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The Journal of Immunology, 2003, 171: 2279-2286.
Copyright © 2003 by The American Association of Immunologists

CD4+ Th Cells Resembling Regulatory T Cells That Inhibit Chronic Colitis Differentiate in the Absence of Interactions Between CD4 and Class II MHC 1

Timothy L. Denning*,{dagger}, Hai Qi{ddagger}, Rolf König*, Kevin G. Scott§, Makoto Naganuma§ and Peter B. Ernst2,§

Departments of * Microbiology and Immunology, {dagger} Pediatrics, and {ddagger} Pathology, University of Texas Medical Branch, Galveston, TX 77555; and § Digestive Health Center of Excellence and Department of Internal Medicine, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulatory CD4+ Th cells can prevent many autoimmune diseases; however, the factors selecting for these cells remain poorly defined. In transgenic mice with a mutation in the CD4 binding region on class II MHC, the disruption of CD4-class II interactions selected for CD4+ Th cells that expressed surface markers and cytokines associated with regulatory Th cells. Th cells from these mice were enriched for CD45RBlow as well as CD25+, while they expressed high levels of the transcription factor associated with regulatory T cells, Foxp3, and cytokines, including IL-4, IL-10, and IFN-{gamma} mRNA and protein. These regulatory Th cells inhibited the function of APCs via IL-10 production, and adoptive transfer of these cells prevented weight loss and inflammation in a model of colitis. CD4+ regulatory Th cells emerged only when interactions between CD4 and class II MHC were deficient on cells of nonhemopoietic origin. These data support a novel model controlling the differentiation of regulatory Th cells and suggest that interactions between CD4 and class II MHC may a useful target for re-educating T cells as a treatment for inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The thymus positively selects for T cells that confer immunity and negatively selects autoreactive T cells that could mediate autoimmune diseases (1). However, some autoreactive T cells escape to the periphery, and it is recognized that self-reactive Th cells exist as regulatory T cells that prevent autoimmune diseases (2).

Regulatory T cells are absent in mice lacking the genes encoding the class II MHC (3), which leads to the onset of colitis (4). Recently, Jordan et al. (5) showed that high affinity TCR/self peptide-MHC interactions in the thymus selected for CD25+ regulatory T cells displaying immunosuppressive function. Moreover, the expression of class II MHC on thymic cortical epithelial cells was sufficient for the development of CD4+CD25+ regulatory T cells that suppressed T cell responses in vitro and inhibited colitis in vivo (3). Thus, recognition of self peptides presented to the TCR is necessary for the ontogeny of regulatory T cells.

While the role of class II MHC-peptide recognition by TCR has been established, it is not known whether the MHC-peptide complex activates regulatory T cells directly through the TCR or in collaboration with interactions via CD4. In this report we investigated the function of CD4+ Th cells that developed in the absence of interactions between class II MHC and the CD4 coreceptor using a transgenic mouse carrying a mutation in the major CD4 binding region on the class II MHC {beta}-chain (referred to as M A{beta}k3 mice) (6). The regulatory Th cell phenotype was defined by the expression of Foxp3, CD45RB, and CD25 as well as the cytokine profile and capacity to inhibit inflammatory effector T cells, particularly in vivo (7). Thus, presentation of self Ags to T cells differentiating into regulatory Th cells requires class II MHC, but not interactions with CD4.


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

C57BL/6 (B6) and C57BL/6 SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME). M A{beta}k mice express a transgenic class II MHC {beta}-chain (A{beta}k) that is mutated at the CD4 binding region of the {beta}2 domain (6) such that the A{alpha}bA{beta}k heterodimer is unable to interact with CD4 (8), while control mice express wild-type A{beta}k (9). Transgenic and control mice were maintained in a conventional animal care facility at the University of Texas Medical Branch (Galveston, TX). All procedures were approved by the animal research committee.

Cells, media, reagents, Abs, and flow cytometry

CD4+ cells were isolated by positive selection using Dynabeads M-450 coated with anti-mouse CD4 mAb and DETACHaBEAD (both from Dynal Biotech, Oslo, Norway) from lymph nodes and spleens before being stimulated with plate-bound anti-CD3 (10 µg/ml) and anti-CD28 (1 µg/ml) (10). While optimizing the methods, it was observed that positive selection yielded more cells and in greater purity than negative selection, but did not change the response of these T cells in the assays used in this study. This is consistent with the experience of others (11). T cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (all from Life Technologies, Grand Island, NY), 1x nonessential amino acids, and 50 µM 2-ME (Sigma-Aldrich, St. Louis, MO).

