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Th1, Th2, and Th17 Effector T Cell-Induced Autoimmune Gastritis Differs in Pathological Pattern and in Susceptibility to Suppression by Regulatory T Cells¹

Georg H. Stummvoll^*, Richard J. DiPaolo^*, Eva N. Huter^*, Todd S. Davidson^*, Deborah Glass^*, Jerrold M. Ward and Ethan M. Shevach^2,*

^* Laboratory of Immunology, Cellular Immunology Section and Comparative Medicine Branch, Infectious Disease Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Th cells can be subdivided into IFN--secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H⁺K⁺-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The Th cells are considered major players in the response against infectious organisms as well as in promoting systemic and organ-specific autoimmune diseases. To convey their full function, Th cells secrete a variety of cytokines and can be subdivided into three different types based on their cytokine signature: IFN--secreting Th1; IL-4- and IL-5-secreting Th2 (1); and IL-17- producing Th17 cells (2, 3). During the past two decades, the Th1/Th2 paradigm prevailed, and these two cell types were implicated in the pathogenesis of different autoimmune diseases. For instance, Th1 cells were thought to promote the pathology in Crohn’s disease (4), whereas Th2 mediated asthma (5). However, recent evidence suggests that a better understanding of the pathogenesis of autoimmune diseases requires that the dichotomous view of Th differentiation must be broadened (2). IL-17-producing Th17 cells appear to play a critical pathogenic role in autoimmunity, promoting organ damage in experimental autoimmune encephalitis (6), which has long been regarded as a typical Th1-mediated disease (3). In addition, Th17 cells appear to be key effector T cells in a variety of human autoimmune diseases including multiple sclerosis, rheumatoid arthritis, respiratory disease, systemic lupus erythematosus, psoriasis, systemic sclerosis, and chronic inflammatory bowel disease (7). Nevertheless, the individual contributions of each of the effector T cell types to autoimmune disease remain difficult to dissect.

Autoimmune gastritis (AIG)³ is one of the few spontaneous animal models of organ-specific autoimmune disease in which the target Ag, the proton pump of the gastric parietal cell, the H⁺K⁺-ATPase, has been identified (8, 9). In addition, murine AIG represents an animal model for pernicious anemia in humans in which T and B cell responses also target the H⁺K⁺-ATPase (10, 11, 12). We have generated a TCR-transgenic (Tg) mouse (TxA23) the T cells of which express a TCR from a clone derived from a 3-day-thymectomized mouse that developed AIG. TxA23 T cells recognize a peptide from the H⁺K⁺-ATPase -chain and spontaneously develop severe AIG. Naive T cells from TxA23 mice transfer AIG into immunodeficient recipients, and the development of AIG can be prevented by cotransfer of polyclonal naturally occurring CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Treg) (13, 14). Suppression correlated with a failure of the transferred autoreactive T cells to produce IFN- and to differentiate into Th1 effector T cells (14).

The first aim of these studies was to assess the capacity of Th1, Th2, and Th17 effector cells derived from naive TxA23 T cells to induce AIG after transfer to immunodeficient recipients. As our previous studies focused on the capacity of polyclonal Tregs to suppress the induction of AIG by naive TxA23 cells, the second aim of this study was to determine the susceptibility of each of the three fully differentiated effector T cell types to suppression by polyclonal Tregs in vivo. Although Tregs have been shown to suppress cytokine production in vitro and IFN- production in vivo (14), we also examined whether Tregs could modulate cytokine production by fully differentiated effector cells in vivo. We demonstrate that each effector cell population induces AIG with a distinctive pathology. Polyclonal Tregs readily suppressed the induction of disease mediated by Th1 effectors, whereas Th2-mediated disease was more resistant to suppression. In contrast, disease induced by Th17 cells was the most aggressive and was susceptible to suppression only at early time points after disease induction.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice and reagents

Female BALB/c and BALB/c-nu/nu (4–8 wk old) were purchased from the National Cancer Institute animal facility and housed under specific pathogen-free conditions. TxA23 TCR-Tg mice have been described previously (15). All mice were maintained in our animal facility and cared for in accordance with institutional guidelines. H⁺K⁺-ATPase -chain peptides, PITAKAIAASVG, aa 630–641, were synthesized by the National Institute of Allergy and Infectious Diseases Peptide Synthesis Unit.

