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The Journal of Immunology, 2001, 167: 1693-1702.
Copyright © 2001 by The American Association of Immunologists

Increased Entry into the IFN-{gamma} 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

Division of Rheumatology and Immunology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN-{gamma}-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-{gamma} intracellular/extracellular protein and mRNA responses compared with B6.G7 T cells as measured by intracellular cytokine analysis, time resolved fluorometry, and RNase protection assays. The increased IFN-{gamma} production was not due to an increase in the amount of IFN-{gamma} produced per cell but to an increase in the number of NOD CD4+ T cells entering the IFN-{gamma}-producing pathway. The increased IFN-{gamma} 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-{gamma} mRNA, an increase in IL-4 mRNA production, and a significantly decreased IFN-{gamma}:IL-4 mRNA transcript ratio compared with NOD cells. CD4+ T cells from C57BL6 mice also showed significantly decreased IFN-{gamma} production compared with CD4+ T cells from NOD.H2b MHC-congenic mice (which have an H2b MHC region introgressed onto an NOD non-MHC background). Therefore, the NOD non-MHC background predisposes to a quantitatively increased IFN-{gamma} response, independent of MHC class II-mediated T cell repertoire selection, even when compared with a prototypical Th1 strain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nonobese diabetic (NOD)3 mice spontaneously develop pancreatic islet lymphocytic infiltration (insulitis) and autoimmune diabetes (1). Genome scanning and subsequent congenic analysis have demonstrated the genetic complexity of the disease process, with at least 20 insulin-dependent diabetes (Idd) genetic loci contributing in a complex manner to cause diabetes (2, 3, 4, 5). Multiple studies have demonstrated that diabetes in NOD mice is autoimmune, with a prominent role for the unique MHC class II molecule (I-Ag7), CD4+ and CD8+ T cells, and B cells (6, 7, 8, 9, 10, 11, 12, 13, 14). T cell epitopes from glutamic acid decarboxylase (GAD) and insulin have been mapped, and T cells reactive to these autoantigens have been isolated (15, 16, 17, 18). Much of the literature points to a strong role for Th1-like CD4+ immune responses in pathogenesis and a decreased Th2 response; a variety of interventions have shown that diverting the immune response in NOD toward Th2 may prevent disease (although pancreatic IL-10 production makes diabetes worse) (19, 20, 21, 22, 23, 24, 25). There is evidence of immune regulatory defects, as well; e.g., in cotransfer experiments, the presence of certain cell populations can stop the transfer of diabetes mediated by autoreactive CD4+ and CD8+ T cells (26, 27, 28). B cells and CD87 T cells seem necessary to develop the disease in vivo (29, 30, 31). Defective thymic T cell activation, defective APC activation, and defective thymic negative selection of T cells have also been shown to contribute to NOD autoimmunity (32, 33, 34, 35, 36).

Despite this large body of evidence, how Idd loci cause (or prevent) diabetes and insulitis remains unclear. Several groups have shown that introgression of protective Idd loci from nonautoimmune strains such as B6 or B10 (producing NOD-congenic strains) can decrease or prevent diabetes and insulitis. The introgression of Idd3 and Idd10 reduces diabetes incidence to ~3% (37, 38). Idd9 alone on the NOD background reduces diabetes incidence to 5% (39). The combination of Idd9, Idd3, and Idd10 eliminates diabetes and virtually eliminates insulitis (39). The strongest linkage in the original genome scan was to the H2g7 locus; NOD MHC F1-congenic mice, carrying all NOD genes except for one copy of a non-NOD MHC interval, have an 80-fold decrease in diabetes (40). Many publications have examined the role of the MHC class II molecule in the autoimmune process, suggesting that it may bind specific autoantigens for presentation to autoreactive lymphocytes and that, due to its defective peptide binding characteristics, I-Ag7 may be inefficient at thymic negative selection (36, 41, 42, 43, 44). Conversely, the NOD MHC region alone is not sufficient for disease, because B6.G7 mice (carrying all non-NOD genes except for the NOD MHC interval) and other NOD MHC-congenic mice do not develop diabetes.