Anti-CD3{epsilon} (145-2C11), anti-CD28 (37.51), anti-Thy1.2 (30-H12), anti-class II MHC (M5/114), OptEIA mouse IL-10 ELISA set, purified rat anti-mouse IL-10 (JES5-16E3), PE-anti-CD45RB (16A), FITC-anti-CD4 (L3T4), PE-anti-CD25 (3C7), PE-anti-IL-10, FITC-anti-IL-2, and isotype control Abs were purchased from BD PharMingen (San Diego, CA). Anti-TGF-{beta} Ab (1D11) was purchased from R&D Systems (Minneapolis, MN). Cells were incubated with mitomycin C (Sigma-Aldrich), recombinant murine IL-2 (Genzyme, Cambridge, MA), or recombinant murine IFN-{gamma} (Roche, Indianapolis, IN). For flow cytometric analysis, cells were assayed using a FACScan (BD Biosciences, Mountain View, CA), and data were analyzed using WinMDI version 2.8 software (The Scripps Institute, La Jolla, CA).

Expression of cytokines and Foxp3

The relative level of cytokine mRNA and secreted protein was assessed using kits (BD PharMingen) according to the manufacturer’s instructions. Total RNA was extracted from CD4+ T cells using TRIzol (Life Technologies) (12), and mRNA expression was measured using the RNase protection assay kit (mCK1; BD PharMingen). The cytometric bead array (BD PharMingen) was used to assess the relative levels of IL-2, IL-4, IL-5, IFN-{gamma}, and TNF-{alpha} in the supernatants of activated T cells (13). Beads were analyzed using cytometric bead array software (BD Biosciences) and WinMDI. IL-10 was assessed using the OptEIA mouse IL-10 ELISA set (BD PharMingen), and TGF-{beta} was detected using the TGF-{beta}1 Emax ImmunoAssay System from Promega (Madison, WI). For detection of intracellular cytokines, purified CD4+ T cells were stimulated with anti-CD3 and anti-CD28, as described above, for 72 h, rested in fresh medium for 2 days, and then restimulated with anti-CD3 and anti-CD28 for 6 h in the presence of monensin (2 µM; Sigma-Aldrich). The expression of mRNA for Foxp3, a transcription factor that has been reported to be associated with the selection of regulatory T cells (14, 15) was also assessed. Total RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA), and yield was estimated spectrophotometrically. RNA was reverse transcribed using the Superscript kit (Invitrogen, San Diego, CA) random hexamer protocol. Foxp3 levels were measured by dual-labeled probe, real-time PCR using a Smart Cycler (Cepheid, CA). The PCR contained 0.3 µM of each primer, 0.2 µM probe, 3 mM MgCl2, and 0.75 U of platinum Taq polymerase (Invitrogen). The primer sequences were designed to bracket an intron to avoid amplification of genomic DNA (15). Their sequences were as follows; Foxp3 primers, 5'-CCC AGG AAA GAC AGC AAC CTT-3' and 5'-TTC TCA CAA CCA GGC CAC TTG-3'; and Foxp3 probe, 5'-TETATC CTA CCC ACT GCT GGC AAA TGG AGT C-3' (Integrated DNA Technologies, Coralville, IA). PCR cycling conditions consisted of 95°C for 300 s, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Critical threshold values were compared against a standard curve to estimate starting amounts of mRNA, and the relative expression of Foxp3 mRNA between samples was estimated by normalizing these values against 18S rRNA critical threshold values generated using a preoptimized 18S rRNA primers and probe set (PE Applied Biosystems, Foster City, CA).

Assessment of anti-inflammatory effects of T cells

RAW264.7 macrophages (5 x 105; H-2d; American Type Culture Collection, Manassas, VA) were added to 12-well plates in a total volume of 1 ml. Supernatants derived from stimulated allogeneic CD4+ T cells (H-2b) were added to a final concentration of 20% in the presence or the absence of the indicated Abs at 1 µg/ml. In some experiments recombinant murine IFN-{gamma} (100 U/ml) was added as a positive control. After 48 h of culture, cells were harvested and stained with anti-class II MHC Ab or isotype control and analyzed by flow cytometry as described previously (16).

Proliferation assays

CD4+ T cells (5 x 104) were cocultured in 96-well plates with B6 APC (105) and 1 µg/ml of anti-CD3{epsilon} in the presence or the absence or recombinant murine IL-2 (10 U/ml). APCs were prepared by erythrocyte lysis of total splenocytes with ACK buffer. Splenocytes were incubated with anti-CD4 magnetic beads (Dynal Biotech) for 30 min on ice and subsequently passed through a magnetic VS+ column (Dynal Biotech). The CD4- APCs (flow-through) were treated with mitomycin C (100 µg/ml) for 30 min at 37°C and washed three times. Cocultures were pulsed with [3H]thymidine for the final 18 h, harvested on an automatic harvester, and counted using a beta counter (17). The background counts of tritiated thymidine incorporation by APCs alone were 345.33 ± 86.22.

