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Phenotypic Characterization and Genetic Determination of T Cell Populations Associated with Lupus Susceptibility Locus Sle1a

Permanent Link: http://ufdc.ufl.edu/UFE0022839/00001

Material Information

Title: Phenotypic Characterization and Genetic Determination of T Cell Populations Associated with Lupus Susceptibility Locus Sle1a
Physical Description: 1 online resource (112 p.)
Language: english
Creator: Cuda, Carla
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acid, lupus, retinoic, sle, sle1a, treg
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Patients suffering from systemic lupus erythematosus (SLE) present with several manifestations, one of which being a decrease in the number of circulating regulatory T cells (Tregs). However, it has been shown that the suppressive capacity of the remaining population is maintained. SLE onset involves a combination of factors including genetic predisposition relating to the occurrence of susceptibility loci as well as environmental triggers. The goal of our lab is to locate genes involved in susceptibility by studying a congenic murine model of SLE containing three susceptibility loci from the NZM2410 spontaneous murine model bred onto a normal B6 background. The NZM2410 susceptibility loci are referred to as Sle1, Sle2, and Sle3 corresponding to chromosomes 1, 4 and 7, and combine to confer the same phenotype as the NZM2410. Major lupus susceptibility locus Sle1, which comprises three independent loci, Sle1a, Sle1b, and Sle1c, has been shown to enhance activation levels and effector functions of CD4-expressing T cells, reduce the size of the Treg subset, which express CD4, CD25 and CD62L, as well as decrease expression of the transcription factor Foxp3 among this population preceding autoantibody production. These phenotypes can be accounted for by Sle1a in a T cell-intrinsic manner. Upon further analysis of Sle1a expression, we found that although a decrease in the Treg population was observed, these cells appeared normal in terms of expression of markers associated with the regulatory phenotype. Both in vivo and in vitro functional studies indicated that these remaining Tregs also maintained their suppressive capacity on a per-cell basis suggesting that Sle1a controls the number of Tregs rather than their function. In addition, these in vitro and in vivo suppression assays showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as well as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic cells. Examination of truncated regions of Sle1a, called Sle1a.1 and Sle1a.2, resulted in intermediate phenotypes, indicating the presence of at least two genes within Sle1a synergistically interacting to confer the observed increase in activation of CD4-expressing T cells and decrease in Treg population. According to Ensembl Release 40 (www.ensembl.org), only one gene is present in the Sle1a.1 region of murine chromosome 1, which encodes for the protein pre-B cell leukemia transcription factor 1 (Pbx1). Little is known about the role of Pbx1 in immune cells and only two isoforms have been previously described, Pbx1-a and Pbx1-b. We found no alteration in gene expression or coding sequence between normal B6 and Sle1a.1 T cells, but rather found a novel isoform, Pbx1-d, expressed only in Sle1a.1 T cells. Sequence analysis revealed that Pbx1-d lacks the DNA binding domain associated with Pbx1-a and Pbx1-b. Microarray analysis revealed an alteration in the retinoic acid signaling pathway in Sle1a.1 CD4+ T cells. Treatment of B6.Sle1a.1 CD4-expressing cells that do not express CD25 with IL-2, TGF-?, and retinoic acid resulted in a decreased adaptive Treg (aTreg) population when compared to normal B6 CD4-expressing cells lacking CD25 expression. These results indicate that Pbx1 controls the level of Tregs in the periphery in a retinoic acid-dependent fashion.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Carla Cuda.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Morel, Laurence.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022839:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022839/00001

Material Information

Title: Phenotypic Characterization and Genetic Determination of T Cell Populations Associated with Lupus Susceptibility Locus Sle1a
Physical Description: 1 online resource (112 p.)
Language: english
Creator: Cuda, Carla
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acid, lupus, retinoic, sle, sle1a, treg
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Patients suffering from systemic lupus erythematosus (SLE) present with several manifestations, one of which being a decrease in the number of circulating regulatory T cells (Tregs). However, it has been shown that the suppressive capacity of the remaining population is maintained. SLE onset involves a combination of factors including genetic predisposition relating to the occurrence of susceptibility loci as well as environmental triggers. The goal of our lab is to locate genes involved in susceptibility by studying a congenic murine model of SLE containing three susceptibility loci from the NZM2410 spontaneous murine model bred onto a normal B6 background. The NZM2410 susceptibility loci are referred to as Sle1, Sle2, and Sle3 corresponding to chromosomes 1, 4 and 7, and combine to confer the same phenotype as the NZM2410. Major lupus susceptibility locus Sle1, which comprises three independent loci, Sle1a, Sle1b, and Sle1c, has been shown to enhance activation levels and effector functions of CD4-expressing T cells, reduce the size of the Treg subset, which express CD4, CD25 and CD62L, as well as decrease expression of the transcription factor Foxp3 among this population preceding autoantibody production. These phenotypes can be accounted for by Sle1a in a T cell-intrinsic manner. Upon further analysis of Sle1a expression, we found that although a decrease in the Treg population was observed, these cells appeared normal in terms of expression of markers associated with the regulatory phenotype. Both in vivo and in vitro functional studies indicated that these remaining Tregs also maintained their suppressive capacity on a per-cell basis suggesting that Sle1a controls the number of Tregs rather than their function. In addition, these in vitro and in vivo suppression assays showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as well as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic cells. Examination of truncated regions of Sle1a, called Sle1a.1 and Sle1a.2, resulted in intermediate phenotypes, indicating the presence of at least two genes within Sle1a synergistically interacting to confer the observed increase in activation of CD4-expressing T cells and decrease in Treg population. According to Ensembl Release 40 (www.ensembl.org), only one gene is present in the Sle1a.1 region of murine chromosome 1, which encodes for the protein pre-B cell leukemia transcription factor 1 (Pbx1). Little is known about the role of Pbx1 in immune cells and only two isoforms have been previously described, Pbx1-a and Pbx1-b. We found no alteration in gene expression or coding sequence between normal B6 and Sle1a.1 T cells, but rather found a novel isoform, Pbx1-d, expressed only in Sle1a.1 T cells. Sequence analysis revealed that Pbx1-d lacks the DNA binding domain associated with Pbx1-a and Pbx1-b. Microarray analysis revealed an alteration in the retinoic acid signaling pathway in Sle1a.1 CD4+ T cells. Treatment of B6.Sle1a.1 CD4-expressing cells that do not express CD25 with IL-2, TGF-?, and retinoic acid resulted in a decreased adaptive Treg (aTreg) population when compared to normal B6 CD4-expressing cells lacking CD25 expression. These results indicate that Pbx1 controls the level of Tregs in the periphery in a retinoic acid-dependent fashion.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Carla Cuda.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Morel, Laurence.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022839:00001


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1 PHENOTYPIC CHARACTERI ZATION AND GENETIC D ETERMINATION OF T CELL POPULATIONS ASSOCIATED WITH LUPUS SUSCEPTIBILITY LOCUS SLE1A By CARLA MARIE CUDA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Carla Marie Cuda

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3 To my Grandparents, my biggest fans, my un failing support. You are both greatly missed. Youre smart, just like your grandfather.

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4 ACKNOWLEDGMENTS I would like to thank m y parents for always being there, encouraging me, pushing me, and believing in me. I thank my family members for their comfort. I thank my friends for their support. I thank my committee members for their continued advice, and I thank my mentor for her guidance. I would like to acknowledge our collaborators at the University of Florida, including Dr. Eric S. Sobel for his help w ith the analysis of the bone ma rrow chimera data, Dr. Byron P. Croker for his analysis of the colitis tissue samp les, Dr. Shiwu Li for his molecular work with Pbx1 Dr. Zhiwei Xu for the microarray, and Dr. He nry Baker for his analysis of the microarray data. I would also like to acknowledge our coll aborators outside the Un iversity of Florida, including Dr. Igor Dozmorov at the Oklahom a Medical Research F oundation for further analyzing the microarray data, and Dr. Vijay Kuchroo at Harvard Medical School for giving us their Foxp3 KI mice. I also thank Leilani Zeumer and Xuekun Su for excellent animal care, Dr. Pui Lee for his advice with DC phenotyping, and the members of the Mo rel laboratory for stimulating discussions.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................... .............11 CHAP TER 1 INTRODUCTION .................................................................................................................. 13 2 LITERATURE REVIEW .......................................................................................................16 Systemic Lupus Erythematosus in Humans ........................................................................... 16 Murine Models of Systemic Lupus Erythematosus ................................................................ 18 Identification of Susceptibility Loci .......................................................................................19 Tregs and Systemic Lupus Erythematosus ............................................................................. 22 Retinoic Acid and the Immune System .................................................................................. 26 Pbx1 and the Immune System ................................................................................................ 27 3 MURINE LUPUS SUSCEPTIBILITY LOCUS SLE1A CONTROLS R EGULATORY T CELL NUMBER AND FUNCTION THROUGH MULTIPE MECHANISMS ............... 30 Introduction .................................................................................................................. ...........30 Materials and Methods ...........................................................................................................32 Mice .................................................................................................................................32 Flow Cytometry ...............................................................................................................32 Suppression Assays .........................................................................................................33 Generation of DCs and DC Phenotyping ........................................................................34 Bone Marrow Chimeras ..................................................................................................34 Statistical Analysis .......................................................................................................... 35 Results .....................................................................................................................................35 Sle1 and Sle1a are Asso ciated with Increased Levels of Activated T Cells and Decreased Levels of Tregs ........................................................................................... 35 Sle1a Tregs Require a Higher Treg :Teff Ratio to Support Inhibition to a Level Equivalent to B6 Tregs ................................................................................................37 Sle1a Expression Increases DC Activation .....................................................................37 Sle1a Expression Affects the Ability of Bo th DCs to Support Treg Suppression and Teffs to be Inhibited ..................................................................................................... 38 Sle1a Expression Intrinsically Affects CD4+ T Cell Phenotypes .................................... 39 Discussion .................................................................................................................... ...........40

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6 4 THE CONTROL OF REGULATORY T CELL NUMBE R AND FUNCTION BY MURINE LUPUS SUSCEPTIBILITY LOCUS SLE1A REQUIRES THE SYNERGISTIC EFFECT OF SUBLOCI SLE1A.1 AND SLE1A.2 .......................................51 Introduction .................................................................................................................. ...........51 Materials and Methods ...........................................................................................................51 Mice .................................................................................................................................51 Sle1a Map ........................................................................................................................ 52 Flow Cytometry ...............................................................................................................52 Suppression Assays .........................................................................................................52 Bone Marrow Chimeras ..................................................................................................53 Statistical Analysis .......................................................................................................... 53 Results .....................................................................................................................................54 More than One Gene Contributes to Sle1a Phenotypes .................................................. 54 Sle1a Requires Both Sle1a.1 and Sle1a.2 Expression for the Increased L evel of T Cell Activation and Decreased Level of Tregs ............................................................55 Sle1a.1 and Sle1a.2 Tregs Can Support Inhibition to a Level E quivalent to B6 Tregs In Vitro ...............................................................................................................56 Sle1a.1 and Sle1a.2 Expression Affects the Ability of Both DCs to Support Treg Suppression and Teffs to be Inhibited, but to a Lesser Extent than Observed for Sle1a .............................................................................................................................56 Sle1a.1 or Sle1a.2 Expression Intrinsically Affects CD4+ T Cell Phenotypes ................ 58 5 MURINE LUPUS SUSCEPTIBILITY LOCUS SLE1A.1 C ONTROLS RETINOIC ACID-ENHANCED TGF-BETA-INDUCED REGULATORY T CELL EXPANSION ..... 67 Introduction .................................................................................................................. ...........67 Materials and Methods ...........................................................................................................67 Mice .................................................................................................................................67 Sle1a.1 Map and Pbx1 Prim ers ....................................................................................... 67 Flow Cytometry ...............................................................................................................68 Apoptotic Cell-Induced Production of IL-10 by Peritoneal Macrophages .....................68 Microarray Analysis for Diffe rential Gene Expression ...................................................69 Induction of CD4+ Foxp3+ Tregs from CD4+ CD25T Cells .......................................... 70 Results .....................................................................................................................................70 Sle1a.1 Contains Only O ne Gene .................................................................................... 70 Sle1a.1 Expression Leads to a Decreased Ratio of CD4+ Foxp3+ to Total CD4+ T Cells ......................................................................................................................... ....71 Alternative Pbx1 Isoforms ............................................................................................... 71 Expression of Sle1a.1 did not Affect Apoptotic C e ll-Induced Production of IL-10 by Macrophages ...........................................................................................................72 Microarray Analysis of Differential Gene Expression Influenced by the NZW allele of Pbx1 .........................................................................................................................73 Expression of Sle1a.1 Results in Def ective RA-Enhanced TGF-Induced Production of Adaptive Tregs ...................................................................................... 74 6 DISCUSSION .................................................................................................................... .....85

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7 REFERENCES .................................................................................................................... ..........94 BIOGRAPHICAL SKETCH .......................................................................................................112

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8 LIST OF TABLES Table page 4-1 Genes contained within the Sle1a in terval according to Ensembl Release 40 .................. 61 5-1 Microarray data obtained from B6 and B6.Sle1a.1 -deriv ed CD4+ T cells ........................81

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9 LIST OF FIGURES Figure page 3-1 Sle1a resu lts in increased CD4+ T cell activation and a diminished Treg compartment ... 44 3-2 Sle1a does not affect expression of markers a ssociated with the regulatory phenotype ... 45 3-3 Sle1a Tregs are deficient in inhibiting proliferation at low Treg:Teff ratios ..................... 45 3-4 Sle1a expression activates dendritic cells .......................................................................... 46 3-5 Sle1a expression in Tregs, Teffs, or APCs aff ects the extent of the inhibition of Teff proliferation........................................................................................................................47 3-6 Sle1a expression in either Tregs or T effs aff ects the extent of disease in an adoptive transfer model ....................................................................................................................48 3-7 Quantification of the effects of CD4+ CD25Teff and CD4+ CD25+ Treg transfers into B6.Rag1-/mice ........................................................................................................... 49 3-8 Sle1a expression affects CD4+ function in a cell-intrinsic manner ................................... 50 4-1 Map of Sle1a ......................................................................................................................60 4-2 Sle1a requires both Sle1a.1 and Sle1a.2 for increased CD4+ T cell ICOS expression and diminished Treg compartment .................................................................................... 62 4-3 Sle1a.1 and Sle1a.2 Tregs can support inhibition to a level equivalent to B 6 Tregs in vitro ....................................................................................................................................63 4-4 Sle1a.1 or Sle1a.2 expression in Teffs or APCs affect s the extent of the inhibition of Teff proliferation ............................................................................................................ ....64 4-5 Expression of Sle1a Sle1a.1 or Sle1a.2 in either Tregs or Tef fs affects the extent of disease in an adoptive transfer model ................................................................................ 65 4-6 Expression of either Sle1a.1 or Sle1a.2 affects CD4+ function in a cell-intrinsic manner................................................................................................................................66 5-1 Genes present in the Sle1a.1 interval .................................................................................76 5-2 Sle1a.1 expression results in a decreased ratio of CD4+ Foxp3+ to total CD4+ T cells .....77 5-3 The NZW allele of Pbx1 results in a two novel isofor ms .................................................. 78 5-4 Depiction of exons associated with both known and novel isofor ms of Pbx1 ..................79

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10 5-5 Expression of the NZW allele of Pbx1 in m acrophages does not alter their IL-10 production in response to apoptotic cells ...........................................................................80 5-6 Expression of the NZW allele of Pbx1 in CD4+ T cells leads to differentially expressed genes involved in the RA-signaling pathway ....................................................82 5-7 Pathway analysis of differe ntially expressed genes in CD4+ T cells isolated from B6 and B6.Sle1a.1 mice .......................................................................................................... 83 5-8 The NZW allele of Pbx1 leads to a defect in RA-enhanced TGF-induced expansion of aTregs in vitro ................................................................................................................84 6-1 The proposed mechanism of Pbx1s role in the connection of the retinoic acid and TGFsignaling pathways in a T cell ................................................................................93

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHENOTYPIC CHARACTERI ZATION AND GENETIC D ETERMINATION OF T CELL POPULATIONS ASSOCIATED WITH LUPUS SUSCEPTIBILITY LOCUS SLE1A By Carla Marie Cuda December 2008 Chair: Laurence Marguerite Morel Major: Medical Sciences--Immunology and Microbiology Patients suffering from systemic lupus er ythematosus (SLE) present with several manifestations, one of which being a decrease in the number of circul ating regulatory T cells (Tregs). However, it has been shown that th e suppressive capacity of the remaining population is maintained. SLE onset involves a combination of factors including genetic predisposition relating to the occurrence of susceptibility loci as well as environmental triggers. The goal of our lab is to locate genes invol ved in susceptibility by studying a congenic murine model of SLE containing three susceptibility loci from the NZM2410 spontane ous murine model bred onto a normal B6 background. The NZM2410 suscep tibility loci are referred to as Sle1 Sle2 and Sle3 corresponding to chromosomes 1, 4 and 7, and co mbine to confer the same phenotype as the NZM2410. Major lupus susceptibility locus Sle1 which comprises three independent loci, Sle1a Sle1b and Sle1c has been shown to enhance activatio n levels and effector functions of CD4-expressing T cells, reduce the size of the Treg subset which express CD4, CD25 and CD62L, as well as decrease expression of the transcription factor Foxp3 among this population preceding autoantibody production. These phenotypes can be accounted for by Sle1a in a T cellintrinsic manner. Upon further analysis of Sle1a expression, we found th at although a decrease in the Treg population was observe d, these cells appeared normal in terms of expression of

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12 markers associated with the regulatory phenotype. Both in vivo and in vitro functional studies indicated that these remaining Tregs also main tained their suppressive capacity on a per-cell basis suggesting that Sle1a controls the number of Tregs rather than their function. In addition, these in vitro and in vivo suppression assays showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as we ll as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic ce lls. Examination of truncated regions of Sle1a, called Sle1a.1 and Sle1a.2 resulted in intermediate phenotypes, indicating the presence of at leas t two genes within Sle1a synergistically interacti ng to confer the observed increase in activation of CD 4-expressing T cells and decr ease in Treg population. According to Ensembl Release 40 (www.ensembl .org), only one gene is present in the Sle1a.1 region of murine chromosome 1, which en codes for the protein pre-B cell leukemia transcription factor 1 (Pbx1). Little is known about the role of Pbx1 in immune cells and only two isoforms have been prev iously described, Pbx1-a and Pb x1-b. We found no alteration in gene expression or coding sequence between normal B6 and Sle1a.1 T cells, but rather found a novel isoform, Pbx1-d, expressed only in Sle1a.1 T cells. Sequence analysis revealed that Pbx1d lacks the DNA binding domain associated with Pbx1-a and Pbx1-b. Microarray analysis revealed an alteration in the re tinoic acid signaling pathway in Sle1a.1 CD4+ T cells. Treatment of B6.Sle1a.1 CD4-expressing cells that do not express CD25 with IL-2, TGF, and retinoic acid resulted in a decreased adaptive Treg (aTreg) population when compared to normal B6 CD4-expressing cells lacking CD25 expression. These results indi cate that Pbx1 controls the level of Tregs in the periphery in a retinoic acid-dependent fashion.

