Genetic and functional analysis of susceptibility to lupus in B6.NZMc7 congenic and subcongenic mice

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Genetic and functional analysis of susceptibility to lupus in B6.NZMc7 congenic and subcongenic mice
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GENETIC AND FUNCTIONAL ANALYSIS OF SUSCEPTIBILITY
TO LUPUS IN B6.NZMc7 CONGENIC AND SUBCONGENIC MICE












By

YING YU















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


UNIVERSITY OF FLORIDA


1998































This dissertation is dedicated
to my daughters
Jenny Yu
Julia Yu













ACKNOWLEDGMENTS


I would like to thank the chairman of my committee, Dr. Edward Wakeland, for

his valuable instruction and encouragement in helping me finish my project. I am also

grateful to my committee members, Drs. Mark Atkinson, Phil Laipis, Joel Schiffenbauer,

and Jin Xiong She, for their helpful discussion and guidance.

I also thank Drs. Laurence Morel and Chandra Mohan, for their initiation of the

project and experiments and helpful advice. I thank Dr. Mohan for help with my

dissertation and discussion. I also appreciate help from Dr. Byron Croker with histology.

I thank Jocelyn Tulsian for daily mouse care. I thank Karen Achey for help with

sequencing TGF-B. I would like to extend my special thanks to Drs. sally Litherland and

Christy Myrick for help with my dissertation.

I have enjoyed my friendship with other members of the Wakeland lab, especially

Nana Tian, Christy Myrick, Alicia Deng, Ping Yang, Guangling Wang, Eric Liang and

Kim Blenman. They have enriched my life immeasurably for the past six years. I want to

thank my mother for her encouragement and support. Lastly, I am grateful to my

husband, Fangshu Yu, for his understanding and encouraging, and for his help and love

throughout the years.














TABLE OF CONTENTS


p....


ACKNOWLEDGMENTS ...................................................................... iii

ABSTRACT ....................................................................................................................... vi

CHAPTERS

1 INTRODUCTION ............................................................................................... 1

Systemic Lupus Erythematosus (SLE) Overview ................................................ 1
The Causes of Human Systemic Lupus Erythematosus ................................. 2
New Zealand Black (NZB), New Zealand White (NZW), and New
Zealand Mixed (NZM) Mice .................................................................... 5
Genetic Analysis of Susceptibility to SLE in Murine Models ....................... 6
The Contribution of MHC and Non-MHC Genes to Murine Lupus
Susceptibility .......................................................................................... 10
Inheritance of SLE Susceptibility as a Threshold Liability ............................... 11
B Cell Activation and Tolerance in Lupus ................................................... 12
The Role of CD4 T Cells in the Pathogenesis of SLE ................................. 14
Autoantibody Production and Lupus Nephritis ............................................ 16
Rationale for This Study ............................................................................... 17

2 MATERIALS AND METHODS ................................................................. 20

M ice ................................................................................................................... 20
Genotypic Analysis ...................................................................................... 21
Measurement of Antibodies .......................................................................... 22
Flow Cytometric Assay .................................................................................. 23
T Cell Proliferation Assay ............................................................................ 24
Immunization ............................................................................................... 25
Histology ...................................................................................................... 26
Sequencing TGF-fl Coding Region ............................................................... 26
Statistics ........................................................................................................ 27










3 GENETIC DISSECTION OF PATHOGENESIS OF SLE: SLE3 ON
MURINE CHROMOSOME 7 LEADS TO A BROAD, LOW-
GRAD E AUTOIM M UNITY ................................................................. 29

Introduction .................................................................................................... 29
Results ................................................................................................................ 32
Discussion ...................................................................................................... 39

4 GENETIC DISSECTION OF SLE PATHOGENESIS:
SLE3 HAS TWO SEPARATE LOCI THAT INTERACT
SYNERGISTICALLY ............................................................................. 71

Introduction .................................................................................................... 71
Results ................................................................................................................ 74
Discussion ...................................................................................................... 78

5 SUM M ARY ..................................................................................................... 107

LIST OF REFEREN CES ................................................................................................. 112

BIOGRAPHICAL SKETCH ........................................................................................... 127













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

GENETIC AND FUNCTIONAL ANALYSIS OF SUSCEPTIBILITY
TO LUPUS IN B6.NZMc7 CONGENIC AND SUBCONGENIC MICE


By

Ying Yu

December, 1998


Chairman: Edward K. Wakeland, Ph. D.
Major Department: Pathology, Immunology and Laboratory Medicine


This study dissects the complex traits of lupus by constructing and analyzing

B6.NZMc7 congenic and subcongenic mice that bear the whole or part of NZM2410-

derived lupus susceptibility interval Sle3.

B6.NZMc7 mice had high serum IgG and IgM levels and generated spontaneous

IgM antibodies against a variety of antigens, suggesting polyclonal B cell activation.

These mice produced IgG anti-nuclear antibodies (ANA) starting at an early age. The

autoantibody titers were lower than parental NZM2410 and penetrance of this phenotype

was approximately 70%. There was no difference between male and female mice in IgG

ANA production. The humoral autoimmune phenotypes were also expressed in

heterozygous mice with delayed onset and decreased penetrance, suggesting that SLE

susceptibility is inherited in a dominant and allele-dose dependent fashion.

vi









One important finding is that Sle3, by itself, can lead to the development of

glomerulonephritis (GN). B6.NZMc7 mice developed chronic GN at a late age with about

40% penetrance. The presence of anti-dsDNA and GN was not strongly correlated. Two

out of nine GN positive animals did not produce IgG anti-DNA or anti-chromatin

autoantibodies. Other specific autoantibodies may be related to GN, and this needs further

investigation.

The underlying immunological abnormalities were also examined in these mice.

Young B6.NZMc7 mice (1-3 month) were used with age- and sex-matched C57BL/6J

(B6) mice as controls. B6.NZMc7 mice exhibited a high percentage of CD4 T cells and

an increased CD4:CD8 T cell ratio in both the spleen and lymph node. A high proportion

of CD4 T cells were activated and expressed the activation marker CD69. These CD4 T

cells expanded prominently in vitro when stimulated with anti-CD3. B6.NZMc7 mice

also showed heightened lymphocyte proliferative responses to anti-CD3 and IL-2 and

exhibited reduced activation-induced T cell death. Consistent with these results in vitro,

B6.NZMc7 mice exhibited high immune responsiveness in vivo when they were

challenged with the T-dependent antigen DNP-KLH. These results suggest that Sle3

mainly impacts T cell activation, proliferation, and apoptosis.

Two subcogenic mouse strains were also generated, one carrying the centromeric

end (B6.NZMc7c) and the other carrying the telomeric end (B6.NZMc7t) of Sle3. The

mice from both the subcongenic strains had polyclonal B cell activation, however, they

produced very low levels of IgG ANA. Penetrance of IgG ANA production is about 20%

in the subcongenic mice, compared with 70% in B6.NZMc7. These results indicate both









loci are required in IgG ANA production. Only B6.NZMc7t mice showed T cell

hyperactivity, which suggests that the gene affecting T cell function is located on the

telomeric end of Sle3. Both strains developed chronic GN, but severity is less compared

to B6.NZMc7. These results suggest that at least two susceptibility genes are present in

the Sle3 interval and that these two genes interact synergistically to cause the

autoimmune phenotypes expressed in B6.NZMc7.














CHAPTER 1
INTRODUCTION


Systemic Lupus Erythematosus (SLE) Overview



SLE is a common, chronic autoimmune disease. It is estimated that over half to

three quarters of a million people suffer from it in the United States. The clinical features

of SLE are very unique. It is a multisystemic disease with the potential to involve

multiple organs, including the skin, joints, serosal surfaces, kidney, heart, lungs, and

central nervous system. The clinical manifestations are extremely diverse depending

largely on which organ system, or systems, is affected. It is variable ranging from skin

rash and joint pain with a mild systemic illness to a fulminant presentation with life-

threatening involvement of the important organs such as kidney (reviewed in refs.1, 2).

SLE occurs mostly in females, and the female to male ratio is about 9:1.

The production of autoantibodies against a spectrum of nuclear antigens is the

hallmark of SLE both in humans and mice. The deposition of circulating antigen-

antibody complexes along the vascular basement membranes of various target organs is

one of the notable pathologic features. However, the mere presence of the autoantibody

does not necessarily lead to autoimmune disease, other immunologic abnormalities are

needed to develop full-blown clinical autoimmune disease.









No single cause has yet been identified, although genetic, environmental and

stochastic factors contribute to the altered immune state (1). The major focus of SLE

research is to understand its cause. It is hoped that the basic and clinical research will

elucidate a plausible etiology, will give direction to disease prevention, and will provide

more specific treatment of the disease.



The Causes of Human Systemic Lupus Ervthematosus



It is well documented that genetic factors are key elements involved in

susceptibility to SLE (3-7, reviewed in Refs 8-9). SLE is concordant in 57% of

monozygotic twin and 5% of dizygotic twins (10) which strongly supports the hypothesis

of a multifactorial etiology with incomplete penetrance, but the characteristics of the

inherited susceptibility factors are not known. Available data indicate the involvement of

multiple genes (10-18). The expression of such genes could involve variables influencing

immune responses, immune regulation, complement, and/or immunoglobulin levels.

Studies of SLE in humans have found several genetic markers associated with

SLE: major histocompatibility complex (MHC) class I allele, B8; MHC class II alleles,

DR2, DR3, DQwl; MHC class III alleles, C4A null phenotype, C2 deficiency.

A significant association of SLE with a particular MHC allele has been

determined. Increased frequencies of MHC class II DR2 or DR3 alleles are associated

with SLE in Caucasian patients (15-18). Further studies indicated the extended haplotype

Al-B8-DR3 is associated with SLE susceptibility (19). Griffing et al. demonstrated that










67% of SLE patients with anti-dsDNA antibodies were DR3 (20). The role of MHC class

II antigens in the pathogenesis of SLE is unknown. Two potential mechanisms are of

particular interest: (a) selective presentation of foreign or self-antigenic peptides by the

polymorphic regions of MHC class II molecules; (b) negative and positive selection of T

cells by MHC class II molecules during maturation in the thymus.

Inherited deficiencies of several complement components have been associated

with lupus-like illnesses. The most common is a deficiency of the second and fourth

components of complement (C2, C4) (11-14). The class III, or complement, region of the

human MHC is located between the class I (B, C, A) and class II (DP, DQ, DR) regions

on the short arm of chromosome 6. The four complement structural genes in this cluster

are C4A, C4B, factor B, and C2. These four components are required for activation of the

complement pathways, C4 and C2 for the classical and factor B for the alternative

pathway. One of the major biological functions of these early components of the classical

pathway, C4 and C2, is to process immune complexes. Complement system dysfunction

may contribute to impaired clearance of immune complexes (21-23).

Non-MHC linked genes were also suggested from studies of two very interesting

families with multiple instances of undifferentiated autoimmune disease and no relation

with any HLA haplotype (24). A comprehensive linkage analysis has not yet been

reported in humans to show which specific chromosomes and regions are involved. The

nature of non-MHC genes in SLE is unknown.

Lupus is a polygenic disease with added contributions from environmental and

stochastic variance. The fact that the concordance rate in identical twins is not 100% can









be used to estimate environmental and stochastic contribution (25-27). Inheritance and

the expression of susceptibility genes predispose individuals to the development of

phenotypes, but phenotypic expression requires triggering from environmental and

stochastic events. Several environmental factors including ultraviolet light, bacterial and

viral infections, and drugs are capable of inducing or exacerbating lupus. Random events

in the development of the immune system, such as the generation of T cell receptor

(TCR) repertoire, also play a role in the generation of SLE-like syndrome. It is speculated

that these factors may act through different mechanisms to alter immune function.

Susceptibility to SLE is inherited as a complex trait characterized by incomplete

concordance of phenotype and genotype. Several features of the disease make genetic

analysis of SLE susceptibility in humans very difficult. First, in a family, not all

individuals who have the same inherited susceptibility alleles manifest disease

(incomplete penetrance). Second, in different families, different sets of susceptibility

alleles may produce identical phenotypes (genetic heterogeneity). Third, several

susceptibility alleles may be required for fully expressing certain phenotypes (polygenic

inheritance).

Development of animal models for this disease can be advantagous for elucidating

the pathogenic mechanisms of the disease and will help studies in human SLE. Animals

can be bred in large quantities and manipulated for genetic analysis. They can also be

maintained in the same facility, thereby minimizing environmental factors.