Induction of colitis in SCID mice

CD4+CD45RBhigh splenic and lymph node T cells were obtained by RBC lysis and magnetic bead depletion using lineage-specific rat mAbs in supernatants (17). The remaining cells were stained with anti-CD4-FITC and anti-CD45RB-PE and sorted into CD4+CD45RBhigh or CD4+CD45RBlow T cells using a FACSVantage (BD Biosciences). The sorted CD4+CD45RBhigh T cells were >98% pure upon reanalysis. The CD4+CD45RBhigh cells (4 x 105) were injected via the i.p. route into B6 SCID mice alone or with 4 x 105 M A{beta}k CD4+ T cells. SCID mice were weighed and monitored weekly after T cell transfers. Mice were euthanized 16 wk post-transfer, and colonic tissue was prepared for microscopic assessment of colitis (12). At this time, 100% of recipient mice receiving CD4+CD45RBhigh cells alone developed clinical signs of colitis, including piloerection, hunch, weight loss, and loose stool, while all mice receiving CD4+CD45RBhigh cells plus M A{beta}k CD4+ T cells remained healthy and continued to gain weight.

Bone marrow chimeras

Hemopoietic chimeras were prepared as described previously (18). Age- and sex-matched hosts were lethally irradiated (1000 rad total, 160 rad/min) using a 137Cs source. The next day, 15–20 x 106 bone marrow cells depleted of T cells by magnetic depletion using anti-Thy1.2 Ab and sheep anti-rat magnetic beads were injected into the tail veins of irradiated mice. Mice were euthanized 12 wk after reconstitution, and CD4+ T cells were purified from lymph nodes and spleen.

Statistical analysis

The mean values for the different parameters were compared using Student’s t test or one-way ANOVA as indicated. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD4+ T cells purified from M A{beta}k mice express a unique cytokine profile

To understand the contribution of interactions between CD4 and class II MHC to Th cell function, we compared CD4+ T cells from the M A{beta}k mice with a defect in CD4-class II MHC interactions to those from B6 mice (6). After 48 h of stimulation with plate-bound anti-CD3/28, M A{beta}k CD4+ T cells expressed increased levels of mRNA for IL-4, IL-10, IL-13, and IFN-{gamma} compared with B6 CD4+ T cells (Fig. 1A). In contrast, CD4+ T cells from M A{beta}k mice were highly refractory in their ability to synthesize mRNA for IL-2.



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  		FIGURE 1. CD4+ T cells purified from M A{beta}k mice display a cytokine profile resembling that of regulatory T cells. A, Purified CD4+ Th cells from B6 or M A{beta}k mice were stimulated with anti-CD3/28. RNA was analyzed by RNase protection assay. Lane 1, B6 CD4+ RNA extracted 48 h poststimulation. Lane 2, M A{beta}k CD4+ RNA extracted 48 h poststimulation. Lane 3, B6 CD4+ RNA extracted 72 h poststimulation. Lane 4, M A{beta}k CD4+ RNA extracted 72 h poststimulation. The far left lane contains unprotected probe that aligns, as indicted with connecting lines, to bands in lanes 1–4. The housekeeping genes L32 and GAPDH are detectable at the bottom of the image. The results shown are representative of observations made from three separate experiments. B, Purified CD4+ Th cells from B6 or M A{beta}k mice were stimulated with anti-CD3/28. Supernatants were harvested at the indicated time points and analyzed using a cytometric bead array. Specific cell supernatant cytokine levels were calculated by regression from a standard curve. Levels of B6 CD4+-derived cytokines ({blacksquare}) and M A{beta}k CD4+-derived cytokines () are shown for the indicated cytokines at various times of stimulation from a representative experiment. The IL-10 response was detected by conventional ELISA. The results shown are representative of observations made from two (cytometric bead array) or three (ELISA) separate experiments, with three replicates in each experiment. C, Purified CD4+ Th cells from B6 or M A{beta}k mice were stimulated with anti-CD3/28 as described in Materials and Methods and assayed for intracellular cytokine production by flow cytometry. The results shown are representative of observations made from three separate experiments.

 
To confirm the differences in cytokine expression at the protein level, T cells were stimulated for 24, 48, and 72 h before supernatants were examined using a cytometric bead array (13), conventional ELISA, and intracellular cytokine staining. CD4+ T cells from M A{beta}k mice secreted higher levels of IL-4, IFN-{gamma}, and especially IL-10 in the absence of IL-2 (Fig. 1B). The mean TGF-{beta} values for M A{beta}k mice were 1190 pg/ml at 24 h, but were undetectable at 48 and 72 h. The mean TGF-{beta} values for control mice were 750, 39, and 34 pg/ml for the respective time points. Cytokine expression patterns in B6 and wild-type A{beta}k mice were comparable (data not shown). M A{beta}k CD4+ T cells were highly enriched for cells secreting IL-10 in the absence of IL-2 (Fig. 1C), as detected by intracellular cytokine staining.