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13 CHAPTER 1 INTRODUCTION System ic lupus erythematosus is a chronic au toimmune disease with varied pathogenicity from mild forms to fatal end organ damage. It manifests itself in auto-antibody production directed primarily against nuclear antigens (ANAs). The most serious clinical outcome results from immune complex deposition in the kidney, which eventually leads to autoimmune lupus glomerulonephritis (GN), and finally complete ki dney failure. Genetic predisposition has been implicated in susceptibility to SLE in the past 20 years. For inherited di seases, the variation in disease expression resulting from ge netic factors reflects variation in genome structure, called a polymorphism (1). The difference in genome stru cture can be assumed to be the cause, either directly or indirectly, for the change in function observed. Alte rations in genome structure can occur in many ways, such as an alteration in prot ein structure encoded by a gene or an alteration in the level of expression of a gene (1). An individual polymorphism can then be inherited from parent to offspring according to Mendelian laws. By identifying functional variations associated with SLE, we can make advancements in unders tanding the root causes of disease development and progression by pinpointing fundamental disease mechanisms. Spontaneous murine models of SLE offer insights into genetic susceptibility, as disease development has been unequivocally dependent on genetic predisposition. Our lab focuses on congenic dissection of the NZM2410 spontaneous murine model of SLE. The NZM2410 strain is a recombinant inbred line derived from th e NZB and NZW strains (2-4). In short, by converting a polygenic system, the NZM2410 strain in our case, into a group of monogenic strains, each carrying a suscepti bility locus on a resistant gene tic background, one can analyze, both genetically and functionally, these monogenic stra ins for traits to associate with a particular genetic defect. The NZM2410 susceptib ility loci are referred to as Sle1 Sle2 and Sle3

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14 corresponding to chromosomes 1, 4 and 7, and co mbine to confer the same phenotype as the NZM2410 (3). Major lupus susceptibility locus Sle1 associated with multiple murine models of SLE as well as human patients (5-11) comprises three independent loci, Sle1a Sle1b and Sle1c (12), and has been shown to enhance activa tion levels and effect or functions of CD4+ T cells, reduce the size of the CD4+ CD25+ CD62L+ Treg subset, as well as decrease expression of the transcription factor Foxp3 among this population preceding autoantibody production (13). It was initially proposed to study one of thr ee susceptibility loci contained within the Sle1 susceptibility locus, Sle1a by characterizing its contribution to SLE disease as well as further refining the genetic map in an effort to narrow down the list of genes within the Sle1a locus potentially involved in SLE susceptibility and pat hogenesis. A review of the literature will be presented, followed by the first chapter of results. In this first chapter, we will examine the entire Sle1a region's effect and show that its expression affects a genetic pathway regulating production of Tregs and responses to Tregs in a ma nner that is not restricted to tolerance to nuclear antigens. We will accomplish this by characterizing the phenotypes of the T cells, effector and regulatory, as well as the antigen-presen ting cells (APCs), in both in vitro and in vivo settings. The identification of the Sle1a gene(s) will therefore uncover a novel and broad pathway by which autoreactive T cells are regulated by Tregs. In the second results chapter, we will further examine Sle1a to refine the genetic map in order to locate genes responsible fo r previously observed phenotypes. Sle1a contains at least two susceptibility loci, and by examining the effect s of expression of these truncated regions of Sle1a we can observe phenotypes that will either co-segregate with the parental strain ( Sle1a ) or require the synergistic involvement of the othe r locus. We will accomplish this by performing the same experiments from the first results ch apter in mice containing truncated regions of Sle1a

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15 called Sle1a.1 and Sle1a.2, and show that none of the phenotypes observed for the entire Sle1a region can be fully explained by expression of either of the individual subloci alone, indicating that the synergistic effect of th e two subloci is a requirement for Sle1a -mediated action. Finally, in the third results chapter, we will further examine one of Sle1a 's subloci, Sle1a.1 to elucidate its involvement in SLE pathogenesis. We will show that this locus contains only one gene, Pbx1 which encodes for pre-B cell leukemia transc ription factor, and that the NZW allele of this gene not only leads to alternate isofor ms of the protein, but leads to alterations in expression of a number of other genes using microarray data analysis. We will carry out functional studies to elucidat e a potential mechanis m of Pbx1's action and hypothesize that expression of the NZW allele of Pbx1 leads to decreased produc tion of adaptive regulatory T cells (aTregs), thereby contributi ng to the observed phenotype of de creased Tregs attributable to expression of the entire Sle1a locus.

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16 CHAPTER 2 LITERATURE REVIEW Systemic Lupus Erythematosus in Humans An estim ated 1.5-2 million Americans currently suffer from some form of SLE according to the Lupus Foundation of America, with this disease most notably affecting women between the ages of 15 and 45. SLE onset involves a combination of fact ors including genetic predisposition relating to the occurrence of susceptibility loci as well as environmental triggers. This disease has a concordance rate of 2-5% for dizygotic twins compared to 24-58% for monozygotic twins, leading to a 10 -fold difference (14). Siblings of SLE-affected individuals have also been shown to be 20 times more likel y to develop SLE than those without affected siblings (15). There is also wide disease va riation among different ethnic populations regarding disease prevalence with a 3-4 times greater prev alence in African-American or Afro-Caribbean populations compared with Caucasia ns in the same location (16,17). Linkage analysis and association analysis have been the two techniques employed to study the genetics of human lupus (18). Linkage st udies look for the segreg ation of linkage of particular genetic markers with disease in the family members of SLE-affected individuals. These markers are generally linked to disease by causing variants due to their relative proximity on the genome and the capability of being inherited t ogether at meiosis rather than being functional themselves (1). If many families presen t with this same marker, it can be inferred that there is a disease causing variant located within that particular area of the genome, called a linkage region (1). Association studies involve the selection of a candidate gene marker, with a variation already known and implicated in disease susceptibility (1). The frequencies of these variations or polymorphisms are assessed betwee n SLE-affected individuals and appropriate controls (19). If a polymorphism has a greate r than expected frequency in SLE-affected

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17 individuals as compared to controls, it can be in ferred that this variant is disease-causing. Until recently it was a problem that only a small number of markers could be studied because a hypothesized gene or set of genes was required to begin study (1). Fortunately, in the past six years, advances in technology have made it po ssible to type thousands of single nucleotide polymorphisms (SNPs) across the whole genome at the same time (20,21). Recent Genome Wide Association scans have imp licated several genes as contribu tors to SLE susceptibility (2225) TNFAIP3 encodes for TNFAIP3, a key regulator of NFB signaling through ubiquitin modification of adaptor proteins RIP and TRAF6 downstream of TNF and Toll-like receptors, respectively (26,27). BANK1 is specifically expressed in B cell s and has a potential role in B cell receptor-mediated activation (28,29). IRF5 encodes for IFN regulatory factor 5 and is a transcription factor downstream of the t ype I IFN and Toll-like receptors (30-33). ITGAM encodes for CD11b, combines with the 2 chain to form a leukocyte integrin, CR3, that is important for adherence to neutrophils and monocy tes to stimulated endothelium, and is also a receptor for the complement component C3 degradation product, iC3b (34). While many linkage regions have been id entified by performing multiple whole genome scans in several cohorts of lupus families, an issue arises becau se these regions are not found in all studies. It is therefore necessary to follo w the approach of analyz ing the results using a rigorous significance cut-off (35) or pooling results from multip le studies in a meta-analysis (36,37) to eliminate false positives and find priority regions on which to focus (1). However, linkage has proven to be rather poor in identif ying candidate genes due to in SLE due to inconsistencies between studies cau sed by a number of issues (1). The first is that there are many types of lupus. The American College of Rheumatology requires only 4 of 11 criteria to be diagnosed with SLE (38), and this may cause in consistencies in what genes lead to particular

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18 symptoms or phenotypes. The second issue is gene tic heterogeneity. It may be that different SLE susceptibility genes act in different ethn ic groups. It has been shown in mice that susceptibility genes act in different ways de pending on the genetic back ground as a result of modifying genes elsewhere in the genome (39). The third, and most interesting in our case, is that detection of genes that onl y modestly affect the phenotype of interest is difficult using linkage analysis (40). Of interest for our st udy is the idea of the "common variants/common disease" hypothesis by Becker, which implicates multiple genetic loci interacting with one another in an additive manner leading to overall disease susceptibility, wh ile individually having only small effects (41). These i ssues lead to the necessity of other methods to elucidate genes involved in susceptibility to SLE. Murine Models of Systemic Lupus Erythematosus A num ber of inbred mouse models which develop SLE-like disorders have been used to further our understanding of SLE genetics. However, caution must be taken when attempting to locate human candidate genes from mouse st udies. Although 99% of murine genes are considered to have a homologous human gene, it cannot be assumed that the gene functions in the same manner and must be investigated, if pos sible, in a human setting to definitively prove its importance (42). There are th ree general types of mouse models used at present to define genetic susceptibility to SLE: gene manipulation-derived, induced, and spontaneous. While these models share the common features of AN As and autoantibodyan d/or immune-complex mediated end-organ disease, they differ in se verity, autoantibody prof ile, sex predominance and clinical manifestations (43). Gene-manipulation-derived models include the Fc RIIb-deficient mice (44), while induced-disease models include the procainamide, prista ne, idiotype, mercuryinduced autoimmunity (DBA/ 2, resistance) (45) and the Mycobacterium bovis -induced systemic autoimmunity in NOD mice (46,47), mapping studies ha ve only been performed in the latter two

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19 (47,48). Induced models have provided insigh ts as to the relationship between genetic susceptibility and environmental f actors (43). The NZB, NZW, MRLFaslpr, and the BXSB strains are the most commonly st udied among the spontaneous models. Extensive studies have also been carried out on recomb inant inbred lines derived from the NZB and NZW strains, the NZM2328 strain and, in the case of our lab, the NZM2410 strain (2-4). In order to study these mice to locate potential candidate gene s, one can follow either the reverse or forward genetics a pproaches. Reverse genetics in volves genetic manipulation via transgenes or site-directed mutagenesis (gene knockout) of known genes in nonautoimmune strains, thereby revealing specific gene alte rations which are capable of promoting SLE development (43). One can also employ this strategy to identify modifier genes that can reduce susceptibility by genetically manipulating S LE-prone strains by backcrossing of mainly knockout genes (43). Forward genetics involves identification of genes based solely on chromosomal region by initially mapping traits to chromosomal regions or loci, generating and characterizing locus-containing interval congenic mice, subsequently narrowing the interval by screening congenic mice with smaller-sized interval s, and then finally the screening of candidate genes (43). This is the method employed by our la b. This approach may lead to difficulties in identifying susceptibility genes should strong li nkage disequilibrium exist in the region of interest and in verifying the ro le of even the most appealing of candidate genes (43). These difficulties can only be worse in the case of hum an SLE, highlighting mouse models as an important complementary tool for confirmi ng the significance of gene variation. Identification of Susceptibility Loci Since publication of a landm ark paper detailing the linkage analysis of type I diabetes in the NOD mouse (49), the desire to locate susceptibility genes for murine models of autoimmune diseases has grown. Our lab has focused its atte ntion on the identification of SLE-susceptibility

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20 loci in the NZM2410 spontaneous murine mode l using genome-wide linkage analysis (or Quantitative Trait Locus mapping) in an effort to discover the ge nes involved in susceptibility. The NZM2410 susceptibility loci are referred to as Sle1 Sle2 and Sle3 corresponding to chromosomes 1, 4 and 7 (3), and combine in the B6. Sle1Sle2Sle3 triple congenic strain to confer the same phenotype as the NZM2410 (50,51). By studying the single, double and triple congenic strains containing either one, tw o or all three susceptibility loci from the NZM2410 bred onto a normal B6 background, distinct phenotypes as well as necessary interac tions between multiple loci for full disease development have been obs erved. In both humans and mice, one of the hallmarks of SLE is the loss of tolerance to nuc lear antigens, most notably chromatin and antidsDNA (10), leading to the production of nuclear antigen specific autoantibodies or ANAs. This particular phenotype has been linked to the Sle1 locus. The Sle1 locus has been identified as having the strongest statistical linkage to clinical disease and nephritis (3), it maps to a genom ic region (telomeric chromosome 1 and 1q23-1q42) that has been linked to other mouse models ( 52) as well as human S LE patients (53), and has been implicated as the root of a multi-step pr ocess leading to SLE pat hogenesis and disease onset based on the following results (53). This locus leads to a selectiv e loss of tolerance to chromatin, with the production of serum IgG antibodies preferentially targeting H2A/H2B/DNA subnucleosomes as early as 5 months of age, w ith a penetrance of more than 75% (54,55). Expression of Sle1 has been shown to be necessary fo r nephritis development in the NZM2410 model (4,51). In addition, a highly penetrant clinical pathology was observed when Sle1 was coexpressed with various single mutations or other Sle -susceptibility loci (44,50,51,56). Mixed bone marrow chimeras have shown that Sle1 expression leads to increased expression of activation markers, cytokine production and gene ration of histone-specific T cells, with the

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21 activation of T cells occu rring independent of B cell presence i ndicating an intrinsic T cell defect (54,57). Based on results from Sle1 recombinants, the production of ANAs maps to at least three independent loci, referred to as Sle1a Sle1b and Sle1c (12). The genetic regions Sle1b and Sle1c currently possess candidate genes, while no candidate gene exists at present for the Sle1a region (58,59). It was proposed that Sle1b is allelic with a cluster of seven genes from the SLAM/CD2 fa mily, with the stronge st candidate being Ly108 (59). It has now been shown that the normal isoform of Ly108 has the capacity to sensitize immature B cells to deletion and RAG re-expression, indicating that Ly108 is a potential regulator of tolerance checkpoints, censoring self-reactive B cells and thereby safeguarding against autoimmunity (60). Ho wever, the NZW allele of Ly108 leads to an alternative isoform lacking this regulator y capacity (60). Cr2 encodes for the compleme nt receptors type 1 and 2 (CR1/CR2) and is a strong candidate gene for Sle1c due to a mutation disrupting its function as a B cell receptor (58). The following studies concentrate on the Sle1a sub-congenic region of Sle1 by examining a strain carrying a truncated region of the cen tromeric end of Sle1a termed B6. Sle1a.1 and a strain which includes the telomeric end of Sle1a termed B6. Sle1a.2 to further characterize this important locus in an attemp t to elucidate its role in the production of autoreactive T cells corresponding to a reduction in Tregs. Expression of Sle1a in T cells has been shown to lead to levels of activation as well as cytokine production similar to that of the entire Sle1 locus (13). Adoptively tran sferred T cells from both Sle1 and Sle1a were shown to be capable of providing help to chromatin-specific B ce lls (13). It was inferr ed that expression of Sle1a alone was necessary and sufficient to induce au toreactive T cells capable of providing help to chromatin-specific B cells to produce ANAs (13) It was also observed that expression of

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22 Sle1a was associated with a reduced level of CD4+ CD25+ Tregs preceding autoantibody production, potentially suggesting a causal relations hip with autoreactive T cell generation (13). Tregs and Systemic Lupus Erythematosus T cells p lay an important role in the developm ent of SLE, both in mice and in humans (61). Normally, central deletion by negati ve selection of T cells reactive to self-peptide occurs in the thymus. This prevents interactions of these auto reactive T cells and self-a ntigen in the periphery, thereby averting an autoimmune state. Should th ese cells escape deleti on, mechanisms exist in the periphery to keep these interactions in check, including extrinsic regulation by Tregs and intrinsic regulation inherently programmed into the cells themselves based on life spans, etc. However, in certain cases, these checks are not fu lly functional and autoimmunity results. In the case of murine models, the disease state cannot be initiated without the help of T cells, and these cells can also be directly pathogenic, forming part of the cellular inf iltrates in skin and interstitial areas of the kidney. Although it is evident that T cells are necessary for di sease, the degree to which intrinsic defects in these cells c ontribute to disease re mains unclear (62). According to Sakaguchi, nearly 30 years ago studies done by Nish izuka & Sakakura (63) and Penhale et al. (64) started the current interest in T cell-me diated control of self-reactive T cells as being a key dominant mechanism involve d in self-tolerance (65). And so began the interest in Tregs, which we re initially defined as CD4+ CD25+ T cells. Substantial evidence exists showing the indispensable role of Tregs in the maintenance of natural self-tolerance and negative control of immune responses capable of leading to pathogenesis (65). Landmark experiments done by Sakaguchi re vealed that re moval of CD4+ CD25+ T cells elicited autoimmune disease as well as enhancing immu ne responses to non-self antigens through the development of autoimmune CD4+ T cells resulting in the brea kdown of B cell se lf-tolerance as