The cellular and immunological effects of non-MHC loci, however, are largely unknown. Fox and Danska (45) analyzed insulitis in NOD and the related nonobese diabetes-resistant (NOR) strain, which shares the MHC but differs at non-MHC loci, and found that in the NOR mice APCs could infiltrate the islet but T cells could not, indicating non-MHC regulation of T cell function. In addition, NOR islets showed decreased Th1 cytokine transcripts. Scott et al. (46) showed that non-MHC polymorphisms could affect differentiation to a Th2 phenotype in a TCR-transgenic model. The large body of literature pointing to immune abnormalities in NOD mice suggests that Idd disease susceptibility loci mediate specific immunological functions along an immune pathway which, in the presence of a critical number of susceptibility alleles, would result in loss of tolerance and autoimmunity. In this paper, we used NOD, C57BL/6, B6.G7 mice (C57BL/6-congenic mice with an introgressed NOD MHC interval; the B6.G7 mice do not develop diabetes, although they develop minor pancreatic lymphocytic infiltration; Ref. 47) and NOD.H2b congenic mice (which have the H2b MHC region introgressed onto the NOD non-MHC background and do not develop diabetes; Ref. 48) to analyze non-MHC control of T cell function. Our results indicate that non-MHC NOD background genes predispose to a significantly enhanced Th1 response independent of MHC-dependent T cell repertoire selection events.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

NOD/Lt, B6.NODc17 (hereafter referred to as B6.G7), NOD.H2b, and C57BL/6 (hereafter called B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The B6.G7 mouse was originally produced and characterized in the laboratory of E. Wakeland (47). The mice were bred and maintained under specific-pathogen-free conditions in the animal facility of University of Pittsburgh Medical Center, Pittsburgh, PA. Mice were used at age of 8–12 wk. Unless otherwise specified, 8-wk-old mice were used.

Ags and mitogens

GAD524–543 and hen egg lysozyme (HEL11–23) peptides were synthesized and HPLC purified at the Molecular Biology and Genetics core facility of the University of Pittsburgh School of Medicine. Con A was obtained from Pharmacia (Piscataway, NJ). PMA, ionomycin, PHA, and saponin were obtained from Sigma (St. Louis, MO).

Immunization and preparation of cell culture

Groups of NOD and B6.G7 mice (n = 2–3) were primed at the base of the tail with peptide/CFA emulsion; 8–10 days later, draining lymph node (LN) cells were isolated under aseptic conditions and stimulated in vitro with Ags or mitogen. For Con A stimulation, naive inguinal LN and spleen cells were collected. The cells were washed and resuspended at 1 x 106 cells/ml in complete medium consisting of RPMI 1640 supplemented with 10% (w/v) FCS, 1 mM L-alanylglutamine (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), 1 mM sodium pyruvate (Life Technologies), and 50 µM 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 4 µg/ml. For Ag-specific T cell assays, the cell suspension at 5 x 105–1 x 106/200 µl was placed in 96-well flat-bottom plates and GAD524–543 or HEL11–23 was added to indicated final concentrations. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere. Supernatants were collected 3–8 days after the stimulation, and the cells were incubated with or without 5 ng/ml PMA (Sigma) and 0.5 µg/ml ionomycin (Sigma) for 3.5 h (37°C, 5% CO2).

Flow cytometric intracellular cytokine analysis

For intracellular cytokine analysis, after the first 1.5 h of the 3.5-h PMA-ionomycin incubation, brefeldin A (final concentration, 10 µg/ml) (Epicentre Technologies, Madison, WI) was added to the culture. At the end of 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) (Caltag, San Francisco, CA) in staining buffer (2% FBS, 0.1% sodium azide in PBS). Cells were fixed for 20 min at 4°C with 2% paraformaldehyde (Sigma) in PBS and then permeabilized for 10 min at room temperature with 0.5% saponin (Sigma) in PBS. Cells were stained in permeabilization buffer for 20 min at 4°C with PE-conjugated anti-IFN-{gamma} Ab (XMG1.2, rat IgG1; BD PharMingen, San Diego, CA) and FITC-conjugated anti-IL-10 Ab (JES5-16E3, rat IgG2b; BD PharMingen). In some cases, anti-IL-4 Ab (BVD4-1D11, rat IgG2b; BD PharMingen) was used instead of anti-IL-10 Ab. The cells were also stained in the same manner with FITC-conjugated rat IgG2b isotype control Ig (BD PharMingen) and PE-conjugated rat IgG1 isotype control Ig (BD PharMingen). Flow cytometric analyses were performed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Wilcoxan statistical analysis of the results was performed using StatView 4.0 (Abacus Concepts, Berkeley, CA).