M A{beta}k CD4+ cells exhibit the phenotype of regulatory Th cells

Since M A{beta}k CD4+ T cells resembled regulatory T cells based on the production of high levels of IL-10 (10, 19), we investigated their expression of surface CD45RB and CD25 as well as mRNA for Foxp3. CD4+ T cells from M A{beta}k mice were enriched for cells expressing CD45RBlow and CD25 compared with B6 CD4+ T cells (Fig. 2A). The relative amount of mRNA for Foxp3 was increased significantly in CD4+ T cells from M A{beta}k mice compared with controls (Fig. 2B). Fractionation of the T cells showed that Foxp3 expression was virtually lacking in CD8+ T cells (negative control), but was enriched significantly in the CD45RBlow subset of CD4+ T cells, particularly those from M A{beta}k mice. Consistent with the lack of IL-2, M A{beta}k CD4+ T cells stimulated with plate-bound anti-CD3/28 proliferated modestly (data not shown). To assess whether M A{beta}k CD4+ T cells were anergic, Th cells were cultured with mitomycin C-treated APCs and anti-CD3 in the presence or the absence of exogenous IL-2 (Fig. 2C). CD4+ Th cells from B6 mice proliferated strongly in response to anti-CD3 stimulation alone, and this proliferation was augmented slightly with the addition of exogenous IL-2. In contrast, M A{beta}k CD4+ T cells did not proliferate in response to anti-CD3 stimulation unless provided with exogenous IL-2, which stimulated a level of proliferation that exceeded that observed during stimulation of B6 CD4+ T cells with anti-CD3 and IL-2. Thus, M A{beta}k CD4+ T cells were anergic and had exaggerated responses to IL-2, further supporting the observation that M A{beta}k CD4+ T cells expressed higher levels of IL-2R{alpha} (CD25).



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  		FIGURE 2. M A{beta}k CD4+ cells resemble regulatory CD4+ Th cells. A, Purified B6 or M A{beta}k CD4+ T cells were stained with PE-conjugated anti-CD45RB or anti-CD25. M1 indicates the percentage of cells expressing low levels of CD45RB (CD45RBlow) or high levels of CD25. B, T cells were enriched from B6 ({blacksquare}) or M A{beta}k () mice, fractionated into subsets and assessed for the expression of mRNA for Foxp3 as described in Materials and Methods. The relative amount of specific mRNA was estimated using dual-labeled probes, and the results are expressed as the ratio of Foxp3 to 18S. The results shown are representative of observations made from three separate experiments. C, CD4+ cells (5 x 104) from B6 ({blacksquare}) or M A{beta}k () mice were cultured with mitomycin C-treated APCs and soluble anti-CD3{epsilon} with or without rIL-2, pulsed with [3H]thymidine, and harvested. Results are the mean counts per minute ± SE of three replicates and are representative of three independent experiments. *, p < 0.05 compared with the other bar within a given stimulus group.

 
M A{beta}k CD4+-derived IL-10 inhibits IFN-{gamma}-induced class II MHC expression

Since activated M A{beta}k CD4+ T cells exhibited a phenotype similar to that of regulatory T cell populations (19), we examined the ability of supernatants derived from the cultures described in Fig. 1B, to modulate the expression of class II MHC by macrophages. B6 supernatants induced the expression of class II MHC (geometric mean fluorescence intensity (MFI), 167.43) above constitutive expression (MFI, 1.47), while supernatants from M A{beta}k CD4+ T cell cultures induced lower levels of class II MHC (MFI, 62.11; Fig. 3). IL-10 was a critical regulatory factor in M A{beta}k supernatants, as neutralizing anti-IL-10 Ab, but not isotype control Ab, increased class II MHC expression to levels comparable to those observed in cells treated with IFN-{gamma} or B6 supernatants. Importantly, neutralizing anti-TGF-{beta} Ab failed to reverse the regulatory effects of 72 h M A{beta}k supernatants. The ability of supernatants from both B6 and M A{beta}k mice to induce class II MHC on RAW264.7 macrophages was due almost exclusively to IFN-{gamma}, as Abs to IFN-{gamma} completely prevented supernatant-induced class II MHC expression. Regulation of CD86 (B7.2) by these cell supernatants showed patterns similar to observations of class II MHC (data not shown).



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  		FIGURE 3. M A{beta}k CD4+-derived IL-10 regulates macrophage class II MHC expression. RAW264.7 macrophage cells were cultured with medium alone or containing 100 U/ml or recombinant murine IFN-{gamma} (upper left panel) or supernatant from C57BL/6 (B6) and M A{beta}k (upper middle panel) CD4+ T cells stimulated with anti-CD3/28. In the other panels, T cell culture supernatants were added in the presence of isotype control or neutralizing Abs to TGF-{beta}, IL-10, or IFN-{gamma} as indicated. RAW264.7 cells were stained with PE-conjugated anti-class II MHC and analyzed using flow cytometry.