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23 well (66). Tregs are detectable in the periphery of normal mice at day 3 post-birth, and rapidly increase to the adult level (5 -10%) around 3 weeks of age (67). The mode by which Tregs perform their supp ressive function involves recognition of and stimulation by self-antigen to control self-reactive T cells in the normal internal environment either by cell-cell contact or by cytokine-media ted pathways, and are capable of suppressing not only T cells with the same antigen specificity, but also those specific for other antigens once activated (68). Foxp3 encodes for Scurfin, a member of the for khead/winged-helix family of transcription factors, which has been implicated in the onset of autoimmune disease when defective (69). Loss-of-function mutations of this transcription factor leads to impaired Treg development, the lethal X-linked lymphoproliferative disorder of th e naturally arising scurfy mouse strain (70-73), and the homologous disorder in humans, IPEX or 'immune dysregulation, polyendocrinopathy, enteropathy, X-linked' syndrome (74-76). This molecule has also been implicated in the development and function of Tregs due to studies which revealed that transduction of Foxp3 induced expression of CD25, CTLA-4, CD103, and GITR, which are closely related to the function of Tregs, as well as suppressing proliferation in vitro of other T cells and preventing autoimmune development in vivo thus giving rise to the idea that Foxp3 is the master control gene for the development and function of Tregs ( 77). Studies have shown that Treg effector function but not necessarily li neage commitment requires the expression of functional Foxp3 protein (78), and that continue d expression is needed to maintain the transcriptional and functional program established du ring Treg cell development (79) Interestingly, it was found that Foxp3 expression leads to di stinct transcriptional programs for those cells produced in the thymus compared to those developing in the periphery (80). High expression of CD62L on

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24 Tregs has also been implicated as important due to tissue-homing capabilities (81). Based on these and other studies, a specific panel of markers will be used to analyze phenotypic differences of Tregs among the chosen mice strains. Tregs are not only produced in the thymus, called natural Treg s (nTregs), but several types of in vivo and in vitro -induced Tregs have been described, ca lled inducible Tregs (iTregs). The difference between these populations and nTregs is that the iTregs are dependent on cytokines. What is unknown is whether thes e populations arise from a common precursor. There are the Tr1 cells, which produce high levels of immunosuppressive IL-10, can be generated by chronic activation in the presence of IL-10 in both mice and humans, a nd are able to prevent autoimmune colitis in an experimental model (82,83). The T h3 cells were observed afte r oral or intravenous antigen application and produce TGF(84). Similar to the Tr1 cells, these cells can mediate their suppressive capacity through IL-10 secretion as well in a cel l-contact independent fashion (84). Induction of alloantigen-specific Tregs can be accomplished by immature DCs expressing low levels of costimulatory molecules in vitro (85). The Tregs produ ced under these conditions are similar to Tr1 cells in that they produce high levels of IL-10, but act like nTregs that are cellcontact dependent. The human CD4+ CD25+ natural-like Tregs act in a cytokine-independent manner, but can drive CD4+ CD25T cells to develop a cytokine-dependent immunosuppressive capacity (86). These natural-like Tregs are formed by rendering CD4+ T cells from peripheral blood anergic with alloantigeneic st imulation in the presence of TGF. CD4+ Foxp3nave T cells have been shown to be capable of conversion to a CD4+ Foxp3+ Treg population, called adaptive Tregs (aTregs), which present with the same phenotypic and suppressive characteristics as nTregs both in vivo and in vitro (87-91). It has also been s hown that this conversion not only requires IL-2, but is dependent on TGF(87,92,93). These aTregs have been used a means of

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25 inducing tolerance in a number of settings, including inflammatory bowel disease and allotransplantation models (8890,94). Clinical use can perhaps be achieved by improving the potency of these aTreg treatments. While the loss of Tregs can result in organ-sp ecific autoimmune dis eases (81,95), little is known about Tregs in the systemic autoimmune diseas e, SLE. The role of Tregs in prevention of systemic autoimmunity has been shown in double transgenic models expressing a TCR and its cognate antigen (96-98). The generation of antig en-specific Tregs was found to be necessary in the (SWRxNZB)F1 model for tolera nce induction to nuclear antigen with histone peptide (99). Spontaneous models for lupus are less unde rstood, however. The NZM2328 lupus model showed no spontaneous global deficiency upon an alysis of neonatal thymectomy experiments (100), mixed results have been shown for ot her models (100,101). One study showed that adoptive transfer of Tregs can ameliorate disease phenotypes induced by SLE (102), although these Tregs were exogenously expanded. Our lab has shown that introduction unmanipulated CD4+ CD25+ Tregs isolated from the B6. Sle1Sle2Sle3 mice into B6. Sle1Sle2Sle3 recipients before the onset of autoantibody production can re duce all disease phenotypes, indicating that the number of Treg plays an important role in systemic autoimmunity. Studies initially done in human SLE reported decreased percentages of CD4+ CD25+ T cells in the peripheral blood of SLE patients as compared to cont rols (103). However, results from these studies were inconclusive due to disc repancies in accurately discriminating the Treg population from activated T cells since Treg s in humans are restricted to the CD25high fraction. nTregs were shown to be lower in number and functionally impaired during active SLE in several studies (104-107), with Foxp3+ Tregs shown to be present in reduced numbers in pediatric patients with SLE (108). Of in terest for our study is the result that CD4+ CD25high T

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26 cells isolated from SLE patients shared the sa me phenotypic and functional characteristics with normal Tregs from healthy controls (106), sugges ting that the number of Tregs and not their function was an important aspect of disease pr ogression. This same study proposed that the SLE Tregs were present at lower numbe rs due to increased sensitivity to Fas-mediated apoptosis. Another study also showed that there were reduced numbers of suppressive CD4+ CD25high Tregs in the peripheral blood of patients with active SLE, and th at these cells presented with decreased levels of Foxp3 as well as decreased suppressive function in vitro (109). However, Tregs in the peripheral blood of patients with inactive SLE were essentially normal. This same study showed that Tregs from the patients with ac tive SLE were also capable of being restored to the normal phenotype in vitro indicating that the possibility to reverse the deficit of Tregs was a possibility, as shown by another group (110). Retinoic Acid and the Immune System Retinoic acid (RA), the most activ e metabolite of vitamin A, and its derivatives regulate a variety of cellular functions including development, prolif eration, differentiation, immune function and death in multiple cell types via two specific families of nuclear receptors functioning as ligand-inducible tr anscription factors, the retinoic acid rece ptors (RAR) and the retinoic X receptors (RXR) (111-114). In the immune system, RA plays important roles in the functiona l regulation of many immune cell types (115). RA has been shown to prevent activation-induced cell death of T cells and inhibits Th1 while enhancing Th2 responses (116,117). RA enhances cytotoxicity and T cell proliferation, most likely mediated by enha ncing IL-2 secretion and signaling in T cells (118,119). In vivo, RA suppresses inflammatory responses and tissue damage in addition to ameliorating symptoms in a variety of autoi mmune diseases in animal models, including experimental autoimmune encephalomyelitis, rheumatoid arthritis, type I diabetes, inflammatory

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27 bowel disease, and SLE (120-124). RA treatment has been shown to ameliorate symptoms in both the NZB/WF1 and MRLFaslpr murine models of SLE through intraperitoneal injection or dietary supplement, respectiv ely (121,125,126), although the mech anism by which it mediates this protection has not been fully elucidated. This RA-mediated protection has been thought to be due to inhibition of Th1 responses. RA produced by CD103+ dendritic cells (DCs) has recently been implicated in inducing aTregs in the gut (127-130) while inhibiting Th17 differentiation (129). Apparently, RA has a dual ro le in the maintenance of tolerance in that it favors induction of Treg cells and has the capacity to either block or enhance Th17 differentiation depending on its co ncentration (131). How this ac tion is mediated has not been addressed but is of great interest in eluc idating the mechanism by which RA suppresses autoimmunity. The Noelle group showed that a unique populati on of aTregs can be generated with RA treatment in vitro that is capable of homing to the small intestine lamina propria in vivo (127). RA was shown to potently synergize with TGFin driving Foxp3 induction, even in the presence of high levels of co-stimulation, there by increasing the frequency of these aTregs as well their durability (127). RA's effect has be en shown to be independent of IL-2, signal transducer and activator of transcription 5 (Stat5) and Stat 3 (132). RA has been implicated in enhancing TGFsignaling by increasing the expressi on and phosphorylation of Smad3, which then binds with Smad4, and this complex can then translocate to the nucleus, thereby resulting in increased Foxp3 expression (133) and th erefore and increa se in Tregs. Pbx1 and the Immune System Pre-B cell leukem ia transcription factor 1 (P bx1) is a member of the TALE (three-aminoacid-loop extension) family of homeodomain prot eins. Pbx1 was first implicated in pre-B cell leukemia in 1990 as a chromosome 1:19 transloc ation (134,135) which resulted in an E2A-Pbx1

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28 fusion protein (homeodomain of Pbx1 and the tr anscriptional activation domain of E2A) that alters normal transcriptional re gulation by Pbx1 (114). Pbx1 contains the following domains: the meis binding domain (MIM), the nuclear loca lization signal (NLS), the hox binding domain (HCM), the PBC homeodomain (PBC-A,B), and the homeo DNA binding domain (HD). Pbx1 can interacts with multiple Hox proteins and this complex can enhance both Hox DNA-binding specificity and affin ity (136-139). Both Meis and Prep, additional TALE proteins, can bind the Pbx1-Hox dimer, resulting in a trimer ic complex capable of further transcriptional regulation due to its higher DNA binding specificity (140-143). Both Meis and Prep associate with Pbx1 in the cytoplasm and induce a confor mational change in Pbx1, exposing the nuclear localization signal, and subsequently causing translocation of the dimeric protein complex to the nucleus (144). Pbx1 and Meis have also been implicated as essentia l cofactors for optimal binding of MyoD to DNA, thereby demonstrating another activity for these cofactors independent from Hox-related transcription (145 ). Treatment with RA has been shown to expand both Meis and Pbx1 expressi on in various cell types (114,146,147). Pbx1 has been implicated in B cell development and has been described as a necessary factor in very early B cell commitment (148). It was demonstrated in mice that Pbx1 plays a key role in pancreatic development, and that it is an important cofactor for the master regulator of pancreatic development and function, Pdx1 (149). Based on a study done by the Ma group and published in 2007, Pbx1, and more specifically Pbx1-b, was shown to be a physiologically critical mediator of apoptotic cell-induced IL-10 gene transcription and IL-10 cytokine production by macrophages, with its transcri ptional role found to be uncoupled from phagocytosis (150).

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29 There is 100% amino acid sequence homology between mouse and human Pbx1. It has been shown to be a strong biological candidate gene for type 2 diabetes at the chromosome 1q21q24 susceptibility locus in humans (151). A small case-control study was done in which 20 Pbx1 variants were evaluated for association with type 2 diabetes in Utah Caucasians, with only three variants shown to be nominally associated, as we re haplotypes involving intron 2 variants (152).

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30 CHAPTER 3 MURINE LUPUS SUSCEPTIBILITY LOCUS SLE1A CONTROLS R EGULATORY T CELL NUMBER AND FUNCTION THROUG H MULTIPE MECHANISMS Introduction 1Dominant suppression through regulato ry T cells, and specifically CD4+ CD25+ T cells (Tregs) expressing the Foxp3 transcription factor has now been accepted as a major mechanism by which self-tolerance is maintained. A decrease in Treg numbers or function has been directly associated with autoimmune pathogenesis in mu ltiple diseases and their associated mouse models (153,154). Reduced numbers of Tregs ( 106,108) as well as defective Treg function (109) have more recently been described in lupus patients. Tregs play a key role in maintaining tolerance to DNA in a transgenic mouse model (96) In spontaneous models of lupus, tolerance induction was dependent on the generation of Foxp3+ Tregs in (NZB X SWR)F1 (99) and (NZB X NZW)F1 (BWF1) mice (155), and Treg transfers showed a significantly prot ective effect in BWF1 mice (102). Other murine studies have documented a Treg protective effect for autoantibody production, but not for end-organ pathology (101,156). While these studies have documented a role for Tregs in controlling at leas t some aspects of lupus pathogenesis, they did not determine the mechanisms responsible for the observed Treg deficiency in either number or function. We have used NZM2410-derived congenic strains to address these questions. The major lupus susceptibility locus Sle1 controls tolerance to nuclear an tigens (10,55) a nd intrinsically affects both B and T cells (57,157). Multiple loci contribute to the Sle1 phenotype (12), and we have shown Sle1a and Sle1c are largely responsible for the generation of autoreactive T cells, 1 Reprinted with permission from The Association of American Immunologists, Inc. Copyright 2007. Cuda, C. M., S. Wan, E. S. Sobel, B. P. Cr oker, and L. Morel. 2007. Murine lupus susceptibility locus Sle1a controls regulatory T cell number and function through multiple mechanisms. J Immunol 179:7439.

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31 with Sle1a alone accounting for CD4+ T cell phenotypes equivalent to that of the entire Sle1 locus (13). CD4+ T cells expressing Sle1a show significantly increase d levels of activation and proliferation, as well as increased production of cytokines. Furthermore, purified Sle1a CD4+ T cells are able to induce in vivo the production of anti -nuclear antibodies (Abs) from either Sle1 or normal B cells (13). Finally, Sle1a is associated with a reduction of CD4+ CD25+ CD62L+ Foxp3+ Treg numbers (13). Conversely, the B6. Sle1.Sle2.Sle3 (B6.TC) strain, which reconstitutes the full autoimmune pathogenesis with the three ma jor NZM2410 susceptibility loci (51), produces dendritic cells (DCs ) that prevent Treg inhibitory functions on effector T cells (Teffs) (158). Production of high amounts of IL-6 by B6.TC DCs is a major mechanism by which this interference occurred, and we have shown that this phenotype maps to Sle1 (158). In this study, we examined th e functional consequences of Sle1a expression on Tregs and cells directly interacting with them. Treg function can be affected by multiple factors, including their number and intrinsic function. Many studies have reported a critical role of accessory cells, especially DCs, for optimal Treg developmen t and function (159), and imaging studies have clearly shown that Tregs exert their regulatory function thr ough direct contact with DCs (160,161). Teffs can also be resistant to suppression, as was shown in the MRL/lpr model of lupus (162). The complexity of a regulatory sy stem in which these three cellular compartments play a critical role requires a model in which each the compartment can be assayed independently in a syngeneic / autologous fash ion (163). The NZM2410 congenic strains, which share over 96% of their C57BL/6 (B6) genome, offer such a model. By comparing the B6. Sle1a congenics to B6 controls, we first confirmed that Sle1a results in a redu ced subset of CD4+ CD25+ CD62L+ Foxp3+ cells. Sle1a Tregs, however, appeared nor mal regarding expression of markers commonly associated with the regulato ry phenotype, and were capable of normal

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32 regulatory activity at hi gh Treg:Teff ratios. Sle1a also induced an increase d level of activation in CD4+ T cells and DCs, and both of these compartments significantly interfered with Treg regulatory function. Finally, we showed that the activated CD4+ T cell phenotypes and reduced Treg numbers required Sle1a expression in these T cells, s uggesting that th e generation of autoreactive T cells results from additive intrinsic defects in both Sle1a expressing CD4+ T cells and DCs. Overall, these results identify Sle1a as a locus playing a major role in T cell tolerance through Treg regulation by multiple mechanisms. Materials and Methods Mice C57BL/6J (B6), C57BL/6J-Cg-IghaThy1aGpila/J (B6. Thy1a), and B6.129S7Rag1tm1Mom/J (B6.Rag-/-) mice were originally obtained from The Jackson Laboratory. The B6.NZM2410Sle1 (B6.Sle1 ) congenic strain contains a 37-cM NZ M2410-derived interval defined by the D1MIT101 and D1MIT155 marker s (164). The B6.NZM2410Sle1a (B6.Sle1a ) sub-congenic line represents a 2.96 Mb interval between and excluding D1MIT370 and D1MIT147 (12,13). Unless specified, experiments were conducted w ith 8-12 month old female congenic mice and B6 matched controls. This age is past the induc tion of anti-nuclear Abs and autoreactive cells in most B6.Sle1 and B6.Sle1a mice (12,55). All mice were bred and maintained at the University of Florida in specific pathogen-free conditions. All experiments were conducted according to protocols approved at the University of Florid a Institutional Animal Care and Use Committee. Flow Cytometry Briefly, cells were first blocked on ice with staining buffer (PBS, 5% horse serum and 0.09% sodium azide) supplemented with 10% rabb it serum and pretreated with anti-CD16/CD32 (2.4G2) to block FcR-mediated binding. Cells were then stained with pre-titrated amounts of the following FITC-, PE-, allophycocyanin-, or biotin-conjugated Abs: CD4 (RM4), CD69