New Zealand Black (NZB). New Zealand White (NZW), and New Zealand Mixed
(NZM) Mice



Several inbred mouse strains that spontaneously develop a disease similar to

human SLE have been important models (1). Over the past three decades, extensive

studies of these murine models, particularly in the area of molecular genetics, have

provided a great deal of information to help our understanding of the disease. They have

also lead us to a better understanding of human SLE and brought us closer to the ultimate

goal of unraveling the mystery of autoimmune diseases.

The mouse strains that have been extensively studied include (NZB X NZW)F1,

(NZB X SWR)F1, BXSB, and MRL-lpr/lpr (28). These strains produce high levels of

antinuclear antibodies (ANAs) and develop lupus-like glomerulonephritis (GN). The New

Zealand black (NZB) (H-2d) strain was initially bred for coat color by M. Bielschowsky

at the University of Otago Medical School in New Zealand (29). The genetic background

of this strain is unknown. NZB mice develop a disease state characterized by an

autoimmune hemolytic anemia (28, 30-31). The lupus-like nephritis is less severe in NZB

mice than in its hybrids. NZB mice produce ANAs, but not high levels of IgG antibodies

to double stranded DNA (dsDNA) or histones. The New Zealand White (NZW) (H-2z)

strain was developed by Hall at the Otago Medical School Animal Breeding Station (31),

and its genetic background is also unknown. NZW mice are, for the most part,

phenotypically normal. (NZB X NZW)F1 hybrid mice, first described by Helyer and

Howie (32), develope florid renal dysfunction and failure similar to human lupus










nephritis. The hybrid mice also develop autoantibodies to a number of nuclear

components including ssDNA, dsDNA, histone and chromatin. The F 1 hybrids of NZB

and NZW provided the first experimental model of human SLE and have been studied

extensively by a large number of investigators. Other two murine strains that also

spontaneously develop a SLE-like syndrome are MRL and BXSB. They were developed

by Murphy and Roths at the Jackson Laboratory (33-34). Addition of these strains has

greatly enhanced the potential value of murine SLE as an experimental model and has

broadened our understanding of autoimmunity.

The New Zealand Mixed (NZM)(H-2z) strains were produced and characterized

by Rudofsky (35). They are a collection of inbred strains derived from selective

inbreeding of progeny from a cross between NZB and NZW mice. These strains have

inherited various genomic segments from the two parental strains, express a board

spectrum of SLE-related autoimmune phenotypes and show different susceptibilities to

lupus. One strain, NZM2410, produces high levels of IgG autoantibodies to nuclear

antigens as early as 4-6 months of age and develops severe lupus-like renal disease in

both sexes.



Genetic Analysis of Susceptibility to SLE in Murine Models




Analysis of autoimmune phenotypes and lupus-like renal disease in (NZB X

NZW) Fl, F2 and backcrosses between NZB and NZW mice illustrates the genetic










complexity and polygenic inheritance of the disease. Earlier genetic studies had

limitations and were not able to assign chromosomal locations of susceptibility genes,

however, these classic studies still provide valuable data. Braverman (36) crossed NZB

and NZW and observed varying severities of renal histopathological changes in the F2

mice, which suggested the involvement of multiple genes from both NZB and NZW.

Knight & Adams (37-38) performed (NZB X NZW)F1 X NZW and (NZB X NZW)F1 X

NZW backcrosses and concluded that NZB contributes a single dominant gene and NZW

contributes two genes to the development of severe renal disease and death. These

investigators also concluded that one of the two NZW genes might be linked to the MHC.

Shirai and colleagues studied the genetics of (NZB X NZW)F1 traits using backcross

analyses and suggested that two dominant genes from NZB and one dominant gene from

NZW contribute to the formation of gp70-anti-gp7O immune complexes (39). Kotzin and

colleagues reported one dominant gene from NZW contributes to renal disease and

suggested a strong correlation between the NZW MHC haplotype, H-2z, and the

phenotype (40-41). Hirose and coworkers extended these observations by constructing

NZB (H-2d) and NZW (H-2z) congenic strains with the reciprocal H-2 and comparing

(NZB.H-2z X NZW)F 1 and (NZB X NZW.H-2d) F I to original (NZB X NZW)F 1. Their

results suggested that heterozygosity at the MHC might be required for disease

development (42). From these studies, two to three genes from NZB and one to two genes

from NZW were estimated to contribute to the disease. The number of genes involved

could be dependant on the particular trait or traits examined as well as the genetic

background.










Recent advances in molecular biology techniques have provided new means to

screen the whole genome for susceptibility loci and to assess the involvement of available

candidate genes. The identification of large numbers of highly polymorphic simple

sequence repeats spaced throughout the genome provide the basis for mapping studies

(43-45). The polymerase chain reaction (PCR) method makes it possible to genotype

large numbers of animals (46), and the development of sophisticated computer programs

makes data analysis quick and accurate. These techniques permit the identification of

multiple loci contributing to a given trait without requiring preexisting knowledge of

candidate loci or their positions. Several groups have used these tools to perform genome-

wide searches and to identify susceptibility loci responsible for lupus nephritis and a

variety of lupus-associated autoimmune phenotypes.

Studies of the (MRL-Ipr X CAST/EI)F1 X MRL-lpr backcross mice identified

two loci associated with renal disease, Lrdml on chromosome 7 and Lrdm2 on

chromosome 12 (47). The study of (NZB X NZW)F2 intercross mice by Kono et al. (48)

identified eight susceptibility loci (Lbwl to -8 on chromosomes 17, 4-7, 18, 1, and

11 respectively) associated with anti-chromatin autoantibody production (Lbwl, Lbw7,

Lbw8), GN (Lbw], Lbw2, Lbw6), and/or mortality (Lbwl-6). Drake et al. investigated

NZB contribution to lupus-like renal disease. Their work with (NZB X NZW)Fl X NZB

backcross showed that a single NZB locus (nbal) on chromosome 4 was associated with

renal disease and death (49). This group also performed genetic analysis in (NZB X

SM/J)F1 X NZW mice and found six NZB loci (on chromosomes 1, 4, 7, 10, 13, 19)

contributing to renal disease (50). They confirmed two loci previously identified (nbal on










chromosome 4, and nba2 on chromosome 1) and detected two new loci (on chromosomes

11 and 14) in a (BALB.H2Z X NZB)F1 X NZB cross (51).

Morel et al. (52) identified three susceptibility intervals and the MHC locus

associated with GN in the NZB/W-derived NZM2410 lupus-prone strain backcrossed to

B6. The three susceptibility loci are Slel on the telomeric end of chromosome 1, Sle2 on

the centromeric end of chromosome 4, and Sle3 on the centromeric end of chromosome 7.

Genetic analysis indicated several ways in which susceptibility genes could influence

overall disease threshold liability in lupus. First, each of these susceptibility alleles

independently contributed to the overall GN susceptibility in an additive fashion, which is

commonly described as a threshold of genetic liability. Second, several susceptibility

alleles are needed to express high frequency of GN, but none of these alleles are

absolutely required. This is an important feature of a polygenic disease. Third, although

the mice express an identical array of susceptibility genes, the proportion of NZM2410

mice developing GN and penetrance of GN increased with age, indicating that the

expression of complex traits requires "triggering events" (53).

In summary, genetic mapping studies have identified 22 susceptibility loci that are

linked to autoimmune phenotypes of lupus. These studies illustrate the complexity of the

genetics of SLE. Within each cross, some loci were associated with a single distinct

phenotype, suggesting they may act on different pathways and contribute specific

component phenotypes to disease susceptibility. Some loci contributed to multiple

phenotypes, suggesting they play roles in a common pathway linked to multiple

phenotypes. Interestingly, susceptibility loci on chromosome 1, 4, and 7 have been









reported by three groups in different crosses, indicating these susceptibility genes may be

common to the lupus pathogenesis in NZB and NZW strains. The number and location of

genes detected are different depending on which affected strains and resistant strains are

used and how they are crossed. This difference may represent the epistatic interactions of

susceptibility genes, resistance genes, and background genes. Clear demonstration of the

nature of each locus and the relationship between loci would require the production of

congenic strains carrying different single loci and polycongenic strains.



The Contribution of MHC and Non-MHC Genes to Murine Lupus Susceptibility




The contribution of certain MHC alleles to lupus susceptibility has been well

documented both in human and mouse models. Such an association is reasonable because

autoimmune diseases are T cell dependent and all T cell mediated responses are MHC

restricted. The MHC may affect predisposition to autoimmune disease by shaping the T

cell receptor repertoire, selecting and presenting peptide to T cells. In studies with crosses

of NZB X NZW or NZB X SWR mice, heterozygosity at the MHC (H-2/ or H-2/q)

conferred greater susceptibility to lupus than homozygous parental haplotypes (54-57).

Antibodies to class II molecules can prevent the development of the disease (58). Further

support for MHC class II as a disease predisposing molecule derives from a report in

which a well-studied I-AmI2 3 chain mutation was bred onto the NZB background (59).

NZB mice congenic for this class II mutation (NZB.H-2m'2) developed anti-dsDNA









antibodies and lupus-like renal disease, similar to (NZB X NZW)F1 mice, whereas NZB

mice congenic for H-2b (NZB.H-2b) did not express any signs of autoimmune disease.

The I-AP sequences of H-2"2 and H-2b differ only three amino acids in the peptide-

binding groove at positions 67, 70, and 71. This three amino acid substitution within the

MHC class II molecule is capable of altering the NZB phenotype to one of severe lupus-

like renal disease. It has been hypothesized that the novel hybrid MHC class II molecules

generated by a and P chains from different haplotype to pair contribute to autoimmunity

(60).

Although the MHC has a major impact on lupus susceptibility, it is not required

and insufficient by itself for the development of the disease. Association of H-2Z with the

disease is imperfect: 14% of heterozygous H-2z backcross mice did not develop the

disease, whereas 12% of homozygous H-2d backcross mice did (61). NZB mice crossed

with normal background BALB/c mice congenic for H-2' do not develop disease (62).

This work illustrates that both MHC and non-MHC genes are required. Genome-wide

searches using microsatellite-defined chromosomal maps have identified multiple non-

MHC loci responsible for predisposing to lupus.



Inheritance of SLE Susceptibility as a Threshold Liability



The SLE is a polygenic disease requiring multiple genes. How multiple genes

interact to cause a disease susceptibility phenotype is not known. Data from a (NZM2410

X B6)F1 X NZM2410 backcross showed that the increase in GN frequency correlated









with an increase in the number of susceptibility loci carried by individual progeny (52).

These results suggest that the disease is inherited as a threshold of genetic liability. This

conclusion is supported by work done by other groups using different backcrosses to

study lupus (50), diabetes (63), and experimental allergic encephalomyelitis (64-65). The

threshold liability hypothesis is based on the concept that several disease susceptibility

genes contribute equally to the overall liability of developing disease and that the

probability and onset of an individual developing disease is proportional to the number of

susceptibility genes inherited. We further demonstrated this relationship by analysis of

three congenic strains that contain only one susceptibility locus from NZM2410 (66).

Each congenic strain expressed distinctly different component phenotypes and multiple

genes were necessary to obtain full expression of SLE.



B Cell Activation and Tolerance in Lupus




The primary function of B lymphocytes is to make antibodies against components

of foreign organisms. Self-reactive B cells are eliminated at multiple checkpoints along

the B cell lineage (reviewed in ref. 67). Each of these checkpoints has triggering

thresholds that impose limits on the extent of tolerance. This is a kind of balance system.

On the one side, such limits ensure that a sufficient fraction of the repertoire is preserved

to mount rapid and effective antibody responses to foreign antigens. On the other side, the

same limits increase the risk of autoimmune disease. A variety of events, either










exogenous or endogenous, could tip the delicate balance so that harmful autoreactivity is

the outcome. If this imbalance is permanent, chronic autoimmune phenomena such as

SLE may occur.

The fundamental event in the pathogenesis of lupus is B cell hyperactivity, which

is responsible for the hypergammaglobulinemia, spontaneous polyclonal antibody

production, and secretion of various autoantibodies (68). Polyclonal B cell activation is

the earliest and most common immunological abnormality of NZB, (NZB X NZW)F1,

and other lupus-prone strains preceding the pathogenic autoimmune response (69-71).

The pathogenic autoantibody-producing B cells are driven by self-antigens (reviewed in

ref. 72). This subset of B cells are clonally expanded and their immunoglobulin genes are

modified by somatic mutation. IgG autoantibody production in lupus is selective for

certain self-antigens and there is strong evidence that B cells are intrinsically defective in

murine lupus-prone mice. SCID mice populated with long-term pre-B cells from (NZB X

NZW)F1 mice, but not those populated with (B6 X DBA/2)Fl pre-B cells, developed

hypergammaglobulinemia and increased IgM and IgG autoantibodies to nuclear antigens,

and 20% of these animals also developed mild nephritis (73). Further studies using pre-B

cell lines derived from the parental NZB and NZW strains suggest that B-lineage cells

from both NZB and NZW parental strains manifest abnormalities associated with the

development of lupus-like disease (74).

