and a time when the host is undergoing intense immune responses to parasite antigens (5, 18, 19). Parasite-Induced Immunopotentiation Effect on macrophage activity. Although the role of macrophages during Trichinella-infection has not been fully established, there are several quantitative and functional changes in peritoneal macrophages that warrant mention. Wing et al. (31) observed that the number of mononuclear cells in the peritoneal cavaties of Trichinella-infected mice increased as much as eightfold over uninfected control mice. This increase in peritoneal exudate cells reached a maximum 18 days after infection and coincided with the infiltration of macrophages and lymphocytes into the intestinal wall and elimination of adult worms from the gut. While the absolute number of macrophages and lymphocytes increased in the peritoneum following infection, the relative proportions remained the same. In studies of macrophage function, it was found that infection with!. spiralis results in a nonspecific activation of the host reticuloendothelial system. The increased activity of macrophages during

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8 trichinosis is similar, but not identical, to the immunopot entiating activity of BCG and parvum. Wing et al. (31) found tha t macrophages from Trichinella-infected mice and parvum-injected mice both inhibited DNA synthesis of EL-4 tumor cells in vitro and had comparable cytocidal activity as determined by 5lCr release assays. However, whereas macrophages parvum-injected mice, or mice infected with Toxoplasma gondii, inhibited the intracellular replication of !. gondii, macrophages from Trichinella-infected mice did not. It is possible that different subsets of macrophages are responsible for activity a gainst tumor cells and cells infected with an intracellular parasite such as !. gondii, and while both subsets are activated by parvum or infection with T. gondii, only the former subset is activated in trichinosis. Infection with T. spiralis also appears to enhance the activity of the fixed reticuloendothelial system. Trichinella-infected mice exhibit an increased rate of clearance of intravenously injected col-loidal carbon 14 to 28 days after infection (32). Effect on tumor growth. Infection with 1. spiralis is reported to lead to an increased resistance to transplantable tumors and a reduction in the incidence of spontaneous tumors. Lubiniecki and Cypess (33) studied the effect of !. spiralis infection on the incubation time and survival time of mice given an ascitic sarcoma. They found that the tumor developed more slowly in mice infected for 28 days with 1. spiralis and the mice survived for longer periods of time. Mice infected for 56 days showed no differences in tumor development relative to control mice. The authors suggested that the!. spiralis infection activated macrophages to be more efficient killers of tumor cells.

PAGE 19

Molinari and Ebersole (34) found an even more dramatic effect on the development of a melanoma following infection with !. spiralis. Control mice developed tumors by Day 28 following tumor challenge and all died within 60 days, while none of the Trichinella-infected mice exhibited any signs of neoplasia. Effect on other infections. Mice infected with!. spiralis for 7 to 21 days are less susceptible to intravenous challenge with Listeria monocytogenes. Trichinella-infected mice had approximately an eightfold higher LD50 and fewer viable bacteria could be isolated from their livers (35). !. spiral is has also been reported to exert effects against infections with Trypanosoma lewisi (36) and Leishmania tropica (37) • Effect on delayed-type hypersensitivity. Molinari et al. (38, 39 40) studied the effect of trichinosis on delayed-type hypersensitivity (DTH) responses to BCG in mice, as measured by footpad swelling. Mice were sensitized with live or heat-killed BCG and subsequently challenged with Old Tuberculin (OT). The response to OT was found to be dependent on i) the route of BCG injection, ii) the timing of BCG sensitization relative to the initiation of Trichinella infection and iii) whether the BCG was live or heat-killed. 9 When mice were sensitized with BCG at varying times after infection with I. spiralis there was an initial suppression of the DTH response which, by 21 days after helminth-infection, converted to a stimulation of DTH responsiveness. The route of sensitization had no effect when using live BCG. When using heat-killed BCG, however, there was no in-itial suppression of the DTH response and the subsequent stimulation of DTH responsiveness occurred only when the sensitizing BCG was injected intravenously or intraperitoneally, not when injected subcutaneously.

PAGE 20

When mice were sensitized to BCG 14 days before infection with T. spira1is,the DTH response was suppressed 14 days after he1minthinfection but, again, it converted to an enhanced response 20 to 85 days after Trichinella-infection. 10 Jones (41) examined the DTH response to sheep erythrocytes and oxaza1one in mice 20 days postinfection spira1is. In contrast to the enhanced response observed to BCG, there was little or no effect on the DTH response to those antigens. Interestingly, the response of parasitized, but unsensitized, mice sometimes had significantly greater responses to challenge with the sheep erythrocytes and oxaza1one. Effect on responses to B-ce11 mitogens. Ljungstrom (42) used three po1yc1ona1 B-ce11 activators to examine the effect which Trichinella-infection has on B-ce11s which are at different stages of maturation. Purified protein derivative (PPD), lipopolysaccharide (LPS) and dextran sulfate (DxS) were used to stimulate mature, intermediate and immature B-ce11s respectively. No significant alteration in the response of intermediate or mature B-ce11s from parasitized mice was observed. Immature B-ce11s, however, were more reactive in parasitized mice than uninfected controls during the late muscular stage of infection. Effect on antibody responses to T-independent antigens. The ability of Trichinella-infected mice to produce antibody in response to T-independent antigens was assessed using polyvinyl pyrro1idone (PVP) and DNP52-Fico11. Serum antibody specific for PVP was only slightly, but consistently, higher 1, 2 and 4 weeks postinfection in A/Sn mice, which are "high responders" to PVP. CBA mice, which are "low responders" to PVP, showed a more pronounced, and significant, elevation of the antibody response to PVP (43), Jones (41) observed that the antibody

PAGE 21

11 response to DNP52-Ficoll, as measured by the number of antibody forming cells in Jerne plaque assays, was significantly enhanced 20 days after Trichinella-infection following either in vivo or in vitro immunization. Parasite-Induced Immunosuppression In contrast to the reports of enhanced reactivity where the major effector cells are probably macrophages or B-lymphocytes, there is a pronounced suppression of several functions which are dependent on Tlymphocytes. Effect on virus infections. Suitably timed challenge with Japanese B encephalitis (JBE) virus results in a higher mortality rate and decreased survival times of Trichinella-infected mice (44). Although no increase in the duration or magnitude of JBE viremia was observed, mice infected for 7 to 28 days spiralis had reduced serum antibody titers to JBE. Also, Kilham and Oliver (45) observed that rats infected with encephalomyocarditis virus 10 days after infection with spiralis had higher death rates than unparasitized rats challenged with the same virus. Effect on antibody responses to T-dependent antigens. Suppression of the antibody response to sheep red blood cells (SRBC) is perhaps the most throughly investigated, and best understood, alteration of host immune responsiveness during trichinosis. Mice immunized with SRBC during the intestinal stage (Day 7) of trichinosis exhibit no change in the antibody response either at the humoral or the single cell level (43,46,47,48). When immunized during the second week of infection they have either normal or reduced antibody responses, depending on the strain of mouse, route of immunization and dose of SRBC used. During the early muscular stage (Day 20), both the humoral antibody level and the number

PAGE 22

12 of spleen cells producing anti-SRBC antibodies a r e r ed uced (43,44,46,47 48). Spleen cells from 20-day infected mice are also less responsive to immunization with SRBC in vitro (47). By the late muscular stage (Day 30) the response to SRBC is essentially normal. Jones al. (47) determined that the transient suppression of humoral immunity to SRBC is mediated by suppressor T-cells (TS) which can be found in the spleens of infected mice. Interestingly, while the humoral response to SRBC was reduced in the spleen, the response of lymph node lymphocytes was enhanced. The antibody response to SRBC can also be diminished, even eliminated, by the injection of solubilized extract of T. spiralis, without affecting the antibody response to the T-independent antigen PVP (48). Trichinosis was also found to depress both the systemic and local antibody responses to orally administered cholera toxin (49). Local antibody production was evaluated by determining the amount of antibody synthesized in vitro by tissue-cultured intestine. The intestines from uninfected mice produced significant levels of both IgA and IgG in vitro, while no antibody synthesis could be detected in the cultured intestine from parasitized mice. The suppressive effect was more pronounced during the intestinal stage of infection. Effect on passive cutaneous anaphylaxis (PCA). Munoz and Cole (50) demonstrated that mice infected with !. spiralis were relatively unresponsive to passive cutaneous anaphylaxis following injection of egg albumin and anti-egg albumin (IgGl and IgE) antibodies. Presumably the Trichinella infection induces the formation of IgE antibodies which bind to mast cells and prevent the binding of passively transferred antigen-specfic IgE.

PAGE 23

13 Effect on responses to T-cell mitogens. Conflicting results exist in the literature relating to the responsiveness of lymphocytes from Trichinella-infected mice to the polyclonal T-cell activators concanavalin A (Con A) and phytohemagglutinin (PHA). A majority of studies indicate that the response of splenic lymphocytes to Con A is significantly reduced 14 to 20 days after infection (42,47,51,52), while the response to PHA is reported to be only slightly depressed (42,47) or not affected at all (51). Effect on graft versus host and allograft rejection responses. Svet-Moldavsky et al. (53) and Chimyshkyan et al. (54) showed that spleen cells from mice infected with!. spiralis for 22 to 72 days were impaired in their.ability to induce a graft versus host reaction, and that mice infected for 20 to 40 days were suppressed in their ability to reject skin allografts. Using split heart allografts, Ljungstrom and Huldt (43) confirmed the observation that transplant rejection is impaired in mice infected with!. spiralis. In summary, infection with!. spiralis alters the ability of the host to respond immunologically to a wide range of antigens. These alterations are complex, and the underlying basis of most of them has not been determined. Mechanisms of Altered Immune Responses Numerous mechanisms have been proposed which may explain the alterations of immune responsiveness induced by infections with T. spiralis, but in most cases these mechanisms are hypothetical only. In any case, each altered immune response which is identified most likely reflects the summation of numerous synergistic and antagonistic factors.