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33 (H1.2F3), CD25 (7D4), CD62L (MEL-14), GI TR (DTA-1), CD103, ICOS (CD278 clone 7E.17G9), B220 (RA3-6B2), CD3 (145-2C11), CD11b (M1/70), CD11c (HL3), CD19 (1D3), CD40 (HM40-3), CD62L, CD80 (16-10A1), CD86 (GL1), I-Ab (AF6-120.1), NK1.1 (PK126), TER119, mPDCA-1 (Miltenyi Biotec ) or isotype controls. A ll antibodies were from BD Biosciences unless otherwise specified. A combination of PE-conjugated anti-CD3, CD19, NK1.1, and TER119 antibodies were used to exclude CD11clow T cells, B cells, natural killer cells and erythroblasts, respectiv ely. Biotin-conjugated Abs were revealed using streptavidinPerCP Cy5.5 (BD Biosciences). Intracellular expression of CD152 (CTLA-4) and IL-10 was analyzed in fixed permeabilized cells with Cytofix/Cytoperm Plus kit (BD Pharmingen). For IL10 expression, splenocytes were cultured in the presence of anti-CD3 and anti-CD28 (1 g/ml) for 72 hours, and intracellular IL-10 levels in CD4+ ICOS+ cells were assessed by flow cytometry. IL-10 was also measured in the cu lture supernatant using an OptEIA Mouse IL-10 ELISA kit (BD Pharmingen) according to manufact urer's instructions. Foxp3 expression was determined using an intracellular Foxp3-PE stai ning kit (eBioscience). Cell staining was analyzed using a FACSCalibur (BD Biosciences) At least 50,000 events were acquired per sample, and dead cells were excluded based on scatter characteristics. Positive staining was determined as equal to or greater than the top 5% of the isotype control. Suppression Assays CD4(antigen presenting cell [APC]), CD4+ CD25Teff and CD4+ CD25+ Treg populations were purified from splenocyt es with magnetic beads using the CD4+ CD25+ Treg cell kit according to the manufacturers instruct ions (Miltenyi Biotech), and cultured in 96-well flat-bottomed plates in the presence of 1 g/ml of anti-CD3 to assess in vitro suppression levels of Tregs. Teffs and Tregs FACS analysis c onsistently showed >90% purity. The number of Teffs was kept constant at 5 x 105 cells per well, whereas the number of Tregs was titrated using

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34 four-fold dilutions. Cultures were main tained for 54 hours before pulsing with 1 Ci/well of [3H]-thymidine for an additional 18 hours. Cells were then collected onto fiber filtermats with a PHD cell harvester (Cambridge Technology) and counted using a Beta-scintillation counter. To assess the suppressive function of Tregs in vivo CD4+ CD25Teff and CD4+ CD25+ Treg populations from 2 month old female donor mice were purified with magnetic beads and transferred into age-matched female B6.Rag-/mice by injection into th e tail vein. Recipients received 4 x 105 B6 or B6.Sle1a Teffs in the presence or absence of B6 or B6. Sle1a 1 x 105 Tregs. After injection, mice were monitored for cl inical signs of colitis for up to 8 weeks, and body weight was monitored weekl y. Mice that lost 15% or mo re of body weight, or showing overt clinical signs of disease were sacrificed. Routine col on, stomach and kidney histology was performed to compare B6 and B6. Sle1a Teffs and Tregs functions and scored blindly in a semiquantitative fashion. Colon multiplicative score (0 81) was calculated by multiplying the thickness score by the infiltrate score in both the mucosa and the muscularis. The kidney additive score (0-4) was computed by adding 1 to the infiltrate score for the presence of granulomas. Generation of DCs and DC Phenotyping DCs were generated from bone m arrow (BM) with GM-CSF and IL-4 (R&D Systems) as previously described (158). To assess activat ion levels and cytokine production, BM-derived CD11c+ DCs were cultured for 24 hours with LPS (Sigma) at 1 g/ml. The supernatants were harvested and stored at 80 oC until assayed with commercial ELISA kits (BD Pharmingen). Bone Marrow Chimeras Chi meras were prepared as previously describe d (157). In brief, 6-8 week old female B6 mice were lethally irradiated with two doses of 525 Rad irradiation (4 hours apart) in a Gammacell 40 137Cs apparatus (MDS Nordion). Donor BM cells were depleted of mature T cells

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35 using CD5 Microbeads (Miltenyi Bi otech). Production of mixed BM chimeras was performed at a 1:1 ratio for the B6. Thy1a and B6.Sle1a strains. Ten million cells were given to each mouse by tail vein injection. Chimeric mice were maintained for 8 weeks, and lymphocytes were analyzed by flow cytometry to evaluate proliferat ion, activation, and Treg levels. The B6. Thy1a and B6. Sle1a origin of the T cells was determined with CD90.1 ( Thy1a ) and CD90.2 (Thy1b). CD4+ cellular proliferation was measured by staining splenocytes with 2.5 M CFSE (Molecular Probes) prior to stimulation with anti-CD3 (1 g/ml) and anti-CD28 (0.5 g/ml) and culture for 48 hours in a 37 C, 5% CO2 incubator. Activation was meas ured by staining lymphocytes with CD4, and CD69 after 12 hours of anti-CD3 and anti-CD28 stimulation. Treg levels were measured by staining lymphocytes with CD4, CD25, and CD62L prior to culture. Statistical Analysis Unpaired t test statistics ( oneor two-tailed as indicated) were used to co mpare the phenotypes of the B6. Sle1 and B6.Sle1a strains with that of B6. Comparisons for BM chimeras were made with paired two-tailed Students t te sts after verification that the data was normally distributed with GraphPad Prism 4. Nonparametr ic Mann-Whitney tests were used when the data were not normally distri buted. Comparisons for colon and kidney pathology were made with one-way ANOVA tests. Each in vitro e xperiment was performed at least twice with reproducible results. Results Sle1 and Sle1a are Ass ociated with Increased Levels of Activated T Cells and Decreased Levels of Tregs Previous results indicated that Sle1 is associated with a sign ificantly increased number of activated CD4+ T cells (57,165) as well as a decreased number of CD4+ CD25+ Tregs, and that this phenotype mapped to Sle1a and to a lesser extent to Sle1c (13,166). We confirmed these

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36 results by showing that congenic mice expressing Sle1 or Sle1a showed a significant increase in activated CD4+ CD69+ or CD4+ CD44+ T cells, and a significant decrease in nave CD4+ CD62L+ CD44T cells as compared to B6 (data not show n). In addition, we show here that both Sle1 and Sle1a CD4+ T cells showed a significantly increased expression of ICOS (Fig. 3-1A), which is a co-stimulatory molecule that is pivotal for T-B interactions and highly expressed on follicular helper T cells (167) We further analyzed CD4+ ICOS+ cells by culturing total splenocytes in the presence of anti-CD3 and anti-CD 28 to assess intracellular levels and secreted IL-10. We observed a trend of increased levels of CD4+ ICOS+ IL-10+ cells as well as production of IL-10 in the culture supernatant associated with Sle1a but not to a statistically significant degree (dat a not shown). B6. Sle1a congenic mice also showed significantly decreased percentages of CD4+ CD25+ CD62Lhi (Fig. 3-1B), with significantly less CD4+ CD25+ cells expressing CD62L, indicating that this locus induced a higher proportion of recently activated cells CD4+ CD25+ cells as opposed to Tregs. These findings were confirmed by intracellular expression of Foxp3 (Fig. 3-1C and D). It is of note that CD4+ CD25+ CD62Lhi cells have lost the Foxp3hi peak in the B6. Sle1 and B6. Sle1a mice, suggesting that this population contains less functional Foxp3+ Tregs in these mice than in B6. Similar results were obtained for younger mice ranging from 5 to 7 months of age (data not shown). Overall, these results confirm that Sle1a expression increases the number of activated T cells and diminishes the Foxp3+ Treg compartment. However, the expression of mark ers commonly associated with Tregs, namely GITR, CD103, and CTLA-4, was not affected by Sle1a expression in both CD4+ CD25+ (Fig. 32A) and CD4+ CD25+ CD62L+ populations (Fig. 3-2B ), suggesting that Sle1a Tregs may be functional, although reduced in numbers.

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37 Sle1a Tregs Require a Higher Treg:Teff Ratio to Support Inhibition to a Level E quivalent to B6 Tregs We assessed Sle1a Treg function using standard suppression assays in which the proliferation of CD4+ CD25Teffs was measured in response to anti-CD3 stimulation in the presence of APCs and graded ratios of Tregs. In these assays, the only variable was the Treg origin, B6 or B6. Sle1a while all other cells were of B6 orig in. As shown in Fig. 3-3, there was no difference between the inhibitory capability of Sle1a and B6 Tregs at a 1:1 Treg:Teff ratio. A significantly diminished inhibitory function appear ed however at 1:4 and 1:16. At this latter ratio, inhibition by Sle1a Tregs was no longer observed, and in some cases increased proliferation was observed with Sle1a Treg addition, as we have previously reported for B6.TC Tregs (158). This titration result is consistent with the CD4+ CD25+ population containing a smaller proportion of functional Tregs in B6. Sle1a mice. Sle1a Expression Increases DC Activation We have recently shown that B6.TC DCs di splay an abnorm al phenotype and hinder Treg function in an IL-6 dependent manner (158). Furthermore, elevated IL-6 production coupled with Treg inhibition mapped to Sle1 We show here that the Sle1a mediates an expansion of CD11c+ CD11b+ B220myeloid mDCs in the spleen (F ig. 3-4A) and lymph nodes (data not shown). Plasmacytoid pDCs gated as CD11c+ B220+ (Fig. 3-4A), but not as B220+ PDCA-1+ (data not shown), were also modestly expanded in B6. Sle1a spleens. In addition, Sle1a DCs displayed a significantly increase d expression of activation mark ers as shown for CD86 (Fig. 34C) and CD80 (Fig. 3-4D) that is similar to that of Sle1 DCs. These ex-vivo phenotypes were age-dependent as they reached statistical signifi cance only in old mice. Increased levels of activation markers such as CD40 and CD86 (Fi g. 3-4B), or CD80 and class II MHC (data not shown), and increased production of IL-6 (Fi g. 3-4E) and IL-12 (Fig. 3-4F) were readily

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38 obtained by LPS stimulation of DC s derived from either young (2-3 months old) or old B6.Sle1a BM. These levels were similar to what we have previously described for B6. Sle1 Overall, these results show that Sle1a induces an age-dependent DC accumulation in secondary lymphoid organs, and that these DCs produce more inflammato ry cytokines than that of the B6 controls. We have previously reported that Sle1 increases activation of peri pheral B cells (157,168). Here we show that Sle1a splenic B cells also expressed a si gnificantly higher level of CD19, CD80 and CD86 in old mice (data not shown). Overall, these results show that Sle1a increases activation not only in CD4+ T cells but also in DCs and B cells. Sle1a Expression Affects the Ability of Both DCs to Support Treg Suppression and Teffs to be Inhibited Given that Sl e1a expression affects all cellular compartments in a suppression assay, namely Tregs, Teffs and APCs, we investigated the consequences of Sle1a expression independently in each of these cellular compar tments on the ability of Tregs to suppress Teff proliferation (Fig. 3-5). As seen earlier, expression of Sle1a in Tregs had a significant effect on Treg function at a low Treg:Teff ratio. Interestingly, Sle1a expression in Teffs significantly hindered the action of Tregs, alt hough this effect was no longer si gnificant at the 1:16 Treg:Teff ratio. Expression of Sle1a in APCs significantly prevented inhi bition at all three ratios, and even induced enhanced proliferation at the 1:16 Treg:T eff ratio. This latter effect was observed with DCs from B6.TC mice (158) suggesting that the Sle1a locus plays a major role in the DC defective functions in this model. In conclusion, Sle1a expression in either one of the 3 members of the suppression assay significantly impacts the ab ility of Tregs to supp ress Teff proliferation. We also assessed in vivo the effect of Sle1a expression on effector and regulatory CD4+ T cell function in a rapid model of disease adapte d from the experimental colitis model (169). B6.Rag-/mice received 4 x 105 CD4+ CD25Teffs from either 2 month old B6 or B6. Sle1a in the

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39 presence or absence of 1 x 105 CD4+ CD25+ Tregs from B6 or B6. Sle1a mice. As expected, B6 Teffs induced weight loss and colitis, which was abrogated by the presence of B6 Tregs (Fig. 3-6 and 3-7A and B). Sle1a -expressing Tregs did not suppress B6 Teff function as well as B6 Tregs, possibly due to a lower ratio of func tional Tregs within the injected CD4+ CD25+ population. We also observed that the Sle1a -expressing Teffs are resistant to suppression by either B6 or Sle1a -Tregs. In addition to lymphocytic infiltrates, Sle1a Teffs resulted in the presence of elevated numbers of polymorphonuclear neutrophils (PMN) in the lesions leading to cryptitis (Fig. 3-6C2). Finally the combination of Sle1a Teffs and Tregs led to the most severe pathology with the presence of giant cells (Fig. 3-6D2). In terestingly, similar results were observed in the kidneys (Fig. 3-7C and data not shown), but not in the stomach. Teffs induced interstitial inflammation and granulomas in B6.Rag kidne ys, which were suppressed by B6 Tregs only when the Teffs were of B6 origin. As in the colon, Sle1a Teffs were also associated with giant cells in the kidneys, indicating a greater level of inflammation. Overall, this in vivo experiment confirmed that the Sle1a CD4+ CD25+ population is less effective at suppressing Teff functions and that Sle1a Teffs are resistant to Treg suppression. Sle1a Expression Intrinsically Affects CD4+ T Cell Phenotypes The results presented above showed that Sle1a expression affects the function of multiple hematopoeitic cell compartments, which prompted us to examine whether Sle1a expression was required for CD4+ T cells to show the functional defects reported above. To address this question, we produced mixed bone marrow chimer as by injecting T celldepleted bone marrow cells from either normal B6. Thy1a or B6.Sle1a ( Thy1b) donor mice into lethally irradiated B6 hosts. As shown in Fig. 3-6, the increas ed proliferation a nd activation of CD4+ T cells, as well as the decreased percentage of Tregs were completely reproduced by Sle1a bone-marrowderived cells (compare B6. Thy1a B6 and B6.Sle1a B6). More interestingly, in mixed

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40 chimeras containing both Sle1a -expressing and normal CD4+ T cells, only those T cells expressing Sle1a displayed enhanced pro liferation, as measured by in vitro CFSE dilution (Fig. 3-8A), and activation, as measured by CD69 e xpression (Fig. 3-8B). Corresponding histograms show expression of levels of both CFSE on gated CD4+ T cells (Fig. 3-8A and B, respectively). Conversely, the percentage of CD62L+ Treg was significantly lower in Sle1a -expressing T cells than in B6 and can be visualized in the co rresponding histogram depi cting CD62L levels on CD4+ CD25+ gated cells (Fig. 3-8C). Taken together, these resu lts show unambiguously that Sle1a results in intrinsically activated CD4+ T cells. Sle1a expression in non-hematopoeitic cells is not required for induction of these phenotypes. The abnormal phenotypes are not transferable to bystander normal T cells, excluding Sle1a being mediated through a soluble factor. Discussion We have shown here that expression of Sle1a is sufficient to induce increased activation levels of CD4+ T cells, DCs and B cells, as well as to down-regulate Treg levels. We also show that Sle1a CD4+ T cells express increased levels of the co-stimulation marker ICOS, which has been shown to play a critical role in T cell he lp to B cells, especially in germinal centers (170,171). Elevated ICOS expre ssion on T cells from lupus patients has now been reported in three independent studies (172-174) These last two studies repor ted that ICOS stimulation of lupus T cells significantly enha nced anti-dsDNA Ab production from autologous B cells, which is equivalent to what we have shown for Sle1a T cells, which were able to induce anti-chromatin production in both autologous Sle1a -expressing B cells and normal B cells (13). These results also suggest that Sle1a confers a T cell phenotype that is found in lupus patients, which further validates the need to identify the identity of the Sle1a gene(s). Future experiments should address the specific role of ICOS in this process. High levels of ICOS have been associated with IL-10 secretion by CD4+ T cells (175), and IL-10 production by CD4+ T cells is significantly

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41 increased in the NZM2410 model (176). There was however no consistent increase of IL-10 production by Sle1a CD4+ T cells, suggesting that another mechanism may be involved. Sle1a induces a reduction in the size of the Treg compartmen t, but these cells express normal levels of CTLA-4, CD103, and GITR, mol ecules which have been commonly associated with the regulatory phenotype. In add ition, at the higher ratios of Treg:Teff, Sle1a -expressing Tregs are fully capable of suppressing the prolif eration of B6 Teffs on a per-cell basis in the presence of B6 APCs. However, at lower ratios of Treg:Teff, this suppressive capability is decreased, consistent with a reduced propor tion of functional Tr egs within the CD4+ CD25+ T cell population of the B6. Sle1a mice. In addition to in vitro suppression assays, we also performed adoptive transfers adapted from the experimental model of colitis to test the in vivo effect of Sle1a on Treg and Teff functions in a rapid model of disease. Results from the in vivo study confirmed our in vitro data. We cannot exclude, however that Sle1a also affects Treg inhibitory functions. Indeed, a recent construct with a non-func tional Foxp3 has demonstrated that the expression of Treg signa ture makers can develop normally in cells that completely lack inhibitory functions (177). A definitive answer to that question will require breeding of Sle1a to a Foxp3 reporter construct, whic h we are currently pursuing. While we have shown that the Sle1a -expressing Tregs are capa ble of suppression, in situations where either the Teffs or the APCs express Sle1a, the suppressive capability of normal B6 Tregs is significantly re duced, suggesting that the Sle1a locus confers a resistance to suppression of Teff proliferation and that the APCs are playing a role in this phenomenon. It is of note that the APC pop ulation used in our in vitro suppression assays contains not only DCs but B cells as well. We have previously shown the effects of Sle1a DCs on Treg suppression (19), however, Sle1a affects both of these cell types. This indicates a potential role of activated

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42 B cells on Treg function, and is an avenue to be studied further. A similar Teff resistance has been previously reported in another model of lupus (162), but it is not clear at this point whether this resistance is the mere consequence of hypera ctivation, or a result of involvement with a specific mechanism. Cbl-b deficiency results in a resistan ce to Treg regulation involving TGF, and this mutation also induces an increased level of activation in effector T cells (178). To our knowledge, no other mechanisms of resistance to Tregs have been reported and additional experiments will be necessary to determine how Sle1a confers this resistance in CD4+ T cells. We have previously shown that DCs from th e NZM2410 triple congenic strain B6.TC prevent Tregs from performing their inhi bitory functions, primarily thr ough the production of IL-6 (158). We report here that Sle1a -expressing DCs present the same phenotype of high IL-6 production and Treg inhibition, indicating that this locus plays a major role in the overall DC phenotype of lupus-prone mice. Interestingly, the type-1 diabetes prone NOD mice, which have a reduced number of Tregs (179,180), also produce APCs th at fail to fully support Treg functions (180). These results suggest that defective support or active inhibition of Treg functions by DCs may be a common mechanism affecting autoimmune pathogenesis. Mixed BM chimeras have shown here that th e increased proliferation and activation of Sle1a -expressing T cells, as well as the reduced Sle1a Treg level require that Sle1a be expressed in these T cells. These results differ from what might have been predicted from the in vitro reconstitution experiments s hown in Figure 3-5, where B6. Sle1a -derived APCs inhibited Treg function. The BM chimera results do not mean that Sle1a exclusively affects CD4+ T cells. In an analogous set of experiments, BM chimeras showed that T cell activ ation and autoreactivity mediated by Sle3 were the indirect result of Sle3 expression within the myeloid compartment (62,181). It is therefore possible that the Sle1a -induced intrinsic defects in CD4+ T cells are

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43 indirectly responsible for the DC and B cell abnormalities. Alternatively, the Sle1a gene(s) may control a pathway present in all three cellular comp artments. In any event, indirect or direct activation of DCs by Sle1a was not sufficient to convey extrinsic changes to B6-derived CD4+ T cells in vivo. The exact cause for these differences is unclear, and highlights the need to confirm in vitro findings with in vivo studies. Additional mixed BM chimeras will be necessary to address whether Sle1a expression in these DCs and B cells is necessary for production of the activated phenotypes. Autoreactive T cells are a feature common to many autoimmune diseases for which a genetic basis has been demonstrated, yet only very few genes have been identified as responsible for this phenotype (182). In addition to Cbl-b discussed above (178), null alleles of Gadd45a (183) or E2f2 (184) result in a lower threshold for T cell activation culminating in autoimmune pathogenesis, while null alleles in Ctla4 (185) and Foxp3 (186) result in massive inflammatory and autoimmune responses through the disruption of the Treg compartment. More recently, a natural polymorphism in the Il2 gene has been identified as responsible for the diabetes susceptibility locus Idd3 in the NOD mouse, also through an impairment of Treg function (187). The Sle1a interval does not contain any gene with obvious function in T cells. Our in vitro results showed that Sle1a confers an autoimmune phenotype to CD4+ T cells in the colon, which is not typically associated with lupus pathogenesis. This indicates that Sle1a affects a genetic pathway regulating production of Tregs and responses to Tregs in a manner that is not restricted to tolerance to nuclear antigens. The identification of the Sle1a gene(s) will therefore uncover a novel and broad pathway by which autoreac tive T cells are re gulated by Tregs.