It is understandable that errors in central or peripheral tolerance at the T or B cell

level can cause systemic autoimmune disease. However there is scant evidence for

defects in B cell tolerance in New Zealand mice (75). Studies indicate that potentially










pathogenic B cells are part of the peripheral repertoire and they can be expanded by graft-

versus-host reactions (76-77). Normal animals can generate autoantibody responses after

immunization with self-peptides (78-80). Thus, defects in central B cell tolerance are

unlikely to be necessary to allow for lupus-like autoantibody production. On the other

hand, defects in the regulation of B cell apoptosis have the potential to lead to

development of autoreactive B cells and increase autoantibody production. There are

some reports of a defect in B cell apoptosis induction in New Zealand mice (81-82). The

importance of this factor in pathogenesis of the disease needs further study.



The Role of CD4 T Cells in the Pathogenesis of SLE




The association of SLE with particular MHC class I alleles, class switching, and

affinity maturation of autoantibody production in this disease strongly suggest that CD4

T cells are important in pathogenesis of the disease. Evidence for the involvement of CD4

T cells includes: the autoinimune condition of (NZB X NZW)Fl mice is improved

following treatment with anti-CD4 antibodies (83); both T and B cells are needed in order

to induce anti-dsDNA IgG synthesis (84); the incidence of lupus nephritis is delayed and

reduced after blocking T cell-B cell interaction (85). Certain CD4 T cells have been

shown to drive pathogenic B cells to selectively secrete anti-DNA autoantibodies in

murine models and in humans (86-89). The major subclass of pathogenic anti-DNA

autoantibodies is IgG2a and IgG2b which is also consistent with CD4 T cell help.










Although an anti-DNA response is the hallmark of lupus, efforts to induce such

autoantibodies by immunization with native (n) DNA have generally been unsuccessful.

The determinants of autoreactive CD4 T cells may reside on different molecules. CD4 T

cell clones specific for anti-nDNA antibody induction were shown to proliferate in the

presence of nucleosomes but not their constituent nDNA or histones (90). These CD4 T

cells may be specific for nucleosomes. Thus, for anti-nDNA antibody induction, CD4 T

cells may provide intermolecular help to anti-DNA specific B cells via the binding of

nucleosomes or other protein-DNA complexes.

Loss of central or peripheral T cell tolerance will cause systemic autoimmune

disease. Studies in lupus mice have suggested that high affinity responses to self-antigens

are tolerated in the thymus and normal thymic deletion of superantigen reactive V3-

bearing T cells have been observed (91-92). T cells specific for dominant determinants of

self-antigens are deleted efficiently in thymus, but T cells directed to cryptic self-

determinants may escape tolerance induction and be part of the normal peripheral T cell

repertoire (reviewed in ref. 93). Therefore, abnormal presentation of cryptic self-

determinants in the periphery or a defect in peripheral T cell tolerance in lupus could

activate autoimmune T cells.










Autoantibody Production and Lupus Nephritis




The hallmark of SLE in human and murine models is elevated serum levels of

antibodies to nuclear antigens. There is substantial evidence that anti-dsDNA antibodies

play an important role in the pathogenesis of renal disease in lupus mice. First, anti-DNA

autoantibodies are found concentrated in kidney elutes. Second, monoclonal anti-DNA

antibodies derived from lupus prone mice form glomerular immune deposits and induce

different structural and functional abnormalities in vivo after transfer into normal or

preautoimmune mice (94). Third, mouse IgG anti-DNA autoantibodies can bind to

isolated perfused kidney and induce kidney dysfunction (95). Fourth, genetic studies of

lupus mice have shown a direct relationship between anti-DNA antibody level, GN, and

mortality (96). However, not all anti-DNA autoantibodies are pathogenic. The cationic

charge, isotype, and cryoprecipitability will determine the pathogenic potential of anti-

DNA antibodies (97).

The pathogenic anti-DNA antibodies induce end-organ damage by tw

mechanisms (98). One is that anti-DNA antobodies directly or indirectly bind to the

glomerulus via heparan sulfates, the major glycosaminoglycan constituent of the

glomerular basement membrane (99-10 1). Another is that anti-DNA antobodies first form

immune complexes and then deposit onto anionic sites on glomerular basement

membrane (102-104).









Although most studies have focused only on anti-DNA antibodies in the cause of

lupus nephritis, considerable evidence indicates that autoantibodies to non-DNA antigens

are also important. Genetic studies have suggested a pathogenic role for autoantibodies to

the endogenous retroviral glycoprotein (gp70) and gp70-anti-gp7O immune complexes in

the (NZB X NZW)Fl model (105). Relatively large amounts of the retroviral

glycoprotein gp70 circulate in the blood of both lupus and non-lupus mice, but only lupus

strains spontaneously produce autoantibodies against this molecule and contain gp70-

anti-gp70 complexes in their serum (106). However, studies on sublines of MRL/lpr mice

specifically bred for low gp70 levels indicated that autoimmune disease, including GN, is

essentially unchanged (107). Thus, although gp70-anti-gp7O deposits may be involved in

the pathogenesis of lupus nephritis, they appear not to be necessary.



Rationale for This Study



SLE is a common, chronic disease. It is estimated that over half to three quarters

of a million people suffer from it in the United States alone, while in other parts of the

globe the numbers are even higher (108). The cause of SLE is unknown. The central

thrust of SLE research is to understand its cause, especially genetic predisposition. If

genes contributing to the susceptibility of SLE can been identified: (a) people with high

risk could be screened and those with susceptibility genes could be started on

preventative therapies, (b) diagnosis will be more easy and accurate, (c) treatment will be










specific according to individual genetic defects, and (d) it will help us understand the

fundamental immunology of self and non-self discrimination.

Because the inheritance of SLE is complex, identification of genes involved in

SLE susceptibility in outbred human populations will be very difficult. Animal models,

such as (NZB X NZW)F1, readily allow experimental crosses to identify susceptibility

genes. Genetic studies in murine models will help to identify SLE susceptibility genes in

humans by comparing the syntenic regions of human and mouse chromosomes and by

providing information about pathogenic pathways.

Success has been made in the identification of genes responsible for simple

Mendelian traits wherein identification is fairly straightforward: linkage analysis,

positional cloning, and detection of mutation (109). Yet many of the most important

medical conditions including heart disease, hypertension, diabetes, asthma, SLE, and

cancer are inherited as complex polygenic traits. The geneticist's challenge is now to

tease apart the multifactorial causes of these diseases.

Our lab focuses on studies of SLE using the lupus-prone murine model

NZM2410. The approaches and strategy used are reviewed by Wakeland (53). We have

identified three intervals (Slel on chromosome 1, Sle2 on chromosome 4, and Sle3 on

chromosome 7) and MHC locus, which confer strong susceptibility to GN (52).

Questions to be answered include what phenotypes each locus contributes, what is the

nature of each susceptibility gene, and how different loci interact with each other. We

analyzed congenic mice to address these questions. This research focuses on Sle3, the

lupus susceptibility interval on murine chromosome 7. B6.NZMc7 is one of four










congenic mouse strains. It carries the Sle3 interval on a resistant B6 background. Genetic

and phenotypic analysis was performed in B6.NZMc7 mice, including serologic studies,

cellular immunology studies, and histology studies (Chapter 3) to define the contribution

of Sle3 by itself and to explore the underlying mechanism by which Sle3 contributes to

lupus. Sle3 is a large interval (about 38 cM) and linkage studies suggest two susceptibility

loci may present in this interval. To explore this possibility, we produced two

subcongenic mouse strains bearing the centromeric end (B6.NZMc7c) or the telomeric

end (B6.NZMc7t) of Sle3 on a resistant B6 background (Chapter 4). These subcongenic

lines were characterized serologically, immunologically, and histologically for the

disease.














CHAPTER 2
MATERIALS AND METHODS


Mice



All mice used in this study were bred and maintained under conventional

conditions in our colony in the University of Florida Department of Animal Resources.

Breeding pairs of C57BL/6J (B6) were originally obtained from the Jackson Laboratory

and NZM2410 breeders were obtained from the Wadsworth Center for Laboratory and

Research, New York State Department of Health (Albany, New York). Both male and

female mice were used for all experiments. Animals were age- and sex-matched for all

experiments.

The B6.NZMc7 congenic strain bearing the Sle3 susceptibility interval on

chromosome 7 was one of four congenic strains generated (110). Figure 2-1 is a

schematic picture showing the breeding strategy used for the production of congenic

mice. Briefly, B6 was chosen as a resistant strain. NZM2410 was backcrossed six

successive times to B6. At each generation, progeny were selected using three simple

sequence repeats (SSR) markers (Ckmm, D7Mit69, D7Mit7O) on chromosome 7 to

maintain the Sle3 interval and two to three other markers per chromosome to minimize

background genes contamination. Finally, the mice were intercrossed to obtain the

homozygous Sle3 interval.







21

The B6.NZMc7c and B6.NZMc7t subcongenic mouse strains were produced by

crossing B6.NZMc7 heterozygotes to B6 to generate a series of recombinants.

Genotyping was performed in these recombinant progeny using SSR markers covering

the Sle3 interval. Two subintervals were chosed, one contains the centromeric end of Sle3

(from ckmm to D7Mit1 54), and other contains the telomeric end of Sle3 (from D7Mit69

to D7Mit 147). Progeny bearing these two subintervals were crossed to B6 again to

generate more animals with these specific recombinations. Finally, mice bearing these

heterozygous intervals were intercrossed to produce mice that were homozygous for these

smaller intervals.



Genotvpic Analysis



Genomic DNA was isolated from mouse tail biopsies by standard

phenol:chloroform extraction. Primers flanking SSR were purchased from Research

Genetics (Huntsville, Ala.) or synthesized at the University of Florida. Polymerase chain

reaction (PCR) technique was used for genotyping. Genomic DNA (100ng) was

amplified in 20 ul reactions with 200 nM paired primers, 0.2 mM dNTPs, and 0.75 U of

Taq DNA polymerase in a standard incubation buffer containing 50 mM MgCI2.

Amplifications were carried out in a Perkin-Elmer/Cetus 9600 thermal cycler under the

following conditions: I cycle at 940 C for 2 min; 35 cycles at 94o for 30 sec, 50-620 C for

30 sec, and 720 for 30 sec; 1 cycle at 72 0 C for 7 min. The PCR products were detected

using 5% agarose gels containing ethidium bromide. Optimal annealing temperature was







22

determined for each primer pair. Each mouse was scored as NZM2410 homozygous, B6

homozygous, or heterozygous for each locus genotyped.



Measurement of Antibodies



Serum was collected bimonthly starting at two mo. of age by tail bleeding. Serum

was tested against a panel of antigens including dsDNA, ssDNA, chromatin or

histone/DNA complex, histone, DNP-KLH, and thyroglobulin using ELISA as previously

described (66,90). Briefly, Immulon II plates (Dynatech, Chantilly, VA) were coated with

each of following antigens: calf thymus dsDNA (50ug/ml); ssDNA (50ug/ml); chromatin

(lug/ml) (prepared as previous described in ref. 111); total histone (1Oug/ml); bovine

thyroglobulin (10ug/ml); DNP-KLH (lug/ml (Sigma Chemical Co., St. Louis, MO). The

plates were blocked with blocking buffer (3%BSA, 0.1%gelatin, 3mM EDTA in PBS).

Serum from test animals was diluted 1:100 and incubated on these plates for two hours.

Specific antibodies were detected using alkaline phosphatase-conjugated goat anti-mouse

IgG or IgM (Boehringer Mannheim) and pNPP substrate. Optical density (OD) values

were obtained with an automated spectrophotometer at 405 nM. All samples were run in

duplicate. To allow for interassay comparisons, OD values were converted to a relative

unit scale (U/ml) in reference to a standard curve obtained with serial dilutions of a

NZM2410 positive serum added in each ELISA plate. Alternatively, the results are

expressed as individual percentage (%OD) (i.e., the ratio of the absorbance from the test

sample divided by the absorbance of 3 mo B6 pooled serum x 100) (112). Positive









sample serum was defined as having antibody titers above the mean titers plus two

standard deviation of five 10-12 mo-old B6 samples on the same plate.