 
M A{beta}k CD4+ T cells prevent wasting and colitis

Although CD45RBlow and CD25 expression are associated with regulatory T cells, proof that M A{beta}k CD4+ T cells functioned as regulatory T cells was obtained by examining their ability to prevent inflammatory responses in vivo. Confirming previous results, the injection of B6 SCID mice with 4 x 105 B6 CD4+CD45RBhigh T cells resulted in weight loss (Fig. 4A) associated with diarrhea and rectal prolapse (20). Histological analysis of colonic sections from these mice revealed mucosal thickening, crypt elongation, epithelial hyperplasia, goblet cell depletion, and marked infiltration with inflammatory cells (Fig. 4B). Cotransfer of 4 x 105 M A{beta}k CD4 prevented CD4+CD45RBhigh-induced wasting and histological signs of inflammation (Fig. 4, A and B), similar to CD4+CD45RBlow cells (21). These results agreed with the in vitro observations that M A{beta}k CD4+ T cells inhibited the proinflammatory effects of effector T cells (Fig. 3) and showed that M A{beta}k CD4+ T cells functioned as regulatory T cells in vivo similarly to CD4+CD45RBlow (21, 22), CD4+CD25+ (23, 24), and Tr1 (10) cells.



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  		FIGURE 4. M A{beta}k CD4+ T cells prevent wasting and colitis in SCID mice. A, SCID mice were injected with no cells (-), CD4+CD45RBhigh cells alone (RBhigh), or CD4+CD45RBhigh cells together with M A{beta}k CD4+ T cells (RBhigh + M A{beta}k CD4). Data shown are the mean weight relative to initial weight for four or five individual mice per group on the day of sacrifice ± SE. *, p < 0.05 comparing SCID mice injected with CD4+CD45RBhigh cells to either of the other two groups using one-way ANOVA. B, H&E histology of colonic sections from individual animals that received no cells (upper panel), B6 CD4+CD45RBhigh cells (middle panel), or B6 CD4+CD45RBhigh cells together with M A{beta}k CD4+ T cells (bottom panel). The line indicates the relative thickness of the mucosa at identical magnification. Note the prevention of extensive mononuclear cell infiltrates (fine arrow) mucosal hyperplasia, goblet cell depletion, and crypt abscesses (large arrow) by M A{beta}k CD4+ T cells.

 
Development of regulatory CD4+ T cells in M A{beta}k mice is dependent upon nonhemopoietic class II MHC

Since CD4+CD25+ regulatory T cells are believed to acquire regulatory function within the thymus (3), we investigated whether the unique phenotype and function of M A{beta}k CD4+ T cells were due to impaired interactions between class II MHC molecules on hemopoietic vs nonhemopoietic cells. Irradiated B6 or M A{beta}k mice were reconstituted with Thy1.2-depleted bone marrow cells derived from either B6 or M A{beta}k donors. Analysis of class II MHC in chimeric mice verified the expression of donor, but not recipient, class II MHC on all hemopoietic cells, indicating that hemopoietic cells were exclusively of donor origin (data not shown). In B6 mice receiving M A{beta}k bone marrow (M A{beta}k>B6), lymph node T cells exhibited a normal distribution of CD4+ and CD8+ populations compared with normal B6 mice or irradiated B6 mice reconstituted with B6 bone marrow (data not shown). In contrast, when irradiated M A{beta}k mice were reconstituted with B6 marrow, they retained a very small population of CD4+ lymph node T cells (~6%) that expressed the regulatory phenotype comparable to that observed in unmanipulated M A{beta}k mice. Interestingly, enrichment for CD45RBlow (Fig. 5A) and CD25 (Fig. 5B) expression and the pattern of cytokine secretion (Fig. 6) by CD4+ T cells were dependent on M A{beta}k nonhemopoietic cells. If irradiated B6 mice were reconstituted with M A{beta}k bone marrow (M A{beta}k >B6), the T cells expressed the normal phenotype (Fig. 5, A and B, and Fig. 6) compared with the enrichment for regulatory T cells in irradiated M A{beta}k mice reconstituted with B6 bone marrow. Collectively, these data suggest that mutation of the CD4 binding region of class II MHC on cells of nonhemopoietic origin is sufficient for the development of CD4+ Th cells that are enriched for CD45RBlow and CD25 expression and for regulatory function.



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  		FIGURE 5. CD45RB and CD25 expression on purified CD4+ lymph node cells from bone marrow chimeric mice. A, CD45RB expression was analyzed by flow cytometry on purified CD4+ lymph node Th cells from irradiated B6 mice reconstituted with B6 bone marrow (B6>B6), irradiated B6 mice reconstituted with M A{beta}k bone marrow (M A{beta}k>B6), or irradiated M A{beta}k mice reconstituted with B6 bone marrow (B6>M A{beta}k). The percentages of CD45RBlow cells are indicated in gate M1. B, CD25 expression was analyzed by flow cytometry on purified CD4+ lymph node Th cells from (B6>B6), (M A{beta}k>B6), or (B6>M A{beta}k). The percentages of CD25+ cells are indicated in gate M1.