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44 Figure 3-1. Sle1a results in increased CD4+ T cell activation and a diminished Treg compartment. Splenocytes fr om 8-12 month old B6, B6. Sle1 and B6.Sle1a mice were labeled for surface CD4, ICOS, CD25, and CD62L and intracellular Foxp3 expression and analyzed by fluorescence-activ ated cell sorting. Each point represents an individual animal. Representative gating on a B6 mouse is shown in the left-hand column (marker for A, and rectangular gate for B-D), and representative expression levels in all three strains are shown in the right-hand column. The light grey filled histograms show isotype controls, dark grey filled histograms s how B6 values, while thick and thin black lines represent B6.Sle1 and B6.Sle1a respectively. All comparisons were performed with B6 values. Two-tailed t tests: *: p<0.05, **: p<0.01, ***: p<0.001.

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45 Figure 3-2. Sle1a does not affect expression of mark ers associated with the regulatory phenotype. CD4+ CD25+ (A) or CD4+ CD25+ CD62L+ (B) splenocytes from B6 and B6. Sle1a mice were compared for GITR, CD103 or intracellular CTLA-4 expression. Representative histograms of at least 5 mice per strain are shown. The percentage of positive cells (based on the isotype control shown by the grey histograms) is indicated above each marker. Figure 3-3. Sle1a Tregs are deficient in inhibiting proliferation at low Treg:Teff ratios. Inhibition of proliferation assays were se t up with B6-derived APCs and Teffs, and either B6 (black bars) or B6.Sle1a (white bars) Tregs at the indicated ratio. A) Representative assay comparing prolif eration in the presence of B6 or Sle1a Tregs (6 mice per strain). Means and standard errors, and results of one-tailed t tests between the 0:1 assays and the various Treg:Teff ratios for each strain. B) Normalized percentage inhibition of proliferation of th e 0:1 assays at the va rious Treg:Teff ratios for each strain combined from three differe nt assays (15 mice per strain). Means and standard errors, and results of one-tailed t tests between the two strains for each Treg:Teff ratio. *: p<0.05, **: p<0.01, ***: p<0.001.

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46 Figure 3-4. Sle1a expression activates dendritic cells. A) pDCs and mDCs were significantly expanded in the spleen of B6. Sle1a mice as compared to B6. B) CD40 and CD86 were significantly up-regulated in B6. Sle1a BM-derived DCs in response to LPS. Splenic DCs were significantly more activated in B6. Sle1 and B6.Sle1a than in B6 mice, as shown by CD86 (C) and CD80 (D) expression. DCs derived from B6. Sle1 or B6. Sle1a BM produced significantly more IL-6 (E) and IL-12 (F) than B6 after LPS exposure. Each point represents an i ndividual animal. All comparisons were performed with B6 values. Two-tailed t tests: *: p<0.05, **: p<0.01, ***: p<0.001.

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47 Figure 3-5. Sle1a expression in Tregs, Teffs, or APCs aff ects the extent of the inhibition of Teff proliferation. The inhibition of CD4+ CD25+ Teff proliferation in presence of 1:1, 1:4, or 1:16 Treg:Teff ratios is expressed as a pe rcentage of the proliferation induced in the absence of Tregs for each condition. The origin, B6 or B6. Sle1a of Tregs, Teffs, and APCs is indicated under each column. The graphs show the means and standard errors of three independent assays with 3-4 mice per strain at 6 m onths of age in each assay. Results of one-tailed t tests between each condition and the "all B6 condition" are indicated for each Treg:Teff ratio. +, #, *: p<0.05, ++, ##, **: p<0.01, +++, ###, ***: p<0.001.

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48 Figure 3-6. Sle1a expression in either Tregs or Teffs aff ects the extent of disease in an adoptive transfer model. CD4+ CD25Teff and CD4+ CD25+ Treg populations from 2 mo old female donor mice were transferred into age-matched female B6.Rag1-/recipient mice. Representative colon histology: All parts of the composite labeled 1 are the same magnification (0.5 mm) and all part s labeled 2 (0.1 mm) are 4 times those labeled 1. Each two sub-figures with the same letter (e.g. A1 & 2) are from the same animal. A1 & 2) B6 Teffs and B6 Tregs. Th e figure is representa tive of the control group with normal thickness and minimal lym phocytic infiltrate in the lamina propria (A2, circle, center). B1 & 2) B6 Teffs and Sle1a Tregs. The figure is representative of about 2-fold increase in thickness (B1). Ther e is a notable increase in lamina propria thickness and mononuclear cell infiltrate. There are also increases in epithelial infiltrating lymphocytes, epithelial a poptosis and mitosis (B2). C1 & 2) Sle1a Teffs and B6 Tregs. This figure is representative of an additional increase in thickness (C1). In addition to the lymphocytes and other findings noted above, increased PMNs are present in the lamina propria and glands (cry ptitis and crypt absce ss, C2, center). D1 & 2) Sle1a Teffs and Sle1a Tregs. This is the greatest overall thickness with inflammation extending into the muscular is propria (D1, bottom). There is an increase in PMN and lymphocytes through out the mucosa. Occasional multinucleated giant cells are present (D2, circle).

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49 Figure 3-7. Quantification of the effects of CD4+ CD25Teff and CD4+ CD25+ Treg transfers into B6.Rag1-/mice. A) Maximum weekly percen tage of body weight loss up to 8 weeks after transfer. The box and whisker plot shows the medians, 25 and 75 percentiles and minima and maxima fo r each group. B) Multiplicative colitis pathology score. C) Additive kidney pathol ogy score (infiltrate score + giant cell presence). In B and C, the strain of orig in of Teff and Tregs is indicated on the X axes, with 0 indicating the ab sence of Tregs. ANOVA: *: p<0.05.

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50 Figure 3-8. Sle1a expression affects CD4+ function in a cell-intrinsic manner. B6 hosts were reconstituted with B6. Thy1a and / or B6. Sle1a BM. Connected samples indicate values for CD4+ T cells expressing the Thy1a (CD90.1-gated) or Thy1b in B6.Sle1a mice (CD90.2-gated) alleles within the same mouse. Controls are represented by B6. Thy1a B6 and B6. Sle1a B6 single-strain BM transfers. A) In vitro anti-CD3 induced proliferation measured as the percen tage of CD4+ CFSElow lymphocytes with representative histog ram showing CFSE expression on gated CD4+ T cells. B) Activation measured as the percentage of CD4+ CD69+ lymphocytes with representative histogram s howing CD69 expression on gated CD4+ T cells. C) Treg levels, measured as the percentage of CD4+ CD25+ splenocytes expressing CD62L with representative histogram showing CD 62L expression. Each point represents an individual animal. Two-tailed t tests: *: p<0.05, **: p<0.01. Data representative of two independent set of BM chimeras with 5 mixed chimeras in each.

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51 CHAPTER 4 THE CONTROL OF REGULATORY T C ELL NUMBE R AND FUNCTION BY MURINE LUPUS SUSCEPTIBILITY LOCUS SLE1A REQUIRES THE SYNERGISTIC EFFECT OF SUBLOCI SLE1A.1 AND SLE1A.2 Introduction In the previo us chapter, we presented results which showed that expression of Sle1a results in increased levels of activation of T and B ce lls as well as DCs. This overall increased activation was accompanied by a decreased Treg compartment. These phenotypes were shown to be due to a T cell-intrinsic de fect. On a per-cell basis, the Sle1a -expressing Tregs were capable of normal suppressive function, a lthough there were less of them, but the Sle1a expressing Teffs were resist ant to suppression and the Sle1a -expressing APCs play a role in this mechanism. Now that we have phenotypes attributable to Sle1a expression, it is of interest to dissect out which phenotypes co-segrega te with the truncated regions of Sle1a Sle1a.1 and Sle1a.2 Once we can determine the regions to which specific Sle1a phenotypes map, we can narrow down the genes leading to SLE susceptibility. Materials and Methods Mice The production of B6. Sle1aNZW/NZW (B6.Sle1a ) was described as B6. Sle1a(15-353) (12) and B6. Sle1a.2NZW/NZW (B6.Sle1a.2) mice as B6. Sle1(111-148) (188). B6. Sle1a.1NZW/NZW (B6.Sle1a.1 ) was obtained as a recomb inant interval from B6. Sle1a (Fig. 4-1). C57BL/6J (B6), C57BL/6J-Cg-IghaThy1aGpila/J (B6. Thy1a), and B6.129S7Rag1tm1Mom/J (B6.Rag-/-) mice were originally obtained from The Jackson Labor atory (Bar Harbor, ME). Unless specified, experiments were conducted with 8-12mont h old female congenic mice and B6 matched controls. This age is past the induction of anti-nuclear Abs and autoreactive cells in most B6. Sle1 and B6.Sle1 sub-congenic mice (12,55). All mice we re bred and maintained at the

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52 University of Florida in specific pathogen-fr ee conditions. All experiments were conducted according to protocols approved by the University of Florida Institutional Animal Care and Use Committee. Sle1a Map PCR genotyping with m icrosatellite markers was performed as previously described (12) and SNP genotyping was perfor med by direct sequencing. Flow Cytometry Briefly, cells were first blocked on ice with staining buffer (PBS, 5% horse serum and 0.09% sodium azide) supplemented with 10% rabb it serum and pretreated with anti-CD16/CD32 (2.4G2). Cells were then stai ned with pre-titrated amounts of the following FITC-, PE-, allophycocyanin-, or biotin-c onjugated Abs: CD4 (RM4), CD69 (H1.2F3), CD25 (7D4), CD62L (MEL-14), ICOS (CD278 clone 7E.17G9), or isotype controls. All antibodies were from BD Biosciences (San Jose, CA) unless otherw ise specified. Biotin -conjugated Abs were revealed by streptavidin-PercP-Cy5a (BD Pharmi ngen). Foxp3 expression was determined using an intracellular Foxp3-PE staining kit (eBioscience, San Diego, CA). Cell staining was analyzed using a FACScalibur (Becton Dickinson Immunocyt ometry Systems, San Jose, CA). At least 30,000 events were acquired per sample, and dead cells were excluded based on scatter characteristics. Positive staining was determined as equal to or greater than the top 5% of the isotype control. Suppression Assays CD4(APC), CD4+ CD25Teff, and CD4+ CD25+ Treg populations were purified from splenocytes with magnetic beads using the CD4+ CD25+ Treg cell kit according to the manufacturers instructions (Miltenyi Biotec). FACS analysis of Tregs and Teffs consistently showed >90% purity. In vitro suppression assays were performe d as previously described (189)

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53 in the presence of 1g/ml anti-CD3. The num ber of Teffs was kept constant at 5 105 cells/well, whereas the number of Tregs was ti trated using 4-fold dilutions. To assess the suppressive function of Tregs in vivo, 4 105 CD4+ CD25Teff and CD4+ CD25+ Treg populations from 6 month old male donor mice were transferred into 2 month old male B6.Rag-/mice in the presence or absence of 1 105 Tregs as previously descri bed (189). After injection, mice were monitored for clinical signs of colitis for up to 8 weeks and body weight was monitored weekly. Mice that lost 15% or more of body weight or showed overt clinical signs of disease were sacrificed. Bone Marrow Chimeras Chi meras were prepared as previously describe d (157). In brief, 6-8 week old female B6 mice were lethally irradiated with two doses of 525 Rad irradiation (4 hours apart) in a Gammacell 40 137Cs apparatus (MDS Nordion). Donor BM cells were depleted of mature T cells using CD5 Microbeads (Miltenyi Bi otech). Production of mixed BM chimeras was performed at a 1:1 ratio for the B6. Thy1a and B6.Sle1a.1 or B6.Sle1a.2 mice. Ten million cells were given to each mouse by tail vein injection. Chimeric mice were maintained for 8 weeks, and lymphocytes were analyzed by flow cytometry to evaluate T cell activation and Treg levels. Activation was measured by staining lymphocytes with CD4, CD90.1 ( Thy1a), CD90.2 ( Thy1b), and CD69 12 hours after stimulation with anti-CD3 (1 g/ml) and anti-CD28 (0.5 g/ml) in a 37 C, 5% CO2 incubator. Treg levels were measured by staining lymphocytes with CD4, CD25, CD62L and either CD90.1 or CD90.2 prior to culture. Statistical Analysis Statistical analyses were perform ed usi ng the GraphPad Prism 4 Software. Nonparametric tests and multiple-test corrections were used as appropriate and as indicated for each experiment. Unpaired t test stat istics (one or two-tailed as i ndicated) were used to compare the

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54 phenotypes of the B6. Sle1a B6.Sle1a.1 and B6.Sle1a.2 strains with that of B6. Comparisons for BM chimeras were made with paired two-tailed St udents t tests after verification that the data were normally distributed with GraphPad Prism 4. Nonparametric Mann-Whitney U tests were used when the data were not normally distributed. Comparisons for colon and kidney pathology were made with one-way ANOVA tests. Each in vitro experiment was performed at least twice with reproducible results. Statis tical significance was obtained when p 0.05, and is indicated in figures as : p 0.05, ** : p 0.01, and *** : p 0.001. Results More than One Gene Contributes to Sle1a Phenotypes We have used the three congenic recom binan t strains shown in Fig. 4-1 to refine the location of the gene(s) responsible for the Sle1a phenotypes. The entire Sle1a interval is covered by the combination of the Sle1a.1 and Sle1a.2 intervals. There is a potential overlap between the two intervals between rs30711102 a nd rs31028646. In addition, the Sle1a.2 interval extends on the telomeric end beyond the Sle1a interval, resulting in B6. Sle1a and B6. Sle1a.2 having the B6 and NZW allele at the Fcgr2b gene, respectively (188). Ther e are a number of genes included within the Sle1a region, as shown in Table 4-1, with the telomeric end showing a more dense set of genes. Some of the genes present with in the interval D1MIT15 to D1MIT353, or the Sle1a interval, include Pbx1 encoding for pre-B cell leukemia transcription factor, Cdca1, known to be involved in the cell cycle, Rgs5 and Rgs4 regulators of G proteins i nvolved in angiogenesis and cardiac development, Hsd17b7 or hydroxysteroid (17-beta) dehydrogenase 7, a steroidconverting enzyme that modulates steroid function, Ddr2 or discoidin domain receptor 2, which aids in regulation of cell proliferation and expression of MMP-1 and MMP-2, Uap1 or UDP Nacetylglucosamine pyrophoshorylase 1, known to be involved in metabolic processes, Uhmk1 or

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55 U2AF homology motif kinase 1, and Sh2d1b or Eat2, which binds SLAM-family receptors with high affinity. Sle1a Requires Both Sle1a.1 and Sle1a.2 Expression for the Increased Level of T Cell Activation and Decreased Level of Tregs Based upon work done by other m embers of the lab (13), it was revealed that Sle1a expression significantly enhances antinuclear autoAb pr oduction and that Sle1a increases the number of autoreactive B cells that respond to alloreactive T cell help, indicating that Sle1a contributes to lupus pathogenesi s. However, neither of these phenotypes completely maps to either subloci, suggesting to us that both Sle1a.1 and Sle1a.2 are involved with the phenotypes observed for the entire Sle1a interval. We have pr eviously reported that Sle1 is associated with a significantly increased number of activated CD4+ T cells as well as a decreased number of CD4+ CD25+ Tregs, and that this phenotype mapped to Sle1a and to a lesser extent to Sle1c (13). In addition, we have reported that Sle1a CD4+ T cells showed a significant increase in expression of ICOS (189), a co-stimulatory molecule shown to be pivotal for T-B cell interactions and highly expressed on follicular helper T cells ( 167). Here we show that expression of Sle1a.1 but not Sle1a.2 leads to a significant increase in ICOS expression ex vivo (Fig. 4-2A). We did not observe this increase for either Sle1a.1 or Sle1a.2 when assessing CD69 expression (data not shown), although we have observe d that expression of either Sle1a.1 or Sle1a.2 leads to a significantly decreased ratio of nave to memory CD4+ T cells (data not shown). The B6. Sle1a mice also showed significantly decreased percentages of CD4+ CD25+ cells expressing CD62L, indicating that this locus induced a highe r proportion of recently activated cells CD4+ CD25+ cells as opposed to Tregs (189). However, the results shown in Fig. 42B indicate that this decrease in Tregs does not map to either the Sle1a.1 or Sle1a.2 locus. These findings were confirmed by assessment of intracellular expressi on of Foxp3 (Fig. 4-2C and D). Similar results