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14 Parasite-Produced Immunosuppressive Factors Faubert and Tanner (55) provide evidence that serum from Trichinella-infected mice may contain parasite-derived immunosuppressive factors. Sera from infected mice agglutinates and kills homologous lymphoc ytes in vitro beginning 7 d ays after infection. These leukoagglutinating and cytotoxic activities reached a maximum 30 days after infection and declined thereafter. Transfer of sera from infected mice also prolongs the survival of allografts in normal recipients. Based on the kinetics of appearance of these activities and the fact that saline extracts of T. spiral is possess both the leukoagglutinating and leukotoxic activities, the authors have concluded that these activities are due to parasite produced substances and not antibody. Others have also shown that saline extracts of I. spiralis are immunosuppressive. Barriga (56) has shown that normal mice injected with a saline extract exhibited depressed antibody responses to the Tdependent antigen SRBC, depressed responses to the mitogens LPS and Con A and a delayed allograft rejection response. Saline extracts of T. spiralis are also reported to suppress the formation of sheep red cell rosette forming cells (57). Altered Structure of Lymphoid Organs Changes in the structure, or architecture, of lymphoid organs (e.g., splenomegaly, lymphadenopathy) may affect the circulation of antigens, or the trafficking of lymphoid cells through these organs and affect the cell-cell interactions requisite for mounting an immune response. Faubert and Tanner (58) showed that the increase in size of lymph nodes in mice following infection was a T-cell dependent phenomenon

PAGE 25

15 since the increase in size did not occur in thymectomized animals. Molinari al. (40) described changes in the microscopic anatomy of the thymus during trichinosis in mice, including an increase in the total cell number and distinct anatomical changes in the thymic cortex and medulla. In contrast to the increase in cell numbers in the thymus observed by Molinari al. (40), Ljungstrom and Huldt (43) reported that the thymic cortex is depleted of lymphocytes, with some infected mice being almost completely devoid of lymphocytes in the cortex. Ljungstrom and Sundqvist (59) and Jones (41) reported that although trichinosis in mice results in splenomegaly, there is no change in the proportion of B-cells and T-cells. Immunosuppressive Serum Factors A number of factors have been identified that are found normally in the serum, or are induced following infection, which are capable of suppressing in vitro tests of cellular immunity. Although some of these factors act specifically and depress reactivity only to antigens of the infecting agent, most act nonspecifically and depress the response of sensitized lymphocytes to unrelated antigens. C-reactive protein. C-reactive protein (CRP) is a trace component of serum that is not readily demonstrable in normal serum but which increases in concentration during the acute phase of febrile illnesses (60). The detection of CRP has long served as an indicator of the presence and degree of inflammatory activity. CRP is known to bind to some, but not all, T-lymphocytes and to suppress their activity. CRP can suppress the proliferative response of T-cells in the mixed lymphocyte response (61,62,63), the generation of cytotoxic T-cells in vitro

PAGE 26

(63), antigen-induced blastogenesis (64) and mitogen-induced MIF production (64). 16 Histamine. It has been shown that some, but not all, murine Tcells have receptors for histamine. These receptors are of the H-2 type and are found on T-cells which inhibit antigen-induced MIF production (65,66,67), antigen-induced blastogenesis (65,66,67), cytotoxic activity against allogeneic cells (68) and the antibody response to SRBC (69). Bekish (70) demonstrated that there is a considerable increase in the free histamine level in the liver, muscle and blood in rodent trichinosis. The author suggests that the rise in histamine level in the tissues and circulation may be an important factor in promoting the migration of Trichinella larvae through the capillary beds since histamine causes the dilatation of capillary vessels and increases their permeability. It is possible that the free histamine also binds to the H-2 receptor-bearing lymphocytes and modulates those functions mentioned above. Antibody-Mediated Alterations Antibody, either alone or complexed with antigen, is capable of both enhancing and suppressing immune responses. Perhaps the simplest explanation of the suppressive effect of antibody is that antibody which is bound to antigen may block any subsequent recognition of that antigen by lymphocytes. Such "blocking antibodies" have been shown to play an important role in the survival of tumor cells in immunocompetent hosts (71,72). It has been demonstrated in some systems that antibodymediated suppression requires intact Fc domains in addition to the antigen binding domains (73). The mechanism(s) o f this t ype of suppression is not understood, although speculation exists that it is mediated

PAGE 27

17 by soluble factors released by Fc-bearing lymphocytes following the binding of antigen-antibody complexes. Antigen-antibody complexes may also enhance immune responses. Dennert (74) found that complexes formed between IgM and SRBC enhanced the antibody response, relative to SRBC alone, both in vivo and in vitro. Dennert suggested that the IgM-SRBC complexes resulted in a more efficient antigen localization in vivo. Anti-idiotypic antibodies may also play an important role in the regu-lation of immune responses, both humoral and cellular (75,76). Antigenic Competition Antigenic competition is the phenomenon whereby the injection of one antigen may inhibit the immune response to another, unrelated anti-gen. The mechanism of antigenic competition is not clear, but it is evidently dependent on T-cells since it involves only T-dependent anti-gens (77). Antigenic competition has been suggested as a possible ex-planation of reduced immune responsiveness during trichinosis (46,47). Suppressor Cells One of the major areas of research in immunology today is the regulation of immune responses. It is well documented that distinct subpopulations of T-cells function not only as effector cells, but also as regulator cells. Some T-cells function as helper cells and others function as suppressor cells. Suppressor T-cells may be activated non-specifically by mitogens such as Con A (78) to produce soluble factors which can mediate suppression in the absence of suppressor cells. Whether Con A induces suppressor cells that are truly nonspecific, or whether it stimulates a great number of antigen-specific clones is not clear. Antigen-specific suppression (reviewed in 79) consists of sup-pression of B-cells, or T-cells, by T-cells. Although suppression may

PAGE 28

18 be exerted directly on B-cells, it appears that the effect is more likely in reducing the activity of helper T-cells, which in turn reduces the response of the B-cells. In only one case has the underlying basis of a Trichinella-induced alteration been determined. In that singular case, it was found that suppressor T-cells were responsible for the reduced antibody response to SRBC during infection (47). Statement of the Problem It has been shown by others that the alloresponsiveness of the host is impaired during infection spiralis. There is a reduced ability of spleen cells from Trichinella-infected mice to induce a graft versus host reaction (54) and Trichinella-infected mice do not reject allografts as rapidly as uninfected mice (43,53,54). However, the underlying b asis of these impaired cell-mediated responses was not determined. The purpose of this study is to determine the cellular basis of the impaired allograft rejection response during infection with spiralis.

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MATERIALS AND METHODS Mice Female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) 8-14 weeks old were the principal experimental animals used in this study. Female CBA/Ca mice of varying ages were used as the source of allogeneic stimulator cells in the mixed leukocyte reactions. The congeneic strains of mice C57BL/IO, BIO.AQR, BlO.A(2R) and BlO.T(6R) were obtained from the Department of Pathology animal colony. Trichinella Infection Mice were infected with 200!. spiralis larvae via stomach intubation by the method of Larsh and Kent (80). The parasite was originally obtained from Dr. John E. Larsh (Department of Parasitology, University of North Carolina, Chapel Hill, NC) and maintained in this laboratory by serial passage in rats and mice. Leukocyte Preparation Spleens or lymph nodes were removed aseptically and gently pressed through wire screens into cold Dulbecco's phosphate buffered saline (D-PBS) contained in plastic petri dishes. The cells were transferred to sterile plastic centrifuge tubes and allowed to settle for 5 minutes on ice to permit the larger clumps to settle out. The remaining single cell suspension was transferred to a new tube and the cells washed 3 times with cold D-PBS. The red blood cells were lysed using warm (37) NH4Cl and the remaining white cells were washed 3 times with cold D-PBS. 19

PAGE 30

On the final wash the white cells were suspended in complete media (RPMI-1640 supplemented with L-glutamine, 5% heat-inactivated fetal calf serum and antibiotics). Viability was determined by trypan blue exclusion and the cells diluted to the desired concentration with com-plete media. Nylon Wool Filtration of Leukocytes 20 Spleen cells were separated into B-cell and T-cell enriched frac-tions by passage of the cells over nylon wool columns, as first described by Julius al. (81). Briefly, 100 x 106 spleen cells in complete media were filtered over 0.6 grams of nylon wool (LP-l Leuko-Pak Leukocyte Filters, Fenwall Laboratories, Deerfield, IL) packed into 6 ml in a 12 ml syringe. Details of the nylon wool preparation and cell elution technique were as described by Henry (82). The adherant and nonadherant populations were tested for purity by testing their susceptibility to lysis with anti-mouse Ig and anti-Thy-l.2 plus complement and their ability to respond to the mitogens Con A, PHA and LPS. The nonadherant fraction was routinely at least 95% pure T-cells while the adherant fraction was only 75-80% B-cells. Differential Cell Counts Leukocytes were suspended in complete media and pelle ted on microscope slides with a cytocentrifuge (Shandon Scientific Co., Inc., Sewickley, PA). The cytocentrifuge preparations were stained with either May-Gruenwald's stain or Wright's stain. Differential cell counts were performed by counting at least 200 cells per sample. Cytotoxicity Assays Leukocytes were suspended to 5 x 106 cells per ml in complete media and incubated for 30 minutes with antisera at 4C. Folloiwng incubation

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21 with the antisera, the cells were washed once with cold media, comp1e-ment added and the cells were reincubated for 45 minutes at 37C. The amounts of antisera and complement giving optimal results were predeter-mined for each lot of reagents. Following cell lysis, killed cells were removed using a 40% Perco11 solution (Pharmacia Fine Chemicals, Piscataway, NJ) according to the manufacturers' instructions. Rabbit anti-mouse Ig (donated by Dr. Catherine Crandall, Depart-ment of Patho1gy) was prepared by injection of rabbits with an antigen-antibody precipitate of sera from mice infected with Ascaris suum (83). The rabbit anti-mouse Ig was shown to recognize all classes of mouse Ig by immunoelectrophoresis. Anti-Thy-l.2 a110antisera (donated by Dr. Michael Norcross, Department of Pathology) was prepared by repeated immunizations of AKR mice with thymocytes from young C3H mice. Mono-clonal anti-Thy-1.2 antibody was purchased from New England Nuclear, Inc. (Boston, MA). Monoclonal anti-Lyt-1 and anti-Lyt-2 were obtained by culturing hybridoma cell lines 53-7.313 (anti-Lyt-1) and 53-6.72 (anti-Lyt-2) obtained from the Cell Distribution Center, The Salk Insti-tute for Biological Research (San Diego, CA). The spent tissue culture media from the hybridomas was centrifuged and filtered to remove cellular debris and used directly. Guinea pig complement was used with a11oanti-sera and was purchased from GIBCO (Grand Island, NY). Rabbit comp1e-ment (Low-Tox-M rabbit complement) was used with monoclonal antibodies and was purchased from Cedar1ane Laboratories, Ltd. (Accurate Chemical and Scientific Corp., Hicksville, NY). Mitogen Stimulation The proliferative response of lymphocytes to mitogens was assayed in a modified micromethod of Hartzman et a1. (84). Lymphocytes were

