Genetic Background on a Nonobese Diabetic Versus C57BL/6 G7 T Cells

Increased Entry into the IFN-γ Effector Pathway by CD4 + T Cells Selected by I-Ag7 on a Nonobese Diabetic Versus C57BL/6 Genetic Background This information is current as of September 28, 2021. Syuichi Koarada, Yuehong Wu and William M. Ridgway J Immunol 2001; 167:1693-1702; ; doi: 10.4049/jimmunol.167.3.1693 http://www.jimmunol.org/content/167/3/1693 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Increased Entry into the IFN-␥ Effector Pathway by CD4؉ T Cells Selected by I-Ag7 on a Nonobese Diabetic Versus C57BL/6 Genetic Background1

Syuichi Koarada, Yuehong Wu, and William M. Ridgway2

IFN-␥-mediated Th1 effects play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We analyzed functional responses of CD4؉ T cells from NOD and B6.G7 MHC congenic mice, which share the H2g7 MHC region but differ in their non-MHC genetic background. T cells from each strain proliferated equally to panstimulation with T cell lectins as well as to stimulation with glutamic acid decarboxylase 524–543 (self) and hen egg lysozyme 11–23 (foreign) I-Ag7-binding peptide -epitopes. Despite comparable proliferative responses, NOD CD4؉ T cells had significantly increased IFN-␥ intracellular/extra cellular protein and mRNA responses compared with B6.G7 T cells as measured by intracellular cytokine analysis, time resolved Downloaded from fluorometry, and RNase protection assays. The increased IFN-␥ production was not due to an increase in the amount of IFN-␥ produced per cell but to an increase in the number of NOD CD4؉ T cells entering the IFN-␥-producing pathway. The increased IFN-␥ response in NOD mice was not due to increased numbers of activated precursors as measured by activation/memory markers. B6.G7 lymphoid cells demonstrated an absolute decrease in IFN-␥ mRNA, an increase in IL-4 mRNA production, and a significantly decreased IFN-␥:IL-4 mRNA transcript ratio compared with NOD cells. CD4؉ T cells from C57BL6 mice also ؉ b ␥

showed signiﬁcantly decreased IFN- production compared with CD4 T cells from NOD.H2 MHC-congenic mice (which have http://www.jimmunol.org/ an H2b MHC region introgressed onto an NOD non-MHC background). Therefore, the NOD non-MHC background predisposes to a quantitatively increased IFN-␥ response, independent of MHC class II-mediated T cell repertoire selection, even when compared with a prototypical Th1 strain. The Journal of Immunology, 2001, 167: 1693–1702.

onobese diabetic (NOD)3 mice spontaneously develop the presence of certain cell populations can stop the transfer of pancreatic islet lymphocytic inﬁltration (insulitis) and diabetes mediated by autoreactive CD4ϩ and CD8ϩ T cells (26– N autoimmune diabetes (1). Genome scanning and subse- 28). B cells and CD87 T cells seem necessary to develop the dis- quent congenic analysis have demonstrated the genetic complexity ease in vivo (29–31). Defective thymic T cell activation, defective of the disease process, with at least 20 insulin-dependent diabetes APC activation, and defective thymic negative selection of T cells by guest on September 28, 2021 (Idd) genetic loci contributing in a complex manner to cause dia- have also been shown to contribute to NOD autoimmunity betes (2–5). Multiple studies have demonstrated that diabetes in (32–36). NOD mice is autoimmune, with a prominent role for the unique Despite this large body of evidence, how Idd loci cause (or MHC class II molecule (I-Ag7), CD4ϩ and CD8ϩ T cells, and B prevent) diabetes and insulitis remains unclear. Several groups cells (6–14). T cell epitopes from glutamic acid decarboxylase have shown that introgression of protective Idd loci from nonau- (GAD) and insulin have been mapped, and T cells reactive to these toimmune strains such as B6 or B10 (producing NOD-congenic autoantigens have been isolated (15–18). Much of the literature strains) can decrease or prevent diabetes and insulitis. The intro- ϩ points to a strong role for Th1-like CD4 immune responses in gression of Idd3 and Idd10 reduces diabetes incidence to ϳ3% (37, pathogenesis and a decreased Th2 response; a variety of interven- 38). Idd9 alone on the NOD background reduces diabetes inci- tions have shown that diverting the immune response in NOD dence to 5% (39). The combination of Idd9, Idd3, and Idd10 elim- toward Th2 may prevent disease (although pancreatic IL-10 pro- inates diabetes and virtually eliminates insulitis (39). The strongest duction makes diabetes worse) (19–25). There is evidence of im- linkage in the original genome scan was to the H2g7 locus; NOD mune regulatory defects, as well; e.g., in cotransfer experiments, MHC F1-congenic mice, carrying all NOD genes except for one copy of a non-NOD MHC interval, have an 80-fold decrease in