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56 were obtained for 5-7 month old mice (data not s hown). Overall, these results indicate that expression of Sle1a.1 alone can account for the observed increase in ICOS expression on CD4+ T cells, but expression of both Sle1a.1 and Sle1a.2 are required for the decreased ex vivo Foxp3+ Treg compartment associated with Sle1a. Sle1a.1 and Sle1a.2 Tregs Can Support Inhibition to a Level Equivalent to B6 Tregs In Vitro We have previously shown that while the in vitro inhibitory capacity of B6 and Sle1a Tregs was sim ilar at a 1:1 Treg:Teff ratio, a si gnificantly diminished inhibitory function was observed at both the 1: 4 and 1:16 ratios for Sle1a Tregs (189). At this latter ratio, inhibition by Sle1a Tregs was no longer observed, and in some cases increased proliferation was observed with Sle1a Treg addition (189), as we ha ve previously reported fo r B6.TC Tregs (158). This result is consistent with the CD4+ CD25+ population containing a smaller proportion of functional Tregs in B6. Sle1a than in B6 mice. Here we assessed the suppressive capacity of B6. Sle1a.1 and B6. Sle1a.2 Tregs in comparison to B6 in assays in which the only variable was the Treg origin, (B6, B6. Sle1a.1 or B6. Sle1a.2 ), while all other cells we re of B6 origin. As shown in Fig. 4-3, there was no major differen ce between the inhibitory capacity of B6, B6. Sle1a.1 (Fig. 4-3A) and B6.Sle1a.2 (Fig. 4-3B) Tregs at any Treg :Teff ratio, contrary to what we observed for Sle1a Tregs (189). Sle1a.1 and Sle1a.2 Expression Affects the Ability of Both DCs to Support Treg Suppression and Teffs to be Inhibited, but to a Lesser Extent than Observed for Sle1a We have previously shown that expression of Sle1a in any of the three members of the suppression assay, namely Tregs, Teffs and APCs, significantly impacted th e ability of Tregs to suppress Teff proliferation (189). Here we perf ormed the same test to assess the consequences of either Sle1a.1 or Sle1a.2 expression in any of the three members of the suppression assay on Treg function (Fig. 4-4). As shown above and contrary to results for expression of Sle1a,

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57 expression of either Sle1a.1 or Sle1a.2 in Tregs did not significantly affect Treg suppressive capacity. While we previously showed that Sle1a expression in the Teff compartment resulted in an increased resistance to Treg suppression in both the 1:1 and 1:4 Treg:Teff settings (189), expression of either Sle1a.1 (Fig. 4-4A) or Sle1a.2 (Fig. 4-4B) in Teffs resulted in significantly hindered Treg suppression only in lower Tr eg:Teff ratios. Finally, expression of Sle1a in APCs significantly prevented inhibition at all three ratios, and even indu ced enhanced proliferation at the 1:16 Treg:Teff ratio (189). However, expression of either Sle1a.1 or Sle1a.2 in APCs showed only a significant effect on Treg functi on at the 1:16 Treg:Teff ratio. Taken together, expression of both the Sle1a.1 and Sle1a.2 subloci is necessary to observe the phenotype associated with Sle1a, namely that expression in any one of the three members of the suppression assay significantly impa cts the ability of Tregs to suppre ss Teff proliferation. Expression of either Sle1a.1 or Sle1a.2 alone, however, affects the ability of Teffs to resist Treg suppression and that of APC to mediate Treg suppressi on, although to a lower le vel that the entire Sle1a interval We also assessed in vivo the effect of Sle1a Sle1a.1 and Sle1a.2 expression on effector and regulatory CD4+ T cell function in a rapid model of diseas e adapted from the experimental colitis model (169). Using this same model, we previous ly showed that Tregs from 3 month old female B6. Sle1a mice could not suppress colitis onset induced by either B6 or B6. Sle1a Teffs (189). In addition, the Teffs expressing Sle1a were resistant to suppression by both B6 and B6. Sle1a Tregs (189). In this case, B6.Rag-/mice received 4 x 105 CD4+ CD25Teffs from either 6 month old B6 or congenic (B6. Sle1a, B6.Sle1a.1 or B6.Sle1a.2 ) mice in the presence or absence of 1 x 105 CD4+ CD25+ Tregs from B6 or congenic (B6. Sle1a B6.Sle1a.1 or B6.Sle1a.2 ) mice. As expected, B6 Teffs induced colitis, here defined by a 15% loss in body weight, which was

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58 abrogated by the presence of B6 Tregs (Fig. 4-5) Colon and kidney histology is pending for all three experiments. We also obs erved that in all three cases, the congenic-expressing Teffs were more potent inducers of colitis onset than B6 Teffs. Tregs expressing Sle1a (Fig 4-5A), and to a lesser extent those expressing Sle1a.1 (Fig. 4-5B) and Sle1a.2 (Fig 4-5C), did not suppress B6 Teff function as well as B6 Tregs, possibly due to a lower ratio of functional Tregs within the injected CD4+ CD25+ population. While we observe d that those Teffs expressing Sle1a are resistant to suppression by B6 Tregs (Fig. 4-5A ), we did not obtain this result for Teffs expressing either of the individual subloci (Fig. 4-5B and C). This in vivo experiment confirmed our previously published results showing that the Sle1a CD4+ CD25+ population is less effective at suppressing Teff functions and that Sle1a Teffs are resistant to Tr eg suppression, as well as confirming our in vitro findings above that th e effect of the entire Sle1a locus requires expression of both Sle1a.1 and Sle1a.2. Sle1a.1 or Sle1a.2 Expression Intrinsically Affects CD4+ T Cell Phenotypes We have previously shown that although Sle1a expression affects multiple hematopoeitic cell compartments, Sle1a results in intrinsically activated CD4+ T cells and its expression in nonhematopoeitic cells is not required for induction of these phenotypes (189). Here we used the same mixed bone marrow chimera approach for both subloci Sle1a.1 and Sle1a.2 and observed similar results to that of Sle1a (Fig. 4-6). The increas ed activation of CD4+ T cells (Fig. 4-6A) and the decreased percentage of Tregs (Fig. 4-6B) were again completely reproduced by Sle1a.1 and Sle1a.2 bone-marrow-derived cells, and in the mi xed bone marrow chimeras containing both congenic and normal CD4+ T cells, only those T cells expressing Sle1a.1 or Sle1a.2 displayed enhanced activation and a d ecreased proportion of CD62L+ Tregs. Interestingly, the increased level of activation and decreased level of Tregs were exaggerated to a level of significance when assessed in the lymphopenic environment of the mixed bone marrow chimera assay as compared

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59 to unmanipulated mice as shown in Fig. 4-2. Ta ken together, we concl ude that expression of either Sle1a.1 or Sle1a.2 results in T cell-intr insic phenotypes. The abnormal phenotypes are not transferable to bystander normal T cells, excluding Sle1a.1 or Sle1a.2 being mediated through a soluble factor.

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60 Figure 4-1. Map of Sle1a From top to bottom are shown: a scale in Mb, the location of the microsatellite markers or SNPs mapp ing the interval termini, and the Sle1a, Sle1a.1 and Sle1a.2 intervals, in which the gray rect angles show the regions of known NZW allelic derivation, and the hatched rectangl es on each side indicate the regions of recombination between the NZW and B6 genomes. Map based on Ensembl Release 40.

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61 Table 4-1. Genes contained within the Sle1a interval according to Ensembl Release 40. Start End Sle1a subloci Symbol Description 169,995,407 170,268,179 Sle1a.1 Pbx1 pre B-cell leukemia transcription factor 1 171,290,203 171,290,544 Sle1a.2 XP_922544.2 RefSeq peptide predicted 171,334,986 171,368,103 Sle1a.2 Cdca1 cell division cycle associated 1 171,492,176 171,532,488 Sle1a.2 Rsg5 regulator of G-protein signaling 5 171,578,985 171,584,317 Sle1a.2 Rsg4 regulator of G-protein signaling 4 171,765,614 171,771,328 Sle1a.2 1700084C01Rik RIKEN cDNA 171,786,212 171,805,880 Sle1a.2 Hsd17b7 hydroxysteroid (17-beta) dehydrogenase 7 171,814,321 171,947,236 Sle1a.2 Ddr2 discoidin domain receptor family, member 2 171,978,678 172,011,621 Sle1a.2 Uap1 UDP-N-acetylglucosamine pyrophosphorylase 1 172,035,931 172,052,068 Sle1a.2 Uhmk1 U2AF homology motif (UHM) kinase 1 172,069,546 172,088,262 Sle1a.2 Sh2d1c EAT-2-related transducer, EAT-2b 172,114,051 172,123,444 Sle1a.2 Sh2d1b EAT-2a 172,145,478 172,148,800 Sle1a.2 1700015E13Rik RIKEN cDNA 172,155,147 172,426,524 Sle1a.2 Nos1ap nitric oxide synthase 1 (neuronal) adaptor protein 172,481,505 172,519,464 Sle1a.2 Olfml2b olfactomedin-like 2B 172,543,889 172,704,443 Sle1a.2 Atf6 activating transcription factor 6 172,710,173 172,722,215 Sle1a.2 Dusp12 dual specificity phosphatase 12 172,743,948 172,749,816 Sle1a.2 Fcrlb Fc receptor-like B 172,754,576 172,764,268 Sle1a.2 Fcrla Fc receptor-like A 172,797,233 172,813,222 Sle1a.2 Fcgr2b Fc receptor IgG low affinity IIb

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62 Figure 4-2. Sle1a requires both Sle1a.1 and Sle1a.2 for increased CD4+ T cell ICOS expression and diminished Treg compartmen t. Splenocytes from B6, B6.Sle1a B6.Sle1a.1 and B6. Sle1a.2 mice were labeled for surface CD4, ICOS (A), CD25, and CD62L (B) and intracellular Foxp3 (C-D) expr ession and analyzed by FACS Each point represents an 8-12 month old individual animal. Representative gatings on a B6 sample are shown in the left-hand column (marker fo r A and rectangular gate for B-D) and representative histogra ms for all four strains are show n in the right-hand column. The light gray-filled histograms show isotype controls, dark gray-filled histograms show B6 values, while thick, thin and dashed black lines represent B6. Sle1a B6.Sle1a.1 and B6.Sle1a.2 respectively. All comparisons were performed with B6 values. Onetailed t tests: *: p<0.05, **: p<0.01, ***: p<0.001.

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63 Figure 4-3. Sle1a.1 and Sle1a.2 Tregs can support inhibition to a level equivalent to B6 Tregs in vitro. Inhibition of proliferation assays were set up with B6-derived APCs and Teffs, and either B6 (black bars), B6. Sle1a.1 (white bars) or B6. Sle1a.2 (gray bars) Tregs at the indicated ratio. A) Representative assay comparing proliferation in the presence of B6, B6.Sle1a.1 or B6.Sle1a.2 Tregs (4 mice per strain). Means and SEs are results of one-tailed t tests between the 0:1 assays and th e various Treg:Teff ratios for each strain. B) Normalized percentage inhibition of proliferation of th e 0:1 assays at the various Treg:Teff ratios for each strain comb ined from two different assays (8 mice per strain). Means and SEs are results of one-tailed t tests between the three strains for each Treg:Teff ratio. *: p<0.05, **: p<0.01, ***: p<0.001.

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64 Figure 4-4. Sle1a.1 or Sle1a.2 expression in Teffs or APCs affect s the extent of the inhibition of Teff proliferation. The inhibition of CD4+ CD25+ Teff proliferation in presence of 1:1, 1:4, or 1:16 Treg:Teff ratio s is expressed as a percen tage of the proliferation induced in the absence of Tregs for each condition. The origin, B6, B6. Sle1a (A), or B6. Sle1a.2 (B) of Tregs, Teffs, and APCs is indicated under each column. The graphs show the means and standard errors of two independent assays with 3-4 mice per strain, age 6 months in each assay. Results of one-tailed t tests between each condition and the "all B6 condition" ar e indicated for each Treg:Teff ratio. +, #, *: p<0.05, ++, ##, **: p<0.01, +++, ###, ***: p<0.001.

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65 Figure 4-5. Expression of Sle1a Sle1a.1 or Sle1a.2 in either Tregs or Teffs affects the extent of disease in an adoptive transfer model. CD4+ CD25Teff and CD4+ CD25+ Treg populations from 6 month old male donor mice were transferred by tail-vain injection into 2 month old male B6.Rag1-/recipient mice. Lines are representative of the percentage of mice per group (n=4) with less than 15% body weight (BW) loss over a 10 week period post-injection. Individual experiments for Sle1a (A), Sle1a.1 (B), and Sle1a.2 (C) donors are shown. Line depictions are as follows: B6 Teffs only black line / black filled box, congenic Teffs only gray line / gray filled box, B6 Tregs:B6 Teffs black line / black open box, B6 Teffs :congenic Tregs black line / gray open box, B6 Tregs:congenic Teffs gray lin e / black open box. Colon and kidney histology is pending for all three experiments.

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66 Figure 4-6. Expression of either Sle1a.1 or Sle1a.2 affects CD4+ function in a cell-intrinsic manner. B6 hosts were reconstituted with B6. Thy1a and/or B6.Sle1a subloci (Thy1b allele) bone marrow. Connected samples indicate values for CD4+ T cells expressing the Thy1a (CD90.1-gated) or Thy1b (CD90.2-gated) alleles w ithin the same mouse. Controls are represented by B6. Thy1a B6 and B6. Sle1a.1 or B6 .Sle1a.2 B6 single-strain bone marrow transfers. A) Ac tivation measured as the percentage of CD4+ CD69+ lymphocytes with representative histogram showing CD69 expression on gated CD4+ T cells. B) Treg levels, measured as the percentage of CD4+ CD25+ splenocytes expressing CD62L with representative histogram showing CD62L expression. Each point represents an individual animal. Two-tailed t tests: *: p<0.05, **: p<0.01, ***: p<0.001.

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67 CHAPTER 5 MURINE LUPUS SUSCEPTIBILITY LOCUS SLE1A.1 C ONTROLS RETINOIC ACIDENHANCED TGF-BETA-INDUCED REGULATORY T CELL EXPANSION Introduction In the previo us chapter, we presented resu lts which indicated that the phenotypes resulting from expression of Sle1a require the synergistic effect of both of its subloci, Sle1a.1 and Sle1a.2 due to the intermediate phenotypes observed for eac h. From this, we can infer that both subloci contain genes necessary for SLE susceptibility, an d can now progress to the assessment of which genes are directly involved since the regions of susceptibility ha ve now been narrowed. We will now focus on lupus susceptibility locus Sle1a.1 due to its small size of less than 1 Mb. Materials and Methods Mice B6. Sle1a.1NZW/NZW (B6.Sle1a.1) was obtained as a recombinant interval from B6. Sle1a (Fig. 4-1). C57BL/6J (B6) mice were origina lly obtained from The Jackson Laboratory (Bar Harbor, ME). ). B6.FOXP3-eGFP mice were obt ained from the Kuchroo group and derived as described (190). We derived the B6. Sle1a.1 .FOXP3-eGFP mice by breeding our B6.Sle1a.1 to Kuchroo's B6.FOXP3-eGFP mice using standard congenic breeding techniques. All mice were bred and maintained at the University of Fl orida in specific pat hogen-free conditions. All experiments were conducted according to prot ocols approved by the University of Florida Institutional Animal Care and Use Committee. Sle1a.1 Map and Pbx1 Primers PCR genotyping with m icrosatellite markers was performed as previously described (12) and SNP genotyping was performed by direct sequencing. Genes were found using Ensembl Release 40 (www.ensembl.org). There are 10 ex ons associated with Pbx1. Primers were designed around exons 5-8, in order to visualize isoform Pbx1-a from Pbx1-b, as Pbx1-b lacks

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68 exon 7, which does not appear to a ffect its function. Our collabor ator, Dr. Shiwu Li designed the following primers, 5' GAA GTG CGG CAT CA C AGT CTC3' from exon 5 and 5' ACT GTA CAT CTG ACT GGC TGC 3' from exon 8, to assess expression levels of multiple isoforms of Pbx1. CD4+ T cells from 5 month old female B6 and B6. Sle1a.1 mice were isolated by negative selection using the SpinSep for Mouse CD4+ T cell enrichment protocol (Stemcell Technologies). B cells from the same mice were isolated by negative selection using the B cell purification kit from Miltenyi Biotec. FACS analysis of the resulting CD4+ and B cell populations consistently showed >90% purity. Flow Cytometry Briefly, cells were first blocked on ice with staining buffer (PBS, 5% horse serum and 0.09% sodium azide) supplemented with 10% rabb it serum and pretreated with anti-CD16/CD32 (2.4G2). Cells were then stained with a pr e-titrated amount of PE-conjugated CD4 (RM4) from BD Biosciences (San Jose, CA). Foxp3 expression was determined by visualizing GFP expression. Cell staining was analyzed using a FACScalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA). At least 30,000 events were acquired per sample, and dead cells were excluded based on scatter char acteristics. Positive staining was determined as equal to or greater than the top 5% of the isotype control. Apoptotic Cell-Induced Production of IL-10 by Peritoneal Macrophages Peritoneal m acrophages were obtained by inject ion of 2.5 ml of thi oglycollate medium (3%) intraperitoneally into female B6 and B6. Sle1a.1 mice at 6 months of age (n=4). Macrophages were harvested 4 days later by peritoneal lavage. Induction of apoptosis was as described previously (191). Briefly, female B6 thymocytes at a concentration of 2 x 106 cells/ml were cultured with 15 g/ml staurosporine (Cayman Chemical, Ann Arbor, MI) for 8 hours at 37C in 5% CO2 to induce a population of early-apoptotic cells. The percentage of early