PAGE 32

cultured with varying concentrations of concanavalin A (Con A; Miles Laboratories, Inc., Kankakee, IL), phytohemagglutinin (PHA; PHA-P, Difco Laboratories, Detroit, MI) and lipopolysaccharide (LPS; LPS-W, typhimurium, Difco Labs.). Five hundred thousand cells in complete media were cultured in quadruplicate cultures for 72 hours in 96-well U-bottom micro titer plates (Linbro Chemical Co., Inc., New Haven, CO) at 37C, 5% CO2 with 0.5 of 3H-TdR (thymidine methyl 3-H, sterile aqueous, 1.9 CMM, Schwarz/Mann, Orangeburg, NY) added per well for the final 24 hours of culture. The cells were collected mechanically (Hiller Harvester, Otto Hiller Co., Madison, WI) using distilled water and the radioactivity measured in a liquid scintillation counter. The LPS was boiled for one hour in PBS (pH 8.0) before use. Mixed Leukocyte Reaction, 22 Unidirectional mixed leukocyte reactions were performed essentially by the procedure described by Rich and Rich (85). Routinely,S x 105 or 2.5 x 105 C57BL/6 (H_2 b ) responder spleen cells were incubated with 5 x 105 mitomycin C-treated CBA/Ca (H_2 k ) stimulator cells in 96-well U-bottom micro titer plates. Quadruplicate cultures were incubated for 96 hours at 37C, 5% CO2 with 0.5 3H-TdR added per well for the final 24 hours of culture. The cells were collected and the radioactiv-ity measured as above (see Mitogen Stimulation). In studies of suppressor activity, 2.5 x 105 responder cells and 2.5 x 105 mitomycin C-treated "regulator" cells from mice infected with spiralis, or uninfected controls, were cultured with 5 x 105 mitomycin C-treated stimulator cells. The MLR was quantitated by comparing the incorporation of 3H-TdR by responder cells following stimulation with allogeneic cells to that

PAGE 33

23 of identical cultures stimulated with syngeneic cells. Complete media for the MLR was the same as for the mitogen stimulation with the addition -5 of 5 x 10 M 2-mercaptoethanol. MLR Supernatant Fluid Culture media from mixed leukocyte reactions of spleen cells from Trichinella-infected mice responding to allogeneic stimulation for 24 hours was rendered cell free by centrifugation (400 x g, 10 minutes) and filtration through 0.22 Millipore filters. This supernatant fluid was tested for suppressor activity at a 1:1 dilution in MLR cultures of leukocytes from normal, uninfected mice. Cell-Mediated Lympholysis Assay The ability of lymphocytes from Trichinella-infected mice to devel-op cytolytic effector cells (TC) was evaluated in vitro as described by Grabstein (86). Briefly, mixed leukocyte cultures were established in 30 ml volumes (75 x 106 C57BL/6 responder cells plus 75 x 106 mitomycin C-treated stimulator cells) in 75 mm3 plastic tissue culture flasks (Falcon #3024; Becton, Dickinson and Co., Cockeysville, MD). Aliquots d . ' . d d f .... 5lC were remove at var10US t1mes an teste or cytOtOX1C act1v1ty 1n r release assays. Two million L-929 (H_2 k ) target cells were incubated with 0.1 ml (Na)25lcr04 (sodium chromate in sterile saline, 1 mCi/ml, New England Nuclear, Boston, MA) for 60 minutes and gently washed 3 times with cold media. Varying numbers of cells were incubated with 104 5lCr-labeled L-929 target cells for 4 hours at 37C, 5% CO2 in 96-well U-bottom microtiter plates. The contents of each well were then resuspended by the forceful addition of 0.05 ml media, and the cells pelleted by gently centrifuging the microtiter plates. Equal volumes of supernatant fluid were removed from each well and quantitated in a gamma counter. Background release was determined by incubating

PAGE 34

24 51 Cr-labeled target cells alone. Maximum release was determined by add-ing 0.1 ml of 1 % Nonidet P40 (NP40; Sigma Chemical Co., St. Louis, MO) 51 to Cr-labeled target cells. The percentage of specific release was calculated using the following formula: experimental cpm -background cpm % specific cytotoxicity -------------------------------x 100% total release cpm -background cpm Delayed-Type Hypersensitivity Assays Delayed-type hypersensitivity to allogeneic cells was assessed in two ways: 1) footpad swelling in normal and Trichinella-infected mice, and 2) by adoptive transfer of spleen cells from allosensitized control and infected mice into the footpads of uninfected mice. The method used was essentially that of Loveland etal. (87). Control and Trichinellainfected mice were sensitized by subcutaneous injection of 3 x 107 allo-geneic (CBA/Ca) spleen cells, followed 6 days later by footpad challenge with 5 x 106 allogeneic spleen cells in a volume of 0.05 ml. In the adoptive transfer experiment 4 x 106 spleen cells from allosensitized control and Trichinella-infected mice were transferred with 4 x 106 allogeneic or syngeneic spleen cells into the footpads of normal, unin-fected C57BL/6 mice. The 24-hour footpad swelling was measured to the nearest 0.01 mm using a dial gauge micrometer (No. 25-441; L.S. Starrett Co., Athol, MA). Skin Grafts Graft beds were prepared aseptically in recipient mice maintained under Nembutal anesthesia. 2 Back skin (approximately 1.0 cm ) was removed aseptically from donor mice and put into place in the graft bed. The graft was sutured into place using a 6-0 silk fitted with an FS-3 needle

PAGE 35

(Ethicon, Inc., Somerville, NJ). Skin graft recipients were caged individually. Graft rejection was read a s total graf t necrosis. Statistical Analysis Data were calculated to give mean a nd standard deviations, and statistical significance determined by Students' t-test. Analysis of delayed-type hypersensitivity experiments was performed using the Fisher Exact test with a footpad swelling of 0.3 mm or greater considered to be significant. 25

PAGE 36

RESULTS Alteration of Blastogenic Responses Mixed Leukocyte Responses The ability of spleen cells a nd lymph node cells from C57BL/6 mice infected with Trichinella spiralis to respond in the one-way mixed leukocyte response (MLR) was assessed. The optimal conditions for the MLR were determined in preliminary experiments. Spleen cells As seen in Table 1, spleen cells from C57BL/6 (H-2 b ) mice infected 2l() days previously with 200 spiralis larvae did not respond as well to allogeneic stimulation with CBA/Ca (H-2 k ) spleen cells as did spleen cells from uninfected mice. Because the magnitude of the MLR response varies greatly between experiments, the results for each e xperiment were e xpressed as the percentage of the control MLR (calculated as described in Materials and Methods). The results of Table 1, presented in graphic form in Figure 2, show that the response of spleen cells from infected mice was approximately 40 % of the control response. As shown in Table 2, the results were qualitatively unaffected by the incubation time of the cultures. Experiments were performed using different concentrations of splenocytes to rule out the possibility that the reduction in MLR responsiveness of the Trichinella-infected mice was due to suboptimal concentrations of responder cells being used. Figure 3 shows that the reduced MLR response of spleen cells from infected mice was observed over a wide 26

PAGE 37

EXP. NO. 1 2 3 4 5 6 7 8 9 10 TABLE 1 THE MIXED LEUKOCYTE RESPONSE OF SPLEEN CELLS RESPONDERa CELL TYPE Control Infected Control Infected Control Infected Control Infected Control Infected Control Infected Control Infected Control Infected Control Infected Control Infected STIMULATOR CELL (CPM SD)b CBA/Ca C57BL/6 22,9l5 1,931 14,590 1,201 41,933 5,501 26,422 1,324 41,666 2,839 29,732 1,117 64,392 4,570 45,788 4,468 33,816 3,275 16,892 2,206 11,117 3,906 4,710 753 10,692 2,286 9,380 1,113 13,884 3,307 12,479 1,293 39,360 4,878 21,571 2,866 47,655 3,460 24,821 3,583 3,473 4,149 466 623 9,750 554 12,174 1,840 9,256 499 10,868 1,376 12,793 1,562 9,653 1,548 10,406 1,211 10,288 1,574 2,798 4,075 989 558 1,142 658 7,803 1,459 4,206 2,858 641 884 11,347 1,658 7,210 1,210 8,983 1,562 6,704 1,604 % CONTROLc P VALUE RESPONSE 53.7 P< 0.005 44.3 P< 0.005 58.5 P< 0.005 70.0 P< 0.005 28.2 P< 0.005 7.6 P< 0.005 16.5 P< 0.005 100.0 NS 51.3 P< 0.005 46.8 P< 0.005 27 a. Responder cell were 5 x 105 splenocytes from uninfected (control) or Trichinella-infected C57BL/6 mice at 21 days of infection. b. Stimulator cells were 5 x 105 mitomycin C-treated splenocytes from allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice. c. The % control response was calculated as: cpm, infected mice (allogeneic response -syngeneic response) X 100% cpm, control mice (allogeneic response -syngeneic response) Spleen cells from 3 to 7 mice were pooled for group in each experiment.

PAGE 38

100 >il (f) z 0 p.., (f) >il s:1 H 0 50 H Z 0 u r.>.. 0 CONTROL MICE -r-INFECTED MICE SOURCE OF RESPONDER CELLS FIGURE 2. A graphic representation of the data in Table 1 (The MLR response of spleen cells from uninfected and Trichinella-infected C57BL/6 mice). See Table 1 for calculation of percentage of control MLR response. 28

PAGE 39

29 TABLE 2 THE INFLUENCE OF INCUBATION TIME ON THE MLR RESPONSE OF SPLEEN CELLS DAYS RESPONDERa STIMULATOR CELL (CPM SD) b % CONTROLc IN VITRO CELL TYPE CBA/Ca C57BL/6 RESPONSE 3 Control 20,695 1,925 10,540 1,707 Infected 13,156 1,906 6,132 1,278 69.1 4 Control 65,004 7,723 14,358 1,141 Infected 25,824 2,204 7,389 294 36.1 5 Control 35,396 4,034 12,490 3,168 Infected 19,976 1,672 7,740 2,215 56.5 a. Responder cells were 5 x 105 splenocytes from uninfected (control) or Trichinella-infected C57BL/6 mice at 21 days of infection. b. Stimulator cells were 5 x 105 mitomycin C-treated splenocytes from allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice. c. The percentage of control response was calculated as described in Table 1. Data are from: one of three experiments g1v1ng identical results; spleen cells from 3-7 mice were pooled for each group in each of the experiments.