Division of Rheumatology and Immunology, Department of Medicine, University of diabetes (40). Many publications have examined the role of the Pittsburgh School of Medicine, Pittsburgh, PA 15261 MHC class II molecule in the autoimmune process, suggesting that Received for publication November 16, 2000. Accepted for publication May it may bind specific autoantigens for presentation to autoreactive 21, 2001. lymphocytes and that, due to its defective peptide binding charac- The costs of publication of this article were defrayed in part by the payment of page teristics, I-Ag7 may be inefficient at thymic negative selection (36, charges. This article must therefore be hereby marked advertisement in accordance 41–44). Conversely, the NOD MHC region alone is not sufficient with 18 U.S.C. Section 1734 solely to indicate this fact. for disease, because B6.G7 mice (carrying all non-NOD genes 1 W.M.R. is supported by the Juvenile Diabetes Foundation, a Pfizer Scholars Grant, and the Competitive Medical Research Fund of the University of Pittsburgh School of except for the NOD MHC interval) and other NOD MHC-con- Medicine. genic mice do not develop diabetes. 2 Address correspondence and reprint requests to Dr. William M. Ridgway, S725 The cellular and immunological effects of non-MHC loci, how- Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, ever, are largely unknown. Fox and Danska (45) analyzed insulitis PA 15261. E-mail address: [email protected] in NOD and the related nonobese diabetes-resistant (NOR) strain, 3 Abbreviations used in this paper: NOD, nonobese diabetic; GAD, glutamic acid decarboxylase; HEL, hen egg lysozyme; RPA, RNase protection assay; LN, lymph which shares the MHC but differs at non-MHC loci, and found that node; NOR, nonobese diabetes-resistant. in the NOR mice APCs could infiltrate the islet but T cells could