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69 apoptotic cells was quantified by flow cyto metry analysis by using Annexin-V and 7-AAD staining according to the manufactur ers instructions and was rou tinely 50-60%. These apoptotic cells were then cultured, according to a previous protocol (150), with the thioglycollate-induced peritoneal macrophages elicite d from male B6 and B6. Sle1a.1 mice at a ratio of 2:1 apoptotic cells to macrophages for 12 hours at 37C in 5% CO2 and analyzed for IL-10 production in the culture supernatant using an OptEIA Mouse IL-10 ELISA kit (BD Pharmingen) according to manufacturer's instructions. Microarray Analysis for Diffe rential Gene Expression CD4+ T cells from 5 month old female B6 and B6. Sle1a.1 mice were isolated by negative selection using the SpinSep for Mouse CD4+ T cell enrichment protocol (Stemcell Technologies). FACS analysis of the resulting CD4+ population consistently showed >90% purity. Our collaborator, Dr. Zh iwei Xu used the RNeasy Mini Kit (Qiagen) to isolate RNA from 3 x 106 B6 and B6. Sle1a.1 CD4+ T cells. cDNA was made, am plified, and labeled with biotin to prepare for hybridization to the Affymetrix Mouse Genome 430 2.0 Array using the Ovation Biotin RNA Amplification and Labe ling System (NuGEN T echnologies, Inc.). Microarray data obtained were normalized with the Affymetrix Microarray Suite (MAS 5.0), based on the housekeeping gene expression profile. Expression values were adjusted to the intensity of the expression value of the 100 housekeeping genes. Supervised analysis was performed by Dr. Henry Baker with a p < 0.001. Dr. Igor Dozmorov further analyzed the microarray data using the following methods. Id entification of differentially expressed genes was carried out with use associa tive analysis presented elsewhere (192). Cross-validation of the selections involved using a jacknife procedur e for characterization of the robustness or reproducibility (R) of the differen tially expressed genes selection. The comparative analysis was repeated for the two groups of samples with exclusion every time of one sample from each

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70 group. For two groups with n and m replicates, there is n x m possible comparisons. Genes selected as differentially expressed in each of these comparisons (selected n x m times) were ranked as having 100% of reproducibility. Analysis of associations of the obtained selections with known biologically important signaling pa thways and molecule s was carried out by Ingenuity Pathway Analysis (IPA)-web-bas ed programs (www.ingenuity.com) Induction of CD4+ Foxp3+ Tregs from CD4+ CD25T Cells CD4+ CD25cells were purified from 3 month old female B6 and B6.Sle1a.1 splenocytes with magnetic beads using the CD4+ CD25+ Treg cell kit according to the manufacturers instructions (Milte nyi Biotec). CD4+ CD25cells (5 x 105 cells/ml) from both B6 and B6. Sle1a.1 mice were stimulated with 1 g/ml anti-CD3 and 10 g/ml anti-CD28 plate-bound antibody in the presence of 100 U IL-2 (Peprotech), 20 ng/ml TGF(Peprotech), and 5 nM RA (SigmaAldrich) and culture d at 37C in 5% CO2 for 5 days. Cell were then labeled for surface CD4 and intracellular Foxp3 expression and analyzed by fluorescence-activated cell sorting. Results Sle1a.1 Contains Only One Gene We have used the three congenic recombinan t strains shown in Fig. 5-1 to refine the location of the gene(s) responsible for the Sle1a phenotypes. The entire Sle1a interval is covered by the combination of the Sle1a.1 and Sle1a.2 intervals. There is a short overlap between the two intervals between rs30711102 and rs31028646. In addition, the Sle1a.2 interval extends on the telomeric end beyond the Sle1a interval, resulting in B6. Sle1a and B6.Sle1a.2 having the B6 and NZW allele at the Fcgr2b gene, respectively (188). Based on information retrieved from Ensembl Release 40 (www.ensembl.org), th ere is only one gene present in the Sle1a.1 locus, Pbx1 which encodes for pre-B cell leukemia transcri ption factor (Pbx1), and this gene is not present in the Sle1a.2 region (Fig. 5-1).

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71 Sle1a.1 Expression Leads to a Decreased Ratio of CD4+ Foxp3+ to Total CD4+ T Cells B6.FOXP3-eGFP mice were obtained from the Kuchroo group and derived as described (190). These mice express GFP whenever Foxp3 is expressed, and are a us eful tool to analyze the Treg population. The B6.Sle1a.1 .FOXP3-eGFP mice were derived by breeding our B6. Sle1a.1 mice to Kuchroo's B6.FOXP3-eGFP mi ce using standard congenic breeding techniques in order to more accurately study the Treg population in the c ontext of expression of the NZW allele of Pbx1 Splenocytes from 3 month old B6.FOXP3-eGFP and B6. Sle1a.1 .FOXP3-eGFP mice were labeled for su rface CD4 expression and analyzed by fluorescence-activated cell sorting. As shown in Fig. 5-2, there is a signif icantly decreased ratio of CD4+ Foxp3+ T cells to total CD4+ T cells in the B6. Sle1a.1 .FOXP3-eGFP mice as compared to the B6.FOXP3-eGFP mice. Interestingly, by analyzing the Treg populati on in this fashion, we reached a level of significance, in dicating that the presence of ac tivated cells within the Treg population of the B6. Sle1a mice was indeed a confounding factor since we did not see this level of significance for the Treg population as defi ned by expression of CD4, CD25 and CD62L in Fig. 4-1. Further analysis is in the process of being completed due to the small number of samples we currently have as we ll as the fact that these mice are much younger compared to the 8-12 month old mice we an alyzed in Fig. 4-1. Alternative Pbx1 Isoforms Since Pbx1 was the only gene present in the Sle1a.1 locus, we needed to determ ine how the NZW allele of this gene differed from the B6 allele. Both sequen ce analysis at the cDNA level and preliminary Affymetrix mi croarray data for expression of Pbx1 obtained from CD4+ T cells revealed no difference betw een the B6 and NZW allele of Pbx1 We wanted to assess the expression level of Pbx1 in B cells, as it has alrea dy been described as a necessary factor in very early B cell commitment (148) as well as CD4+ T cells, since we have previously shown in

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72 Chapter 4 that expression of Sle1a.1 results in CD4+ T cell-intrinsic defects. There are 10 exons associated with Pbx1. Primers were designed by our collaborator, Dr. Shiwu Li, that would be capable of detecting both of the known isofor ms of Pbx1, Pbx1-a and Pbx1-b. Primers were designed around exons 5-8, in order to distingu ish isoform Pbx1-a from Pbx1-b, as Pbx1-b lacks exon 7, which does not appear to a ffect its function. Interestingl y, using these primers, we found two novel isoforms. Sequence analysis was pe rformed on all bands to confirm that that correspond to the inferred Pbx1 isoforms base d on size. Fig. 5-3 shows PCR products using primers designed for Pbx1 with corresponding exon / intron st ructure shown in Fig.5-4. Known isoforms Pbx1-a and Pbx1-b, 391 bp and 278 bp, resp ectively, are only found in B cells of both strains. Two novel isoforms were observ ed, Pbx1-c, 167 bp, found in both B and CD4+ T cells of both strains and Pbx1-d, 118 bp, found in both B and T cells, but only in the B6.Sle1a.1 strain. Pbx1-c lacks exon 7 and and only a portion of exon 6, while Pbx1-d lacks both exons 6 and 7 entirely (Fig 5-4). Pbx1 contains the followi ng domains: the meis binding domain (MIM), the nuclear localization signal (NLS), the hox binding domain (HCM), the PBC homeodomain (PBC-A,B), and the homeo DNA binding domain (HD). Based on sequence analysis, Pbx1-c appears to lack the HCM, potentially rende ring it incapable of binding Hox, while Pbx1-d appears to lack both the HCM and the HD, renderi ng this isoform incapable of binding both Hox and DNA. Since this isoform presumably ca nnot bind DNA, we predicted that its function would be impaired in the cells in which it is expressed. Expression of Sle1a.1 did not Affect Apoptotic Cell-I nduced Production of IL-10 by Macrophages Based on a study done by the Ma group and published in 2007, Pbx1, and m ore specifically Pbx1-b, was shown to be a physiologi cally critical mediator of apoptotic cellinduced IL-10 gene transcription and IL-10 cytokine production by macrophages with its

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73 transcriptional role found to be uncoupled from phagocytosis ( 150). Because expression of the NZW allele of Pbx1 leads to the presence of alternativ e Pbx1 isoforms in immune cells, we hypothesized that the Sle1a.1 -expressing macrophages would produ ce less IL-10 in response to stimulation with apoptotic cells. Thymocytes were isolated from B6 mice and stimulated with staurosporine, a potent inhibitor of protein kinases known to induce apoptosis, and subsequently cultured for 12 hours with thioglycollate-eli cited peritoneal macrophages from B6 and B6. Sle1a.1 mice. IL-10 production by the macrophages in the culture supernatant was measured by ELISA. As shown in Fig. 5-5, we f ound no difference between the normal and Sle1a.1 expressing macrophages regarding IL-10 production in response to apoptotic cells. Our collaborator, Dr. Li, assessed which isoforms of Pbx1 were present in both B6 and B6. Sle1a.1 derived peritoneal macrophages and found that macrophages from B6 mice expressed only Pbx1b while macrophages from B6. Sle1a.1 mice expressed Pbx1-a and Pbx1-b, with more of the Pbx1-b isoform present. We concluded that sinc e both of these isoforms are capable of normal function, the production of IL-10 by macrophages from either strain would be normal as well. Our results confirmed this hypothesis. Microarray Analysis of Differential Gene Exp ression Influenced by the NZ W allele of Pbx1 The NZW allele of Pbx1 led to alternative and novel isof orms of the transcription factor, and it was of interest to find out the effect of this alteration in a more global sense. By subjecting cDNA prepared from B6 and B6.Sle1a.1 CD4+ T cells to Affymetrix microarray hybridization and analysis and then evaluating differential gene expression, we were able to gain a better understanding of how the NZW allele of Pbx1 globally affected this cell population. The Affymetrix Mouse Genome 430 2.0 Array is th e most comprehensive whole mouse genome expression array, with analysis of over 39,000 transcripts on a singl e array. Table 5-1 shows a partial list of differentially expr essed genes with a confidence leve l of greater than 50 that the

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74 results are robust and reproducible, labeled as the R value. Of the first 80 genes that were found to be differentially expressed between B6 and B6. Sle1a.1 CD4+ T cells, 16 were involved in retinoic acid signaling. Fig 5-6 shows a heat map based on the expression of 14 of these genes known to be regulated by retinoic acid. The B6. Sle1a.1 CD4+ T cells appear to have an overall lower expression of this set of genes involved in retinoic acid signaling compared to B6 CD4+ T cells. Analysis of associations of the obtai ned selections with known biologically important signaling pathways and molecule s was carried out by Ingenuity Pathway Analysis (IPA) webbased programs by our collaborator, Dr. Igor Dozmorov. As shown in Fig. 5-7, the RA-signaling pathway potentially has a di rect effect on Pbx1. By examining cl usters of distinct yet integrated pathways, we were able to form th e hypothesis that the NZW allele of Pbx1 may lead to a difference in the capability of th e resulting isoform of Pbx1, Pbx1-d, to function as a mediator of RA-signaling. Expression of Sle1a.1 Results in Defective RA-Enhanced TGF-Induced Production of Adaptive Tregs We have presented that the NZW allele of the Pbx1 gene leads to an a lternative isoform of the Pbx1 transcription factor th at is predicted to lack th e DNA binding domain, presumably rendering the protein nonfuncti onal thereby leading to a dom inant negative mutation. CD4+ T cells from the B6.Sle1a.1 mice which possess this potential ly nonfunctional isoform of Pbx1 were shown to have differential expression of genes involved in the RA-signaling pathway. Since both the Noelle and Kuchroo groups prev iously showed that RA can enhance TGFinduced production of aTregs (127,190), we wanted to assess whether the CD4+ CD25T cells from the B6. Sle1a.1 mice reacted differently to treatment with RA than normal B6 CD4+ CD25T cells. CD4+ CD25T cells isolated from both B6 and B6. Sle1a.1 mice were cultured with antiCD3, anti-CD28, and IL-2 in the presence or absence of TGFRA for 5 days and then stained

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75 for surface CD4 and intracellular Foxp3 expression. We observed no Foxp3 induction in the presence of only IL-2 and there was no difference between B6 and B6. Sle1a.1 in the percentage of CD4+ Foxp3+ cells when TGFwas present (Fig. 5-8A). RA alone cannot induce Foxp3 expression in the presence of IL-2, but when TGFis present, we see an increase in the CD4+ Foxp3+ population for B6, with B6. Sle1a.1 not showing as substantial an increase. Figs. 5-8B and C show the significant decrease in both the percent increase (Fig 5-8B) and fold increase (Fig. 5-8C) values with RA addition for the Sle1a.1 -expressing cells, indicating that there is a defect in the RA signaling path way that participates in TGF-induced Foxp3 expression. We believe this is mediated by Pbx1, and we predic t that the potential l ack of DNA binding by the Pbx1-d isoform contributes to th is defect in RA-enhanced TGF-induced production of aTregs.

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76 Figure 5-1. Genes present in the Sle1a.1 interval From top to bottom are shown: a scale in Mb, the location of the microsatellite markers or SNPs mapping the interval termini, and the Sle1a, Sle1a.1 and Sle1a.2 intervals, in which the gray rectangles show the regions of known NZW allelic derivation, and the hatched rectangles on each side indicate the regions of re combination between the NZW and B6 genomes. Map based on Ensemble Release 40. Pale blue box i ndicates the only gene present within the Sle1a.1 region.

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77 Figure 5-2. Sle1a.1 expression results in a decreased ratio of CD4+ Foxp3+ to total CD4+ T cells. Splenocytes from 3 month old B6.FOXP3-eGFP and B6. Sle1a.1 .FOXP3-eGFP mice were labeled for surface CD4 expression and analyzed by fluorescence-activated cell sorting. Each point represents an individua l animal. Representative plots for CD4 and GFP expression are shown on the left. Ratios (r) are derived from the % CD4+ GFP+ T cells (blue rectangle) divided by the % total CD4+ T cells (yellow rectangle) and are shown on the right. Two-tailed t test: *: p<0.05.

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78 Figure 5-3. The NZW allele of Pbx1 results in a two novel isof orms. PCR was performed on B cells and CD4+ T cells isolated from 5 month old female B6 and B6. Sle1a.1 mice using Pbx1 primers designed by Dr. Shiwu Li. Known isoforms Pbx1-a, 391 bp, and Pbx1-b, 278 bp, and are only found in B cells of both strains. Two novel isoforms were observed, Pbx1-c, 167 bp, found in both B and CD4+ T cells for both strains and Pbx1-d, 118 bp, found in both B and T cells, but only in the B6. Sle1a.1 strain.

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79 Figure 5-4. Depiction of exons associated with both known and novel isoforms of Pbx1. Primers were designed by our collaborator for Pbx1 around exons 5-8 to be able to visualize the known isoforms, Pbx1-a and Pbx1-b, as Pbx1-b lacks exon 7. Two novel isoforms were observed using these primers and seque nce analysis was done to confirm that Pbx1-c and Pbx1-d contained the exons s hown above. Gray boxes indicate exons potentially not contained or differe nt among the known and novel isoforms.

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80 Figure 5-5. Expression of the NZW allele of Pbx1 in macrophages does not alter their IL-10 production in response to apoptot ic cells. A total of 0.5 x 105 B6 or B6. Sle1a.1 thioglycollate-elicited peritoneal macropha ges were stimulated with B6-derived apoptotic cells at a 2:1 ratio of apoptotic cells to macrophages for 12 hours and analyzed for IL-10 production. Graph is re presentative of three experiments with 4 mice per group at 6 months of age.

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81 Table 5-1. Microarray data obtained from B6 and B6. Sle1a.1 -derived CD4+ T cells. Strain B6 B6. Sle1a.1 Affymetrix ID Gene Mean SD Mean SD R 1436134_at Scn2b 39 10 0 0 100 1442946_at Atxn7 48 12 2 0 100 1438855_x_at Tnfaip2 155 36 58 12 100 1451608_a_at Tspan33 57 5 24 10 100 1416105_at Nnt 58 15 20 4 100 1416708_a_at Gramd1a 51 3 20 1 100 1419839_x_at Prpf19 117 27 56 11 100 1437614_x_at Zdhhc14 61 14 23 7 100 1438711_at Pklr 77 12 34 9 100 1449635_at Prpf19 114 25 52 12 100 1451453_at Dapk2 65 20 14 3 100 1418634_at Notch1 26 10 76 19 100 1427301_at cd48 31 15 83 12 100 1456103_at Pml 57 21 17 6 86 1434184_s_at Map4k4 36 14 85 19 86 1450543_at Myo1h 38 10 83 10 86 1423389_at Smad7 28 13 65 12 86 1435912_at Ubxd7 29 6 66 12 86 1424631_a_at Ighg 16 3 140 108 86 1416326_at Crip1 36 10 71 18 86 1429184_at Gvin1 122 20 225 26 86 1444003_at Lincr 62 12 30 9 71 1435679_at Optn 77 18 38 8 71 1436847_s_at Cdca8 49 10 21 1 71 1428842_a_at Ngfrap1 59 7 31 6 71 1435930_at Zfp291 85 9 43 8 71 1419406_a_at Bcl11a 71 27 37 5 71 1428942_at mt2 72 34 16 5 71 1433761_at Pde4dip 42 18 98 22 71 1418970_a_at Bcl10 52 18 116 19 57 1424227_at Polr3h 58 13 29 3 57 1417621_at Nfatc1 71 23 138 34 57 1436886_x_at Xab2 55 11 28 4 57 1437142_a_at Pigo 83 18 43 7 57 1417376_a_at Cadm1 64 23 32 6 57 1436934_s_at Aco2 87 26 43 6 57 1457669_x_at Rfc2 71 23 30 7 57 1430029_a_at Tspan31 34 10 84 25 57

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82 Figure 5-6. Expression of the NZW allele of Pbx1 in CD4+ T cells leads to differentially expressed genes involved in the RA-s ignaling pathway. Heat map based on Affymetrix microarray data from the B6 and B6. Sle1a.1 -derived CD4+ T cells.