PAGE 40

10 • CONTROL r-. • ("") I 0 8 ,...; ;:E: • p.. u '-" Z • 0 / H 6 0 0 • u Z H INFECTED Z 4 /O _____ H H H :r:: ("") 2 o O.S 1 2 3 S RESPONDER CELLS FIGURE 3. The influence of varying ratios of responder cells to stimulator cells on the MLR. Responder cells were sp1enocytes from uninfected CS7BL/6 mice (control) or CS7BL/6 mice infected 21 days previously with 200 T. spira1is larvae (infected). The cpm [3H] incorporation equals the cpm following stimulation with S x 10 allogeneic (CBA/Ca) spleen cells minus the cpm following stimulation with S x lOS syngeneic spleen cells. The results are from one of two experiments giving identical results; three mice were used in each group for each experiment. 30

PAGE 41

31 range of responder cell to stimulator cell ratios. Based on these results, all remaining experiments were performed using either 0.25 x 666 10 or 0.5 x 10 responder cells and 0.5 x 10 stimulator cells. As seen in Figure 4, the HLR was qualitatively similar when using either 6 6 0.25 x 10 or 0.5 x 10 responder cells. To rule out the possibility that the reduced response of the infected mice was due to a decrease in the relative proportion of T-cells in the spleens, MLR assays were performed using nylon wool-enriched splenic T-cells from normal and infected mice. As seen in Table 3, purified T-cells from infected mice were less reactive in the MLR than T-cells from uninfected mice. To evaluate the MLR response of defined subsets of T-cells, assays were performed using spleen cells from normal and Trichinella-infected B10.AQR mice responding in the MLR to spleen cells from con-genic strains of mice. As seen in Figure 5, the T-cells from infected mice which respond in the MLR to H-2K plus H-2D region differences were even more suppressed, relative to cells from uninfected mice, than those cells which respond to entire H-2 differences or H-21 plus H-2S region differences. Lymph node cells To ascertain whether the reduced MLR response of splenocytes was part of a general suppression, or restricted to the spleen, mixed leukocyte reactions were performed using lymph node cells. As seen in Figure 6, the MLR response of leukocytes from the axillary and brachial lymph nodes of Trichinella-infected mice was reduced approxi-mately to the same degree as splenic leukocytes. Nylon wool-enriched

PAGE 42

100 100 >Ll U) z 0 -r-p.., U) >Ll p::: p::: 50 50 H 0 E-4 Z 0 u 0 N -CONTROL INFECTED CONTROL INFECTED 2.5 x 105 RESPONDER CELLS 5.0 x 105 RESPONDER CELLS FIGURE 4. Comparison of the MLR response using 2.5 x 105 and 5.0 x 105 responder cells. Responder cells were sp1enocytes from uninfected C57BL/6 mice (control) or C57BL/6 mice infected 21 days previously with 200 I. spira1is larvae (infected). Stimulator cells were 5.0 x 105 allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 1 for calculation of percentage of control MLR response. The results of ten experiments, with 3-7 mice per experiment, are averaged for each graph. W N

PAGE 43

EXP. NO. 1 2 3 4 5 a. TABLE 3 THE MLR RESPONSE OF NYLON WOOL-ENRICHED SPLENIC T-CELLS RESPONDERa CELL TYPE Control Infected Control Infected Control Infected Control Infected Control Infected STIMULATOR CELL (CPM SD)b CBA/Ca C57BL/6 12,501 1,748 9,031 1,330 32,664 4,603 15,452 2,066 18,645 1,486 13,167 2,129 46,833 5,494 27,563 2,840 22,391 2,149 17,330 1,533 2,840 363 208 128 1,579 274 1,604 371 1,041 842 1,543 478 4,431 233 3,913 711 3,609 670 4,497 1,208 % CONTROLc RESPONSE 89.7 44.6 66.0 55.8 68.3 Responder cells were 5 x 105 nylon wool-nonadherant spleen cells from uninfected (control) and Trichinella-infected C57BL/6 mice at 21 days of infection. b. Stimulator cells were 5 x 105 mitomycin C-treated allogeneic (CBA/Ca) or syngeneic (C57BL/6) splenocytes. c. The percentage of control response was calculated as described in Table 1. Spleen cells from 3-7 mice were pooled for each group in each experiment. 33

PAGE 44

U) :z 0 p.. U) H 0 E-< :z 0 u 0 iN! 100 80 60 40 . 20 • H-2 K+D MLR STIMULUS 34 I+S FIGURE 5. The MLR response of uninfected (control) and Trichinellainfected B10.AQR mice. Stimulator cells were splenocytes from C57BL/10 (entire H-2 difference; H-2), B10.A(2R) (H-2K plus H-2D differences; K+D) and B10.T(6R) (H-2I plus H-2S differences; I+S) mice. Responder cells from infected mice were obtained 21 days after infection. The response of spleen cells from infected mice was compared to the response of spleen cells from uninfected mice following stimulation with the same congeneic strain of mouse. See Table 1 for calculation of percentage of control MLR response. The data is the average of two experiments; three mice were used in each group for each experiment.

PAGE 45

U) z 0 p... U) p::: ...:l E-t Z 0 u 0 100 50 . -CONTROL INFECTED INFECTED (POOLED) (AXILLARY) (BRACHIAL) SOURCE OF RESPONDING LYMPH NODE CELLS 35 FIGURE 6. The MLR response of lymph node cells. Responder cells were 2.5 x 105 pooled lymph node cells from uninfected C57BL/6 mice (control) or from the axillary and brachial lymph nodes of C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (infected). Stimulator cells were 5.0 x 105 mitomycin C-treated allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 1 for calculation of percentage of control MLR response. The results of three experiements are averaged; 3 to 7 mice were used in each group for each experiment.

PAGE 46

36 lymph node T-cell populations from infected mice were also less reactive in the MLR than enriched T-ce11s from normal, uninfected mice (Figure 7). Mitogen Responses The ability of spleen cells from C57BL/6 mice infected with T. spira1is for 21 days to respond to the T-cell mitogens Con A and PHA was assessed. For each experiment a dose response was performed using at least three concentrations of mitogen. The response of spleen cells from infected mice to Con A was reduced relative to controls (Figure 8), while the response to PHA was unaltered (Figure 9). Kinetics of Altered Blastogenic Responses To determine when during infection the MLR and mitogen responses of spleen cells from Trichinella-infected mice was reduced, a timecourse study was performed. As seen in Figure 10, the MLR response of splenocytes from infected mice was maximally reduced at 21 days of infection and the response to Con A (Figure 11) was reduced maximally at 14 days of infection; both responses returned to normal by 3 to 4 months. The response to PHA (Figure 11) was not significantly altered at any time during infection. A comparison of the altered MLR and Con A responses (Figure 12) shows that the kinetics of the reduced MLR and Con A responses were nearly identical. Mechansim of Altered Blastogenic Responses To determine whether the suppressed MLR response of spleen cells from infected mice was due to i) depletion of T-cells in the spleens of infected animals, ii) lysis of the allogeneic stimulator cells in the assays or iii) an active suppression of the MLR responder cells, the experiments described below were done.

PAGE 47

(I) z 0 p.., (I) .....:l 0 E-< Z 0 u r... 0 100 50 CONTROL MICE INFECTED MICE SOURCE OF RESPONDING LYMPH-NODE CELLS 37 FIGURE 7. The MLR response of nylon wool-enriched lymph node T-cells. Responder cells were 2.5 x 105 pooled lymph node T-cells from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected 21 days previously with 2001. spiral is larvae (INFECTED). Stimulator cells are 5.0 x 105 allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 1 for calculation of percentage of control MLR response. The results presented are from a single e xperiment.

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80 r-. C""l I 0 ..-l 60 p... u '-' Z 0 H H < H p::: 0 40 0 u Z H Z H H 20 ::c C""l • o 0.25 • INFECTED 0.5 CONTROL • 1 l1g Con A / WELL o 2 FIGURE 8L The Con A response of spleen cells. Responder cells were 5.0 x 105 spleen cells from uninfected C57BL/6 mice (CONTROL) or Cs7BL/6 mice infected 21 days previously with spiral is larvae (INFECTED). This graph is a representative of 5 experiments, all of which gave the same qualitative result. 38

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z o H o o u Z H Z H E-I 40 20 10 CONTROL o * : :>< INFECTED • • 0.25 0.5 1 2 J.lg PHA / WELL FIGURE 9. The PHA response of spleen cells. Responder cells were 5.0 x 105 spleen cells from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected 21 days previously with 200 T. spiralis 39 larvae (INFECTED). This graph is a respresentative of 5 experiments; all of which gave the same qualitative result.

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w U'.l Z o p.., U'.l ta p:: s3 H o u o 100 80 60 40 20 7 .. CONTROL e (8) 14 • (15) l'i 21 INFECTED • • (8) 40 63 • /(2) 84 DAYS OF TRICHINELLA SPIRALIS INFECTION • (2) l3l FIGURE 10. The MLR response of spleen cells during infection with T. spiralis. Responder cells were 5 x 105 splenocytes from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected for various times with spiralis larvae (INFECTED). Stimulator cells were 5 x 105 allogeneic (CBA/Ca) or syngeneic spleen cells. Numbers in parenthesis indicate the number of experiments performed at eact time point. See Table 1 for calculation of percentage of control MLR response. o

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CJ) z o p... CJ) 100 80 60 H H Z o u >:t.. o 40 20 e\ • 7 14 . .-----• • Can A o 0 PHA 21 40 63 84 131 DAYS OF TRICHINELLA SPIRALIS INFECTION FIGURE 11. The T-cell mitogen responses of spleen cells during infection with T. spiralis. Responder cells were 5 x 105 spleoncytes from uninfected C57BL/6 mice (control) or C57BL/6 mice infected for various times with 200 T. spiralis larvae. See Table 1 for calculation of % of control response. r'

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• 100 t ------------____ 0 • hO 80 -I \ .--./ 60 i \ \ • / .... CIl Z 0 p.. CIl H E-I • z 40 0 u • • Con A r:r., 0 • N 20 0 MLR 7 14 21 40 63 84 131 DAYS OF TRICHINELLA SPIRALIS INFECTION FIGURE 12. A comparison of the reduced MLR and Con A responses during infection with T. spira1is. Data are the same as that presented in Figures 10 and 11. +: N

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43 To determine if there was a decrease in the proportion of T-cells in the spleens of infected animals, the percentage of T-cells, and Bcells, in the spleens of normal and infected mice was determined in cytotoxic assays using anti-Thy-I.2 and anti-Ig antisera. As seen in Table 4, the relative proportion of B-cells and T-cells was the same following infection, even though there was an increase in the absolute number of leukocytes in the spleens of infected mice. To determine whether the decreased MLR response was due to an increased lysis of the allogeneic stimulator cells, two experiments were performed. In the first, mitomycin C-treated spleen cells from normal and Trichinella-infected C57BL/6 mice were used as stimulators in the MLR, with CBA/Ca spleen cells as the responders. As seen in Table 5, the MLR responses of normal CBA/Ca spleen cells following stimulation with spleen cells from normal and infected C57BL/6 mice were equal. In the second experiment, spleen cells from normal and infected C57BL/6 mice were incubated with 5lCr-labeled CBA/Ca stimulator cells and the amount of lysis of stimulator cells determined after 4 hours. As seen in Tahle 6, there was no difference in the release of SICr from the stimulator cells following incubation with spleen cells from normal and Trichinella-infected mice. These results indicated that the reduced MLR response was not due to a more rapid lysis of stimulator cells in cultures containing splenocytes from infected mice. Experiments were then done to determine whether the reduced MLR response of spleen cells from infected mice was due to active suppression. Mitomycin C-treated spleen cells from normal and Trichinella-infected mice were added as third party regulator cells to MLR assays of normal spleen cells. As seen in Table 7, mitomycin C-treated regulator cells

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TABLE 4 SPLEEN CELL POPULATION DURING INFECTION WITH TRICHINELLA SPIRALIS TRICHINELLA-a UNINFECTED MICE INFECTED MICE TOTAL CELLS PER SPLEEN 55.5 x 10 6 67.8 x 106 b CELL TYPE % % Mononuclear cells 100c 95c Eosinophils OC 5 c B-lymphocytes 52 3 d 48 3 d T-lymphocytes 29 7 d 23 5 d a. Spleen cells from Trichinella-infected C57BL/6 mice at 21 days of infection. b. Total spleen cell counts are the average of 5 experiments. The increase in total cell count during infection is significant to P< 0.05. c. Differential cell counts are the average of two experiments, using spleen cells pooled from 4 mice in each group. d. No significant difference between control and infected groups. 44