Serum total IgG and IgM were measured using a sandwich ELISA. Briefly,

Immulon I plates (Dynatech, Chantilly, VA) were coated with capture antibodies, anti-

mouse IgM and IgG (Boehringer Mannheim), and blocked with blocking buffer described

above. IgG or IgM standards (Cappel Research) and 1:100 dilution of serum were added

for two hours. Detection of antibodies was as described above.



Flow Cytometric Assay



Flow cytometric analysis (FACS) was performed as described previously (85). All

primary antibodies were obtained from Pharmingen (San Diego, CA) and used at pre-

titrated dilution. Single cell suspensions of the spleen and lymph nodes were prepared by

gently mincing the tissues in a serum-free DMEM using the frosted ends of two sterile

glass slide. Lysis of erythrocytes was made in hypotonic, buffered ammonium chloride

solution. Bone marrow cells were obtained by washing the cells out of the femur with a

needle mounted on a 1-ml syringe. Peritoneal cells were obtained by lavage of the

peritoneal cavity with the serum-free DMEM using a 3-ml syringe. Cell surface Fc

receptors were first blocked with staining medium (PBS, 5% horse serum, 0.05% azide)

containing 10% normal rabbit serum. Cells were then stained on ice with optimized

amounts of FITC, phycoerythrin, or biotin-conjugated primary monoclonal antibodies

diluted in staining medium for 30 min. Following two washes, biotin conjugated









antibodies were revealed using streptavidin-PE (Gibco BRL, Grand Island, NY) or

streptavidin-Quantum Red (Sigma Chemicals, St.Louis, MO). Cell staining was analyzed

using a FACScan (Becton-Dickinson, San Jose, CA). Dead cells were excluded on the

basis of scatter characteristics and propidium iodide uptake and 10,000 events were

acquired per sample. Data were analyzed using Lysis II software (Becton-Dickinson).

Anti-IgM-PE was obtained from Catag (South San Francisco, CA). PE-coupled

anti-I-A (M5/114) was purchased from Boehringer Mannheim (Indianapolis, IN). The

following dye- or -biotin-coupled antibodies were obtained from PharMingen (San

Diego, CA): CD4 (RM4-5), CD5 (53-7.3), CD8 (Ly-2), CD25 (7D4), CD80/B7-1 (16-

1OAI).



T Cell Proliferation Assay



1 x 106 erythrocytes-depleted spleen cells/well were cultured in 200 pl

Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated

horse serum, 0.1mM nonessential amino acids, lmM sodium pyruvate, 10 mM Hepes,

0.05 mM 2-ME, lOug/ml gentamicin, and anti-bacterial/anti-mycotic (ABAM) in flat-

bottomed 96-well tissue culture plates (Costar Corp.). Cells were stimulated with anti-

CD3 (1Oug/ml, lug/ml, or 0.1ug/ml) or rIL-2 (20U/ml, 1OU/ml, or 5Umi) for 24h, 72h,

120h. 1 tiCi of [3H] thymidine in 25.d culture medium was added to each well at 18h

before harvesting. Incorporated thymidine was measured by using standard liquid










scintillation counting techniques. Mean standard error of mean (SEM) of triplicate

samples was presented.

To assess CD4 and CD8 T cell proliferation in vitro, splenocytes were cultured at

1 x 106 cells/ 200ul DMEM as described above with anti-CD3 (lug/ml) for 24h, 48h, 72h.

The cells were counted, stained with FITC-anti-CD4 and PE-anti-CD8. FACS analysis

was performed as described above. The number of CD4 and CD8 T cells was determined

by total counted cells /ml multiplied by the percentage of CD4 and CD8 T cells.




Immunization



B6 and B6.NZMc7 mice (Four to six per group, 2mo old, sex matched) were

injected intraperitoneally with DNP-KLH (100 ug/mouse; Calbiochem, La Jolla, CA) in

complete Freund's adjuvant (CFA) and boosted two weeks later with same dose of DNP-

KLH in incomplete Freund's adjuvant. Serum was collected before immunization (dO),

14 days after first immunization (d14), and 7 and 30 days after the booster (d21, d51).

Immunized mice were assessed for serum anti-hapten titers using ELISA. Serum was

diluted 500, 2500, 12500, 62500 fold in PBS containing 2% BSA and incubated on DNP-

BSA (Calbiochem, La Jolla, CA) coated Immulon II plates. Detection of antibodies was

as described above. Antibody activity is expressed as OD.










Histology



Mice were sacrificed at 12mo of age. Thymus, lung, heart, liver, lymph node,

spleen, and kidney were fixed in 10% neutral buffered formalin and embedded in

paraffin. Sections were cut, placed on slides, and stained with hemotoxylin, eosin, and

periodic acid-Schiff. Multiple sections from control and experimental strains were

examined by light microscopy for indication of inflammation and tissue damage in a

blind fashion. Glomerulonephritis (GN) was scored from 0 to 4, in which 1 = < 10% of

glomeruli affected; 2 = 11-25% of glomeruli affected; 3 = 26 50% of glomeruli affected;

and 4 = >50% of glomeruli affected. Control B6 mice had scores of 0 and 1. Glomerular

lesions were classified as mesangiopathic (M) and capillary hyaline (H). The H lesion

was considered more severe than the M lesion.



Sequencing TGF-A3 Coding Region



Total RNA was extracted from liver of B6.NZMc7 and B6 mice using a total

RNA isolation Kit (Ambion, Austin, Texas). cDNA was obtained by reverse-

transcriptase-PCR using random primers and total RNA. For sequencing the TGF-fi gene,

five pairs of overlapping primers were designed according to published mouse sequence

from Genebank to amplify 1.6kb of cDNA. Two mice were used from each strain and

PCR was performed as described above. Five segments of PCR products from each

mouse were cloned using a TA cloning kit (Invitrogen Corp., San Diego, CA).







27

Sequencing of two independent clones from each segment was performed in both

directions on an AB 1373 automated sequencer (Applied Biosystems, Foster City, CA).



Statistics



A nonpaired Student's t-test was used to determine statistical significance of

quantitative data and a chi-square test was used for qualitative data.











Production of Congenic Strains


C57BI/6


bb
C57BI/6


N1 (50% B6)




N2 (75% B6)



N3 (87.5% B6)




N4 (93.8% B6)


nb bb C57BI/6




b bb C57BI/6




nb bb




nb


N7 (99.2% B6)


nb


nb


I! I ''



nn nn nb nb bb


Congenic Strain

Figure 2-1. Illustration of the breeding strategy used in the construction of congenic
mouse strains (N=breeding generation; m=NZM2410 allele; b=B6 allele). In
parentheses is the average percentage of the mouse genome that should be B6-derived.


NZM


nn













CHAPTER 3
GENETIC DISSECTION OF PATHOGENESIS OF SLE: SLE3 ON MURINE
CHROMOSOME 7 LEADS TO A BROAD, LOW-GRADE AUTOIMMUNITY



Introduction



SLE is a prototypic autoimmune disease characterized by the involvement of

multiple organ systems and females preponderance. The disease is strikingly

heterogeneous with regard to organ patterns, severity, clinical course, genetic variable,

and immunologic expressions (reviewed in refs. 1, 2). Although the etiology of SLE is

unclear, a combination of genetic factors, environmental factors, and stochastic events are

required to produce disease (reviewed in refs. 8, 9). The disease is a consequence of

processes involving loss of immune tolerance, pathogenic autoantibody production, and

tissue destruction mediated by direct autoantibody binding and/or the deposition of

immune complexes. The production of autoantibodies against nuclear antigens is the

hallmark of SLE.

The analysis of etiopathogenesis of SLE in humans is very difficult due to

polygenic control of the disease, low penetrance, heterogeneity, and non-organ specific

nature of this disease. Several inbred mouse strains that spontaneously develop disease

similar to human SLE have been important models for elucidating the pathogenesis of the

disease (28). (NZB X NZW)F l is the first and most intensively studied model. More than









90% of female (NZB X NZW)F1 mice develop severe lupus nephritis and die of renal

failure (30). These mice also produce high level autoantibodies against a number of

nuclear components. The New Zealand Mixed (NZM) strains are newer inbred strains

derived from selective inbreeding of progeny from crosses between NZB and NZW mice

(35). A total of 27 NZM strains were produced. These strains have inherited various

genomic segments from the two parental strains and they are all homozygous for the

NZW histocompatibility (H-2) haplotype (Ku, Au, Sz, DM). The genetic differences among

these strains lead to phenotypic differences in disease expression. The NZM2410 strain

produces high levels of IgG autoantibodies to nuclear antigens as early as four to six

months of age and develops severe lupus-like renal disease in both sexes.

Dr. Morel performed interval mapping in a (NZM2410 X B6) X NZM2410

backcross using highly polymorphic simple sequence repeats (SSRs) as genetic markers.

Three susceptibility intervals and MHC locus on chromosome 17 were identified as

conferring susceptibility to acute GN. The three intervals are Slel, on the telomeric end of

chromosome 1; Sle2, on the centromeric end of chromosome 4; and Sie3, on the

centromeric end of chromosome 7 (52). Similar regions have also been found to confer

SLE susceptibility in (NZB X NZW)F1 (48,50). Genetic analysis suggested that the

disease susceptibility is inherited as a multifactorial threshold liability. First, none of

these four loci are required for the development of acute GN. Second, each susceptibility

locus independently contributes to the overall incidence of GN with a relative small

effect. Third, at least three susceptibility alleles are required in order to express a high

level of GN.







31

Although interval mapping has provided information about the numbers and

chromosomal locations of the genes responsible for autoimmunity in NZM2410, it has

provided little about the nature of the component phenotypes contributed by each

susceptibility gene and it is not productive for finely mapping genes. The approach we

used following interval mapping was to move the four susceptibility intervals from

NZM2410 to the resistant strain B6 background using a speed congenic technique

(110,113). This congenic approach, which originated from the Nobel Prize-winning

studies of histocompatibility by George Snell (114-115), is designed to dissect a complex

trait into its component genetic elements and allow for the fine mapping,, and

identification and characterization of each gene. This approach has been used in the

genetic analysis of insulin-dependent diabetes in the NOD mouse, where either resistance

intervals were bred to a susceptible background (116-118) or susceptible intervals were

bred to a resistant background (119).

We recently produced a collection of B6 congenic strains bearing the individual

lupus susceptibility intervals from NZM24 10. Here we report the detailed analysis of the

B6.NZMc7 congenic strain and the component phenotypes expressed by Sle3. Sle3 is

likely a major susceptibility locus leading to pathogenesis of lupus because this locus

leads to two major phenotypes, ANA production and GN, and because this locus was

found in multiple SLE-related crosses (reviewed in ref. 120). To understand the role of

Sle3 in the pathogenesis of SLE, we examined spontaneous IgM and IgG responses to

self-nuclear antigens and foreign antigens in these mice from 2 to 12 months of age, and

examined in vivo immune responses to antigenic challenge in 2 month old B6.NZMc7.










We performed the surface phenotypic and functional analyses of lymphoid cells from the

spleen, peritoneal cavity, bone marrow, and lymph node in these mice to determine the

nature of possible lymphocyte defects leading to autoantibody production. The results

indicate that Sle3 profoundly affects T cell activation, proliferation, and subset

distribution at an early age, which may lead to production of polyclonal IgM and IgG and

anti-nuclear autoantibodies. T cell changes in B6.NZMc7 mice compared to B6 are

heightened CD4:CD8 T cell ratio, increased CD69+CD4 T cells in both the spleen and

lymph node, increased lymphocyte proliferation and CD4 T cell expansion in response to

anti-CD3 stimulation in vitro, and increased in vivo immune response to T-dependent

antigens. B cells from the spleen and lymph node of B6.NZMc7 mice did not differ from

those of B6 in number, B cells subset (B 1 vs. B2 cells), or function (B cell proliferation

in vitro). B6.NZMc7 mice showed some characteristics of B cell hyperactivity including

elevated serum IgM levels, polyclonal IgM, and high expression of some B cell surface

markers at a late age. Finally, Sle3 leads to autoantibody-mediated GN.



Results




Thirty-six B6.NZMc7 congenic mice were initially obtained. These mice differed

genetically from B6 at the three selecting loci for the Sle3 interval, an unknown number

of unselected flanking loci, and unlinked contaminating loci (probability 0.78%). To

define the NZM2410 interval and to establish a congenic line, thirteen marker loci on







33

chromosome 7 were used to map the termini of the congenic interval and to identify

recombination. The mice were grouped according to their genotype (Figure 3-1). Fifteen

mice from group 1 that carry about 30 cM of chromosome 7 from NZM2410 were chosen

as the founders of the congenic strain and used to continue breeding. These mice and their

progeny were used for phenotypic studies. The autoimmune phenotypes expressed in both

homozygous and heterozygous B6.NZMc7 mice were identified by comparison with age-

matched B6 controls. All animals were housed in identical conditions and bled bimonthly

beginning at 2 months of age for autoantibody analysis..