 


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  		FIGURE 6. Cytokine expression by CD4+ Th cells from bone marrow chimeras. Purified CD4+ Th cells from irradiated B6 mice reconstituted with B6 bone marrow (B6>B6), irradiated B6 mice reconstituted with M A{beta}k bone marrow (M A{beta}k>B6), or irradiated M A{beta}k mice reconstituted with B6 bone marrow (B6>M A{beta}k) were stimulated with anti-CD3/28. Supernatants were harvested after 72 h and were analyzed using the cytometric bead array. The IL-10 response was detected by conventional ELISA. The results shown are representative of observations made from two (cytometric bead array) or three (ELISA) separate experiments with three replicates in each experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data show that CD4+ T cells with properties and functions resembling those of regulatory T cells (7, 25) developed when interactions between nonhemopoietic class II MHC and CD4 were disrupted. While no single, unequivocal marker or cytokine panel that defines regulatory T cells exists (7), CD4+ T cells from the M A{beta}k transgenic mouse can be considered regulatory, since they express patterns of cytokines (low IL-2 and increased IFN-{gamma} and IL-10) similar to other regulatory T cells (10, 25), are enriched for cells expressing CD45RBlow and CD25high, express significantly higher levels of Foxp3 (15, 26), and inhibit inflammatory responses in vitro and in vivo (25). It is remarkable that M A{beta}k mice have extremely low levels of CD4+ T cells, but regulatory T cells expressing IL-10, CD45RBlow, and CD25high are actually enriched at least 2-fold compared with those in control mice. It is not likely that these changes merely reflect activation, since IL-2 was markedly reduced in these T cells.

While several criteria can be used to ascribe regulatory function to a Th cell, the various experimental conditions used by investigators make it difficult to know how many regulatory T cell populations exist and whether all types are selected in a similar fashion. For example, T cells with the function of regulatory T cells have been generated by long-term culture in the presence of IL-10 (10). These regulatory T cells (Tr1 cells) resembled the Th cells from M A{beta}k mice in that they had reduced IL-2 production, but enhanced IL-5, IL-10, and IFN-{gamma} (10). While the expression of IFN-{gamma} and IL-10 appears paradoxical, regulatory T cells have been described to produce inflammatory cytokines, such as IFN-{gamma} and TNF-{alpha} (25). IFN-{gamma} has been implicated in oral tolerance (27), which would be consistent with anti-inflammatory functions, particularly if it is present in association with IL-10 and TGF-{beta}. In view of the variation in host flora, cytokine milieu, and other factors that modulate gene expression and define the context in which T cells differentiate and function, it is unlikely that a single cytokine panel will define regulatory T cells.

Within the context of the entire cytokine response mediated by regulatory T cells, IL-10 contributes significantly to the anti-inflammatory effects of regulatory T cells (28). Based on the detection of cytoplasmic cytokines, IL-10 was expressed by >10% of the activated T cells from M A{beta}k transgenic mice in the absence of IL-2 (Fig. 1C). After secondary stimulation, this increased to 30%, compared with 15% in control mice (data not shown). Other cytokines, including TGF-{beta}, may contribute significantly to the anti-inflammatory effects of regulatory T cells (29, 30). However, neutralizing TGF-{beta} Ab did not reverse the regulatory effects of M A{beta}k supernatants on macrophages (Fig. 3), which is consistent with the lack of soluble TGF-{beta} detected at this time point. These results do not infer that these cells could not mediate contact-dependent inhibition through surface TGF-{beta} as described by others (30), since this function was not assayed directly.

Although several cytokines and surface markers are associated with regulatory T cells, only recently has a T cell gene product been described that appears necessary and sufficient to select for the development of at least some regulatory T cells. Foxp3, a forkhead/winged helix transcription factor gene, has been shown to program the development and function of regulatory T cells (15, 26). The absence of this gene in mice and humans is associated with severe autoimmune disease that rapidly leads to the death of the host (31). In contrast, overexpression of the gene in transgenic mice decreases T cell responsiveness and results in fewer peripheral T cells (32). If Th cells in M A{beta}k mice are selectively enriched for regulatory T cells, one might predict that these mice would overexpress Foxp3 and have diminished T cell numbers and responsiveness. Previous studies have shown that Th cells in M A{beta}k mice are decreased in number (6), while the data from this report confirm their hyporesponsivenss and their increased expression of Foxp3.

The novel paradigm that emerges from our data is that Th cells are more likely to be imprinted with the phenotype of a regulatory T cell when MHC class II recognition is separated from the interaction with CD4. This idea is supported by the observation that normal bone marrow seeded into irradiated M A{beta}k mice leads to the generation of T cells that resemble regulatory T cells. In addition, high affinity TCR recognition of self Ags (5) presented by cortical class II MHC (3) selects for regulatory T cells. This is supported by the fact that mice deficient in class II MHC lack regulatory T cells (3). As the expression of class II MHC by cortical thymocytes is sufficient to select for regulatory T cells (3), it is possible that this occurs in double-negative T cells within the cortex that could express high affinity TCR and recognize MHC during the earliest stages of T cell differentiation. Alternatively, signaling through CD4 may be deficient in immature thymocytes, resulting in a functional bypass of this second signaling pathway. While our studies do not directly implicate the thymus in development of CD4+ regulatory T cells, the fact that nonhemopoietic cells controlled the development of regulatory T cells is consistent with a role for the thymus. The separation of signaling from CD4 leading to regulatory T cells is in agreement with a report that tetramers consisting of peptide and the {alpha}1 and {beta}1 domains of HLA molecules induce high levels of IL-10 in human Th cells (33). Additional evidence for this concept comes from the induction of regulatory Th cells by treating mice with nondepleting CD4 Abs (34) and the suppression of intestinal inflammation by CD4 mimetics (35). The role of CD4 in controlling selection for T cells with a regulatory phenotype in the periphery vs the thymus remains to be clarified.