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EXP. NO. 1 2 3 a. TABLE 5 SPLEEN CELLS FROM C57BL/6 MICE AS STIMULATORS IN THE MLR ASSAY STIMULATOR CELL (CPM SD)a CBA/Ca UNINFECTED C57BL/6 INFECTED C57BL/6 1,370 286 12,678 1,620 l3,483 286 4,559 2,350 23,667 4,621 24,872 3,309 2,788 4,339 9,l35 1,438 10,198 1,268 Stimulator cells were 5 x 105 mitomycin C-treated spleen cells from uninfected CBA/Ca and C57BL/6 mice, and Trichinella-infected C57BL/6 mice at 21 days of infection. Responder cells were 5 x 105 spleen cells from uninfected CBA/Ca mice. Pooled spleen cells from 3 responder mice were used in each experiment. 45

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TABLE 6 RELEASE OF 51 Cr FROM 51Cr-LABELED CBA/Ca STIMULATOR C ELLS EXP. 1 EXP. 2 EXP. 3 Maximum Release a 2,784 83 8,496 218 1,878 B ackground Release b 507 29 1,220 61 2 24 Uninfected Responders c 436 31 980 39 303 Infected Responders d 456 23 1,140 46 275 a. b. Amount of 51 Cr released following addition of 0.5% NP40. Amount of 51 Cr released spontaneously. 46 94 15 18 11 c. Amount spleen of 51 Cr released following incubation of 51Cr-labeled CBA/Ca cells with spleen cells from uninfected C57BL/6 mice. d. Amount spleen mice. of 51 Cr released following incubation of 51Cr-labeled CBA/Ca cells with spleen cells from Trichinella-infected C57BL/6 Spleen cells from 4-5 mice were pooled for each experiment.

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TABLE 7 ACTIVE SUPPRESSION OF THE MLR RESPONSE IS MEDIATED BY SPLEEN CELLS FROM INFECTED MICE EXP. REGULATORa STIMULATOR CELL (CPM SD) b % CONTROLc NO. CELL TYPE CBA/Ca C57BL/6 RESPONSE 1 None 41,666 2,839 9,256 499 Control 47,710 2,829 6,508 1,703 Infected 27,845 1,762 6,282 609 52.3 2 None 64,392 4,570 12,793 1,526 Control 64,991 1,926 6,296 699 Infected 46,282 1,708 7,141 2,356 66.7 3 None 33,816 3,275 10,405 1,211 Control 30,574 1,628 7,898 887 Infected 22,296 3,305 7,735 1,195 64.2 4 None 11,564 1,369 4,323 906 Control 7,380 416 2,561 354 Infected 3,782 534 2,284 119 31.1 a. Regulator cells were 2.5 x 10 5 mitomycin C-treated spleen cells from uninfected (control) or Trichinella-infected C57BL/6 mice at 21 days of infection. b. Stimulator cells were 5 x 10 5 mitomycin C-treated spleen cells from allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice. c. The percentage of control response was calculated as described in Table 1. 47 Responder cells were 2.5 x 10 5 spleen cells from uninfected C57BL/6 mice. Spleen cells from 3-7 mice were pooled for each group in each experiment.

PAGE 58

from normal mice showed little or no effect on the response of normal spleen cells. Regulator cells from Trichinella-infected mice, however, caused a significant suppression of the response of normal spleen cells. Figure 13 shows that the active suppression had similar kinetics to the reduced response following infection. Experiments similar to those performed to demonstrate active suppression in the were performed to determine whether active suppression was also responsible for the reduced Con A response during infection. Interestingly, in 3 experiments, no evidence for an active suppression mechanism could be demonstrated. 48 The above experiments indicate the presence of a cell in the spleens of infected mice that is capable of suppressing the response, but not the Con A response. To identify the active suppressor cell, spleen cells from infected mice were separated into B-cell and T-cell enriched fractions and each fraction tested for its ability to mediate suppression of the Nylon wool adherant and nonadherant splenocytes were obtained from normal and infected mice and used as regulators in the As seen in Table 8, both the B-cell and T-cell enriched fractions contained suppressor activity. Negative selection utilizing specific antisera plus complement was then used to enrich for B-cells and T-cells. Some cells were treated sequentially with both anti-Thy-1.2 and rabbit anti-mouse Ig plus complement to enrich for non-B, non-T cells ("null" cells). As shown in Figure 14, both B-cell and T-cell enriched fractions again contained suppressor activity, although the T-cell fraction was more suppressive than the B-cell fraction. The "null" cell fraction had only a slight, statistically insignificant, effect on the responder cells.

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• 100 K ------------(4)-\ (2) 80 -I / (5) • tI) (5) \-z 0 P-< tI) 60 p:: H 0 p:: E-I (8) z 0 u 40 0 20 7 14 21 40 63 84 131 DAYS OF TRICHINELLA SPIRALIS INFECTION FIGURE 13. Spleen cell-mediated active suppression of the MLR during infection. The influence of mitomycin C-treated third party regulator spleen cells from C57BL/6 mice infected with 200!. spiralis larvae for various times in a control MLR is compared to the influence of third party regulator cells from uninfected mice. An. equal number of responder cells and regulator cells (2.5 x 10 5 ) was added to 5 x 105 mitomycin C-treated stimulator cells. Numbers in parenthesis indicates the number of experiments performed at each time point. See Table 7 for calculation of percentage of control response. +:\.0

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50 TABLE 8 SUPPRESSION OF THE MLR RESPONSE MEDIATED BY NYLON WOOL-ADHERANT AND NONADHERANT SPLENOCYTES RESPONDERa REGULATORb STIMULATOR CELL (CPM SD)c % CONTROLd CELL TYPE CELL TYPE CBA/Ca C57BL/6 RESPONSE Control None 17,854 1,646 2,849 243 Infected None 10,882 1,503 5,152 563 38.2 Control Control 19,807 1,089 3,423 432 109.2 Control Infected 7,506 779 2,654 570 33.0 Control Infected-nonadherant 7,378 518 2,818 1,575 30.4 Control Infected-adherant 7,262 686 1,841 331 36.2 a. Responder cells were 2.5 x 105 spleen cells from uninfected (control) or Trichinella-infected C57BL/6 mice at 21 days of infection. b. Regulator cells were 2.5 x 105 mitomycin C-treated spleen cells that were unfractionated from uninfected or Trichinella-infected C57BL/6 mice, or nylon woo1-adherant and nonadherant cells from infected mice. c. Stimulator cells were 5 x 105 mitomycin C-treated spleen cells from allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice. d. The percentage of control response was calculated as described in Table 1. Spleen cells were pooled from 4 mice for this experiment. This experiment is on of three which gave qualitatively identical results.

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51 100 80 (/) z 0 p... (/) 60 . H 0 e--z 0 .. u r.x. 0 40 20 . '. : Con-Spl Tsp-Spl Tsp-T Tsp-B Tsp-Null SUPPRESSOR CELL TYPE FIGURE 14. Identification of the MLR suppressor cell by negative selection using specific antisera plus complement. Responder cells were 2.5 x 105 splenocytes from uninfected C57BL/6 mice. Regulator cells were 2.5 x 105 unfractionated spleen cells (Spl), or spleen cells treated with anti-Ig plus complement (T), anti-Thy-l.2 plus complement (B) or both anti-Ig and anti-Thy-l.2 plus complement (Null) from uninfected C57BL/6 mice (Con) or C57BL/6 mice infected 21 days previously with 200 I. spiralis larvae (Tsp). Stimulator cells were 5 x 105 mitomycin C-treated allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 7 for calculation of percentage of control response. Data are the average of three experiments.

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A more precise identification of the suppressor T-cell was accomplished through the use of monoclonal anti-Lyt antibody. As seen if Figure 15, treatment of third party regulator cells with anti-Lyt-2 plus complement removed essentially all suppressor activity, while treatment with anti-Lyt-l plus complement had essentially no effect. These experiments indicated that the suppressor T-cell found in the spleens of infected mice had the Lyt 1-,2/3+ surface phenotype. To ascertain whether suppression could be mediated by soluble factors secreted by the suppressor cells, supernatant fluid was re-moved from the MLR cultures of Trichinella-infected mouse spleen cells responding to allogeneic cells and added to the MLR assays of spleen cells form normal, uninfected mice. As seen in Table 9, supernatant fluids from the MLR cultures of splenocytes from infected mice had no effect on the response of normal spleen cells. Alterations of Cell-Mediated Lympholysis (CML) Activity 52 To determine whether alterations in the effector arm of the allo-graft rejection response occur during infection, spleen cells from normal and Trichinella-infected mice were sensitized in vitro to allogeneic (CBA/Ca) spleen cells and then tested for their ability to 51 mediate target cell lysis in chromium release assays using Cr-labeled L-929 cells as target cells. Maximum cytolytic activity was developed in the MLR after 7 days. As seen in Figure 16, there was no difference in the cytolytic ability of spleen cells from Trichinella-infected mice compared to spleen cells from normal, uninfected mice. Changes in Splenic T-Cell Subsets The relative proportions of T-cell subsets, as defined by the Ly-l and Ly-2 surface antigens, was determined for uninfected C57BL/6 mice

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53 ,.. 100 80 .. Cf.) z 0 p... r Cf.) 60 E-< Z . 0 U 0 40 .. 20 f Con-Spl Tsp-Spl . + + Tsp-Ly-l Tsp-Ly-23 SUPPRESSOR CELL TYPE FIGURE 15. Identification of the MLR suppressor cell by negative selection using anti-Ly antisera plus complement. Responder cells were 2.5 x 105 spleen cells from uninfected C57BL/6 mice. Regulator cells were 2.5 x 105 spleen cells treated with anti-Ly-2 plus complement (Ly-l+), anti-Ly-l plus complement (Ly-23+) ,or unfractionated spleen cells (Spl) from uninfected C57BL/6 mice (Con) or C57BL/6 mice infected 21 days previously with 200 T. spiralis larvae (Tsp). Stimulator cells were 5 x 105 mitomycin C-tr;ated allogeneic (CBA/Ca) or syngeneic spleen cells. See Table 7 for calculation of percentage of control response. Data are the average of three experiments.