T cell purification and differentiation

Naive TxA23 T cells were isolated from the thymus of TxA23 mice expressing the surface marker Thy-1.1. As an enrichment step, thymocytes were depleted of CD8⁺ cells after incubation for 10 min with anti-CD8 beads MACS microbeads (Miltenyi Biotec) using the autoMACS deplete-sensitive program. Finally, cells were sorted for CD4⁺CD25^– cells in our cell sorting facility. Sorting resulted in 99–100% purity of CD4⁺CD25^–CD8^– naive T cells. In some experiments, thymocytes from TxA23 Thy-1.1/Thy-1.2 heterozygous mice were used. Purified TxA23 CD4⁺CD25^– thymocytes were stimulated with plate-bound anti-CD3 and anti-CD28 (2 µg each per well) in 24-well plates (0.25 x 10⁶ cells/well) in complete RPMI medium consisting of RPMI 1640 supplemented with 10% heat-inactivated FCS (Atlanta Biologicals), penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (all from BioSource International). The culture medium was supplemented with specific recombinant cytokines and/or anti-cytokine-mAbs as follows. For Th1 differentiation, recombinant human IL-12 (10 ng/ml) and anti-IL-4 (10 µg/ml; clone 11B11); for Th2 differentiation, rIL-4 (1000 U/ml), anti-IL-12 (10 µg/ml; clone C17.8), and anti-IFN- (10 µg/ml; clone XMG1.2; Ref. 16); for Th17 differentiation, TGFβ (2.5 µg/ml), rIL-6 (10 µl/ml), anti-IL-4, anti-IFN-, anti-IL-12, and anti-IL-2 (clone S4B6; 10 µg/ml). All cell cultures were split 1:2 on day 4 after activation and were further supplemented with rIL-2 (100 U/ml) or with medium alone in the case of Th17 cultures (17). Optimal differentiation of Th17 cells was achieved after 1 wk of differentiation in vitro. To optimize Th1 and Th2 differentiation, cultures were restimulated on day 8 with H⁺K⁺-ATPase--chain_630–641 (10 µg/ml) in combination with APC (1 x 10⁶; T cell-depleted BALB/c splenocytes that had been irradiated with 3000 rad) and cultured for an additional week under the same conditions as described previously.

Purification of Treg

A single-cell suspension of spleen cells from BALB/c mice was stained with PE-anti-CD25 (PC61) for 15 min, washed, incubated for 15 min with anti-PE microbeads, and then isolated using the autoMACS deplete-sensitive program. The positive fraction, which contained the CD25⁺ T cells, was further enriched for CD25⁺ T cells by using MACS LS columns. Purified CD4⁺CD25⁺ T cells were 91.4 ± 2.8% FoxP3⁺ as measured by flow cytometry.

Cell transfer experiments

All cells were washed twice with PBS, and the cultured T cells were diluted into PBS such that an i.p. injection of 0.5 ml/mouse resulted in the transfer of 50,000 TxA23Thy-1.1⁺ T cells.

Flow cytometry

Cell surface stainings were performed according to standard procedures using Abs against CD8, CD19, CD25, L-selectin (CD62L), Thy-1.1, and Thy-1.2 directly conjugated to FITC or PE purchased from BD Pharmingen. Anti-CD4-Tri-Color was purchased from Caltag Laboratories. mAbs for intracellular stainings (anti-IL-2, anti-IL-4, anti-IL-5, anti-IL-10, anti-IL-17, anti-IFN-, anti-TNF-) conjugated to PE or allophycocyanin were purchased from BD Pharmingen. PE- or FITC-labeled mAbs to FoxP3 were from eBioscience. All flow cytometry was performed on a FACScan or FACSCalibur and analyzed using CellQuest software (all from BD Biosciences) and FLOWJo (TreeStar).