B6.NZMc7 mice have hypergammaglobulinemia and spontaneous polyclonal B

cell activation. As the most common immunological abnormalities in lupus mice,

hypergammaglobulinemia and spontaneous polyclonal B cell activation were first

examined using total serum IgM and IgG levels and spontaneous IgM antibody

production to four antigens: histone, ss DNA, bovine thyroglobulin, DNP-KLH.

B6.NZMc7 mice had significantly high levels of total serum IgM (Figure 3-2, panel A)

and IgG (Figure 3-3) compared with B6 at 9 months of age (p< 0.0001). Elevated IgM

levels started at 5 months of age and were found in both homozygous and heterozygous

mice (Figure 3-2, panel B). These mice also spontaneously produced IgM

polyclonal/polyreactive antibodies against the four antigens at 5 months and this

increased with age (Table 3-1). The penetrance of polyclonal IgM is 85% in 9 months

B6.NZMc7 (percentage of positive mice who produced IgM antibodies against two or

more of these four antigens). These mice were also tested for spontaneous IgG antibodies

to the same four antigens, and penetrance was 64% at 9 months of age.







34

B6.NZMc7 mice spontaneously produce IgG anti-nuclear autoantibodies (ANA).

The presence of IgG antibodies against a variety of nuclear antigens is the characteristic

of SLE in both humans and mouse models. NZM2410 produce high levels of ANA at an

early age. To determine if Sle3 by itself can lead to this phenotype and to determine the

antibody characteristics of B6.NZMc7 compared with NZM2410, we quantified IgG

autoantibodies to four nuclear antigens (ssDNA, dsDNA, histone, chromatin or

DNA/histone complex) bimonthly from 2 to 12 month old. B6.NZMc7 produced IgG

autoantibodies against these four nuclear antigens starting at 2 months of age (p<0.01),

especially anti-DNA and anti-chromatin antibodies (Figure 3-4). The titers were much

lower in the congenic mice compared to NZM2410 (data not shown) and the average

titers did not increase with age. This phenotype is dominant with slightly decreased levels

and delayed onset in heterozygous B6.NZMc7. Penetrance of IgG ANA, as shown in

Table 3-2, is about 60-80% and does not typically increase with age unlike what is seen

in B6.NZMcl mice (L Morel, personal communication). After analyzing individual mice,

we found IgG ANA titers in some B6.NZMc7 mice to wax and wane from 2 to 12

months (Figure 3-5). Autoantibody levels did not stay positive for long in these mice and

quickly dropped, which may be due to tissue deposition of antibodies. Waxing and

Waning of IgG ANA has also been reported in many SLE patients (121).

The fact that SLE is preponderant among women during their reproductive life

implies that sex hormones have a role in the pathogenesis of autoinmunity in humans.

Sex hormone manipulation experiments in lupus-prone murine strains also show that

female sex hormones facilitate the development of autoimmunity (122-123). After









detailed analysis of serology data comparing male and female B6.NZMc7 congenic mice,

we found that there was no gender bias in titer or frequency of autoantibodies against any

autoantigens from 5 to 12 months (p>0.05).

B6.NZMc7 mice exhibit a high immune response to DNP-KLH. To test immune

system function before disease onset or in the very early stage of the disease, 2 months

old B6.NZMc7 mice and B6 control mice were challenged in vivo with a T-dependent

antigen, DNP-KLH. IgM and IgG anti-DNP antibodies were measured before

immunization (dO), 14 days after first immunization (d14, primary immune response), 7

days after the booster (d21, secondary immune response), and at d51. B6.NZMc7 mice

showed high primary and secondary IgM anti-DNP immune responses (p < 0.05) (Figure

3-6), high secondary IgG anti-DNP immune responses (p < 0.01) (Figure 3-7), and anti-

DNP antibodies were remained longer compared with B6. This experiment indicates that

the genetic defects in B6.NZMc7 mice lead to a hyper-immune activation status and may

involve both B and T cell compartments.

To define abnormalities in the B and T cell compartments, cell surface molecule

expression, cell function and subset distributions of B and T cell were investigated.

Sle3 affects T cell subopulations. To explore the impact of Sle3 on T cells, cell

subpopulations were first investigated via surface marker expression. In order to define

primary defects, our studies focused on young B6.NZMc7 mice (1-3 month). Summary of

FACS analysis results is presented in Table 3. The results indicate that the CD4:CD8 T

cell ratio was significantly increased in both the spleen and lymph node of B6.NZMc7

mice compared to B6 (Figure 3-8). There was no difference between males and females







36

(p > 0.05). High proportions of spleen CD4 T cells expressed the activation marker CD69

(p < 0.01) indicating the activation of CD4 T cell in the early stages of disease. This

effect was not seen in the lymph nodes. There was no difference in the expression of L-

selectin between B6.NZMc7 and B6 T cells.

Hyper responsiveness of CD4 T cells in vitro. To study CD4 and CD8 cell

response to stimulation in vitro, spleen cells were cultured with anti-CD3 (1 ug/ml) for

various times, stained with anti-CD4 and anti-CD8, and analyzed using flow cytometry.

The results are shown in Figure 3-9. Before culture (data not shown) and at 24 hours post

culture, CD4 and CD8 cell numbers were not significantly different between B6.NZMc7

and B6. However, the absolute numbers of CD4 T cells were higher in B6.NZMc7 mice

than in B6 at 48 and 72 hours of culture. In contrast to CD4 cells, the numbers of CD8

cells were less in B6.NZMc7 mice. The decrease of CD4 cell numbers both in

B6.NZMc7 and B6 mice at 72 hours of culture was observed. This may be due to the low

concentration of anti-CD3 used, since there is no such decrease of CD4 cell numbers in

experiments using 2.5 ug/ml anti-CD3 stimulation combined with anti-CD28 (C Mohan,

personal communication). Figure 3-10 shows 2D dot plots of flow cytometry of this

experiment. Percentage of CD4 cells was higher in B6.NZMc7 than in B6 after 48 hours

of culture (49% vs. 33%). This increased expansion of CD4 T cells in B6.NZMc7 may be

due to a high proportion of activated CD4 T cells, high responsiveness of CD4 T cells, or

reduced apoptosis of CD4 T cells. Indeed, B6.NZMc7 T cells also exhibited reduced

activation-induced T cell death (data not shown).







37

Sle3 impacts T cell proliferation. The extent of lymphocyte proliferation in 2

month B6.NZMc7 mice was also examined using 3H-thymidine incorporation assay in

spleen cell culture with graded doses of anti-CD3 or rIL-2 for 72 hours compared with

B6. As shown in Figure 3-11, B6.NZMc7 mice exhibited heightened proliferative

responses to anti-CD3 and rIL-2. The data suggest that T cells from B6.NZMc7 have an

early CD3-mediated T-cell activation aberration.

Sle3 and B cell compartment. There were no significant differences between

B6.NZMc7 and B6 in B cell numbers and B cell subsets (BI vs. B2 cells). In 9 to12

months old B6.NZMc7 mice, I-A (MHC class II molecular), B7-1 (costimulatory

molecular) expressions on B cell surface were high (data not shown), which indicated that

high proportions of B6.NZMc7 B cells were activated in the late stage of disease. There

was also no significant difference in vitro B cell proliferation responses to LPS and anti-

IgM between B6 and the congenic mice (data not shown).

Sle3 leads to low incidence of glomerulonephritis (GN). B6.NZMc7 mice

survive for at least 12 months with no obvious proteinuria. The kidney histology was

examined in one year old B6.NZMc7 and B6 mice. The total frequency of GN was about

40% in B6.NZMc7 homozygous mice and 7 % in heterozygous mice (scored 2 and up)

(Figure 3-12). Severe GN (scored 3 to 4, more than 25-50% of glomeruli being affected)

was only seen in 20% of the homozygous mice. GN was seen in both males and females

with no sex bias. Two distinct types of GN were present in these animals. One was

hypercellular and the other showed large intracapillary hyaline deposits with minimal









cellularity. As a control, one year old B6 had few pathological changes (scored 1 or less

than 1).

Detailed data analysis was performed to define the role of anti-dsDNA

autoantibodies in the pathogenesis of GN. The results showed no strong relationship

between titers or onset of the antibodies and GN. Two out of nine GN positive animals

were negative for both anti-dsDNA and anti-chromatin autoantibodies.

Histological analysis of the spleen, lymph node, lung, and liver showed lymphoid

hyperplasias with one out of 33 samples having a lymphoma.

Sequence analysis of candidate gene: transforming growth factor-beta (TGF-,) in

B6.NZMc7. TGF-/3 is a potent immunoregulatory molecule. Although it can both

suppress and enhance immune cell functions in vitro (124-126), most evidence suggests

that it is a natural immunosuppressor (127-130). TGF-/3 knockout mice exhibited a

wasting syndrome accompanied by multifocal lymphoid infiltrates in the heart, lungs,

salivary glands, and other organs (131-132). They also showed SLE-like autoimmune

serology (133). TGF-,/is located within the Sle3 interval of murine chromosome 7, which

makes it a good candidate gene. Primers were designed to amplify 1.5kb previously

published mRNA of TGF-/3 coding region (134). RT-PCR showed that TGF-f3 was

expressed in the spleen and kidney (data not shown). The sequence of TGF-fl was not

polymorphic between B6.NZMc7 and B6 (data not shown). No conclusive data was

obtained in expression assays. Further studies are needed to establish if TGF-/3 is

involved in SLE pathogenesis.












Discussion




The genetic basis of lupus is very complex, involving contributions from multiple

genes. To identify these genes, interval mapping was performed, and three susceptibility

intervals and one locus were identified: Slel on chromosome 1, Sle2 on chromosome 4,

Sle3 on chromosome 7, and the MHC locus on chromosome 17 in (NZM2410 X B6)F I X

NZM2410 backcross (52). The phenotypes in the progeny reflect the epistatic interaction

of these multiple genes. We believe that each susceptibility interval plays a unique role in

the initiation of pathogenesis. Separating each susceptibility interval was achieved using

a congenic breeding approach in which each interval bearing individual susceptibility

gene or genes from NZM2410 was moved to the resistant B6 background by repeated

backcross and selection (113). B6 and B6 congenic for Slel, Sle2, or Sle3 have same

MHC and background genes except for the selected genomic segment derived from

NZM2410. We successfully produced four congenic strains and genetically dissected the

phenotypes associated with each susceptibility interval. Slel on murine chromosome 1

leads the loss of tolerance to the H2A/H2B/DNA sub-nucleosome with little or no kidney

damage (135). Sle2 on murine chromosome 4 leads to B cell hyperactivity with no IgG

autoantibodies or nephritis (136). Here we present detailed phenotypic analysis of Sle3 on

murine chromosome 7 and explore the nature of possible lymphoid cell defects causing

the appearance of autoantibodies.







40

B6.NZMc7 showed generalized immune dysfunction. These animals exhibited

hypergammaglobulinemia and polyclonal B cell activation. This phenotype is a most

common immunological abnormality among murine lupus models (137-139). Intrinsic B

cell defects were suggested by the finding of an enhanced in vitro responsiveness of these

B cells to LPS and conventional antigens, and by several cell transfer experiments (73-74,

140-143). The mechanisms underlying polyclonal B cell activation in B6.NZMc7 are not

clear. These mice did not show differences in B cell number, B cell activation or

proliferation in vitro responses to LPS and anti-IgM. Polyclonal B cell activation is

known to accelerate autoimmune disease. This is supported by the strong association of

this phenotype with disease development in the various murine lupus models (70), by the

report that it can accelerate mortality, autoantibody level, and by GN in lupus-prone but

not in normal mice (144).

IgG anti-nuclear autoantibody production is a hallmark of SLE both in humans

and mouse models. Sle3 by itself leads to IgG anti-nuclear autoantibody production

starting at an early age. Autoantibody titers in B6.NZMc7 were lower than in the parental

NZM2410 indicating the requirement of other genes for high level IgG ANA production.

IgG ANA level in B6.NZMc7 was not constant and uniquely waxed and waned from 2 to

12 months of age. The causes of this are unknown. A possible explanation is that the

antibodies and antigens form immune-complexes and deposit in tissues including kidney.