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54 TABLE 9 LACK OF SUPPRESSION BY SUPERNATANT FROM "REDUCED" MLR CULTURES RESPONDER CELLa STIMULATOR CELL (CPl1 SD) b % CONTROLc CBA/Ca C57BL/6 RESPONSE Control 15,095 2,042 6,717 560 Infected 7,981 857 5,119 695 34.2 Control + Supernatant from d Infected Culture 15,549 1,408 6,210 360 111.5 a. Responder cells were 5 x 105 spleen cells from uninfected (control) or Trichinella-infected C57BL/6 mice at 21 days of infection. b. Stimulator cells were 5 x 105 mitomycin C-treated spleen cells from allogeneic (CBA/Ca) or syngeneic (C57BL/6) mice. c. The percentage of control response was calculated as described in Table 1. d. Supernatant from infected cultures is tissue culture media from an MLR assay of spleen cells from Trichinella-infected C57BL/6 mice in response to allogeneic (CBA/Ca) stimulation, removed after 24 hours of incubation and added at a 1:1 dilution (50% final volume). These data are from one of three experiments giving the same qualitative results. Spleens from 3-7 mice were pooled for each group in each experiment.

PAGE 65

Cf.) H Cf.) :>-< H U H H U iJ;:I p.. Cf.) 50 40 30 20 10 o CONTROL INFECTED 0--0--0-0 1 2 4 8 10 25 50 100 EFFECTOR TO TARGET CELL RATIO FIGURE 16. Cell-mediated lympholysis (CML) mediated by spleen cells from Trichinella-infected C57BL/6 mice. Effector cells were sensitized in MLR cultures against CBA/Ca (H-2k) spleen cells and tested for CML ability on 51Cr-labeled L-929 (H-2 k ) target cells. Effector cells were from uninfected C57BL/6 mice (CONTROL) or C57BL/6 mice infected 21 days previously with spiralis larvae (INFECTED). The graph is data from one of three experiments giving nearly identical results. Percent of specific lysis was calculated as described in the Materials and Methods. 55

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56 and for C57BL/6 mice at 21 days of infection with !. spiralis. As seen in Table 10, during infection there was a consistent decrease in the percentage of T-cells that were sensitive to mono clonal anti-Ly-l anti-body plus complement, without change in the percentage of cells sensitive to anti-Ly-2 antibody plus complement. In a single exepriment (experi-ment 3, Table 10) there were fewer cells from infected mice that were susceptible to lysis by simultaneous treatment with both antibodies. Together these data indicate that there is a decrease in the relative + proportion of Ly-l T-cells, but whether this reflects a conversion of + -Ly-l ,2/3 T-cells to blast cells (which cannot be lysed with these antibodies plus complement) or a conversion or Ly-l+,2/3+ T-cells to -+ Ly-l ,2/3 T-cells is not clear. Delayed-Type Hypersensitivity (DTH) to Allogeneic Cells To evaluate the DTH responsiveness of Trichinella-infected mice, uninfected C57BL/6 mice and C57BL/6 mice infected for 21 days with T. spiralis were sensitized with allogeneic (CBA/Ca) spleen cells and challenged 6 days later. The results (Table 11) show that there was no difference in the DTH response, as measured by footpad swelling, of control and infected mice. The DTH responsiveness of spleen cells from infected mice was also tested by injecting challenge cell together with sensitized spleen cells from control and infected mice into the footpads of uninfected mice. The results (Figure 17) were suggestive of a depressed DTH response of spleen cells from infected mice, but were not statistically significant (P = 0.17). Two of four mice re-ceiving sensitized spleen cells from control mice showed significant DTH responses, while none of five animals receiving sensitized spleen cells from infected mice showed significant footpad swelling.

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TABLE 10 CHANGES IN SPLENIC T-CELL SUBSETS EXP. TREATMENT % OF CELLS KILLEDa NO. CONTROL INFECTED 1 C,b 6.3c 7.2 anti-Ly-l + C' 76.1 62.3 anti-Ly-2 + C' 28.8 28.2 anti-Thy-l.2 + C' 84.1 82.7 2 C' 3.8 6.1 anti-Ly-l + C' 63.8 55.1 anti-Ly-2 + C' 34.7 35.2 anti-Thy-1. 2 + C' 81.9 80.8 3 C' 4.5 5.9 anti-Ly-l + C' 71.9 59.1 anti-Ly-2 + C' 29.7 31. 9 anti-Thy-1. 2 + C' 83.4 82.7 anti-Ly-l plus anti-Ly-2 + C' 79.6 68.2 a. Target cells were nylon wool-enriched splenic T-cells from uninfected (control) and Trichinella-infected C57BL/6 mice at 21 days of infection. b. C' is rabbit complement at a final dilution of 1:40. c. At least 300 cells were counted for each determination. 57

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TABLE 11 DTH RESPONSE TO ALLOGENEIC CELLS GROUP STATUS OF RESPONDING MICE FOOTPAD S H ELLI N G (mm SD)a 6-HOUR SHELLIN G 24-HOUR SWELLING 1 Unsensitized, control C57BL/6 mice 0.07 0.04 0.05 0.06 2 Allosensitized, control b C57BL/6 mice 0.09 0.06 0.32 0.11 3 Allosensitized, Trichinella-c infected C57Bl/6 mice 0.06 0.04 0.34 O.lOd a. Footpad swelling was measured after the injection of 5 x 106 allogeneic (CBA/Ca) spleen cells contained in 0.05 ml. b. Allosensitized C57BL/6 mice were injected with 30 x 106 allogeneic (CBA/Ca) spleen cells subcutaneously 6 days prior to footpad challenge. c. Infected C57BL/6 mice were at 21 days of infection at the time of allosensitization. d. There was no significant difference between group 1 and group 2. The measurements of 5 mice were averaged for each group_ 58

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0.5 0.4 ........ '--' 0 Z H H 0.3 H ;3 Cf) p.., E-< 0 0 0.2 0.1 -. -L(1) Control C57BL/6 + CBA/Ca ..... (2) Infected C57BL/6 + CBA/Ca I(3) Control C57BL/6 + C57BL/6 _L... (4) Infected C57BL/6 + C57BL/6 59 T .L (5) CBA/Ca FIGURE 17. Adoptive transfer of DTH responsiveness. Control and Trichinella-infected C57BL/6 mice were sensitized by subcutaneous injection of 30 x 10 6 allogeneic (CBA/Ca) spleen cells. Six days after allosensitization, 4 x 10 6 spleen cells from allosensitized control and infected mice were transferred with 4 x 106 allogeneic (CBA/Ca) or syngeneic spleen cells into the footpads of uninfected C57BL/6 mice. The 24-hour footpad swelling, measured to the nearest 0.01 mm., of 5 mice was averaged for each group. There was no significant difference (P = 0.17) between group 1 and group 2.

PAGE 70

Effect of Adoptively Transferred Cells on Allograft Rejection -+ To determine whether the Ly-l ,2/3 T-cells from infected mice which are capable of actively suppressing the MLR response were also capable of impairing the allograft rejection response, adoptive trans-fer experiments were performed. As predicted (Table 12), there was no difference in the time required for complete graft rejection be-tween mice receiving spleen cells from uninfected mice or Trichinella60 + -infected mice, or splenocytes from infected mice enriched for Ly-l ,2/3 + or Ly-l ,2/3 T-cells.

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1-2. 3. 4. 5. 6. SOURCE OF a DONOR CELLS TABLE 12 EFFECT OF ADOPTIVELY TRANSFERRED CELLS ON ALLOGRAFT REJECTION TIMES OF INDIVIDUAL GRAFT REJECTION (DAYS) Control, Spleen h 16,17,17 Infected, Spleen 14,15,16 Infected, B-cells 14,17,18 Infected, T-cells 14,14,19 Infected, Ly-l + T-cells 14,16,18 Infected, + Ly-2/3 T-cells 14,15,16 AVERAGE TIME OF REJECTION 16.7 15.0 16.3 15.7 16.0 15.0 a. Donor cells (4 x 106 ) from uninfected (control) or Trichinellainfected CS7BL/6 mice were injected I.V. into uninfected CS7BL/6 mice immediately prior to placement of allograft from CBA/Ca mice. h. Transferred cells were spleen cells that were unfractionated (spleen), treated with anti-Thy-l.2 plus complement (B-cells), filtered over nylon (T-cells), treated with anti-Ly-2 plus complement (Ly-l ) or treated with anti-Ly-l (Ly-2+). 61

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DISCUSSION Infection with Trichinella spiralis results in a \vide range of alterations in host immune responsiveness, including suppression and enhancement of both humoral and cellular immune functions. Little is known, however, concerning the underlying mechanisms of these altered immune responses. This research was to determine the mechanism(s) of an impaired cell-mediated immune function, the allograft rejection response, which has been reported in Trichinella infections by others (43,53,54) . At least two points relevant to the observation that mice in-fected with T. spiralis do not reject skin allografts as rapidly as uninfected mice, have been established by this research. First, the + -specific "defect" appears to be in the ability of the Ly-l ,2/3 T-cells to respond to allogeneic cells, and not in the ability of the -+ Ly-l ,2/3 cytotoxic T-lymphocytes (CTL's) to effect lysis of allo-geneic target cells. + Second, the reduced responsiveness of the Ly-l , 2/3-T-cells is at least in part, to the suppressive effect of -+ Ly-l ,2/3 T-cells. Together, these results indicate that the impaired allograft rejection response of Trichinella-infected mice results from an active suppression of those T-cells which are responsible for the recognition of allogeneic cells. The MLR and CML assays were used to study the recognition and killing pahses, respectively, of the allograft rejection response of mice after infection with Trichinella spiralis, with the response of 62

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splenocytes to the T-cell mitogens Con A and PHA used as an additional indicator of altered T-cell function. The basic findings w ere that 63 the response to allogeneic cells in the MLR (Table 1) and the response to Con A (Figure 8) were reduced following infection, while the response to PHA was unaltered (Figure 9) and there was no detectable change in the ability of splenocytes to effect lysis of allogeneic target cells in the CML assay (Figure 16). The reductions in MLR and Con A responsiveness were transient and showed strikingly similar patterns, in terms of duration and magnitude, of suppression (Figure 12). The fact that the response to Con A was significantly reduced (to 30 % of the control response at 14 days of infection), while the response to PHA was not affected, clearly indicates that the infection does not affect all T-cell subsets equally. This observation is in agreement with several other reports of parasite-induced reduction of mitogen responsiveness. Barriga (57) reported similar results after injecting a soluble extract of !. spiralis into mice. He found that spleen cells from mice injected with an extract of T. spiralis showed a "severe" depression in Con A responsiveness and only a "slight" depression in PHA responsiveness. Jones (41) also reported a greater depression of the response to Con A than to PHA following infection with!. spiralis in mice. A similar dichotomy in the reduction of T-cell mitogen responses has been observed in baboons infected with schistosomes. Cottrell al. (88) found that serum from infected baboons dramatically suppressed both the MLR and Con A responses of lymphocytes from uninfected animals, while the inhibition of the PHA response was only marginal.