Intracellular cytokine staining was performed with PE- or allophycocyanin- labeled mAbs as described (17). In brief, cells were stimulated for 4 h with PMA and ionomycin with Golgistop added after 2 h. Cell stimulation was terminated by fixing in 4% formyl saline. Fixed cells were stained in 0.1% saponin permeabilization buffer for 1 h and finally analyzed on a FACSCalibur flow cytometer (BD Biosciences).

In vitro T cell proliferation assay

Single-cell suspensions were prepared from gastric lymph nodes (gLN) or from cell cultures, washed in PBS containing 5% FCS and penicillin-streptomycin (100 U/ml and 10 µg/ml, respectively). The number of TxA23Thy-1.1⁺ cells was determined by cell counts and FACS. TxA23 T cells (5 x 10³) were added to a 96-well U-bottom tissue culture plate with T-depleted irradiated splenocytes (1 x 10⁵; 3000 rad) with or without H⁺K⁺-ATPase--chain_630–641 peptide (50 µg/ml) in complete RPMI. After 72 h, cultures were pulsed with [³H]TdR for 8 h.

Gastric pathology

For each group, at least five stomachs were ligated and inflated by injection of fixative (4% paraformaldehyde). The stomachs were opened by cutting along the greater curvature, flattened with filter paper, and embedded in paraffin. Sections were stained with either H&E or LUNA stain. CD3⁺ lymphocytes in gastric sections were stained with a rabbit anti-CD3 (Dako) at 1/300, using an Ag retrieval solution in a food steamer (Diva Solution; Biocare). The extent of gastritis was graded on a scale of 1–6, depending on the extent of mononuclear cell infiltration and parietal and chief cell destruction (14). General descriptions for scores are as follows: 1.0, scattered lymphocytes throughout submucosa and muscularis; 1.5, one or two small dense blankets of lymphocytes; 2.0, two to four small dense clusters of lymphocytes in the submucosa/mucosa; 3.0, two or three areas with intermediate infiltration spanning one-third of the mucosa; 4.0, large nodules of lymphocytic accumulation spanning one-half to all of the mucosa; 4.5, large nodules of lymphocytic accumulation spanning one-half to all of the mucosa, beginning to show evidence of parietal and chief cell destruction (<25%); 5.0, heavy lymphocytic infiltration throughout the mucosa, parietal and chief cell loss (25–75%), and replacement by foamy cells; and 6.0, total parietal and chief cell loss, no mucosal architecture, and many foamy cells. Thus, grades 1–4 indicate (potentially reversible) inflammation without damage of gastric parietal cells, whereas grades 4.5–6 indicate destructive AIG with loss of parietal cells and, finally, total destruction of the gastric mucosa. Stomach sections were scored blindly by two investigators. Anti-PC Ab were detected by immunofluorescence on cryostat sections of normal BALB/c stomach as previously described (18). The method for isolating cells from stomach tissue has been described previously (19).

ELISAs

Ig ELISAs were performed as described (20). Briefly, plates were coated overnight with 100 µl of anti-mouse IgE, anti-mouse IgG1, and anti-mouse IgG2a (all from BD Pharmingen). The next day, samples (1/50 or 1/500 dilutions of mouse serum or mouse Ig standards; BD Pharmingen) were added and stained with biotin anti-mouse IgE/IgG1/IgG2a (all from BD Pharmingen) and, in a second step, with streptavidin-HRP (BD Pharmingen) (1/4000 dilution in diluents/blocking buffer). Thereafter, tetramethylbenzidine solution (Sigma-Aldrich) and, finally, stop solution (2 N H₂SO₄) were added. Plates were read at 450 nm, and the optical densities of the samples were then plotted on the linear part of the standard curve and multiplied by the dilution factor to calculate Ig concentration in serum samples. Cytokines were measured in supernatants of stimulated cells using cytometric bead assay (Pierce Biotechnology).