One of the most interesting features is that Sle3 leads to development of GN. In

contrast to acute GN in parental NZM2410, B6.NZMc7 developed chronic GN with

relatively low penetrance, and no apparent proteinuria. Two types of GN were observed,







41

one was hypercellular and the other was large intracapillary hyaline deposit with minimal

cellularity, whereas in NZM2410 GN showed a mixture of these two types. These

observations suggest that the gene(s) in the Sle3 interval may play a key role in mediating

development of GN in NZM2410 and the other gene(s) may act to accelerate GN. This

conclusion has been confirmed by the study of double congenic mice containing Slel and

Sle3 (C Mohan, L Morel, unpublished observations). B6.NZMclc7 double congenic mice

developed acute GN with high penetrance, as seen in NZM2410. The genes causing

kidney damage can potentially act on different points of the pathogenic pathways. For

example, genes may enhance the production of pathogenic autoantibodies, impact

clearance of immune complexes, facilitate binding of autoantibodies or immune

complexes to kidney, and increased the sensitivity of the kidney to damage.

It has been reported that anti-DNA antibodies have pathogenic potential (94-97),

however, our study does not find a strong correlation between IgG anti-dsDNA and GN

in B6.NZMc7. Two out of nine GN positive mice fail to produce anti-dsDNA or anti-

chromatin autoantibodies. One possibility is that GN is caused by autoantibodies against

other antigens. Further studies are in progress to search for other autoantibodies such as

antibodies to the endogenous viral protein, gp70 and to small nuclear ribonucleoproteins.

Another possibility is that titers of autoantibodies in these two mice are low and that

tissue deposition of the autoantibodies makes it serum negative in these two mice.

IgG ANA production can be triggered by specific autoantigens and can be the

result of polyclonal B cell activation. There is a report that polyclonal B cell activation

alone does not lead to IgG ANA production (136). Increasing evidence suggests that









autoantibodies appear to be the products of autoantigen-driven immune responses (145-

149) characterized by IgG class switching, somatic mutation, and clonal expansion of

lupus B cells. This pathogenic autoantibody production in both spontaneous and

experimentally induced SLE is T helper (Th) cell dependent and Th cells are

predominantly CD4 positive (84,87-89). The studies of using anti-CD4 monoclonal

antibody to deplete or block the activation of CD4 T cells further demonstrate a

requirement for this subset in lupus development in mouse models (150-153). These T

cells may be polyclonally activated (154 -155) or autoantigen-specific (156-157).

Both B and T cell defects can lead to the overproduction of pathogenic

autoantibodies. This study indicates that Sle3 mainly impacts T cell function. First,

development of autoinmune disease in young B6.NZMc7 mice was accompanied by an

increased percentage of CD4 T cells and an increased CD4:CD8 T cell ratio in both the

spleen and lymph node. However there was no significant difference found in total T cell

numbers between young B6.NZMc7 and B6 mice (data not shown). Second, a high

percentage of CD4 T cells in B6.NZMc7 was activated and expressed activation marker

CD69. When cultured with anti-CD3 in vitro, CD4 T cells expanded preferentially. Total

lymphocytes also showed heightened proliferative responses to anti-CD3 and IL-2

stimulation. Hyper responsiveness was further demonstrated by in vivo T-dependent

antigen DNP-KLH challenge assays. B6.NZMc7 showed heightened primary and

secondary immune responses to DNP. Third, B6.NZMc7 CD4 T cells exhibited reduced

activation-induced T cell death. In addition, preliminary data from bone marrow transfer

assays showed expression of a high CD4:CD8 T cell ratio can be transferred by









B6.NZMc7 bone marrow to B6 recipients (E. Sobel, unpublished observations). Taken

together, these results suggest intrinsic T cell defects are present in B6.NZMc7 congenic

mice.

CD4 T cells can augment IgG ANA production. They can provide cognate help,

which augments antigen-specific B cells via gp39 and CD40 transducing a second signal

for B cell growth and differentiation (85), and non-antigen-specific help, which augment

any bystander B cells through providing B cell growth and differentiation cytokines (87).

The autoantigen-nonspecific T cell help may promote generalized humoral autoinimunity,

such as hypergammaglobulinemia and autoantibody production, but autoantigen-specific

T cell help is required to maturing such autoimmune responses to causing end-organ

disease (158). T cell hyperactivity in B6.NZMc7 may provide both autoantigen-specific

and autoantigen-nonspecific help for autoantibody production and immune complex-

associated end-organ disease.

We can not make a conclusion about B cell abnormalities in this study. Polyclonal

B cell activation, IgG ANA production, and high primary and secondary immune

responses in vivo may indicate possible B cell abnormalities and lead to the loss of

tolerance to nuclear antigens in B6.NZMc7. It is unclear whether these observations are

due to intrinsic B cell defects or normal B cell responses to abnormal environment factors

including T cell hyperactivity, or both. Pre-B cell transfer experiments are in progress and

will help to address this question.

The humoral phenotypes and nephritis were found in both B6.NZMc7

homozygous and heterozygous mice, with delayed onset and less severity as well as







44

decreased penetrance in heterozygotes. This result indicates the mode of inheritance is

dominant and in an allele dose-dependent fashion. Further studies are needed to

determine the mode of inheritance of cellular defects identified here.

This study demonstrates that B6.NZMc7 bearing SIe3 exhibit complex and

multifaceted phenotypes which are different from B6.NZMcl and B6.NZMc4. Sle3

contributes to lupus susceptibility in NZM2410 predominantly by impacting T cell

activation and proliferation leading to the low grade IgG ANA production and lupus

nephritis. The phenotypes in B6.NZMc7 are less pronounced than these in NZM2410.

The relative complex phenotypes may reflect the presence of two or more SLE-

susceptibility genes within this interval. This possibility has been suggested by the

presence of two peaks in genetic linkage studies on backcross mice (52) and further

supported by results from linkage studies in intercross mice (159). To explore this

possibility, we separated the telomeric and centromeric peaks within the Sle3 interval by

generating two subcongenic strains. Detailed analysis of these subcongenic mice is

presented in Chapter 4.

TGF-f is a potential candidate gene located in the SIe3 interval. The coding region

of this gene has been sequenced and it was found that there is no consistent sequence

polymorphism between B6.NZMc7 and B6. Further studies are needed to determine if

there are any differences in expression levels of this gene.









1 2 3 4 5 6 7 8 9
D7MIT178 2.3 0 0 11 )1 1"0 10 EJ 00 0 0 0
CKMM 4 0j N Nm 11 U m1 C ] E C 0
D7MIT76 4.6 ** ** 1)1) 10i:] 101 ImDJI U U 0
D7MIT154 5 U mu mi 11) E 0 13 N
D7MITl14 9.8 mm so N 111 11 ) D 1m ) U N ID EJ
D7MIT155 11.2 NNE E* Ern m U ujj Em* I 10 10 i]
D7MIT25 14.3 0 N E E No mE on *0 Ir 10 1 E i
D7MIT69 17.8 N* mm No EnE U N U E
D7MIT158 18.9 N E N 0 E E E N N N E MEN U N U
P 23.4 mmm mm mm mm m mmm N E
D7MIT70 23.8 E** N 0 N M mom 0 U U
D7MIT31 34.1 *** 100 ml 0 N lU 3)3) N)E3) U[ I 0 I
D7MIT68 50.6 [ [ [ 00 00 00 00 0) OD E r 3I 0

numbers of mice 2 7 6 3 3 1 1 1 1 1 2 2 1 2 1 1 1

Fig 3-1. Genotypes of the 36 original B6.NZMc7 congenic mice. Thirteen markers coving the Sle3 interval,
(indicated on the left) were used to genotype each animal. The distance of each marker to the centrimeric end is
shown in the second column and is expressed in units of centiMorgan (cM). Black boxes represent mice typed as
NZM24 10 homozygotes, white boxes, mice typed as B6 homozygotes, half black boxes represent heterozygous
mice. Mice from group 1 were used as founders for breeding all B6.NZMc7 mice used in this study.


Marker loci cM


Group

























Figure 3-2. Total serum IgG and IgM in 9 month old mice. High levels of serum IgG and IgM were
observed in B6.NZMc7 homozygous mice (black dot) compared with B6 (white triangle). Each dot
represents an individual mouse. Horizontal line is mean value.






















oo3Wa 40.o


o 30D




AA
1A A

AA
0 0

B6 B6.NZMc7hom. B6 B6.NZMcThom.
n=10 a=21 n=1O n-21
























Figure 3-3. Kinetics of total serum IgM levels in B6.NZMc7 homozygous mice. There is no significant
difference between B6.NZMc7 homozygotes (black dot, all groups n=23, 2 mo group, n=13) and
heterozygotes (white dot, n=5-7). Horizontal lines are mean values. Each dot represents an individual
mouse.










600-

500-

400-

300-

S200-

" 100-
0


00
* 0


0


o 0
0: 2


Age (months)


0













Table 3-1. Spontaneous production of IgM antibodies against a panel of four antigens in B6.NZMc7 homozygotes
compared with age-matched B6.

5 mo 7 mo 9 mo 11-12 mo
g B6 B6.NZMc7 B6 B6.NZMc7 B6 B6.NZMc7 B6 B6.NZMc7



ssDNA 10825a 342-+114*b 14232 359104* 141+0.32 356120" 16564 473-157"

Histone 131-34 357116* 14524 26547* 15135 294075* 14358 394136*

Thyroglobulin 170089 605318* 29198 1179749* 417251 1012-684* 397184 1545991"

DNP-KLH 105019 284095* 18341 32084* 21763 374145* 22390 433163*

a All data expressed as mean % OD standard error of mean (SEM). % OD is the ratio of the sample OD divided by the OD
of normal B6 pooled serum x 100 (112). The number of mice tested is 14 B6, and 34 B6.NZMc7.
b represents student's t test significance, p < 0.001.
























Figure 3-4. IgG anti-dsDNA and anti-chromatin autoantibody production in 2 to 12 month old B6.NZMc7
mice. B6.NZMc7 homozygous mice (black dot, all groups n=23, 2 mo group, n=13) produce both anti-
dsDNA and anti-chromatin antibodies starting at 2 mo of age (p<0.001). The phenotypes are also found in
B6.NZMc7 heterozygous mice (white dot, n=8) with slightly low titers. Significant difference was
detected in B6.NZMc7 homozygous and heterozygous mice compared with B6 (white triangle, n=8)
Horizontal lines represent mean values.












A5


4.

3-

2.


I I I I I I -- I


B
140

120

.100.

4so,
C 0
< n
C 0
A.


A


Hi
S
'S


Age (months)


Age (months)


A


0 0
o c


S
I
S
II
0

4?


v 6

0 % I: to
~~ML-10 0


12 2 5 7 9 11


12I I I 7I
12 2 5 7 9 11












Table 3-2. Penetrance of IgG anti-nuclear autoantibody production in B6.NZMc7.


2 mo 5 mo 7 mo 9 mo 11-12 mo

Antigen B6.NZMc7 B6.NZMc7 B6.NZMc7 B6.NZMc7 B6.NZMc7
hom.b het. hom. het. hom. het. hom. het. hom. het.

dsDNA 17%c NDd 37% 10% 45% 17% 39% 28% 74% 65%

ssDNA 33% ND 40% 5% 64% 21% 73% 22% 82% 65%

Chromatin 17% ND 26% 5% 61% 50% 45% 44% 41% 65%

Histone 33% ND 40% 10% 24% 17% 55% 17% 44% 50%

N_ 12 35 20 33 24 33 18 34 20

a Number of mice tested.
b Homozygous (hom.)B6.NZMc7, heterozygous (het.) mice.
c Percentage of positive mice. The mouse is counted as positive if its antibody titers are above the levels of mean plus 2 standard
deviation found in six 12 mo old B6 mice assayed on the same plate.
d ND: not determined.
























Figure 3-5. Waxing and waning nature of anti-ANA production in B6.NZMc7 mice. Each line represents
an individual mouse. A total of 8 mice were selected from 23 mice tested from 2 to 12 months of age for
anti-dsDNA at the same ELISA plate. The horizontal line represents mean plus 2 standard deviation serum
antibody levels present in ten 12 month old B6 mice. Levels of antibody above this line are considered
positive.










d



z

I
* -


11


Age (months)


3-


2-


iEli UUUMMi EM I
























Figure 3-6. Immune responses to T-dependent antigen DNP-KLH in vivo. A) IgM anti-DNP antibody
production before immunization, 14 days after primary immunization, 7 and 30 days after boosted
injection (d21, and d5 1) at serum dilutions of 1:500 in B6.NZmc7 mice (white dots, dashed line) and age-
sex-matched B6 (black dots, solid line). B) IgM anti-DNP antibodies at day 51 at different serum dilutions.
Data are from one of three independent experiments, and each dot is expressed as mean OD plus standard
deviation from five B6.NZMc7 or B6 mice. *, p <0.05; **, p<0.01.