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64 One possible explanation for the reduced Con A response in the absence of any significant reduction in the PHA response is that immune complexes are formed during the course of infection. Suppression of the proliferative response of mouse spleen cells to Con A by immune complexes, in the absence of any effect on the response to PHA, has been reported by Ryan al. (89) and confirmed by Stout and Herzenberg (90). Immune complex-mediated suppression probably results from an interaction of the Fc portion or the immunoglobulin in the complex with an appropriate Fc receptor (FcR) on the T-cell. The differential effect of immune complexes on the Con A and PHA responses may be explained by the observation of Stout and Herzenberg (90), which was that the response to Con A was a characteristic of the FcR+ T-cells, while the removal of + the FcR T-cells had no effect on the response to PHA. Since there is no direct correlation between mitogen responsiveness and the Ly pheno-type of T-cells, it is not possible to relate the reduced Con A response to any particular subset of T-cells, as defined by the Ly surface anti-gens. The fact that spleen cells, and lymph node cells, from Trichinella-infected mice do not respond as well to allogeneic stimulation in the MLR as do control cells is additional evidence that the cell-mediated immune system is impaired during infection. It is well known that the activity of lymphocytes in vitro is greatly influenced by the cellular concentration, and at least one report (91) suggests that most experi-ments showing helper and suppressor activities are simply artifacts of varying cell concentrations. It was found, however, that the response of spleen cells from Trichinella-infected mice was reduced over a wide range of responder cell to stimulator cell ratios (Figure 3), indicating

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65 that the reduced MLR response observed in this study is not an artifact of cell density differences. Cells from infected mice also failed to achieve the magnitude of the response of control cells when incubated for shorter or longer periods of time (Table 2), ruling out the possibility that the reduced response was simply due to a change in the timing of optimal responsiveness. In this study, three possible mechanisms which might explain the reduced MLR responses during!. spiralis infection were considered: 1) a decrease in the relative proportion of T-cells, or MLR-reactive cells, in the spleens following infection, 2) activation of suppressor cells which depress the MLR response and 3) preferential lysis of the allogeneic stimulator cells when incubated with spleen cells from the infected mice. An overall decrease in the proportion of T-cells in the spleen following infection was unlikely. Firstly, there was a marked decrease in the response to Con A with no reduction in the response to PHA. This would not be expected if there was an overall decrease in the frequency of T-cells. Secondly, nylon wool-purified T-cells from infected mice were also less responsive in the MLR than were purified T-cells from uninfected mice (Table 3). By using equal numbers of purified T-cells, it should have been possible to overcome any effect due to a simple decrease in the proportion of T-cells in the spleen during infection. Lastly, there was no change in the percentage of T-cells and B-cells in the spleen, following infection, when quantitated directly in cytotoxic assays using anti-mouse Ig and anti-Thy-I.2 (Table 4). These results are in agreement with Ljungstrom (59) and Jones (41), who

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66 also reported no change in the relative proportions of T-cells and B-cells following infection with!. spiralis in mice. These experiments have clearly ruled out the possibility that the reduced MLR response following infection is simply due to a decrease in the relative proportion of T-cells in the spleen. However, they do not reveal whether any shift in the relative proportions of the various T-cell subsets has occurred. The possibility of a shift in the relative proportions of T-cell subsets occurring during infection was suggested by two observations. First, as already discussed, there was a significant depression of the response to Con A with no apparent effect on the response to PHA. Since no evidence for an active suppression of the Con A response could be demonstrated, the reduced response may reflect a decrease in the frequency of Con A, but not PHA, responsive cells during infection. Second, a similar dichotomy of T-cell responsiveness was observed when using Trichinella-infected BlO.AQR mice as the source of responder cells in the MLR. By using select strains of congeneic mice, it was possible to induce a MLR response to defined regions of the H-2 complex. Once again, it was observed that not all T-cell subsets were affected equally by the infection. The H-2K/D responsive T-cells were significantly less responsive than were the H-2I/S responsive T-cells (Figure 5). + It is fairly well established that in the both the Ly-l and + the Ly-2 T-cells contribute to the proliferative response (92,93,94), however, when there is complete disparity at the MHC, as was the case in the experiments of this dissertation, the proliferating cell type is + + + predominantly the Ly-l ,2/3 T-cell (94,95). The Ly-l ,2/3 T-cells are the major responding cell type when responders and stimulators differ

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67 -+ only at the H-2K or H-2D region (94,95), while the Ly-l ,2/3 T-cells appear to contribute little, if at all, to proliferation in the MLR (95). Thus, the impaired MLR response following infection with T. spiralis may be partially due to a relative depletion of the Ly-l+,2/3+ T-cells, and almost assuredly reflects a hyporesponsiveness of the + -Ly-l ,2/3 T-cell subset. The most direct, yet still inconclusive, evidence for a shift in the relative proportions of T-cell subsets was the quantitation of the percentage of splenic T-cells expressing the Ly-l and Ly-2 surface an-tigens. There was a consistent decrease in the percentage of T-cells expressing the Ly-l antigen, with no change in the percentage of T-cells expressing the Ly-2 antigen (Table 10). This decrease in the frequency + + of Ly-l T-cells could result from a conversion of Ly-l T-cells to blast cells, which cannot be lysed using the monoclonal antibodies with complement; or it may reflect a conversion of the Ly-l+,2/3+ T-cells to -+ Ly-l ,2/3 T-cells, which is consistent with current dogma envisioning the Ly-l+,2/3+ T-cell as a precursor of Ly-l-,2/3+ T-cells (96,97). In third party mixing experiments, mitomycin C-treated spleen cells from Trichinella-infected mice were capable of actively suppressing the MLR response of control spleen cells (Table 7). This indicates that the reduced MLR response of splenocytes from infected mice is also due, at least in part, to an active suppression mechanism. Suppressor cells were enriched by treatment with anti-mouse Ig plus complement (Figure 14) and were completely abrogated by treatment with anti-Ly-2, but not anti-Ly-l, plus complement (Figure 15). Thus the suppressor cell was -+ identified as a Ly-l ,2/3 T-cell.

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68 Another explanation for the reduced MLR response and the active suppression, and which has precedence, is that spleen cells from infected mice may be lysing the allogeneic stimulator cells. Sugarbaker and Natthews (98) have demonstrated that cytotoxic cells can cause a suppres-sion of mixed leukocyte cultures through lysing the stimulator cells. In their experiments, the cytotoxic cells were identified as T-cel1s. To test for the possibility that stimulator cell lysis may be responsible for the reduced MLR response following infection, mitomycin C-treated spleen cells from normal and Trichinella-infected C57BL/6 mice were used as stimulators in the MLR, with CBA/Ca spleen cells as the responders. No difference was noted in the NLR response when the stimulator cells were from normal and infected C57BL/6 mice (Table 5), indicating that spleen cells from infected mice did not lyse allogeneic cells more rapidly than spleen cells from control mice. In more direct experiments, mitomycin C-treated CBA/Ca stimulator cells were internally labelled W'th 51Cr, and the f 1 11 1 . d t . d b amount 0 ator ce e ermlne y measur-51 ing the release of Cr after incubation with responder cells from nor-mal and infected mice. Again, there was no evidence of a more rapid lysis of stimulator cells when incubated with spleen cells from infected mice (Table 6). Cell-mediated 1ympho1ysis (CML) assays comparing spleen cells from normal and Trichinella-infected C57BL/6 mice showed that there was no alteration in the cytolytic activity of splenic T-ce1ls during infection (Figure 16). These results are similar to those of Hathcock (personal communication), who found that spleen cells from Trichinella-infected C57BL/lOSn (H-2 b ) mice, sensitized either in vitro or in vivo, to B10.BR (H-2 k ) spleen cells, showed a slight, but statistically insignificant,

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69 51 increase in the CML activity when tested in Cr release assays on RDM4 k (H-2 ) target cells. These results indicate that there is no effect on the ability of the Ly-l-,2/3+ CTL's to lyse allogeneic target cells following infection. After Bach and Hirschhorn (99) first described the MLR as an in vitro correlate of the allograft rejection response, it became apparent that distinct subsets of T-ce1ls were required to interact in order to generate cytotoxic T-cells (CTL's) in vitro. Bach al. (100) later showed that the cell population comprising the CTL's could be physically separated from the MLR responsive cells using mono1ayers of adherant allogeneic cells. Thus, it became established that distinct populations of T-1ymphocytes were responsible for the recognition and killing phases of cell-mediated immunity. The MLR was accepted as the in vitro corre-late of the recognition phase of allograft rejection, and was primarily + -a function of the Ly-1 ,2/3 T-cel1s, while the CML assay was accepted as the in vitro correlate of the killing phase, and was generally medi-+ ated by Ly-l ,2/3 T-cel1s. One exception to this scheme is that CTL's in mice possessing the Ly_1 a , Ly_lb and Ly_3 b alleles are Ly-l+,2/3+ T-ce1ls (101). In spite of the numerous observations in vivo, and the many experi-ments in vitro, that supported the view that the generation of CTL's in vitro was an appropriate model for studying the cellular events that occur during allograft rejection, there was actually no direct proof that CTL's played any role in the rejection process. In a recent study Loveland a1. (87) have shown that the Ly-2/3+ T-cells, in fact, played no role in the rejection of skin allografts in CBA/H mice that

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70 were thymectomized as adults, lethally irradiated, bone marrow-reconsti-tuted and then repopulated with sensitized lymphocy t e s depleted of + + + + Ly-l , Ly-2 or Ly-l ,2/3 T-cel1s. It was clearly demonstrated that + the rejection of skin allografts was entirely dependent on the Ly-1 , 2/3-T-ce11s, and at the same time, entirely independent on the presence + of Ly-2/3 cytotoxic T-cel1s. This work is supported by that of Hube r and Cantor (102), who demonstrated nearly identical results when trans-ferring nonsensitized lymphocytes into "B mice" that had a surviving skin allograft still in place. -+ The transfer of Ly-1 ,2/3 T-ce11s into the skin graft-bearing animals did not result in any evidence of graft + -rejection, whereas the transfer of Ly-1 ,2/3 T-cel1s did lead to graft rejection. In addition to identifying the Ly-1+,2/3-T-cell as the mediator of graft rejection, Loveland et a1. (87) found that there was a perfect correlation between graft rejection and the DTH response, as measured by footpad swelling. The studies of Loveland a1. (87) cast serious doubt on the validity of the current dogma that graft rejection is effected by cyto-lytic T-ce11s, and that the generation of CTL's in vitro is an appro-priate model to study the graft rejection response. These new data indicate, in fact, that graft rejection is actually another manifestation of delayed-type hypersensitivity. + -The observed suppression of Ly-1 ,2/3 -+ T-cell function in the absence of any alteration of Ly-l ,2/3 T-cell function in this dissertation research is consistent with the hypothesis that graft rejection is mediated by the Ly-1+,2/3-T-ce11s. In light of the fact that skin allograft rejection appears to be + -+ mediated by Ly-1 ,2/3 (DTH) T-ce11s, and not the "classic" Ly-1 ,2/3