Fluorometry

IL-4, IL-10, and IFN-{gamma} were detected by time-resolved fluorometry. The capture Ab 11B11 (anti-IL-4), JES5-2A5 (anti-IL-10), or AN18 (anti-IFN-{gamma}) 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 cytokine standards, recombinant mouse IL-4, IL-10, and IFN-{gamma}, were titrated and incubated for 1 h on the plates, which were then washed; the detection layer (BVD6-24G2-biotin for IL-4, JES5-16E3-biotin for IL-10, or XMG1.2-biotin for IFN-{gamma}) was then added. Bound detection Ab was detected with europium-streptavidin (Wallac, Uppsala, Sweden). The plates were read on a Wallac 1420 Victor time-resolved fluorometer. Data were imported to Excel (Microsoft, Redmond, WA) and analyzed using DeltaSOFT (Biometallics, Princeton, NJ) using a four-parameter fit model to quantify cytokine results in nanograms per milliliter. Wilcoxan statistical analysis of the results was performed using Statview 4.0.

RNase protection assay (RPA)

Total RNA was extracted from LN or splenic cells using the RNeasyminikit (Qiagen, Valencia, CA). The RNA was redissolved in RNase-free water, and yield was estimated by spectrophotometry; equal quantities of RNA were used for analysis. RPA was performed using RiboQuant from BD PharMingen according to the manufacturer’s protocol. The multiprobe template set mCK-1 (containing templates for IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-{gamma}, L32, and GAPDH) was purchased from BD PharMingen. The templates were used to synthesize the [{alpha}-32P]UTP-labeled probes (3000 Ci/mmol, 10 mCi/ml; NEN Life Science Products, Boston, MA) in the presence of a GACU pool using a T7 RNA polymerase (BD PharMingen). Hybridization with 5–15 µg RNA was performed for 12–14 h at 56°C, and the products were digested with RNase A and T1 mixture. The samples were treated by proteinase K in proteinase K buffer with yeast tRNA and then extracted with phenol and chloroform-isoamyl alcohol (50:1) and precipitated in the presence of ammonium acetate. The samples were loaded on acrylamide-urea gel and run at 40 W with 0.5x Tris-borate-EDTA electrophoresis buffer for 2 h. The gel was adsorbed to filter paper, vacuum dried, and then exposed on film (X-AR; Kodak, Rochester, NY) with intensifying screens at -70°C. The films were scanned, and densitometry performed using Quantity One software (BD Biosciences). Absolute RNA levels were calculated using 2- to 4-h exposures of housekeeping gene expression and normalizing using Quantity One software. Wilcoxan statistical analysis of the results was performed using StatView 4.0. The Th1:Th2 cytokine ratios were calculated in the form [(NOD Th1 cytokine)/(B6.G7 TH1 cytokine)]//[(NOD Th2 cytokine)/(B6.G7 Th2 cytokine)], and the results were analyzed by Wilcoxon statistics using StatView 4.0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After exposure to mitogenic stimuli, NOD LN and spleen cells have significantly increased numbers of CD4+IFN-{gamma}+ T cells compared with B6.G7 despite quantitatively similar proliferative responses