A B
3.5*


2.5 ".
o'2 -" _""

1.5





0 14 21 51 -
I in 500 1 in 2500 1 in 12500 1 in 62500


Serum dilton


Time (Days)
























Figure 3-7. Immune responses in vivo to T-dependent antigen DNP-KLH. A) IgG anti-DNP antibody
production before immunization, 14 days after primary immunization, 7 and 30 days after boosted
injection (d21,d51) at serum dilution of 1:12500 in B6.NZMc7 mice (white dots, dashed line) and age-
sex-matched B6 (black dots). B) IgG anti-DNP antibodies at day 51 at different serum dilutions. Data are
from one of three independent experiments, and each dot is expressed as mean OD plus standard deviation
from five B6.NZMc7 or B6 mice. *, p < 0.05; **, p < 0.01











A
B3




2 t








14 21 51 0 I I
I i 500 1 in 2500 1in 12500 1 in 62500
Time (Days) Serum dilution












Table 3-3. Summary of FACS analysis of T cells in B6.NZMc7 mice compared with age matched B6.



1-3 mo mice 1-3 mo mice

Spleen Lymph node
B6 B6.NZMc7 B6 B6.NZMc7
n-26 n-24 n-16 n-9

%CD4 T cells 17.452.97a 20.16+2.7**b 26.104.71 35.676.23***

%CD8 T cells 11.732.48 11.192.22 24.575.89 24.415.83

CD4:CD8 T cell ratio 1.510.16 1.860.42*** 1.12+0.33 1.540.45*

CD69+CD4 T cells 16.49.3 238** 2416 1513


" All data expressed as mean standard deviation (SD).
b represent student t test significance, p < 0.05; **, p < 0.01; ***, p <0.001.
























Figure 3-8. Spleen and lymph node CD4:CD8 T cell ratio in 1 to 3 month B6.NZMc7 mice. There was
no difference between males and females.Data is pooled from 10 independent experiments, for each test,
age- and sex- matched mice were used and B6 mice included. The results are presented as mean plus
standard deviation. *, p < 0.05, ***, p < 0.001.











N=24


N--9


N=26

T


B6 B6.NZMc7


Spleen


N=16

I


B6 B6.NZMc7

Lymph node


C









QO
























Figure 3-9 B6.NZMc7 spleen CD4 and CD8 T cells expansion after anti-CD3 (1 ug/ml) stimulation in
vitro. Black bars are B6, white bars are B6.NZMc7. The mean value of duplicates is presented. Data
represent one of three independent experiments. All mice are age- and sex-matched. Cell numbers were
calculated using FACS data and cell counting.






180,000

150,000

120,000

90,000

60,000

30,000

0


24


CD4 cells


48


CD8 cells


72


I1r


24


48


Culture Time (hours)


;N


72


[-
























Figure 3-10. Plots show increased percentage CD4 cells in splenic lymphocytes cultured with anti-CD3
(1 ug/ml) for 24h, 48h, and 72h from B6.NZMc7 compared with age-matched B6. This experiment is
representative of three independent experiments.









Oh 24h 48h 72h

11 70 42.9- 5f
PM in

1793 ftB6
S 17.93 33.46 .32.44


C D 8 .32.6 *. 346

N i'
B6.NZMc7
~19.75 204 44694





CD4
:.7.2._.': .'. : !'' .0 ",' '- i3 .6 =.
























Figure 3-11. Lymphocyte proliferation assay. Splenocytes 1 x 106 were culture with graded doses of anti-
CD3 (panel A) or rIL-2 (panel B) for 72h. Lymphocyte proliferation was measured by 3H-thymidine
incorporation which added in the last 18 h of culture. Dashed line represents B6.NZMc7, solid line age-
and sex-matched B6. Each bar represents mean cpm (counts per minute)standard error of mean (SEM)
incorporated by triplicate cultures. This experiment is representative of 3 independent experiments.
* p <0.05, ** p < 0.001














** **l
0 0em


0.1 1

Anti-CD3 (ug/ml)


9,0001


M 6,000-


.C ,11

iftft
o.U


* J


5 10
rIL-2 (U/ml)


30,000-

25,000-

20,000-
I-
= 15,000-
lsooo



5,000-

0
























Figure 3-12. The frequency of GN in B6.NZMc7 homozygous (hom) and heterozygous (het) for the Sle3
interval. GN was graded according to percent of glomeruli affected (grade 1 to 4) and the morphological
pattern of nephritis (H= hyaline, M= mesangiopathic).











45%-


z

0 30%

-2GN
1 3+4H GN
15%



0%
B6NZMc7 horn. B6.NZMc7 het.
n=22 n=16














CHAPTER 4
GENETIC DISSECTION OF SLE PATHOGENESIS:
SLE3 HAS TWO SEPARATE LOCI THAT INTERACT SYNERGISTICALLY


Introduction



SLE is a prototypic systemic autoimmune disease characterized by production of

autoantibodies against a spectrum of nuclear antigens and autoantibody-mediated organ

damages especially kidney damage. The cause of disease is unclear. The genetic factors

and environmental factors as well as stochastic factors contribute to the altered immune

state (1). Identification of underlying gene mutations or allelic variants that affect the

function of genes and an understanding of their functional consequences will impact the

diagnosis, treatment, and prevention of SLE.

Several inbred mouse strains that spontaneously develop a disease similar to

human SLE are important models (1). The most-studied is the F1 hybrid of NZB and

NZW. New Zealand Mixed (NZM) are inbred strains derived from selective inbreeding of

progeny from a cross between NZB and NZW (35). A total of 27 NZM strains were

produced. These strains have inherited various genomic segments of the two parental

strains and are homozygous at the histocompatibility (H-2) locus for the NZW haplotype

(Ku, Au, SZ, Dz). The genetic differences among these strains lead to phenotypic

differences in disease expression. NZM2410 is a strain that produces high levels of IgG









autoantibodies to nuclear antigens as early as 4 to 6 months of age and develops severe

lupus-like renal disease in both sexes. A genome scan was performed in the backcross

progeny of NZM2410 and B6 to identify the chromosomal locations of susceptibility

genes (52). Three susceptibility intervals and the MHC locus were found to confer acute

GN susceptibility. The three intervals were: Slel, in the telomeric end of chromosome 1;

Sle2, in the centromeric end of chromosome 4; Sle3, in the centromeric end of

chromosome 7. Similar regions have also been identified in NZB X NZW crosses (48,50,

reviewed in ref. 160). Although these linkage studies have provided important

information about the chromosomal locations of susceptibility genes and the inheritance

of the disease, they tell us little about the role of each gene in the pathogenesis of lupus

and they are not productive for fine mapping. The construction of congenic strains is a

good approach to address these issues. The susceptibility intervals from the susceptible

donor strain are introduced into the genome of the resistant recipient strain by repetitive

backcross of the donor strain to the recipient (113). We recently generated a collection of

B6 congenic strains bearing the individual lupus susceptibility intervals from NZM2410.

Thus, by comparing B6 and the B6 congenic strains differing only for the specific

chromosomal segments, we are able to characterize the phenotypes contributed to disease

pathogenesis by each susceptibility interval. Interestingly, these congenic strains exhibit

different serological and immunological abnormalities and varying susceptibility to

nephritis (66,135-136).

Detailed analysis of B6.NZMc7 bearing the Sle3 interval is presented in Chapter

3. In summary, our results indicate that Sle3 profoundly affects T cell activation,







73


proliferation, differentiation, and apoptosis. B6.NZMc7 mice had increased CD4:CD8 T

cell ratios and increased CD69+CD4+ T cells in both the spleen and lymph node. They

showed heightened lymphocyte proliferation and CD4 T cell expansion in response to

anti-CD3 stimulation in vitro and increased in vivo immune responses to T-dependent

antigens. CD4 T cells exhibited reduced activation-induced cell death. B6.NZMc7 spleen

and lymph node B cells did not differ from B6 in number, B cells subset (BI vs. B2

cells), or functional analysis (B cell proliferation in vitro). However, B6.NZMc7 mice

showed high serum IgM and IgG levels, high polyclonal IgM antibodies, and high

expressions of some B cell surface markers in a late age. Finally, Sle3 lead to

autoantibody-mediated GN. The relatively complex phenotypes in B6.NZMc7 suggest

the presence of two or more SLE-susceptibility loci within this interval. This possibility

has also been suggested by the presence of two peaks in our genetic linkage studies on

backcross (52) and intercross mice (159).

Here we explore this possibility by generating two subcongenic strains bearing the

telomeric or the centromeric peaks of Sle3. The construction of B6.NZMc7c and

B6.NZMc7t subcongenic strains was described in Chapter 2. B6.NZMc7c contains the

centromeric end of Sle3 and B6.NZMc7t contains the telomeric end of Sic3. These strains

were analyzed for their serological phenotypes, evidence of GN, and T cell phenotypes

compared to B6 and the parental B6.NZMc7 strain. Results suggest that Sle3 has at least

two separate susceptibility loci that interact in a synergistic way to cause polyclonal B

cell activation, IgG ANA production, and development of GN. However, increased

CD4:CD8 T cell ratios and increased CD4 T cell expansions in vitro as well as the










heightened T cell proliferations in vitro were seen only in the mice carrying the telomeric

end of Sle3.



Results




Dense linkage map of SSR markers on murine chromosome 7 covering the Sle3

interval. In order to produce the subcongenic mouse strains and finely map susceptibility

loci, a precise map of SSR markers is very important. Ninty-six (NZM2410 X B6)Fl X

NZM2410 backcross and 144 F2 intercross progeny representing approximately 400

meiosis were genotyped using 25 markers covering the Sle3 interval on murine

chromosome 7. The map of SSR markers was constructed using MAPMARKER

computer software (161). As shown in Figure 4-1, the order and genetic distances

between markers that we obtained were mostly in agreement with published data (162).

However, one marker order difference was observed. The marker D7Mit69 was proximal

to D7Mit8l and D7Mitl58 in our map, but distal to these markers in the published map.

We also defined the order of 12 markers (underlined in Figure 4-1), which could not be

separated due to the small number of mice genotyped in the construction of published

map. This SSR marker map is helpful in determining recombination sites during

production of the subcongenic strains.

Generating the subcongenic strains was described in Chapter 2. As shown in

Figure 4-1, B6.NZMc7c contains the centromeric end of Sle3 with NZM2410-derived







75

alleles from marker ckmnm (4 cM) to D7Mit154 (5 cM). B6.NZMc7t contains the

telomeric end of Sle3 with NZM2410-derived alleles from marker D7Mit69 (17.8 cM) to

D7Mitl47 (30.6 cM).

Both loci lead to h)pergammaglobulinemia and spontaneous Dolyclonal B cell

activation. The mice from both subcongenic strains showed generalized immune

hyperactivity like parental B6.NZMc7 mice. They had high total serum IgG and IgM

levels compared with B6 at 9 months of age (Figure 4-2). They showed high polyclonal

IgM antibodies against ssDNA, histone, DNP-KLH, thyroglobulin at 5 months of age

(Figure 4-3) as well as at 7, 9, and 12 months of age (data not shown). However, titers of

polyclonal IgM antibodies were lower than B6.NZMc7. Consistent with low antibody

titer, the penetrance of polyclonal IgM was lower also and there was little polyclonal IgG

antibody production in either of the subcongenic strains (Figure 4-4). These results

suggest that both loci can independently cause a low-grade polyclonal B cell activation,

but they need to interact synergistically in generating the pronounced phenotypes seen in

B6.NZMc7.

Both loci are required for IgG ANA production. B6.NZMc7 mice spontaneously

produce IgG antibodies against a broad spectrum of antigens, including nuclear and non-

nuclear antigens. This phenotype was also examined in mice bearing the subintervals. As

shown in Table 4-1, the frequency of IgG autoantibody production to a panel of four

nuclear antigens was very low. For example, the frequency of anti-dsDNA at 12 months

is about 13% in B6.NZMc7c, and 32% in B6.NZMc7t compared to 74% in B6.NZMc7.

Titers of IgG autoantibodies in both the subcongenic strains were also much lower







76

compared with B6.NZMc7 at 7 months of age (Figure 4-5) as well as at 9 and 12 months

of age (data not shown). In summary, neither locus by itself on B6 background is

sufficient to drive spontaneous IgG ANA production.