Statistical analysis

All group results are expressed as mean ± SD, if not stated otherwise. Student’s t test or Fisher’s exact test (two-tailed) were used for the comparison of group values and discriminatory parameters, where appropriate. For comparing group values that did not follow Gaussian distribution, the Mann-Whitney test (two-tailed) was used. Welch’s correction was applied if variances were significantly different. Pearson and Spearman correlation coefficients were calculated for variables following or not following Gaussian distributions, respectively. p values <0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Generation of Th1, Th2, and Th17 effector populations

Naive CD4⁺Thy1.1⁺ T cells were isolated from thymi of TxA23 mice and cultured under specific priming conditions as described. Before injection, the in vitro differentiated cells were analyzed by intracellular cytokine staining. Th1 cultures were highly enriched for IFN--producing cells (67.2 ± 26.1%, mean ± SD), whereas only a very small percentage produced IL-4 (1.9 ± 2.0%), IL-5 (1.0 ± 1.0%), or IL-17 (1.0 ± 1.3%). Cells cultured under Th2 conditions produced IL-4 (41 ± 13%) and IL-5 (33.4 ± 23.2%), but almost no IFN- (1.6 ± 1.5%) or IL-17 (1.0 ± 0.5%). Cells cultured under Th17 conditions were highly enriched for IL-17 producers (62.5 ± 25.9%) and contained only low percentages of IFN- (1.1 ± 1.5%)-, IL-4 (1.7 ± 0.7%)-, or IL-5 (0.9 ± 0.9%)-producing lymphocytes (Fig. 1A).

View larger version (52K): [in this window] [in a new window]   FIGURE 1. Characteristics of Th1, Th2, and Th17 cell lines. A, Cytokine production by the Th1, Th2, and Th17 cell lines upon restimulation with PMA and ionomycin. B, In vitro T cell proliferation of the cells line stimulated with APC and peptide. C, Expression of V2, CD25, and CD62L on the cell lines before transfer. D, Selective homing of CD4+Thy1.1+ T effectors to the gLN 2 wk after transfer. E, Accumulation of T effectors in the gastric mucosa.

After in vitro differentiation, all T effector population responded to their cognate Ag as measured by proliferation in vitro (Fig. 1B). All groups of T effectors also highly expressed the H⁺K⁺-ATPase- specific V2 receptor, high levels of CD25, but low levels of CD62L (Fig. 1C). After i.p. injection, T effectors preferentially accumulated in the gLN (Fig. 1D). As expected, CD4⁺Thy1.1⁺ cells could also be isolated from the inflamed gastric mucosa and represented a major fraction of all recovered cells (14). A typical example is given for a stomach flush at 6 wk after injection (Fig. 1E).

In vitro differentiated Th1, Th2, and Th17 effectors induce AIG

All three Th cell types transferred AIG into nu/nu recipients. Two weeks after injection, only mild inflammation was seen in all groups, and no parietal cell destruction was observed. After 4 wk, gastritis with destruction of 25% of parietal cells was observed in all groups, but the incidence of destructive gastritis was highest in recipients of Th17 cells (80%), intermediate in mice that received Th2 cells (60%), and significantly less in recipients of Th1 cells (20%). However, after 6 wk, 100% of mice in the Th17 group, 93% in the Th2 group, and 77% in the Th1 group showed high-grade AIG as defined by severe or complete parietal cell loss (score 5–6), epithelial hyperplasia, and massive inflammatory infiltrates (Table I and Fig. 2, A–D). In addition, >50% of the mice in each group were positive for serum Abs directed against parietal cells (anti-PC Abs), a feature typical of murine and human AIG (21).

View larger version (152K): [in this window] [in a new window]   FIGURE 2. Th1, Th2, and Th17 T effector cells induce AIG with distinct histological features. A, Normal glandular stomach from a BALB/C-nu/nu mouse. Six weeks after transfer, Th1 (B), Th2 (C), and Th17 (D) effectors all induce highly destructive AIG (grades 5–6) characterized by epithelial hyperplasia, parietal cell destruction, and inflammatory cell infiltration. Eosinophils were rare and scattered in Th1 infiltrates (E), were present at higher numbers and forming clusters in Th2 infiltrates (E), but were most prominent in Th17-mediated AIG (F). Values for magnifications are approximate due to editing limitations.