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 1694 NON-MHC CONTROL OF NOD T CELL RESPONSES not, indicating non-MHC regulation of T cell function. In addition, cases, anti-IL-4 Ab (BVD4-1D11, rat IgG2b; BD PharMingen) was used NOR islets showed decreased Th1 cytokine transcripts. Scott et al. instead of anti-IL-10 Ab. The cells were also stained in the same manner (46) showed that non-MHC polymorphisms could affect differen- with FITC-conjugated rat IgG2b isotype control Ig (BD PharMingen) and PE-conjugated rat IgG1 isotype control Ig (BD PharMingen). Flow cyto- tiation to a Th2 phenotype in a TCR-transgenic model. The large metric analyses were performed using a FACScan flow cytometer (BD body of literature pointing to immune abnormalities in NOD mice Biosciences, Mountain View, CA). Wilcoxan statistical analysis of the re- suggests that Idd disease susceptibility loci mediate specific im- sults was performed using StatView 4.0 (Abacus Concepts, Berkeley, CA). munological functions along an immune pathway which, in the presence of a critical number of susceptibility alleles, would result Fluorometry in loss of tolerance and autoimmunity. In this paper, we used IL-4, IL-10, and IFN-␥ were detected by time-resolved fluorometry. The NOD, C57BL/6, B6.G7 mice (C57BL/6-congenic mice with an capture Ab 11B11 (anti-IL-4), JES5-2A5 (anti-IL-10), or AN18 (anti- introgressed NOD MHC interval; the B6.G7 mice do not develop IFN-␥) was bound to flat-bottom microtiter plates that were then blocked with 1% BSA in PBS at room temperature for 1 h. Sample supernatants and diabetes, although they develop minor pancreatic lymphocytic in- ␥ b cytokine standards, recombinant mouse IL-4, IL-10, and IFN- , were ti- filtration; Ref. 47) and NOD.H2 congenic mice (which have the trated and incubated for1hontheplates, which were then washed; the b H2 MHC region introgressed onto the NOD non-MHC back- detection layer (BVD6-24G2-biotin for IL-4, JES5-16E3-biotin for IL-10, ground and do not develop diabetes; Ref. 48) to analyze non-MHC or XMG1.2-biotin for IFN-␥) was then added. Bound detection Ab was control of T cell function. Our results indicate that non-MHC NOD detected with europium-streptavidin (Wallac, Uppsala, Sweden). The plates were read on a Wallac 1420 Victor time-resolved fluorometer. Data background genes predispose to a significantly enhanced Th1 re- were imported to Excel (Microsoft, Redmond, WA) and analyzed using sponse independent of MHC-dependent T cell repertoire selection DeltaSOFT (Biometallics, Princeton, NJ) using a four-parameter fit model events. to quantify cytokine results in nanograms per milliliter. Wilcoxan statistical Downloaded from analysis of the results was performed using Statview 4.0. Materials and Methods Mice RNase protection assay (RPA) NOD/Lt, B6.NODc17 (hereafter referred to as B6.G7), NOD.H2b, and Total RNA was extracted from LN or splenic cells using the RNeasy C57BL/6 (hereafter called B6) mice were purchased from The Jackson minikit (Qiagen, Valencia, CA). The RNA was redissolved in RNase-free water, and yield was estimated by spectrophotometry; equal quantities of Laboratory (Bar Harbor, ME). The B6.G7 mouse was originally produced http://www.jimmunol.org/ and characterized in the laboratory of E. Wakeland (47). The mice were RNA were used for analysis. RPA was performed using RiboQuant from bred and maintained under specific-pathogen-free conditions in the animal BD PharMingen according to the manufacturer’s protocol. The multiprobe facility of University of Pittsburgh Medical Center, Pittsburgh, PA. Mice template set mCK-1 (containing templates for IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, ⌱FN-␥, L32, and GAPDH) was purchased from BD were used at age of 8–12 wk. Unless otherwise specified, 8-wk-old mice ␣ 32 were used. PharMingen. The templates were used to synthesize the [ - P]UTP- labeled probes (3000 Ci/mmol, 10 mCi/ml; NEN Life Science Products, Ags and mitogens Boston, MA) in the presence of a GACU pool using a T7 RNA polymerase (BD PharMingen). Hybridization with 5–15 ␮g RNA was performed for GAD524–543 and hen egg lysozyme (HEL11–23) peptides were synthesized 12–14 h at 56°C, and the products were digested with RNase A and T1 and HPLC purified at the Molecular Biology and Genetics core facility of mixture. The samples were treated by proteinase K in proteinase K buffer the University of Pittsburgh School of Medicine. Con A was obtained from with yeast tRNA and then extracted with phenol and chloroform-isoamyl by guest on September 28, 2021 Pharmacia (Piscataway, NJ). PMA, ionomycin, PHA, and saponin were alcohol (50:1) and precipitated in the presence of ammonium acetate. The obtained from Sigma (St. Louis, MO). samples were loaded on acrylamide-urea gel and run at 40 W with 0.5ϫ Tris-borate-EDTA electrophoresis buffer for 2 h. The gel was adsorbed to Immunization and preparation of cell culture filter paper, vacuum dried, and then exposed on film (X-AR; Kodak, Rochester, NY) with intensifying screens at Ϫ70°C. The films were Groups of NOD and B6.G7 mice (n ϭ 2–3) were primed at the base of the scanned, and densitometry performed using Quantity One software (BD tail with peptide/CFA emulsion; 8–10 days later, draining lymph node Biosciences). Absolute RNA levels were calculated using 2- to 4-h expo- (LN) cells were isolated under aseptic conditions and stimulated in vitro sures of housekeeping gene expression and normalizing using Quantity with Ags or mitogen. For Con A stimulation, naive inguinal LN and spleen One software. Wilcoxan statistical analysis of the results was performed cells were collected. The cells were washed and resuspended at 1 ϫ 106 using StatView 4.0. The Th1:Th2 cytokine ratios were calculated in the cells/ml in complete medium consisting of RPMI 1640 supplemented with form [(NOD Th1 cytokine)/(B6.G7 TH1 cytokine)]//[(NOD Th2 cytokine)/ 10% (w/v) FCS, 1 mM L-alanylglutamine (Life Technologies, Gaithers- (B6.G7 Th2 cytokine)], and the results were analyzed by Wilcoxon burg, MD), 100 U/ml penicillin, 100 ␮g/ml streptomycin (Life Technolo- statistics using StatView 4.0. gies), 1 mM sodium pyruvate (Life Technologies), and 50 ␮M 2-ME. For Con A activation, 1 ml of the cell suspension was placed in 24-well plates (BD, Franklin Lakes, NJ), and Con A was added to a final concentration of Results ␮ ϫ 5 ϫ 4 g/ml. For Ag-specific T cell assays, the cell suspension at 5 10 –1 After exposure to mitogenic stimuli, NOD LN and spleen cells 106/200 ␮l was placed in 96-well flat-bottom plates and GAD or 524–543 have significantly increased numbers of CD4ϩIFN-␥ϩ T cells HEL11–23 was added to indicated final concentrations. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere. Supernatants were compared with B6.G7 despite quantitatively similar proliferative collected 3–8 days after the stimulation, and the cells were incubated with responses or without 5 ng/ml PMA (Sigma) and 0.5 ␮g/ml ionomycin (Sigma) for