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83 Figure 5-7. Pathway analysis of di fferentially expressed genes in CD4+ T cells isolated from B6 and B6.Sle1a.1 mice. The red designation indicate s genes with increased expression while the green designation indicates gene s with decreased expression compared to normal B6. Our collaborator, Dr. Igor Dozmor ov, analyzed microarray data from the B6. Sle1a.1 CD4+ T cells using pathway analysis so ftware. Based on these clusters, a connection between Pbx1 and RA was revealed.

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84 Figure 5-8. The NZW allele of Pbx1 leads to a defect in RA-enhanced TGF-induced expansion of aTregs in vitro. CD4+ CD25cells (5 x 105 cells/ml) from both 3 month old female B6 and B6. Sle1a.1 mice were cultured with plate-bound anti-CD3 (1 g/ml) and anti-CD28 (10 ug/ml), IL-2 (100 U) in the presence or absence of TGF(20 ng/ml) RA (5 nM) for 5 days and labeled for surface CD4 and intracellular Foxp3 expression and analyzed by fluorescence-activated cell sorting. A) Representative FACS plot depicting in tracellular staining of Foxp3 expression by CD4+ T cells. Quantification of CD4+ Foxp3+ cells measured as the percent (B) increase with RA addition and fold (C) in crease with RA addition from the base TGF-induced population from 3 experiments. Two-tailed t tests: ***: p<0.001.

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85 CHAPTER 6 DISCUSSION We have shown that expression of Sle1a is suf ficient to induce increased activation levels of CD4+ T cells, DCs and B cells, as well as to down-re gulate Treg levels. We also showed that Sle1a CD4+ T cells express increased levels of the co-stimulation marker ICOS, which has been shown to play a critical role in T cell help to B cells, especial ly in germinal centers (170,171). Elevated ICOS expression on T cells from lupus patients has now been reported in three independent studies (172174). These last two studies reported that ICOS stimulation of lupus T cells significantly enhanced anti-dsDNA Ab production from autologous B cells, which is equivalent to what we have shown for Sle1a T cells, which were able to induce anti-chromatin production in both autologous Sle1a -expressing B cells and normal B cells (13). These results also suggest that Sle1a confers a T cell phenotype that is found in lupus patients, which further validates the need to discover the identity of the Sle1a gene(s). Future experiments should address the specific role of ICOS in this process. High levels of ICOS have been associated with IL-10 secretion by CD4+ T cells (175), and IL-10 production by CD4+ T cells is significantly increased in the NZM2410 model (176). There was however no consistent increase of ex vivo IL-10 production by Sle1a CD4+ T cells, suggesting that another mechanism may be involved. We assessed levels of ICOS expre ssion in the subloci associated with Sle1a and found that while expression of Sle1a.1 led to a significant in crease in ICOS expression, this was not observed for expression of Sle1a.2 While Sle1a expression has also been shown to lead to a significant increase in CD69 expression on CD4+ T cells, we did not observe this for either of the subloci, indicating that neither Sle1a.1 nor Sle1a.2 can fully account for the activation phenotypes associated with Sle1a expression.

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86 Sle1a induces a reduction in the size of the Treg compartmen t, but these cells express normal levels of CTLA-4, CD103, and GITR, mol ecules which have been commonly associated with the regulatory phenotype ( 193-196). When we analyzed B6. Sle1a.1 and B6.Sle1a.2 strains for their Treg populations, we found that although there was a trend toward s a decrease in the CD4+ CD25+ CD62L+ population, it was not to the level of significance when compared with B6 values. Upon analysis of intracel lular expression of Foxp3 among the CD4+ CD25+ and CD4+ CD25+ CD62L+ population, we found that only Sle1a and Sle1a.2 populations expressed significantly decreased levels of this transcription factor know n to commit cells to the Treg lineage. Again, these results indicate that neither sublocus can completely account for the decrease in the Treg compartment associated with Sle1a In addition, at the higher ratios of Treg:Teff, Sle1a -expressing Tregs are fully capable of s uppressing the proliferation of B6 Teffs on a per-cell basis in the presence of B6 APCs. However, at lower ratios of Treg:Teff, this suppressive capability is decreased, consistent with a reduced proportion of functional Tregs within the CD4+ CD25+ T cell population of the B6. Sle1a mice. However, we saw that at all ratios of Treg:Teff, both Sle1a.1 and Sle1a.2 -expressing CD4+ CD25+ Tregs were capable of suppression equal to that of normal B6 CD4+ CD25+ Tregs. In addition to in vitro suppression assays, we also performed adoptive transfers adap ted from the experimental model of colitis to test the in vivo effect of Sle1a on Treg and Teff functions in a ra pid model of disease. Results from the in vivo study confirmed our in vitro data for Sle1a while we saw mixed results for the subloci. The Sle1a.2 -expressing Tregs were less able to prevent colitis onset than normal B6 Tregs, but were not as defective in function as the Sle1a -expressing Tregs. We cannot exclude, however that Sle1a also affects Treg inhibi tory functions. Indeed, a recent construct with a nonfunctional Foxp3 has demonstrated that the expr ession of Treg signature makers can develop

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87 normally in cells that completely lack inhibitory functions (177). A definitive answer to that question will require the full pa nel of experiments involving Sle1a -expressing mice, along with Sle1a.1 and Sle1a.2 -expressing mice, bred to a Foxp3 reporte r construct, which we are currently pursuing. We have found that the B6. Sle1a.1 .FOXP3-eGFP mice at a young age present with a significantly decreased ratio of CD4+ Foxp3+ T cells to total CD4+ T cells, but analysis of this cell populations function is required and is currently being pursued. While we have shown that the Sle1a -expressing Tregs are capa ble of suppression, in situations where either the Teffs or the APCs express Sle1a, the suppressive capability of normal B6 Tregs is significantly re duced, suggesting that the Sle1a locus confers a resistance to suppression of Teff proliferation and that the APCs are playing a role in this phenomenon (189). We performed these same expe riments with cells expressing Sle1a.1 and Sle1a.2 and found similar results. It would appear that expression of Sle1a.1 and Sle1a.2 in Teffs or APCs confers resistance to suppression by normal Tregs, although not to the extent observed for Sle1aexpressing Teffs or APCs. This is yet further evidence validating our hypothesis th at expression of both Sle1a.1 and Sle1a.2 are necessary to observe the full effect of Sle1a expression. It is of note that the APC population used in our in vitro suppression assays contai ns not only DCs but B cells as well. We have prev iously shown the effects of Sle1a DCs on Treg suppression (19), however, Sle1a affects both of these cell types. This indicates a potential role of activated B cells on Treg function, and is an avenue to be stud ied further. A similar Teff resistance has been previously reported in another mode l of lupus (162), but it is not cl ear at this point whether this resistance is the mere consequence of hyperactiva tion, or a result of involvement with a specific mechanism. Cbl-b deficiency results in a resistan ce to Treg regulation involving TGF, and this mutation also induces an increased level of ac tivation in effector T cells (178). To our

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88 knowledge, no other mechanisms of resistance to Tregs have been reported and additional experiments will be necessary to determine how Sle1a confers this resistance in CD4+ T cells. We have previously shown that DCs from the NZM2410 triple congenic strain, B6.TC, prevent Tregs from performing their inhi bitory functions, primarily thr ough the production of IL-6 (158). We report here that Sle1a -expressing DCs present the same phenotype of high IL-6 production and Treg inhibition, indicating that this locus plays a major role in the overall DC phenotype of lupus-prone mice. Interestingly, the type-1 diabetes prone NOD mice, which have a reduced number of Tregs (197,180), also produce APCs th at fail to fully support Treg functions (180). These results suggest that defective support or active inhibition of Treg functions by DCs may be a common mechanism affecting autoimmune pathogenesis. Mixed BM chimeras have shown here that th e increased proliferation and activation of Sle1a -expressing T cells, as well as the reduced Sle1a Treg level require that Sle1a be expressed in these T cells. Similar results were obtained for both Sle1a.1 and Sle1a.2 These results differ from what might have been predicted from the in vitro reconstitution experiments shown in Figure 3-5, where B6. Sle1a -derived APCs inhibited Treg fu nction. The BM chimera results do not mean that Sle1a exclusively affects CD4+ T cells. In an analogous set of experiments, BM chimeras showed that T cell activa tion and autoreactivity mediated by Sle3 were the indirect result of Sle3 expression within the myel oid compartment (62,181). It is therefore possible that the Sle1a -induced intrinsic defects in CD4+ T cells are indirectly responsible for the DC and B cell abnormalities. Alternatively, the Sle1a gene(s) may control a path way present in all three cellular compartments. In any event, i ndirect or direct activation of DCs by Sle1a was not sufficient to convey extrinsic changes to B6-derived CD4+ T cells in vivo. The exact cause for these differences is unclear, and highlights the need to confirm in vitro findings with in vivo

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89 studies. Additional mixed BM chimeras will be necessary to address whether Sle1a expression in these DCs and B cells is necessary fo r production of the activated phenotypes. Autoreactive T cells are a feature common to many autoimmune diseases for which a genetic basis has been demonstrated, yet only very few genes have been identified as responsible for this phenotype (182). In addition to Cbl-b discussed above (178), null alleles of Gadd45a (183) or E2f2 (184) result in a lower threshold for T cell activation culminating in autoimmune pathogenesis, while null alleles in Ctla4 (185) and Foxp3 (198) result in massive inflammatory and autoimmune responses through the disruption of the Treg compartment. More recently, a natural polymorphism in the Il2 gene has been identified as responsible for the diabetes susceptibility locus Idd3 in the NOD mouse, also through an impairment of Treg function (199). The Sle1a interval does not contain any gene with obvious function in T cells. Our in vitro results showed that Sle1a confers an autoimmune phenotype to CD4+ T cells in the colon, which is not typically associated with lupus pathogenesis. This indicates that Sle1a affects a genetic pathway regulating production of Tregs and responses to Tregs in a manner that is not restricted to tolerance to nuclear antigens. The identification of the Sle1a gene(s) will therefore uncover a novel and broad pathway by which autoreac tive T cells are re gulated by Tregs. Using Ensembl Realease 40, we found th at only one gene exists in the Sle1a.1 region of chromosome 1, Pbx1 which encodes for a transcription factor necessary for transcription of multiple genes. There is 100% amino acid se quence homology between mouse and human Pbx1, and it is therefore a possibility that we may find similar results among SLE patients as we found in our SLE mouse model. We intend to pursue this further by analyzing a large cohort of SLE patients with ageand sex-matched controls. It would also be of use for us to group patients with

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90 active SLE versus inactive SLE and correlate the nu mber of Tregs in their peripheral blood to the isoforms of Pbx1 pres ent in their T cells. TGFhas been shown to induce Foxp3 expression. Activin, a member of the TGFfamily of growth factors, is known to be a critical regulator of follicle-stimulating hormone (FSH) expression by activating transcription of the FSHsubunit and stimulating FSH secretion (200-202). Like all members of the TGFfamily, activin signals through serine/threonine kinases, which then phosphorylate intracellular receptor-specific Smad proteins, Smad2 and Smad3 in the case of activin (203). Smad6 or Smad7 has the capacity to block this phosphorylation, thereby blocking tr anslocation to the nucleus (204). In 2004, it was published that three activin-respon sive regions are required for full activin response with smad-binding elements (SBE) present in all (205). One of these elements was found to bind a complex containing Pbx1, Prep1 and Smad4, identifying P bx1 and Prep1 as Smad binding partners and mediators of activin action (205). It has been shown that both Meis and Prep associate with Pbx1 in the cytoplasm and induce a conforma tional change in Pbx1, exposing the nuclear localization signal, and subsequently causing translocation of the dimeric protein complex to the nucleus (144). Treatment with RA has been shown to expand both Meis and Pbx1 expression in various cell types (114,146,147). Since RA was shown to potently synergize with TGFin driving Foxp3 induction (127), it is of interest to elucidate this mechanism. RA has been implicated in enhancing TGFsignaling by increasing the ex pression and phosphorylation of Smad3, resulting in increased Foxp3 expression (133). In our case, we propose that RA-enhanced TGF-induced expression of Foxp3, and therefore the production of aTregs, is mediated by Pbx1. In a nor mal situation (Fig. 6-1A), TGFbinds the serine/threonine kina ses, resulting in the phosphorylat ion of intrace llular receptor-

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91 specific Smad3, which then binds with Smad4, a nd this complex can then translocate to the nucleus. Normal amounts of Smad7 would not bloc k this process. Smad3/4 complexes can bind DNA alone, but with low affinity, so their inter action with additional tr anscription factors is required for target gene regulati on. Since the transcription fact or Pbx1 has been shown to bind with Smad partners, we postulate that the partner is Smad3, and that this interaction leads to enhanced binding to a target gene, perhaps Foxp 3 in this case. RA has been involved in increasing Pbx1 expression via tran scriptional regulation as well st abilization of Pbx1 proteins, and shown to be likely related to an increase in the association between Pbx1 and Meis. The binding of Meis induces a conformational change in Pbx1, expos ing the nuclear localization signal, and subsequently causing translocation of the dimeric protein complex to the nucleus, where it can bind DNA but with low affinity as well. A complex involving Meis/Pbx1 and Smad3/4 may be the connection between the RA and TGFsignaling pathways. In our Sle1a.1-expressing T cells (Fig. 6-1B ), where the NZW allele of Pbx1 is expressed, we have a shown that there is a reduced induction of Foxp3 expression with RA addition to the culture with TGF. Since the NZW allele of Pbx1 leads to expression of an alternative isoform predicted to lack the DNA binding domain, we hypothesize that Pbx1-c, present in both Sle1a.1 and normal T cells can carry out the normal function of binding the Smad3/4 complex to transcribe target genes, but Pbx1-d, found only in the Sle1a.1 -expressing T cells, is unable to do so. Also, as shown in Table 5-1, the Sle1a.1 T cells express significantly more Smad7, a factor known to block the phosphorylation of Smad3, ther eby rendering it incapa ble of binding Smad4 and subsequently translocating to the nucleus. Therefore, ther e is an overall reduction in transcription of target genes in the Sle1a.1 -expressing T cells, leading to a decreased production

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92 of aTregs. It should be noted th at there is the potent ial role for RA in differential splicing of target genes as well, in this case Pbx1 Further studies involving molecu lar protocols are in preparati on to test this hypothesis. Since we have found that Pbx1-d seems to be expressed at higher levels in patients with SLE, we have begun studies involving a human T cell line, the Jurkat T cell line, in order to test the effect of overexpression of the Pbx1-d isoform on T cell function. Our collaborator has made a viral construct of Pbx1-d and infected Jurkat T cells, which do not nor mally contain this truncated isoform. We are currently in the process of performing the aforementioned assay involving RAenhanced TGF-induced production of aTregs using the virally-infected Jurkat T cells compared to those Jurkat T cells infected with a random gene. S hould we observe that there is a decrease in the production of aTregs, we can de finitively say that Pbx1 is involved in mediating RA-enhanced production of aTregs a nd is an integral transcription f actor in this mechanism. We could also potentially test the levels of phosphorylated Sm ad3 between normal B6 and Sle1a.1 expressing T cells, or possi bly test the level of bi nding activity of Smad3 to Pbx1 to see if we can definitively prove that Pbx1 is the factor that links the RAand TGF-signaling pathways.

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93 Figure 6-1. The proposed mechanism of Pbx1s role in the connection of the retinoic acid and TGFsignaling pathways in a T cell. Retinoic acid can enhance TGF-induced signaling with the binding of the Pbx1-c complex (A) to the Smad complex in the nucleus leading to increase d transcription of target genes when bound to DNA, but since Pbx1-d lacks the DNA binding domain, less transcription occurs when this isoform is present (B).

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112 BIOGRAPHICAL SKETCH Having lived all over the country as a young girl Carla chose to atte nd the University of Florida after graduating in Ma y 1999 with honors from Gainesvill es own Eastside High School International Baccalaureate (IB) program. An advanced placement high school biology class led her to choose microbiology and cell ccience as he r major with a minor in chemistry. By her senior year, she was set on pursu ing a Ph.D. in biomedical res earch and applied to schools around the nation offering interdisciplinary program s. She graduated cum laude in the Spring of 2003 with her Bachelors and remained at the Univer sity of Florida to begi n her Ph.D. studies in the Fall of 2003. The College of Medicines Interdisciplinary Pr ogram in Biomedical Research gave her many options as to which discipline to choose. An interesting undergraduate class in immunology as well as a core course in the interdisciplinary program in immunology and microbiology led her to choose a research path in that direction. In the Spring of 2004, after a six week rotation, Dr. Laurence Morel offered her a position as a Graduate Research Assistant in the Department of Pathology, Immunology and La boratory Medicine stud ying a congenic murine model of systemic lupus erythematosus.