It is known that CD4 and class II MHC form a stable interaction that leads to p56lck activation and signal transduction within Th cells (36). In the M A{beta}k mouse, deficient CD4-mediated signaling may select Th cells with higher than normal TCR affinity for self peptide/MHC II and regulatory function. In fact, Th cells selected in the absence of CD4 lack T cells exhibiting low affinity binding to their ligands (28). The TCR repertoire in the M A{beta}k mouse remains to be defined and compared with that in the CD4-deficient mouse.

In summary, with the absence of CD4-class II MHC interactions, only a small percentage of CD4+ Th cells complete positive selection and immigrate into the periphery. Thus, we propose the hypothesis that M A{beta}k CD4+ Th cells are positively selected and bypass elimination via negative selection due to their fundamental state of anergy to conventional signaling via TCR. This model for the selection of regulatory T cells may have important implications for understanding the control of various autoimmune diseases, including those in the digestive tract.


   Acknowledgments
 
We thank Mark Griffin and Kim Palkowetz for technical assistance with flow cytometry, and Richard Eberle for assistance with irradiation studies. Drs. Cohn and Vidrich kindly consulted on the technical aspects of the real-time PCR.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK50980, AI 48173, RR00175, and CHK35741; the Crohn’s and Colitis Foundation of America; and The John Sealy Memorial Endowment Fund Development Grant. T.L.D. was supported by Grant T32AI07626. Back

2 Address correspondence and reprint requests to Dr. Peter B. Ernst, Department of Internal Medicine, University of Virginia, P.O. Box 800708, Charlottesville, VA 20908-0708. E-mail address: pernst{at}virginia.edu Back

3 Abbreviations used in this paper: M A{beta}k, transgenic mice carrying a mutation in the major CD4 binding region on the class II MHC {beta}-chain; MFI, mean fluorescence intensity. Back