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71 CTL's, it is perhaps most important to consider what cells have been found capable of suppressing DTH responses. + -In fact, both Ly-l ,2/3 -+ and Ly-l ,2/3 T-cells have been found capable of suppressing DTH responses. Thompson al. (103) typed the T-cell which suppressed the DTH response of CBA/H mice to SRBC as Ly-l+,2/3-, while Huber al. (104) typed the T-cell which suppressed the DTH response to the same -+ antigen in C57BL/6 mice as Ly-l ,2/3. Not to be outdone, Benacerraf and colleagues (105,106,107) have been characterized the suppression of DTH to the azobenzenearsonate (ABA) determinant in A/J mice, and found + a complex set of interactions involving three sets of T-cells; a Ly-l , + + -+ 2/3 (TSl) T-cell, a Ly-l ,2/3 (T S2 ) T-cell and a Ly-l ,2/3 (TS3) T-cell. The development of suppressor activity is induced by intra-venous injection of ABA-conjugated spleen cells, which induces TSI to secrete a soluble factor (TsFl), which bears both I-J determinants and ABA-antigen binding activity. The TsF l activates TS2 cells to secrete TsF2 , which has anti-idiotypic binding activity and activates the final suppressor cell, the TS3 T-cell. The TS3 T-cell, once activated, acts in a nonspecific manner to suppress the DTH response irrespective of antigen specificity. The identification of the MLR suppressor T-cell in this study as a Ly-l-,2/3+ T-cell is not unexpected, based on findings of suppressed DTH responses and MLR responses reported by others. The most exten-sively studied MLR suppressor T-cell system is that of S.S. Rich, R.R. Rich and their colleagues (108,109,110). They have shown that allosen-sitized T-cells, upon restimulation with allogeneic cells, secrete an antigen-specific molecule which actively suppresses proliferation of lymphocytes in the MLR assay. This molecule lacks conventional Ig

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72 determinants and suppresses the proliferative response in a genetically restricted manner. Unfortunately, the Ly-l phenotype of the suppressor + T-cell has not been determined, although it is known to by Ly-2/3 (personal sornrnunication with S.S. Rich). In a similar situation, Bluestone al. (111) characterized spleen cells from tumor-bearing + mice which suppressed the MLR response as Ly-2/3 T-cells, but again the Ly-l phenotype was not determined. Interestingly, in the MLR suppressor T-cell system of the Riches, the only strain of mouse examined and found not to produce a soluble MLR suppressor factor was the C57BL/6 strain (112). This may account for the inability to detect any evidence for a soluble mediator of MLR suppression in this study (Table 9). If impaired allograft rejection during Trichinella infection does + -result from the suppression of the Ly-l ,2/3 (DTH) T-cells, then it might be expected that infected mice would not mount as strong a DTH response to allogeneic cells as would uninfected mice. A test of this prediction showed, in fact, that the DTH response, as measured by foot-pad swelling, to allogeneic cells appeared to be unaltered (Table 11). However, a suppressed DTH response might be masked by the parasite-induced inflammatory response in the footpads of mice during infection. The DTH reactivity of lymphocytes was then tested by transferring allo-sensitized cells, from infected and uninfected mice, into the footpads of uninfected mice. The purpose of transferring the cells was to remove the DTH response from the environment of the parasite-induced inflarnrna-tory response. Although not statistically significant (P = 0.17), the results (Figure 17) suggested that the DTH response of spleen cells from infected mice was, in fact, suppressed relative to uninfected controls.

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73 While this result seems contrary to the enhanced DTH response of Trichinella-infected mice to BCG reported by Molinari and colleagues (38,39, 40), or the unaltered DTH response to SRBC and oxazalone reported by Jones (41), it is consistent with the hypothesis that impaired allograft rejection results from suppression of the T-cells which mediate DTH responses to alloantigens.

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CONCLUSION An impairment of the mixed leukocyte reactivity and Con A re-sponsiveness, but not the CML activity and FHA responsiveness, was demonstrated during infection with T. spiralis, and the kinetics of the reduced responses characterized. An active, cell-mediated sup-pression of the response, but not the Con A response, was demon-strated. It is hypothesized that the suppression of the Con A re-sponse, in the absense of any effect on the FHA response, reflects the known ability of immune-complexes to preferentially suppress the Con A response. An active suppression of the MLR response during -+ infection was ascribed to the activity of Ly-l ,2/3 T-cells. No evidence for a soluble mediator of MLR suppression was found, imply-ing that cell-to-cell contact is required to effect suppression. The data suggested, but did not prove, that there is a shift in the rela-tive proportions of subsets during infection, with a relative increase in the frequency of Ly-l-,2/3+ T-cells. Together the results indicate that the impaired skin allograft rejection response following infection with Trichinella spiralis results from an active suppression of Ly-l+,2/3-(DTH) T-cell activity -+ by Ly-l ,2/3 suppressor T-cells. Whether this results from an increase in the proportion of Ly-l-,2/3+ T-cells during infection, or from an activation of preexisting Ly-l-,2/3+ T-cells is not established. 74

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40. Molinari, J.A., R.H. Cypess, and B.N. Appel. 1975. Effect of infection with Trichinella spiralis a nd BCG on thymic histology. Int. Arch. Allergy Appl. Immun. 776-783. 78 41. Jones, J.F. 1977. Mechanisms of altered immune responsiveness in mice infected with Trichinella spiralis. Ph.D. dissertation, Uni versity of Florida. 42. Ljungstrom, I. 1980. Studies on the responsiveness of spleen cells to various polyclonal T and B cell activators during Trichinella spiralis infection. Immunol. Parasit. 2: 111-120. 43. Ljungstrom, I., and G. Huldt. 1977. Effects of experimental trichinosis on unrelated humoral and cell-mediated immunity. Acta. Pathol. et Microbiol. Scand. 85: 131-141. 44. Lubinicki, A.S., and R.H. Cypess. 1975. Immunological sequelae of Trichinella spiralis infection in mice: Effect on the antibody responses to sheep erythrocytes and Japanese B encephalitis virus Infect. Immun. 11: 1306-1311. 45. Kilham, L., and L. Oliver. 1961. The promoting effect of trichinosis on encephalomyocarditis (EMC) virus infections in rats. Amer. J. Trop. Med. Hyg. 10: 879-889. 46. Faubert, G.M. 1976. Depression of the plaque-forming cells to sheep red blood cells by the newborn larvae of Trichinella spiralis. Immunol. 30: 485-489. 47. Jones, J.F., C.A. Crandall, and R.B. Crandall. 1976. T-dependent suppression of the primary antibody responses to sheep erythrocytes in mice infected with Trichinella spiralis. 48. Barriga, 0.0. 1975. Selective immunodepression in mice by Trichi nella spiralis extracts and infections. Cell. Immunol. 17: 306-309. 49. Ljungstrom, I., J. Holmgren, G. Huldt, S. Lange, and A-M. Svennerholm. 1980. Changes in intestinal fluid transport and immune responses to enterotoxins due to concomitant parasite infection. Infect. Immun. 30: 734-740. 50. Munoz, J.J., and R.L. Cole. 1977. Effect of Trichinella spiral is infection on passive cutaneous anaphylaxis in mice. Infect. Immun. 15: 84-90. 51. Hall, B.T., J.F. Jones, R.B. Crandall, and C.A. Crandall. 1979. Trichinella spiralis: Correlates in vitro of altered immune responsiveness in mice. Exp. Parasitol-.-47: 305-312. 52. Tanner, C.E., H-C. Lim, and G. Faubert. 1978. Trichinella spiralis: Changes caused in the mouse's thymic, splenic and lymph node cell populations. Exp. Parasitol. 45: 116-127.

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53. Svet-Moldavsky, G.J., G.S. Shaghijan, I.Y. Chernyakhovskaya, D.M. Mkheidze, T . A . Litovchenko, N .N. Ozeretskovskaya, and Z.G. Kadaghidze. 1970. Inhibition of skin allograft rejection in Trichinella-infected mice. Transpl. 2: 69-71. 79 54 . Chimyshkyan, K.L., I.K. Shkvatsabaya, H.SH. Ovumyan, V.A. Babichev, L.P. Trubcheninova, E.V. Sorokina, and G.J. Svet-Moldavsky. 1976. The effect of Trichinella spiralis on graft-versus-host reaction, transplantation immunity and antibody formation. Biomedicine 25 : 176-180. 55. Faubert, G.M., and C.E. Tanner. 1975. Leucoagglutination and cytotoxicity of the serum of infected mice and of extracts of Trichinella spiralis larvae and the capacity of infected mouse sera to prolong skin allografts. Immunol. 28: 1041-1050. 56. Barriga, 0.0. 1978. Depression of cell-mediated immunity following inoculation of Trichinella spiralis extract in the mouse. Immunol. 34: 167-173. 57. Barriga, 0.0. 1978. Modification of immune competence by parasitic infections. I. Responses to mitogens and antigens in mice treated with Trichinella spiralis extract. J. Parasitol. 64: 638-644. 58. Faubert, G., and C.E. Tanner. 1974. Enlargement of lymph nodes during infection with Trichinella spiralis: A preliminary histological study. In "Trichinellosis" (C.W. Kim, Ed.), pp. 353-366. Intext Educational Pub., New York. 59. Ljungstrom, I., and K-G. Sundqvist. 1979. Lymphocyte activation induced by Trichinella spiralis infection reflected as spontaneous DNA synthesis in vitro. Clin. Exp. Immunol. 38: 381-388. 60. Gotschlich, and G.M. Edelman. 1965. C-reactive protein: A molecule composed of subunits. Proc. Natl. Acad. Sci. U.S.A. 54: 558-566. 61. Mortensen, R.F., A.P. Osmand, and H.Gewurz. 1975. Effects of C reactive protein on the lymphoid system. I. Binding to thymusdependent lymphocytes and alteration of their function. J. Exp. Med. 141: 821-839. 62. Ting, J.P.Y., and D.F. Ranney. 1980. Selective suppression of the murine autologous mixed lymphocyte reaction by physiological concentrations of hydrocortisone. Cell. Immunol. 53: 138-150. 63. Mortensen, R.F., and H. Gewurz. 1976. Effects of C-reactive protein on the lymphoid system: II. Inhibition of mixed lymphocyte reactivity and generation of cytotoxic lymphocytes. J. Immunol. 116: 1244-1250.

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BIOGRAPHICAL SKETCH Bruce "Ted" Hall was born on July 15, 1948, in Stroudsburg, Pennsylvania. His parents moved to Santa Maria, California, in 1958 and were kind enough to take him along. He graduated from U.C.L.A. sine laude in 1972 (or maybe it was 1973) and eventually came to the University of Florida. After completing his dissertation defense he will attempt to drive his hunk-a-junk car to Washington, D.C., where he will begin postdoctoral research at the ter Reed Army Institute of Research. 84

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I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of B. Crandall, Ph. D., Chairman Professor of Immunology and Medical Microbiology have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of Catherine A. Crandall, Ph. D. Associate Professor of Pathology have read this study and that in my opinion it standards of scholarly presentation and is fully for the degree of Paul A. Klein, Ph. D. Associate Professor of Pathology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. Assistant Professor of Pathology have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of Profes?or of Immunology and.Medica1 Microbiology This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1981 (f /


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