3.5 h (37°C, 5% CO2). Although NOD and B6.G7 mice share the diabetes-associated MHC class II H-2g7 interval, B6.G7 mice do not develop diabetes. Flow cytometric intracellular cytokine analysis We tested whether peripheral CD4ϩ cells were functionally dif- For intracellular cytokine analysis, after the first 1.5 h of the 3.5-h PMA- ferent under genetic control of B6 Idd loci in the setting of I-Ag7 ionomycin incubation, brefeldin A (final concentration, 10 ␮g/ml) (Epi- selection. Our initial approach used pan-T cell lectin stimulation centre Technologies, Madison, WI) was added to the culture. At the end of with Con A followed by restimulation with PMA-ionomycin. This the incubation, the cells were stained for 20 min at 4°C with Tri-Color (R-phycoerythrin-cyanine 5 tandem)-conjugated anti-CD4 mAb (CT-CD4) assay is designed to convert naive T cells into functionally active (Caltag, San Francisco, CA) in staining buffer (2% FBS, 0.1% sodium cytokine secretors (49). Stimulated NOD peripheral spleen and LN azide in PBS). Cells were fixed for 20 min at 4°C with 2% paraformalde- produced significantly increased numbers of IFN-␥-positive CD4ϩ hyde (Sigma) in PBS and then permeabilized for 10 min at room temper- cells, by intracellular cytokine analysis, compared with B6.G7 ature with 0.5% saponin (Sigma) in PBS. Cells were stained in permeabi- ␥ (Fig. 1). NOD spleen cells showed a significant increase in the lization buffer for 20 min at 4°C with PE-conjugated anti-IFN- Ab ϩ ϩ 5 (XMG1.2, rat IgG1; BD PharMingen, San Diego, CA) and FITC-conju- percentage of IFN-␥ cells, in the number of IFN-␥ cells per 10 ϩ ϩ gated anti-IL-10 Ab (JES5-16E3, rat IgG2b; BD PharMingen). In some cells, and in the percentage of IFN-␥ CD4 cells (Fig. 1B). NOD The Journal of Immunology 1695 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Increased intracellular IFN-␥ production in Con A-PMA-ionomycin-stimulated NOD vs B6.G7 CD4ϩ T cells. A, NOD and B6.G7 LN and spleen cells were cultured with (top half) or without (bottom half)ConAasinMaterials and Methods and then restimulated with PMA-ionomycin and assayed for cytokine production by intracellular cytokine analysis using ﬂow cytometry plus staining for CD4 expression. NOD Con A-PMA-ionomycin- stimulated spleens and lymph nodes demonstrated an increase in both total IFN-␥ϩ cells (top panels) and CD4ϩIFN-␥ϩ cells (bottom panels) compared with B6.G7, in both lymph node (left side) and spleen (right side). One representative of eight experiments is shown. B and C, Complete results of eight Con A-PMA-ionomycin experiments demonstrating signiﬁcantly increased NOD percent IFN-␥ϩ cells, increased IFN-␥ϩ cells normalized per 105 cells, and increased NOD percent CD4ϩIFN-␥ϩ cells (CD4 fractionation performed in six of eight experiments) in both spleen (B) and LN (C). Each symbol represents a different experiment. 1696 NON-MHC CONTROL OF NOD T CELL RESPONSES

FIGURE 2. Increased extracellular IFN-␥ production in Con A/PMA-ionomycin-stimulated NOD vs B6.G7 spleen and LN cells. NOD and B6.G7 spleen (A) and LN (B) cells were cultured and stimulated as in Fig. 1. Equal numbers of NOD spleen and LN cells produced signiﬁcantly more IFN-␥ than did B6.G7 cells (p ϭ 0.025, spleen; p ϭ 0.046, LN). One representative of eight experiments is shown. *, Statisti- cally signiﬁcant difference. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Cytokine mRNA quantitation by RPA in Con A-PMA-ionomycin-stimulated NOD and B6.G7 spleen and LN cells. A, NOD and B6.G7 LN or spleen cells were stimulated as in Figs. 1 and 2. RNA was extracted and subject to RPA as described in Materials and Methods. Lane 1, NOD and B6.G7 Con A-stimulated LN cells; 24 h exposure. Housekeeping genes (L32 and GAPDH; shown beneath the lane at 4 h exposure) were used for RNA quantitation. Lane 2, NOD and B6.G7 Con A-stimulated spleen cells, 24 h exposure with 4 h housekeeping gene expression shown beneath. Gels had the following controls (not shown): unprotected probe sets at 1/200 and 1/400 dilution, control mouse RNA, control yeast tRNA. Gel films were scanned and densitometry was performed using Quantity One software (see Materials and Methods); results were exported to Excel and statistical analysis was performed using StatView. One representative of six experiments is shown. The complete densitometry data set from the six experiments is shown in Table I. B, RNA splenic expression levels from RPA analysis. NOD spleens demonstrate significantly increased IFN-␥ (p ϭ 0.03), increased IL-2, and decreased IL-4 mRNA compared with B6.G7 spleens. The y-axis represents the densitometry reading of the RNA signals, normalized by the intensity of the housekeeping genes (L32 and GAPDH) (see Materials and Methods). The complete densitometry data set from the six experiments is shown in Table I. *, Statistically significant difference. The Journal of Immunology 1697