Although NOD and B6.G7 mice share the diabetes-associated MHC class II H-2g7 interval, B6.G7 mice do not develop diabetes. We tested whether peripheral CD4+ cells were functionally different under genetic control of B6 Idd loci in the setting of I-Ag7 selection. Our initial approach used pan-T cell lectin stimulation with Con A followed by restimulation with PMA-ionomycin. This assay is designed to convert naive T cells into functionally active cytokine secretors (49). Stimulated NOD peripheral spleen and LN produced significantly increased numbers of IFN-{gamma}-positive CD4+ cells, by intracellular cytokine analysis, compared with B6.G7 (Fig. 1Go). NOD spleen cells showed a significant increase in the percentage of IFN-{gamma}+ cells, in the number of IFN-{gamma}+ cells per 105 cells, and in the percentage of IFN-{gamma}+ CD4+ cells (Fig. 1GoB). NOD LN cells from the same experiments showed a comparable significant increase in the percentage of IFN-{gamma}+ cells, in the number of IFN-{gamma}+ cells per 105 cells, and in the percentage of IFN-{gamma}+CD4+ cells (Fig. 1GoC). Naive NOD and B6.G7 spleen and LN cells stimulated only with PMA-ionomycin did not differ in IFN-{gamma} production, which was essentially undetectable (Fig. 1GoA, bottom). Measurement of extracellular IFN-{gamma} protein production by time-resolved fluorometry demonstrated that NOD Con A-stimulated LN and spleen cells also generated significantly increased extracellular IFN-{gamma} compared with B6.G7 cells (Fig. 2Go). The finding of increased numbers of individual IFN-{gamma}+ cells, correlated with the increased IFN-{gamma} protein production, suggested that more NOD cells entered the IFN-{gamma} pathway, rather than that NOD cells made more IFN-{gamma} on a per cell basis than B6.G7 cells.



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  		FIGURE 1. Increased intracellular IFN-{gamma} production in Con A-PMA-ionomycin-stimulated NOD vs B6.G7 CD4+ T cells. A, NOD and B6.G7 LN and spleen cells were cultured with (top half) or without (bottom half) Con A as in Materials 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-{gamma}+ cells (top panels) and CD4+IFN-{gamma}+ 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-{gamma}+ cells, increased IFN-{gamma}+ cells normalized per 105 cells, and increased NOD percent CD4+IFN-{gamma}+ cells (CD4 fractionation performed in six of eight experiments) in both spleen (B) and LN (C). Each symbol represents a different experiment.

 


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  		FIGURE 2. Increased extracellular IFN-{gamma} 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. 1Go. Equal numbers of NOD spleen and LN cells produced significantly more IFN-{gamma} than did B6.G7 cells (p = 0.025, spleen; p = 0.046, LN). One representative of eight experiments is shown. *, Statistically significant difference.

 
Differential mRNA cytokine transcript levels in NOD and B6.G7 spleen and lymph node cells

Regulation of IFN-{gamma} 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-{gamma} mRNA than B6.G7 lymphoid cells (Fig. 3Go and Table IGo). RPA also demonstrated increased production of IL-2 mRNA by NOD vs B6.G7 cells (up to 5.5-fold more; Fig. 3Go and Table IGo). 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. 2Go and Table IGo). B6.G7 did not demonstrate a consistently increased IL-10 mRNA level compared with NOD (Table IGo). NOD spleen cells demonstrated significantly increased IFN-{gamma}:IL-4 and IL-2:IL-4 (Th1:Th2) cytokine mRNA ratios compared with that of B6.G7 (Table IGo) but not a significantly increased IFN-{gamma}:IL-10 ratio (Table IGo). Despite these significant cytokine production differences, NOD and B6.G7 had similar proliferative responses to T cell panstimulation, suggesting that the enhanced capacity of NOD T cells to enter the IFN-{gamma}-producing pathway was unrelated to the capacity to enter the cell cycle (Fig. 4GoA). The results suggest that non-MHC loci control the capacity of NOD peripheral CD4+ T cells to enter the IFN-{gamma}-producing pathway but do not affect the capacity of these cells to enter the cell cycle.



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  		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. 1Go and 2Go. 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 IGo. B, RNA splenic expression levels from RPA analysis. NOD spleens demonstrate significantly increased IFN-{gamma} (p = 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 IGo. *, Statistically significant difference.

 

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  		Table I. Quantitation of RNA levels from RPA experiments1

 


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  		FIGURE 4. Proliferative response to lectins and activation status of NOD and B6.G7 LN cells. A, Naive NOD and B6.G7 LN cells were prepared as in Materials and Methods and stimulated for proliferative response with PHA. Thymidine incorporation was quantified after 96 h as described in Materials and Methods. One representative of three experiments shown. B, NOD, B6, and B6.G7 peripheral lymphoid cells were harvested from naive mice, analyzed for CD4 and CD69 expression by flow cytometry, and gated by forward scatter (FSC) and side scatter (SSC) (left column plots) either as all viable lymphocytes (middle column plots; larger gate from left column) or as blasting (right column plots; smaller gate from left column) subsets. The numbers represent the percentage of positive cells from the gated populations. One representative of two experiments is shown.