Responses of subcongenic mice to DNP-KLH challenge. As described above,

both the subcongenic strains spontaneously produced IgM antibodies to nuclear and non-

nuclear antigens, but they spontaneously produced little IgG autoantibodies. To further

assess immune responses, we challenged these mice in vivo with DNP-KLH. IgM and

IgG anti-DNP antibodies were examined before immunization, primary immune

response, secondary immune response, and duration of the response. In contrast with

B6.NZMc7 mice that produced high IgM anti-DNP antibodies, there was no significant

difference in the IgM anti-DNP antibody levels between the subcongenic strains and B6

(p>0.05). However, the IgM anti-DNP antibodies lasted longer in B6.NZMc7t (p<0.01)

(Figure 4-6, panel A), which could be seen from serum dilution 1:500 to 1:62500 (Figure

4-6, panel B). A high IgG anti-DNP secondary response was observed in B6.NZMc7t

(p<0.01), which was also seen in B6.NZMc7. The IgG anti-DNP antibodies lasted longer

in both the subcongenic strains compared with B6 (p<0.01) (Figure 4-7).

B6.NZMc7t exhibited increased CD4:CD8 T cell ratios. To define T cell

abnormalities in the subcongenic strains, we first examined the T cell surface marker

expression using flow cytometry. Table 4-2 summarizes the results of FACS analysis.

B6.NZMc7t mice showed increased percentages of CD4 T cells and increased CD4:CD8

T cell ratios in both the spleen and lymph node (Figure 4-8), with similar findings in







77

B6.NZMc7 (data not shown). A high percentage of splenic CD4 T cells in B6.NZMc7t

expressed activation marker CD69 (p < 0.01).

CD4 T cells from B6.NZMc7t exhibit high expansion in vitro. We also

examined absolute CD4 and CD8 T cell numbers after splenocytes from both the

subcongenic strains were stimulated with anti-CD3 in vitro. Consistent with increased

CD4 T cells in vivo, the absolute numbers of CD4 T cells were also increased in vitro in

B6.NZMc7t (Figure 4-9, panel A). Although CD4 T cell numbers were higher in

B6.NZMc7t compared with B6 and B6.NZMc7c, CD4 T cell numbers were even more

significantly increased after 48h of culture. A small drop of cell numbers in 72h culture

was noticed in B6.NZMc7 (Chapter 3), and also seen in B6.NZMc7t, probably due to

reasons discussed in Chapter 3. In contrast, CD8 cells were highly expanded in both B6

and B6.NZMc7c compared to B6.NZMc7t (Figure 4-9, panel B).Figure 4-10 shows the

change of percentage CD4 and CD8 T cells in 2D-dot-plot from the same experiment.

These results further demonstrate that the telomeric end of Sle3 impacts T cell function.

The telomeric subinterval also impacts lymphocyte proliferation. T cell

hyperactivity is an important phenotype in B6.NZMc7 mice and is believed to play a

important role in driving B cell autoantibody production. We therefore examined T cell

proliferation in subcongenic mice using a 3H-thymidine incorporation assay. T cells from

B6.NZMc7t exhibited an increased stimulation index (counts per minute in stimulated

cultures divided counts per minute in unstimulated cultures) in response to anti-CD3 (10

ug/ml) and IL-2 (10 U/ml) in vitro (Figure 4-11). T cells had higher responses to anti-

CD3 than to IL-2. The heightened proliferative responses to these stimuli may be due to







78

increased numbers of activated T cells, increased signal transduction, or reduced

apoptosis.

Both loci lead to a low incidence of glomerulonephritis (GN). The subcongenic

mice were sacrificed at 12 months for kidney histology. As shown in Figure 4-12,

surprisingly, the total frequency of GN (scored 2 and above) in both homozygous

subcongenic mice is as high as that in parental B6.NZMc7 (about 45%). However, the

overall severity of GN was less in the subcongenic strains compared with B6.NZMc7.

Both the subcongenic strains exhibited the mesangiopathic glomerular lesions that were

less severe than the hyaline lesions in B6.NZMc7. There was no sex bias in GN

frequency. GN was correlated with the production of autoantibodies against at least one

of four nuclear antigens, but not strongly correlated with the titers and onset of

autoantibody production (data not shown).



Discussion



Although significant volumes of data describing many humoral and cellular

abnormalities in murine lupus mice have been generated in the past three decades, the

genetic defects causing these abnormalities have not been determined. Although genetic

linkage studies have identified at least nine different loci to confer lupus susceptibility

(163), contribution and nature of each locus in the pathogenesis of abnormalities need to

be defined. Our approach to address these questions is via the production and analysis of









congenic strains bearing individual loci. From studies of B6.NZMc7 (Chapter 3), we

found Sle3 on murine chromosome 7 impacted T cell function, led to polyclonal B cell

activation, IgG ANA production, and lupus nephritis. In this study, we further dissected

the complex traits of B6.NZMc7 by generating two subcongenic strains and analyzing

their phenotypes.

We found that two subintervals both loci within Sle3 could lead to spontaneous

polyclonal B cell activation by themselves. These phenotypes are also observed in B6

mice congenic for the Sle2 interval on chromosome 4 (B6.NZMc4) (136). These

observations suggest that polyclonal B cell activation is a basic abnormality in murine

lupus and contributes to the pathogenesis of IgG ANA and lupus nephritis. Since

B6.NZMc4 mice have polyclonal B cell activation and lack IgG ANAs and nephritis,

polyclonal B cell activation alone is not sufficient for their autoimmune phenotypes.

The penetrance of autoantibodies against the four nuclear antigens decreased

dramatically in both subcongenic strains compared with B6.NZMc7, which indicates

requirement of both loci. The total GN frequency in the subcongenic strains is same as in

the parental strain, but the severity is reduced. These results further confirme our

hypothesis that SLE is inherited as a threshold liability (53) requiring multiple

susceptibility genes to cause high penetrance of phenotypes. This feature also makes the

further narrowing of subintervals very difficult.

GN is associated with IgG autoantibody production against four nuclear antigens

in both the subcongenic strains. We believe the conclusion is also true in parental

B6.NZMc7 mice because immunoflorescence assas of B6.NZMc7 kidneys showed the









presence of antibodies and complements in kidney (data not shown). Our experiments can

not define the specificity of autoantibodies causing GN.

The susceptibility genes causing the humoral autoinimune responses in the

subcongenic strains could impact T cell function, B cell function, or both. In a previous

study, we demonstrated that Sle3 primarily impacts T cell function, which may drive

lupus B cell activation and production of autoantibodies. To map this gene(s), we

performed the same sets of experiments on the subcongenic strains. We found that

B6.NZMc7t mice exhibited increased CD4:CD8 T cell ratios, higher IgG immune

responsiveness to the T-dependent antigen, heightened T cell proliferative responses to

anti-CD3 and IL-2. These findings suggest this gene(s) may locate in the telomeric end of

Sle3. B6.NZMc7t mice contain the disease gene(s), but fail to produce the same level of

IgG ANA as B6.NZMc7 indicating requirement of the gene(s) from the centromeric end

of Sle3 to augment these phenotypes.

B6.NZMc7c mice showed no obvious T cell functional aberrations. The

underlying mechanism for polyclonal B cell activation and lower level IgG ANA

production is not clear. It could be due to B cell dysfunction, subtle T cell function

changes, or both. Further detailed studies are needed.

This study successfully dissects the complex traits associated with Sle3 and

identifies the existence of at least two loci in the Sle3 interval. The experimental findings

indicate that both loci are required to generate certain levels of humoral autoinimunity

and severe lupus nephritis, but only the telomeric locus impacts T cell activation,

proliferation, and differentiation.
























Figure 4-1 A map of simple sequence repeat (SSR) markers on murine chromosome 7 covering the Sle3
interval. The map was constructed using MAPMAKER software according to 400 meiosis. NZM2410-
derived susceptibility intervals (white bars) in B6.NZMc7 and the two subcongenic strains are showed,
lines represent B6-derived alleles. The distances from each marker to the centromere were expressed in
cM (centiMorgan).








cM
2.3
4.9-
5



9.8
11.2
11.4
11.8
13.2
14.3


17.8
18.7
18.9
20.1
20.3- -
22
22.2
23.4
23.8

30.6
30.9
31.1


D7Mitl78
ckmm
D7Mit76
D7Mit56
D7Mitl54

D7Mitll4
~D7Mitl55


--"'' D7Mit78 D7Mit79
u)7Mit54
D7Mit25

-D7Mit69
-D7Mit8l
7Mit158 I D7Mit83
--------D7Mit85 D7Mitl45


D7Mitl76

D7Mit70


D7Mitl47
D7Mit93

-------- D7Mit3l


D7Mitl59


D7Mitl8l
D7Mitl62


h B6.NZMc7c







B6.NZMc7


B6.NZMc7t
























Figure 4-2. Total serum IgG and IgM level in 9 month subcongenic strains compared with B6.NZMc7.
Each dot represents individual mouse. Horizontal line is mean value.
















12W0












A


4W



3w


20


E

11W


I


I. I I I
136 B6.NZIW7 IKNEkc D6T JNZbc7t
FI0 v=21 0=24 32


0


w
.11*


AA
A--
AA


I I I

96 BNDOh? B6IQi. k7c B6.NDk7t
3=10 m721 v=24 1=25
























Figure 4-3. Polyclonal IgM antibodies against a panel of four antigens in 5 month old subcongenic strains
compared with B6 and B6.NZMc7. A similar data pattern is also seen in 7, 9, and 12 month old mice from
these four strains with increased titers. Antibody titer is expressed as %OD (detailed definition in Methods
and Materials). Data is presented as mean and standard deviation. p < 0.05, ** p < 0.01, *** p < 0.001.





800 0 B6 n=14
U B6.NZMc7c n=35
EJ B6.NZMc7t n=30
C U B6.NZMc7 n=34
'600-
o **

~400
*** ***


200 *

00
o 0 .....
ssDNA Histone DNP-KLH Thyroglobulin
























Figure 4-4. Penetrance of IgM and IgG polyclonal antibody production in 9 month old subcongenic mice
compared with parental B6.NZMc7. The mouse was counted as positive if it produced antibodies above
the mean plus 2 SD often B6 to any two of four antigens (ssDNA, histone, DNP-KLH, thyroglobulin).








90% l B6.NZMc7c n=24
B6.NZMc7t n=28
U B6.NZMc7 n=33

60%



30%





Polyclonal IgM Polyclonal IgG




00
00












Table 4-1. Penetrance of IgG anti-nuclear autoantibody production in B6.NZMc7c and B6.NZMc7t homozygous mice.


5 mo 7 mo 9 mo 11-12 mo
Antigen
c7c c7t c7c c7t c7c c7t c7c c7t

dsDNA 3%b 9% 8% 24% 0% 21% 13% 32%

ssDNA 12% 12% 4% 8% 8% 11% 13% 27%

dsDNA/histone 3% 3% 15% 8% 25% 18% 8% 23%

Histone 3% 3% 15% 8% 4% 7% 17% 23%

Na 33 34 26 25 24 28 24 22

a Number of mice tested.
b Percentage of positive mice. A mouse is counted as positive if its antibody titers are above the levels of mean plus 2 standard
deviations in six 12 month old B6 mice assayed on same plate.























Figure 4-5. IgG autoantibody production against a panel of four nuclear antigens in 7 month homozygous
and heterozygous subcongenic mice compared with B6.NZMc7 and B6. The data is expressed as % OD
detailedl definition in Methods and Materials). Horizontal lines indicate the mean value of antibody titers.













dsDNA 450 dsDNA/histone
250
400 0
0 o S 0
2 350
0 0 030 0
200300-
150 0

IS 14250.
00
ic o o 2@@s
100O 200- 0
0 0ft 0 0
so + -+-- rg ,,, 7 10o0- o

15 so0
500





3oo ssDNA

0 3 Histone

250

IS0 200 0
e 0
0 00
100. AA 1 100

a0 0

A 00
50 ,0 _ __ _ _

0
B6 B6.NZMc7 B6.NZIc7c B6.NZMk7t
horn. ht. hon. heL hon, het B6 B6.NZMc7 B6.NZMc7c B6.NZMc7t
n-25 n=33 n--24 n=20 n=30 n=21 n=31 horn. het hom het hom. het
n=25 n=33 n-24 n=20 n=30 n=21 n=31
























Figure 4-6. Immune responses to T-dependent antigen DNP-KLH in vivo. A) IgM anti-DNP antibody
production before immunization (dO), 14 days after primary immunization (d14), 7 and 30 days after boost
(d21 and d5 1) at Serum dilution of 1:500 in B6.NZmc7c mice (white dots with dashed line), B6.NZMc7t
mice (white triangles), and age- and sex-matched B6 (black dots). B) IgM anti-DNP antibodies on 5 Idays
at different serum dilutions. Data is from one of three independent experiments, and each dot is expressed
as mean OD and standard deviation of five animals. *, p < 0.05; **, p < 0.01.