Inflammatory infiltrates were found not only in the gastric mucosa but also in the submucosa, ranging from scattered infiltration to nodular foci of predominately CD3⁺ lymphocytes. Heavy submucosal infiltration was rarely seen in Th1-mediated disease, often observed in the Th2 group, and the rule in the Th17 group. Interestingly, the cellular composition of the inflammatory infiltrates in the glandular stomach, the site of parietal cells, consisted of mainly polymorphonuclear granulocytes with many eosinophils and some CD3⁺ lymphocytes in Th17 recipients. In Th2 recipients, the infiltrates were composed of lymphocytes, small clusters of eosinophils, and multinucleated macrophage giant cells, whereas in recipients of Th1 cells, the infiltrates mainly consisted of CD3⁺ lymphocytes, a few scattered eosinophils, but no giant cells (Table I and Fig. 2, E and F). Most forestomachs in all groups showed mild to moderate signs of inflammation, squamous cell hyperplasia in all groups and, only in the Th17 group, eosinophilic infiltration. The spleens of all groups showed signs of increased lymphoid hyperplasia as indicated by germinal center hyperplasia and plasmacytosis (Table I).

Over time, the total number of cells in the gLN increased in all T effector groups, but with varying kinetics. The total number of cells per gLN rapidly increased in the Th17-injected group leading to a peak at 4 wk (11.5 ± 4.5 x 10⁶ cells/gLN) that was 10-fold higher than in recipients of Th1 (1.4 ± 5.4 x 10⁶ cells/gLN) or Th2 (1.5 ± 0.7 x 10⁶ cells/gLN cells). At 6 wk after injection, when almost all mice in all three Th groups had destructive AIG, there were still marked differences in gLN cell numbers with the highest numbers in Th17-injected mice (7.3 ± 1.9 x 10⁶), followed by the Th2 (2.7 ± 1.2 x 10⁶), and the Th1 recipients (1.4 ± 0.4 x 10⁶; Fig. 3A). The increase in total cell number was paralleled by an increase in CD4⁺Thy1.1⁺ TxA23 cells. Recipients of Th17 cells had the highest numbers (Fig. 3B). However, the major cell type in the gLN in all groups consisted of CD19⁺ B cells, which accounted for 68–75% of cells in all groups. No major differences were observed in the frequency of CD11b⁺, CD11c⁺, or Gr-1⁺ cells (data not shown).

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Kinetics of increase in total cell numbers (A) and CD4+Thy-1.1+ effector cells (B) in the gLN following transfer of Th1 (), Th2 (), and Th17 () effectors. Very few effector cells could be detected in the inguinal lymph nodes (ingLN).

Total serum IgE, IgG1, and IgG2a were analyzed in several groups of recipient mice (n = 20 for each group). As expected, serum IgE levels were higher in Th2-injected mice (7.6 ± 9.6 µg/ml) than in Th1-injected mice (2.6 ± 1.8 µg/ml) but were, surprisingly, highest in Th17-mediated disease (12.0 ± 2.0 µg/ml). IgG2a and IgG1 levels varied widely between all experimental groups, and no consistent differences were seen between groups (data not shown).

Suppression of AIG by Tregs

Significant signs of AIG were first seen 4 wk after transfer in recipients of all three effector cell types. The least destructive disease was seen in Th1 recipients, whereas the most aggressive disease was seen in Th17 recipients. Cotransfer of polyclonal Tregs reduced the incidence of high-grade destructive gastritis and disease severity (as indicated by a reduction of the gastritis score) in each group (Fig. 4A). After 6 wk, when most mice had developed severe AIG, Tregs prevented destructive AIG in 84% of Th1-injected and 70% of Th2-injected mice but were not effective in suppressing disease in Th17-mediated AIG (only 9% with nondestructive disease). In addition, Treg significantly reduced disease severity in Th1- and Th2-mediated AIG but not in Th17-injected mice (Fig. 4B). The effects of Tregs on anti-PC Abs were also analyzed at 6 wk after injection. Tregs reduced the percentage of anti-PC Ab⁺ mice in Th1- and Th2-injected mice by more than one-half but had only minimal effects on anti-PC Abs in Th17 recipients (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Inhibition of destructive gastritis by cotransfer of polyclonal Treg 4 wk (A) and 6 wk (B) after transfer. Six weeks after transfer, Tregs reduced the number of irreversibly diseased mice and significantly decreased the mean gastritis score in Th1-AIG (from 5.5 ± 1.1 to 2.0 ± 1.6, p < 0.0001) and in Th2-AIG (from 5.7 ± 1.1 to 2.7 ± 1.9, p < 0.0001) but had no effect on Th17-mediated disease (from 5.9 ± 0.2 to 5.1 ± 1.3, p = NS). Gray areas, significant destructive gastritis. Bars, median gastritis score.