 
It was possible, although the mice studied were only 8 wk old and prediabetic, that the increase in IFN-{gamma}-producing cells in NOD mice was due to increased precursor frequency of activated cells stimulated by nascent NOD autoimmune disease. To examine this possibility, we compared peripheral LN and spleen cells from NOD and B6.G7 mice for activated CD4+ T cells. There was no difference in CD4+CD69+ T cells between the two strains (Fig. 4GoB), nor did NOD mice have more CD4+CD69+ blast cells (Fig. 4GoB). Therefore, the increased IFN-{gamma} response of NOD mice was not due to an increase in activated effector cells detectable in NOD peripheral lymphoid organs. NOD mice have been reported to demonstrate CD4 lymphocytosis, which might affect cytokine levels (5, 50). We demonstrated, however, that the B6.G7 mice shared the lymphocytosis trait with NOD mice, compared with B6 mice (Fig. 5Go). NOD and B6.G7 mice consistently demonstrated ~100% more CD4+ cells in the spleen and LN compared with B6 mice (Fig. 5Go), as well as consistently increased SP CD4+ cells in the thymus (Fig. 5Go). Therefore, the differential cytokine responsiveness of the strains was not due to differing setpoints for peripheral CD4 population levels.



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  		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.

 
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-{gamma} on restimulation (because naive CD4+cells do not produce detectable IFN-{gamma}, 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-{gamma}-positive CD4+ T cells under these conditions (Fig. 1Go, bottom), suggesting that the increased IFN-{gamma} 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-{gamma} production compared with T cells selected by the same MHC on a B6 genetic background. Therefore, we tested CD4+ IFN-{gamma} 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-{gamma}+CD4+ cells compared with B6 mice (Fig. 6Go) and produced significantly increased amounts of extracellular IFN-{gamma} (Fig. 6Go). These studies therefore strongly support the hypothesis that naive CD4+ T cells on an NOD non-MHC genetic background have an enhanced genetically determined propensity toward entering the IFN-{gamma}-producing pathway after stimulation compared with T cells selected by the same MHC but on a B6 non-MHC background.



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  		FIGURE 6. Increased intracellular and extracellular IFN-{gamma} production in Con A-PMA-ionomycin-stimulated NOD.H2b vs B6 CD4+ T cells. Spleen cells were taken from NOD.H2b and B6 mice and stimulated with Con A-PMA-ionomycin as in Fig. 1Go. Intracellular cytokine analysis (A) and extracellular cytokine production (B) were measured as above. NOD.H2b spleens had significantly increased numbers of CD4+IFN-{gamma}+ cells compared with B6 spleens (p = 0.04) and produced significantly more extracellular IFN-{gamma} (p = 0.04). One representative of five experiments shown. *, Statistically significant difference.

 
NOD GAD524–543- and HEL11–23-stimulated CD4+ T cells mount a significantly greater IFN-{gamma} response than B6.G7 CD4+ T cells, despite similar proliferative responses

The finding that pan-T cell stimulation produced a much greater IFN-{gamma} response in NOD than in MHC-matched B6.G7 mice led us to ask whether the strains mounted different CD4+ T cell responses to I-Ag7 binding autoantigens implicated in NOD autoimmune diabetes. GAD has been implicated in the pathogenesis of both human and murine autoimmune diabetes, and GAD524–543 has been established as a major NOD T cell epitope (15, 16). We tested the capacity of priming with GAD524–543 to stimulate proliferative and cytokine responses in NOD and B6.G7 mice. NOD and B6.G7 mice mounted similar proliferative responses to GAD524–543 (consistent with a similar MHC class II I-Ag7-mediated selection of the peripheral TCR repertoire) (Fig. 7GoA). However, GAD-responding cultures generated significantly higher numbers of IFN-{gamma}-positive CD4+ T cells in NOD mice than in B6.G7 mice, consistent with the Con A results above (Fig. 7GoB). It remained possible that the enhanced IFN-{gamma} production by GAD-reactive T cells was due to preactivation of potentially autoreactive GAD-responding T cells (although we could not detect increased numbers of activated cells (Fig. 4GoB). Therefore, we tested the response of NOD and B6.G7 to priming with a foreign Ag, HEL11–23, to which the T cell repertoire should be naive. The NOD and B6.G7 mice mounted similar proliferative responses to HEL11–23 (Fig. 8GoA). Again, however, NOD mounted much higher IFN-{gamma} responses than B6.G7 despite similar proliferative responses in the same cultures (Fig. 8GoB). The results suggest that TCR-mediated as well as lectin-mediated T cell activation produce an enhanced IFN-{gamma} response in NOD vs B6.G7 mice, despite a similar TCR-mediated capacity to recognize the Ag in both strains.