Table I. Quantitation of RNA levels from RPA experimentsa

Expt. 1 Expt. 2 Expt. 3 Expt. 4 Expt. 5 Expt. 6

NOD IFN-␥ 274.24 201.65 1174.84 224.88 372.77 138.41 NOD IL-2 19.78 30.22 450.14 —b 27.93 11.94 NOD IL-4 38.85 8.62 281.42 4.81 14.16 6.34 NOD IL-10 30.23 41.28 273.34 21.67 193.07 13.74

B6.G7 IFN-␥ 196.66 144.58 557.36 94.38 316.43 79.4 B6.G7 IL-2 25.27 14.97 80.65 — 10.64 5.00 B6.G7 IL-4 69.33 68.70 157.71 12.39 42.02 7.6 B6.G7 IL-10 71.31 42.96 47.43 22.47 15.79 2.5

IFN-␥ NOD:B6.G7 ratio ( p ϭ 0.03) 1.39 1.39 2.11 2.38 1.18 1.74 IL-2 NOD:B6.G7 ratio 0.78 2.02 5.58 — 2.62 2.39 IL-4 NOD:B6.G7 ratio 0.56 0.13 1.78 0.39 0.34 0.83 IL-10 NOD:B6.G7 ratio 0.42 1.48 5.76 0.96 12.23 5.49

IFN-␥:IL-4 ratio ( p ϭ 0.03) 2.49 10.6 1.18 6.1 3.4 2.10 IL-2:IL-4 ratio ( p ϭ 0.04) 1.39 15.5 3.13 — 7.7 2.88 IFN-␥:IL-10 ratio ( p ϭ 0.35) 3.3 0.93 0.37 2.48 0.09 0.31 Downloaded from a RPAs were performed on spleen and LN cells from NOD and B6.G7 mice as in Fig. 2. The gels were exposed for 24 h; then the films were scanned and analyzed using Quantity 1 software and Statview (see Materials and Methods). For quantitation of housekeeping RNA expression levels, films were exposed for 1–4 h. Signal intensity over background was calculated for each probe signal and corrected by background gene RNA expression (not shown). The NOD:B6.G7 signal ratios were calculated using the normalized signals from two lanes. Cytokine ratios were calculated by dividing NOD vs B6.G7 IFN-␥ (or IL-2) ratios to NOD vs B6.G7 IL-4 (or IL-10) ratios from rows 9–12 and analyzing the ratios for all experiments using Wilcoxan statistics. A significant difference suggests an increased NOD vs B6.G7 Th1:Th2 ratio for that cytokine set (see Materials and Methods). b —, Not detectable. http://www.jimmunol.org/

LN cells from the same experiments showed a comparable significant increase in the percentage of IFN-␥ϩ cells, in the number of IFN-␥ϩ cells per 105 cells, and in the percentage of IFN-␥ϩCD4ϩ cells (Fig. 1C). Naive NOD and B6.G7 spleen and LN cells stimulated only with PMA-ionomycin did not differ in IFN-␥ production, which was essentially undetectable (Fig. 1A, bottom). Measurement of extracellular IFN-␥ protein production by time- resolved fluorometry demonstrated that NOD Con A-stimulated by guest on September 28, 2021 LN and spleen cells also generated significantly increased extracellular IFN-␥ compared with B6.G7 cells (Fig. 2). The finding of increased numbers of individual IFN-␥ϩ cells, correlated with the increased IFN-␥ protein production, suggested that more NOD cells entered the IFN-␥ pathway, rather than that NOD cells made more IFN-␥ on a per cell basis than B6.G7 cells.

Differential mRNA cytokine transcript levels in NOD and B6.G7 spleen and lymph node cells Regulation of IFN-␥ production is complex and could occur at several checkpoints. Using RPA, we demonstrated that NOD lectin-stimulated spleen and LN cells produce quantitatively significantly more IFN-␥ mRNA than B6.G7 lymphoid cells (Fig. 3 and Table I). RPA also demonstrated increased production of IL-2 mRNA by NOD vs B6.G7 cells (up to 5.5-fold more; Fig. 3 and Table I). At the mRNA level, we detected more IL-4 production (up to 7.7-fold increase) by B6.G7 than by NOD spleen cells (Fig. 2 and Table I). B6.G7 did not demonstrate a consistently increased IL-10 mRNA level compared with NOD (Table I). NOD spleen cells demonstrated significantly increased IFN-␥:IL-4 and IL-2: FIGURE 4. Proliferative response to lectins and activation status of NOD IL-4 (Th1:Th2) cytokine mRNA ratios compared with that of and B6.G7 LN cells. A, Naive NOD and B6.G7 LN cells were prepared as in B6.G7 (Table I) but not a significantly increased IFN-␥:IL-10 ratio Materials and Methods and stimulated for proliferative response with PHA. (Table I). Despite these significant cytokine production differ- Thymidine incorporation was quantified after 96 h as described in Materials ences, NOD and B6.G7 had similar proliferative responses to T and Methods. One representative of three experiments shown. B, NOD, B6, and B6.G7 peripheral lymphoid cells were harvested from naive mice, ana- cell panstimulation, suggesting that the enhanced capacity of NOD ␥ lyzed for CD4 and CD69 expression by flow cytometry, and gated by forward T cells to enter the IFN- -producing pathway was unrelated to the scatter (FSC) and side scatter (SSC) (left column plots) either as all viable capacity to enter the cell cycle (Fig. 4A). The results suggest that ϩ lymphocytes (middle column plots; larger gate from left column) or as blasting non-MHC loci control the capacity of NOD peripheral CD4 T (right column plots; smaller gate from left column) subsets. The numbers rep- cells to enter the IFN-␥-producing pathway but do not affect the resent the percentage of positive cells from the gated populations. One repre- capacity of these cells to enter the cell cycle. sentative of two experiments is shown. 1698 NON-MHC CONTROL OF NOD T CELL RESPONSES