Tregs decrease total cell numbers and numbers of T effectors in the gLN

Cotransfer of Tregs significantly reduced the total number of cells in the gLN and the numbers of the injected CD4⁺Thy1.1⁺ effectors at both 4 and 6 wk after transfer in recipients of Th1 and Th2 effectors (Fig. 5). Although the total cell number and number of CD4⁺Thy1.1⁺ effectors were also reduced in Th17-injected mice at 4 wk, only a minimal, nonsignificant reduction was observed after 6 wk (Fig. 5 and Table II). No effect of Tregs on cell numbers could be observed in the nondraining inguinal LN (not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Cotransfer of Treg ()decreases the total cell number (A) and the numbers of CD4+Thy1.1+ T effectors (B) in the gLN. Please note the different scales for Th1, Th2, and Th17 graphs. *, Significant differences (p < 0.05 for A and p < 0.01 for B, respectively).

In the previous experiments, T effector cells (5 x 10⁴) and Tregs (1 x 10⁶) were injected at a ratio of 1:20. To determine whether a change in the T effector-Treg ratio would result in more effective prevention of AIG, we cotransferred 15,000 or 5,000 of the more resistant to Treg-mediated suppression Th2 and Th17 effectors with 1 x 10⁶ Tregs resulting in T effector-Treg ratios of 1:67 and 1:200, respectively. Destructive gastritis was seen in 70% of the mice following transfer of 15,000 Th2 effectors, but transfer of 5,000 Th2 effectors induced destructive AIG in only a small percentage of mice (30%). Cotransfer of Tregs prevented destructive gastritis in all recipients at 6 wk (Fig. 6A).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Suppression by Tregs depends on the T effector-Treg ratio. Disease scores in mice that received different numbers of Th2 (A) and Th17 (B) effectors with or without a constant number (1 x 106) of Tregs. Black symbols, mice injected with T effectors only; gray symbols, mice coinjected with Treg. Gray areas indicate significant destructive gastritis. Bars, the median gastritis score.

Effector T cells recovered from animals protected by Tregs remain functional in vitro

Although Tregs induced significant suppression of effector cell expansion and destructive AIG following cotransfer of Th1 or Th2 cells, significant numbers of effector T cells could still be detected in protected mice. To determine whether exposure to Treg in vivo modified effector T cell function, cells were recovered from the gLN 6 wk after transfer, and 5,000 CD4⁺Thy1.1⁺ T effectors were rechallenged with their cognate peptide presented by irradiated APCs. Th1, Th2, and Th17 effectors proliferated after restimulation with no differences between cells from animals that received effectors alone or effectors and Tregs (Fig. 7A). Cotransfer of Tregs also had no effect on the capacity of the effector cells to produce cytokines after stimulation with PMA and ionomycin, but changes in the pattern of cytokine secretion were observed. A high percentage (53%) of effectors from the Th1 recipients remained IFN- producers, but a similar percentage (56%) of IFN- producers was seen in Th2 recipients accompanied by a reduction in IL-4 producers (8%) compared with 41% in the starting population. In the Th17 groups, 18% of the effector T cells were IL-17 producers after 6 wk, but 22% also produced IFN- (Fig. 7B), in contrast to very low percentages of IFN- producers in the starting population. Cells derived from both recipients of Th2 and Th17 cells produced low, but similar, levels of IL-5, indicating that the enhanced eosinophilic infiltrates in the Th17 recipients were not secondary to higher levels of IL-5 production by Th17 cells (Fig. 7C).