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  		FIGURE 7. NOD and B6.G7 proliferative and cytokine responses to priming with GAD524–543. NOD and B6.G7 mice (n = 2 or 3) were primed with GAD524–543 in CFA, and responses were assayed as described in Materials and Methods. Cultures were simultaneously set up to assay thymidine incorporation (A) and intracellular cytokine production by CD4+ cells (B). The number of CD4+IFN-{gamma}+ cells after stimulation with 25 µm Ag as described above is expressed per 105 CD4+ cells. One representative of three experiments is shown.

 


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  		FIGURE 8. NOD and B6.G7 proliferative and cytokine responses to priming with HEL11–23. NOD and B6.G7 mice (n = 2 or 3) were primed with HEL11–23 in CFA and responses assayed as described in Materials and Methods. Cultures were set up to simultaneously assay thymidine incorporation (A), IFN-{gamma} extracellular protein production as measured by europium fluorometry (Eu+) (B), and IFN-{gamma} intracellular cytokine production after stimulation with 25 µm Ag as described above (C). The number of IFN-{gamma}+ cells is expressed per 105 cells.

 
NOD and B6.G7 mice demonstrate no quantitative difference in CD4+CD25+ cells

It was possible that the enhanced IFN-{gamma} response in NOD mice was due to the presence of a regulatory cell present in B6.G7 mice (disease preventative) and absent in NOD mice. It has been reported that CD25+CD4+ cells may function as regulatory cells (52, 53, 54). We found no difference in the number of CD4+CD25+ cells between B6.G7 and NOD mice (Fig. 9Go). Therefore, the enhanced IFN-{gamma} response in NOD mice was not due to the lack of these regulatory T cells compared with B6.G7 mice, at least at the time points tested.



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  		FIGURE 9. No difference in CD25 expression on CD4+ 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. 4Go) by size as either all viable lymphocytes (top) or blasting cells (bottom). The experiment was conducted twice, and both results are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that NOD vs B6 non-MHC loci control peripheral T cell capacity to enter the IFN-{gamma}-secreting pathway ( Figs. 1–4GoGoGoGo and 6–8 and Table IGo). More NOD than B6.G7 CD4+ T cells, whether panstimulated with lectins or stimulated by TCR-specific I-Ag7 binding auto or foreign Ags, enter the IFN-{gamma}-producing pathway (Figs. 1Go, 7Go, and 8Go). Equivalent starting populations of NOD CD4+ T cells make more extracellular IFN-{gamma} and produce more IFN-{gamma} mRNA in response to the same stimuli than do B6.G7 CD4+ cells ( Figs. 1–4GoGoGoGo). Figs. 7Go and 8Go show that TCR-mediated entry into the cell cycle as stimulated by the I-Ag7:GAD524–543 or I-Ag7:HEL11–23 complexes is virtually identical in B6.G7 and NOD mice, but that the NOD T cell response generates more IFN-{gamma}. Fig. 6Go demonstrates that the increased IFN {gamma} production is truly a result of the NOD non-MHC background, because these results were replicated in the NOD.H2b compared with the B6 mice. These findings are significant in light of the substantial literature implicating Th1 and IFN-{gamma} pathological effects in NOD autoimmunity (19, 20, 21, 22, 23, 24, 25) and the demonstration that anti-IFN-{gamma} Abs prevent diabetes (55). IL-12 mRNA expression correlated with insulitis (56), IL-12 administration accelerates diabetes (57), and an IL-12 antagonist prevented diabetes by reducing IFN-{gamma} levels (58). Moreover, insulitis onset correlated with IL-18 expression in NOD mice, which maps near the non-MHC Idd2 locus (59). The literature supports the idea suggested here, therefore, that a quantitatively enhanced IFN-{gamma} pathway could be regulated by multiple non-MHC loci to produce an autoimmune diathesis via exaggerated IFN-{gamma} responses. The powerful NOD IFN-{gamma} 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 IFN-{gamma} response than when selected on a BALB/c background (60, 61). B6 has been considered a strong Th1, IFN-{gamma}-reactive genetic background, especially in the leishmaniasis model (62, 63). In this context, the much stronger NOD IFN-{gamma} response than that of B6 appears excessive and potentially deleterious.