FIGURE 5. NOD and B6.G7 mice demonstrate peripheral and central CD4 lymphocytosis compared with B6 mice. NOD, B6.G7, and B6 peripheral spleen, LN, and thymuses were harvested from individual naive mice, gated for viable cells by forward and side scatter, and analyzed for CD4 and CD8 expression. One representative of four experiments is shown. Downloaded from http://www.jimmunol.org/

It was possible, although the mice studied were only 8 wk old peripheral lymphoid organs. NOD mice have been reported to and prediabetic, that the increase in IFN-␥-producing cells in NOD demonstrate CD4 lymphocytosis, which might affect cytokine lev- mice was due to increased precursor frequency of activated cells els (5, 50). We demonstrated, however, that the B6.G7 mice shared stimulated by nascent NOD autoimmune disease. To examine this the lymphocytosis trait with NOD mice, compared with B6 mice possibility, we compared peripheral LN and spleen cells from (Fig. 5). NOD and B6.G7 mice consistently demonstrated ϳ100% NOD and B6.G7 mice for activated CD4ϩ T cells. There was no more CD4ϩ cells in the spleen and LN compared with B6 mice difference in CD4ϩCD69ϩ T cells between the two strains (Fig. (Fig. 5), as well as consistently increased SP CD4ϩ cells in the by guest on September 28, 2021 4B), nor did NOD mice have more CD4ϩCD69ϩ blast cells (Fig. thymus (Fig. 5). Therefore, the differential cytokine responsive- 4B). Therefore, the increased IFN-␥ response of NOD mice was ness of the strains was not due to differing setpoints for peripheral not due to an increase in activated effector cells detectable in NOD CD4 population levels.

FIGURE 6. Increased intracellular and extracellular IFN-␥ production in Con A-PMA-ionomycin-stimulated NOD.H2b vs B6 CD4ϩ T cells. Spleen cells were taken from NOD.H2b and B6 mice and stimulated with Con A-PMA-ionomycin as in Fig. 1. Intracellular cytokine analysis (A) and extracellular cytokine production (B) were measured as above. NOD.H2b spleens had significantly increased numbers of CD4ϩIFN-␥ϩ cells compared with B6 spleens (p ϭ 0.04) and produced significantly more extracellular IFN-␥ (p ϭ 0.04). One representative of five experiments shown. *, Statistically significant difference. The Journal of Immunology 1699