View larger version (35K): [in this window] [in a new window]   FIGURE 7. Proliferative responses to peptide and allophycocyanin (A) and cytokine production by T effectors after PMA-ionomycin restimulation (B) or by cytometric bead assay of supernatants after allophycocyanin and peptide restimulation (C) of recovered from the gLN 6 wk after transfer without () or with Treg ().

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Although TxA23 mice spontaneously develop AIG characterized by a predominant Th1 pattern of cytokine secretion, we were readily able to induce differentiation of thymocytes from the TxA23 mouse to Th1, Th2, and Th17 cell lines. After transfer, all three effector T cell populations preferentially accumulated in the gLN and the gastric mucosa. Several studies have shown that in vitro differentiated Th1 and Th2 effector cells are capable of promoting organ-specific autoimmune disease (23). Th1-mediated inflammatory infiltrates consisted mainly of CD3⁺ T lymphocytes, whereas Th2-mediated infiltrates also contained a large number of granulocytes, primarily eosinophils, and were associated with elevated serum IgE levels. We now show that all three types of T effector cells induced destructive AIG in the vast majority of recipients, but with different histological patterns independent of disease severity. As expected, Th1-mediated infiltrates mainly consisted of CD3⁺ lymphocytes and Th2-mediated infiltrates had a higher number of granulocytes and eosinophils. Surprisingly, the highest number of infiltrating polymorphonuclear leukocytes including a very high percentage of eosinophils was observed in the Th17-injected mice. Disease induced by Th17 cells seemed to be the most aggressive type (24), given that destructive AIG was seen in all animals after 4 wk and only 5000 Th17 effectors could induce destructive AIG in 100% of recipients. This enhanced aggressiveness of the disease induced by Th17 cells may be secondary to their increased proliferative responses in vivo or perhaps to enhanced homing to the target organ. It also remains possible that the expansion of the Th17 cells in vivo and their resistance to suppression by Tregs are in part mediated by environmental factors in the gastric mucosa or in the gLN such as high levels of IL-23 production by the gastric mucosa.

Previous studies of the composition of cellular infiltrates associated with Th17 cells have demonstrated that they were composed of granulocytes, predominantly neutrophils (25, 26). It is unclear whether the predominantly eosinophilic infiltration observed in our experiments is a unique characteristic of Th17-induced AIG or whether studies of other Th17-associated diseases overlooked the presence of eosinophils, because they are hard to distinguish from neutrophils without special staining. It is also possible that the protocol we used to generate Th17 cell lines, which included the use of anti-IL-2, generated the production of other members of the IL-17 family in addition to IL-17A (27), such as IL-17E, which stimulates eotaxin production and thus eosinophilic infiltration. Alternatively, IL-17A itself is able to trigger eotaxin release by other cells in some circumstances, as shown for smooth airway muscle cells (28).

It is also of interest that the cell lines used to induce AIG exhibited variable patterns of stability after transfer and exposure to autoantigen in vivo. All of the lines exhibited the appropriate restricted pattern of cytokine expression when tested before transfer. Th1 cells for the most part maintained their phenotype when isolated from animals 6 wk after transfer, whereas cells isolated from Th2 recipients contained a significant number of IFN--producing cells after 6 wk. It is possible that the IFN--producing cells were derived from noncommitted precursors in the starting population that responded to environmental signals in this AIG model that seems to favor Th1 differentiation (14). Alternatively, it remains possible that some plasticity exists in the committed Th2 population that facilitates conversion to Th1 cells upon receipt of environmental cues as has been shown in certain infectious disease models (29). A significant number of the recovered cells from the Th17-injected mice continued to produce IL-17, but also a similar percentage of IFN--producing cells was recovered; interestingly, as examined in a limited study, many of these cells were IL-17/IFN- double producers (data not shown). Because a significant number of IFN--producing cells are seen in almost all purported Th17-mediated diseases (30), it remains possible that the IFN--producing cells in the Th17 recipients represent Th17 cells that have further differentiated to produce both IL-17 and IFN-. The lack of stability in the cytokine profile over 6 wk in vivo had little effect on the striking differences in the inflammatory infiltrates or on the susceptibility of the animals to suppression by Treg.