We sought to remove the powerful effect of the NOD MHC loci on NOD CD4+ T cell function by using B6.G7 and NOD.H2b MHC-congenic mice. The shared class I and II elements of NOD and B6.G7, or NOD.H2b and B6, mice suggest that thymic selection 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 periphery (35) and Fig. 5Go, which shows a quantitatively similar SP CD4+ thymic and CD4+ peripheral T cell repertoire compared with B6 mice. However, the similarity of the T cell repertoires has not been proved. It is possible that some non-MHC loci change the specific TCR repertoire in these strains despite identical MHC selecting elements. This proposition is testable and efforts are under way in our laboratory to demonstrate the composition of the TCR repertoire in these mice. Our initial analysis of cytokine production at the protein level suggested that the cytokine response in NOD vs B6.G7 mice differed quantitatively rather than qualitatively, i.e., that the B6 loci apparently did not divert the T cell responses from a Th1 (as assayed by IFN-{gamma} protein levels) to a Th2 profile (as assayed by IL-4 and IL-10 protein levels) but simply produced fewer IFN-{gamma} effector cells. The more sensitive RPA assay showed a definite shift in mRNA transcripts from IFN-{gamma} to IL-4 in the B6.G7 compared with the NOD lymphoid cells. The NOD:B6.G7 IFN-{gamma}:IL-4 transcript ratio was significantly higher in NOD than in B6.G7 spleens (Fig. 3GoB and Table IGo). Moreover, the levels of IL-4 mRNA were up to 7.7-fold higher in B6.G7 than in NOD spleens and LN (Fig. 2GoB and Table IGo). At the mRNA level, then, our results support a diversion toward a Th2 phenotype of T cells consistent with a previous TCR-transgenic model showing effects of non-MHC loci on autoimmune diabetes (46). We demonstrated consistently increased IL-2 mRNA transcript in NOD vs B6.G7 cells (Fig. 3Go and Table IGo), whereas the same cells demonstrated comparable proliferation (Fig. 4Go). This is an interesting result because Idd3 may be IL-2 (64, 65). Relatively decreased IL-2 effect on cellular proliferation could therefore represent a mechanism of Idd3-mediated disease susceptibility. This is a subject of ongoing research in our laboratory.

Our results favor the hypothesis that some disease-protective B6 non-MHC loci may divert the functional cytokine response of T cells selected on I-Ag7 and therefore prevent the quantitatively enhanced entry into the IFN-{gamma} effector pathway we have demonstrated in NOD mice, while simultaneously producing more Th2-like T cells with potentially regulatory effects. One way to analyze these abnormalities is to examine the phenotypes demonstrated here in non-MHC-subcongenic NOD strains (2). It may be possible to demonstrate which phenotypes segregate with which loci, an approach that has been successfully undertaken in the murine model of systemic lupus erythematosus (66, 67, 68). The goal of this approach is to use disparate phenotypes as a tool both to help in identifying the genes underlying the Idd loci and to illustrate the complex mechanisms of multigenic control of the immune subsystems involved in disease pathogenesis.


   Footnotes
 
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 Medicine. Back

2 Address correspondence and reprint requests to Dr. William M. Ridgway, S725 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. E-mail address: ridgway2{at}pitt.edu Back

3 Abbreviations used in this paper: NOD, nonobese diabetic; GAD, glutamic acid decarboxylase; HEL, hen egg lysozyme; RPA, RNase protection assay; LN, lymph node; NOR, nonobese diabetes-resistant. Back

Received for publication November 16, 2000. Accepted for publication May 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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