It remained possible that NOD mice had increased numbers of quiescent, autoreactive memory T cells that had been previously activated by autoantigen and therefore could be recruited to produce IFN-␥ on restimulation (because naive CD4ϩcells do not produce detectable IFN-␥, only previously stimulated cells (51)). To exclude this possibility, we performed control assays using a primary stimulation of PMA-ionomycin without priming the naive cells first with Con A. Neither NOD nor B6.G7 naive spleen and LN cells produced significant numbers of IFN-␥-positive CD4ϩ T cells under these conditions (Fig. 1, bottom), suggesting that the increased IFN-␥ response in NOD mice was not due to an increased number of preprimed autoreactive memory cells. It still remained possible that quiescent, undetected autoreactive T cells in the NOD peripheral lymphoid organs contributed to the increased IFN production. We used a different genetic system to exclude this possibility. Our results predicted that T cells selected by a different MHC on an NOD non-MHC background would have increased IFN-␥ production compared with T cells selected by the Downloaded from same MHC on a B6 genetic background. Therefore, we tested CD4ϩ IFN-␥ production by T cells selected by I-Ab on NOD vs B6 non-MHC backgrounds (NOD.H2b vs B6 mice). T cells from NOD.H2b mice demonstrated significantly increased numbers of IFN-␥ϩCD4ϩ cells compared with B6 mice (Fig. 6) and produced significantly increased amounts of extracellular IFN-␥ (Fig. 6). http://www.jimmunol.org/ These studies therefore strongly support the hypothesis that naive CD4ϩ T cells on an NOD non-MHC genetic background have an enhanced genetically determined propensity toward entering the IFN-␥-producing pathway after stimulation compared with T cells selected by the same MHC but on a B6 non-MHC background. FIGURE 7. NOD and B6.G7 proliferative and cytokine responses to ϭ priming with GAD524–543. NOD and B6.G7 mice (n 2 or 3) were primed with GAD in CFA, and responses were assayed as described in ϩ 524–543 NOD GAD524–543- and HEL11–23-stimulated CD4 T cells Materials and Methods. Cultures were simultaneously set up to assay thy- ϩ ϩ mount a significantly greater IFN-␥ response than B6.G7 CD4 midine incorporation (A) and intracellular cytokine production by CD4 by guest on September 28, 2021 ϩ ϩ T cells, despite similar proliferative responses cells (B). The number of CD4 IFN-␥ cells after stimulation with 25 ␮m Ag as described above is expressed per 105 CD4ϩ cells. One representative The finding that pan-T cell stimulation produced a much greater of three experiments is shown. IFN-␥ response in NOD than in MHC-matched B6.G7 mice led us to ask whether the strains mounted different CD4ϩ T cell re- g7 sponses to I-A binding autoantigens implicated in NOD autoim- NOD and B6.G7 mice demonstrate no quantitative difference in mune diabetes. GAD has been implicated in the pathogenesis of CD4ϩCD25ϩ cells both human and murine autoimmune diabetes, and GAD 524–543 It was possible that the enhanced IFN-␥ response in NOD mice has been established as a major NOD T cell epitope (15, 16). We was due to the presence of a regulatory cell present in B6.G7 mice tested the capacity of priming with GAD to stimulate pro- 524–543 (disease preventative) and absent in NOD mice. It has been re- liferative and cytokine responses in NOD and B6.G7 mice. NOD ϩ ϩ ported that CD25 CD4 cells may function as regulatory cells and B6.G7 mice mounted similar proliferative responses to ϩ ϩ (52–54). We found no difference in the number of CD4 CD25 GAD (consistent with a similar MHC class II I-Ag7-medi- 524–543 cells between B6.G7 and NOD mice (Fig. 9). Therefore, the en- ated selection of the peripheral TCR repertoire) (Fig. 7A). How- hanced IFN-␥ response in NOD mice was not due to the lack of ever, GAD-responding cultures generated significantly higher these regulatory T cells compared with B6.G7 mice, at least at the ␥ ϩ numbers of IFN- -positive CD4 T cells in NOD mice than in time points tested. B6.G7 mice, consistent with the Con A results above (Fig. 7B). It remained possible that the enhanced IFN-␥ production by GAD- Discussion reactive T cells was due to preactivation of potentially autoreactive We have demonstrated that NOD vs B6 non-MHC loci control GAD-responding T cells (although we could not detect increased peripheral T cell capacity to enter the IFN-␥-secreting pathway numbers of activated cells (Fig. 4B). Therefore, we tested the re- (Figs. 1–4 and 6–8 and Table I). More NOD than B6.G7 CD4ϩ T sponse of NOD and B6.G7 to priming with a foreign Ag, HEL11–23, cells, whether panstimulated with lectins or stimulated by TCR- to which the T cell repertoire should be naive. The NOD and B6.G7 specific I-Ag7 binding auto or foreign Ags, enter the IFN-␥-pro- mice mounted similar proliferative responses to HEL11–23 (Fig. 8A). ducing pathway (Figs. 1, 7, and 8). Equivalent starting populations Again, however, NOD mounted much higher IFN-␥ responses than of NOD CD4ϩ T cells make more extracellular IFN-␥ and produce B6.G7 despite similar proliferative responses in the same cultures more IFN-␥ mRNA in response to the same stimuli than do B6.G7 (Fig. 8B). The results suggest that TCR-mediated as well as lectin- CD4ϩ cells (Figs. 1–4). Figs. 7 and 8 show that TCR-mediated ␥ g7 mediated T cell activation produce an enhanced IFN- response in entry into the cell cycle as stimulated by the I-A :GAD524–543 or g7 NOD vs B6.G7 mice, despite a similar TCR-mediated capacity to I-A :HEL11–23 complexes is virtually identical in B6.G7 and recognize the Ag in both strains. NOD mice, but that the NOD T cell response generates more 1700 NON-MHC CONTROL OF NOD T CELL RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 9. No difference in CD25 expression on CD4ϩ cells from NOD, B6, and B6.G7 mice. Naive lymphoid cells were collected from NOD, B6, and B6.G7 mice and stained for CD4 and CD25 expression. The cells were gated (as in Fig. 4) by size as either all viable lymphocytes (top) or blasting cells (bottom). The experiment was conducted twice, and both results are shown.