In our previous studies, we demonstrated that the induction of AIG by naive TXA23 CD4⁺ thymocytes could be readily suppressed by polyclonal Tregs from normal BALB/c mice (9). We used the same Treg-T effector ratios in the present study and showed that polyclonal Tregs could markedly suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced AIG only at early time points. We and others have also shown that Ag-specific Tregs are much more potent inhibitors of the induction of organ-specific autoimmunity (22, 31) and have proposed that Ag-specific Tregs prevent disease by inhibiting T cell activation and expansion by acting on autoantigen-presenting dendritic cells (22, 32, 33, 34, 35). It remains unclear how polyclonal Tregs exert their inhibitory effects. Our previous studies with naive T cells demonstrated that they failed to inhibit the initial effector cell expansion in the draining lymph nodes but did have effects on Th1 differentiation. In sharp contrast, in the present studies, significant inhibition of the effector cell expansion was seen when polyclonal Tregs were cotransferred with Th1 and Th2 effectors and the inhibition of expansion was an excellent correlate of protection from destructive AIG. If one assumes that Treg-mediated suppression is independent of the type of effector T cells present, it is possible that Th17 effectors, and to a lesser extent Th2 effectors, are able to overcome suppression by proliferating to a greater extent than Th1 effectors and potentially overcome suppression by outnumbering the Tregs for access to dendritic cells.

There are several important differences between our previous studies with naive effectors and the differentiated effector cells used here. Naive effectors could easily be detected in the gLN as early as 3 days after transfer, and significant expansion could be demonstrated by day 5 (14). In contrast, the fully differentiated effector cells could hardly be detected in the gLN after 1 wk (not shown). This result is not surprising in that the effector populations expressed only low levels of CD62L that is required for entry into lymph nodes. It remains possible that the effector cells were first stimulated by their target Ag in the stomach and then acquired the ability of traffic to the gLN (36). The cotransferred Treg express high levels of CD62L and could easily be detected in all lymph nodes at early time points after transfer (Ref. 14 and data not shown). It remains conceivable that a small percentage of the polyclonal Treg population expressing receptors for Ags derived from the target organ were retained in the gLN, could expand, and could then be prepared to exert inhibitory effects on dendritic cells, resulting in inhibition of the expansion of the susceptible effector populations upon their arrival in the gLN. A primary effect of Treg on T cell expansion is also consistent with our data, demonstrating that the effector cells that can be isolated from the gLN of protected animals are not anergic and are fully competent to proliferate and produce effector cytokines ex vivo. This result is also consistent with studies in the 3-day thymectomy model of oophoritis in which the continuous presence of Treg was required for protection from disease (37). Taken together, these findings are most consistent with a model where Tregs in vivo are not shutting off effector cytokine by T cells directly, but are regulating their expansion by acting on APC.

Cellular biotherapy with Treg is now being considered as a potential therapy for organ-specific autoimmunity. Although most studies in experimental animal models suggest that Ag-specific Tregs act in bystander manner and are the most potent inhibitors of disease, it may be very difficult to isolate Ag-specific Foxp3⁺ T cells from patients. The present studies demonstrating a strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provide an experimental basis for the clinical use of polyclonal Tregs in human disease. It remains to be determined whether the mechanism of action of the polyclonal Treg is similar to or different from those proposed for the Ag-specific Treg.

	Acknowledgments

 
We thank Dr. J. Milner for advice and help with the ELISA, Sarah Tanksley for cell sorting, and the team of the animal facility for helping maintain our mouse colony.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported funds from the intramural program of the National Institute of Allergy and Infectious Diseases and by a Max Kade Foundation Postdoctoral Research Exchange Grant (to G.H.S.).

² Address correspondence and reprint requests to Dr. Ethan M. Shevach, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10 Room 11N315, Bethesda, MD 20892. E-mail address: eshevach{at}niaid.nih.gov

³ Abbreviations used in this paper: AIG, autoimmune gastritis; Tg, transgenic; Treg, regulatory T cell; gLN, gastric lymph node; CD62L, L-selectin.

Received for publication January 8, 2008. Accepted for publication May 25, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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