Received for publication January 23, 2003. Accepted for publication June 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. von Boehmer, H.. 1994. Positive selection of lymphocytes. Cell 76:219.[Medline]
  2. Shevach, E. M.. 2000. Regulatory T cells in autoimmunity. Annu. Rev. Immunol. 18:423.[Medline]
  3. Bensinger, S. J., A. Bandeira, M. S. Jordan, A. J. Caton, T. M. Laufer. 2001. Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4+25+ immunoregulatory T cells. J. Exp. Med. 194:427.[Abstract/Free Full Text]
  4. Mombaerts, P., E. Mizoguchi, M. J. Grusby, L. H. Glimcher, A. K. Bhan, S. Tonegawa. 1993. Spontaneous development of inflammatory bowel disease in T cell receptor mutant mice. Cell 75:275.
  5. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, A. J. Caton. 2001. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2:301.[Medline]
  6. Gilfillan, S., X. Shen, R. Konig. 1998. Selection and function of CD4+ T lymphocytes in transgenic mice expressing mutant MHC class II molecules deficient in their interaction with CD4. J. Immunol. 161:6629.[Abstract/Free Full Text]
  7. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, A. Bandeira. 2001. On the ontogeny and physiology of regulatory T cells. Immunol. Rev. 182:5.[Medline]
  8. Konig, R., X. Shen, R. N. Germain. 1995. Involvement of both major histocompatibility complex class II {alpha} and {beta} chains in CD4 function indicates a role for ordered oligomerization in T cell activation. J. Exp. Med. 182:779.[Abstract/Free Full Text]
  9. Konig, R., L. Y. Huang, R. N. Germain. 1992. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. Nature 356:796.[Medline]
  10. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. deVries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
  11. Friedl, P., P. B. Noble, K. S. Zanker. 1995. T lymphocyte locomotion in a three-dimensional collagen matrix: expression and function of cell adhesion molecules. J. Immunol. 154:4973.[Abstract]
  12. Song, F., K. Ito, T. L. Denning, D. T. Kuninger, J. Papaconstantinou, W. Gourley, G. Klimpel, E. Balish, J. Hokanson, P. B. Ernst. 1999. Expression of the neutrophil chemokine KC in the colon of mice with enterocolitis and by intestinal epithelial cell lines: effects of flora and proinflammatory cytokines. J. Immunol. 162:2275.[Abstract/Free Full Text]
  13. Cook, E. B., J. L. Stahl, L. Lowe, R. Chen, E. Morgan, J. Wilson, R. Varro, A. Chan, F. M. Graziano, N. P. Barney. 2001. Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics. J. Immunol. Methods 254:109.[Medline]
  14. Josephson, K., D. T. McPherson, M. R. Walter. 2001. Purification, crystallization and preliminary x-ray diffraction of a complex between IL-10 and soluble IL-10R1. Acta Crystallogr. D 57:1908.[Medline]
  15. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor. FOXP3. Science 299:1057.[Abstract/Free Full Text]
  16. Denning, T. L., N. A. Campbell, F. Song, R. P. Garofalo, G. R. Klimpel, V. E. Reyes, P. B. Ernst. 2000. Expression of IL-10 receptors on epithelial cells from the murine small and large intestine. Int. Immunol. 12:133.[Abstract/Free Full Text]
  17. Shen, X., B. Hu, P. McPhie, X. Wu, A. Fox, R. N. Germain, R. Konig. 1996. Peptides corresponding to CD4-interacting regions of murine MHC class II molecules modulate immune responses of CD4+ T lymphocytes in vitro and in vivo. J. Immunol. 157:87.[Abstract]
  18. Marguerat, S., H. R. MacDonald, J. P. Kraehenbuhl, J. P. van Meerwijk. 1999. Protection from radiation-induced colitis requires MHC class II antigen expression by cells of hemopoietic origin. J. Immunol. 163:4033.[Abstract/Free Full Text]
  19. Read, S., F. Powrie. 2001. CD4+ regulatory T cells. Curr. Opin. Immunol. 13:644.[Medline]
  20. Elson, C. O.. 1995. Experimental models of inflammatory bowel disease. Gastroenterology 109:1344.[Medline]
  21. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, R. L. Coffman. 1993. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C.B-17 scid mice. Int. Immunol. 5:1461.[Abstract/Free Full Text]
  22. Morrissey, P. J., K. Charrier, S. Braddy, D. Liggitt, J. D. Watson. 1993. CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice: disease development is prevented by cotransfer of purified CD4+ T cells. J. Exp. Med. 178:237.[Abstract/Free Full Text]
  23. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A. Cumano, A. Bandeira. 2001. CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008.[Abstract/Free Full Text]
  24. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
  25. Papiernik, M.. 2001. Natureal CD4+ CD25+ regulatory T cells: their role in the control of superantigen responses. Immunol. Rev. 182:180.[Medline]
  26. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.[Medline]
  27. Lee, H. O., S. D. Miller, S. D. Hurst, L. J. Tan, C. J. Cooper, T. A. Barrett. 2000. Interferon {gamma} induction during oral tolerance reduces T-cell migration to sites of inflammation. Gastroenterology 119:129.[Medline]
  28. Wang, Q., L. Malherbe, D. Zhang, K. Zingler, N. Glaichenhaus, N. Killeen. 2001. CD4 promotes breadth in the TCR repertoire. J. Immunol. 167:4311.[Abstract/Free Full Text]
  29. Fuss, I. J., M. Boirivant, B. Lacy, W. Strober. 2002. The interrelated roles of TGF-{beta} and IL-10 in the regulation of experimental colitis. J. Immunol. 168:900.[Abstract/Free Full Text]
  30. Nakamura, K., A. Kitani, W. Strober. 2001. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor {beta}. J. Exp. Med. 194:629.[Abstract/Free Full Text]
  31. Wildin, R. S., F. Ramsdell, J. Peake, F. Faravelli, J. L. Casanova, N. Buist, E. Levy-Lahad, M. Mazzella, O. Goulet, L. Perroni, et al 2001. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27:18.[Medline]
  32. Khattri, R., D. Kasprowicz, T. Cox, M. Mortrud, M. W. Appleby, M. E. Brunkow, S. F. Ziegler, F. Ramsdell. 2001. The amount of scurfin protein determines peripheral T cell number and responsiveness. J. Immunol. 167:6312.[Abstract/Free Full Text]
  33. Burrows, G. G., Y. K. Chou, C. Wang, J. W. Chang, T. P. Finn, N. E. Culbertson, J. Kim, D. N. Bourdette, D. A. Lewinsohn, D. M. Lewinsohn, et al 2001. Rudimentary TCR signaling triggers default IL-10 secretion by human Th1 cells. J. Immunol. 167:4386.[Abstract/Free Full Text]
  34. Waldmann, H., S. Cobbold. 1998. How do monoclonal antibodies induce tolerance? A role for infectious tolerance. Annu. Rev. Immunol. 16:619.[Medline]
  35. Okamoto, S., M. Watanabe, M. Yamazaki, T. Yajima, T. Hayashi, H. Ishii, M. Mukai, T. Yamada, N. Watanabe, B. A. Jameson, et al 1999. A synthetic mimetic of CD4 is able to suppress disease in a rodent model of immune colitis. Eur. J. Immunol. 29:355.[Medline]
  36. Shaw, A. S., K. E. Amrein, C. Hammond, D. F. Stern, B. M. Sefton, J. K. Rose. 1989. The lck tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain. Cell 59:627.[Medline]



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