ature supports the idea suggested here, therefore, that a quantita- by guest on September 28, 2021 tively enhanced IFN-␥ pathway could be regulated by multiple non-MHC loci to produce an autoimmune diathesis via exagger- ated IFN-␥ responses. The powerful NOD IFN-␥ response demonstrated here is notable, because it is quantitatively greater than that of T cells selected by two MHC molecules on the B6 genetic background. It has previously been shown that T cells selected on a B6 background generate a stronger ⌱FN-␥ response than when selected on a BALB/c background (60, 61). B6 has been consid- ered a strong Th1, IFN-␥-reactive genetic background, especially in the leishmaniasis model (62, 63). In this context, the much FIGURE 8. NOD and B6.G7 proliferative and cytokine responses to stronger NOD IFN-␥ response than that of B6 appears excessive priming with HEL . NOD and B6.G7 mice (n ϭ 2 or 3) were primed 11–23 and potentially deleterious. with HEL in CFA and responses assayed as described in Materials 11–23 We sought to remove the powerful effect of the NOD MHC loci and Methods. Cultures were set up to simultaneously assay thymidine in- ϩ b corporation (A), IFN-␥ extracellular protein production as measured by on NOD CD4 T cell function by using B6.G7 and NOD.H2 europium fluorometry (Euϩ)(B), and IFN-␥ intracellular cytokine produc- MHC-congenic mice. The shared class I and II elements of NOD b tion after stimulation with 25 ␮m Ag as described above (C). The number and B6.G7, or NOD.H2 and B6, mice suggest that thymic selec- of IFN-␥ϩ cells is expressed per 105 cells. tion of the TCR repertoire would be similar in the strains. This is a reasonable hypothesis given the data showing that B6.G7 and NOD mice have a similar number of autoreactive precursors in the IFN-␥. Fig. 6 demonstrates that the increased IFN ␥ production is periphery (35) and Fig. 5, which shows a quantitatively similar SP truly a result of the NOD non-MHC background, because these CD4ϩ thymic and CD4ϩ peripheral T cell repertoire compared results were replicated in the NOD.H2b compared with the B6 with B6 mice. However, the similarity of the T cell repertoires has mice. These findings are significant in light of the substantial lit- not been proved. It is possible that some non-MHC loci change the erature implicating Th1 and IFN-␥ pathological effects in NOD specific TCR repertoire in these strains despite identical MHC se- autoimmunity (19–25) and the demonstration that anti-IFN-␥ Abs lecting elements. This proposition is testable and efforts are under prevent diabetes (55). IL-12 mRNA expression correlated with in- way in our laboratory to demonstrate the composition of the TCR sulitis (56), IL-12 administration accelerates diabetes (57), and an repertoire in these mice. Our initial analysis of cytokine production IL-12 antagonist prevented diabetes by reducing IFN-␥ levels (58). at the protein level suggested that the cytokine response in NOD vs Moreover, insulitis onset correlated with IL-18 expression in NOD B6.G7 mice differed quantitatively rather than qualitatively, i.e., mice, which maps near the non-MHC Idd2 locus (59). The liter- that the B6 loci apparently did not divert the T cell responses from The Journal of Immunology 1701 a Th1 (as assayed by IFN-␥ protein levels) to a Th2 profile (as 14. Ridgway, W. M., M. Fasso, and C. G. Fathman. 1999. A new look at MHC and assayed by IL-4 and IL-10 protein levels) but simply produced autoimmune disease. Science 284:749, 751. 15. Tisch, R., X. D. Yang, S. M. Singer, R. S. Liblau, L. Fugger, and H. O. McDevitt. fewer IFN-␥ effector cells. The more sensitive RPA assay showed 1993. Immune response to glutamic acid decarboxylase correlates with insulitis a definite shift in mRNA transcripts from IFN-␥ to IL-4 in the in non-obese diabetic mice. Nature 366:72. 16. Kaufman, D. L., M. Clare-Salzler, J. Tian, T. Forsthuber, G. S. Ting, B6.G7 compared with the NOD lymphoid cells. The NOD:B6.G7 P. Robinson, M. A. Atkinson, E. E. Sercarz, A. J. Tobin, and P. V. Lehmann. IFN-␥:IL-4 transcript ratio was significantly higher in NOD than in 1993. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in B6.G7 spleens (Fig. 3B and Table I). Moreover, the levels of IL-4 murine insulin-dependent diabetes. Nature 366:69. 17. Wegmann, D. R., R. G. Gill, M. Norbury-Glaser, N. Schloot, and D. Daniel. mRNA were up to 7.7-fold higher in B6.G7 than in NOD spleens 1994. Analysis of the spontaneous T cell response to insulin in NOD mice. and LN (Fig. 2B and Table I). At the mRNA level, then, our results J. 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the CD4 CD25 immunoregulatory T cells that control autoimmune diabetes. section of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell acti- by guest on September 28, 2021 Immunity 12:431. vation, differentiation, and cell death. J. Immunol. 162:6492.