CD4+ T cells from the TCR transgenic TxA23 mouse recognize a peptide from the H/K-ATPase α-chain. When TxA23 CD4+ thymocytes are differentiated into Th1, Th2, and Th17 lines, all three subpopulations induced autoimmune gastritis (AIG) upon transfer into nu/nu recipients. The induction of AIG by naive T cells or by Th1 or Th2 cell lines could be prevented by the cotransfer of polyclonal Foxp3+ T regulatory cells (nTreg), whereas Th17-induced AIG was resistant to suppression. We compared the capacity of different types of Treg to suppress Th17-mediated AIG. Cotransfer of either nTreg or polyclonal TGFβ-induced Treg (iTreg) did not prevent AIG, while cotransfer of TGFβ-induced Ag-specific TxA23 iTreg completely prevented the development of disease. Ag-specific iTreg were able to suppress Th17-mediated disease when injected 6 days after the Th17 effectors. The implications of these results for the use of Treg for the cellular biotherapy of autoimmune disease are discussed.
The concept that organ-specific autoimmune disease is a result of dysregulated autoantigen-specific Th1 responses has recently been broadened by the discovery of IL17-producing helper T cells designated Th17 (1). Th17 cells are involved in the pathogenesis of autoimmune diseases such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, which previously were regarded as exclusively Th1-mediated (2). The cytokine IL-17 has proinflammatory effects and induces tissue damage during the course of various autoimmune diseases (3). We have developed a TCR transgenic mouse model of autoimmune gastritis (AIG)4 in which the TCR transgenic (TxA23) T cells recognize a peptide from the H/K-ATPase α-chain (4) and the transgenic mice spontaneously develop AIG early in life. When naive TxA23 thymocytes are differentiated in culture into Th1, Th2, and Th17 lines, all three T cell subpopulations were capable of inducing AIG upon transfer into immunoincompetent BALB/c nu/nu recipients (5). When compared with in vitro differentiated transgenic Th1 and Th2 cells, Th17 induced a distinct histological pattern of autoimmune gastritis characterized by a highly destructive disease with cellular infiltrates composed primarily of eosinophils, accumulation of transgenic CD4+ T effectors in the gastric mucosa and draining lymph nodes, and high levels of serum IgE (5). Cotransfer of polyclonal Foxp3+ regulatory T cells (nTreg) markedly suppressed disease induced by naive CD4+ T cells (6) or fully differentiated Th1 and Th2 lines (5). In contrast, disease induced by Th17 lines was highly resistant to suppression even at a high regulatory T cell (Treg) to effector ratio (20:1).
The resistance of Th17-induced AIG to suppression by Treg raised the possibility that Th17 cells are intrinsically not responsive to suppression by Treg or that the inflammatory milieu generated by Th17 cells induced the production of cytokines that rendered the effector T cells resistant to suppression or neutralized the suppressive effects of the Treg (7). We (8) and others (9) have previously demonstrated that autoantigen-specific Treg are much more potent suppressors of the induction of autoimmune disease than polyclonal Treg. As it is relatively easy to generate a large number of either polyclonal or Ag-specific activated TGFβ-induced Treg (iTreg) that are potent suppressors of the induction of organ-specific (8) and systemic autoimmune disease (10), we compared these iTreg populations for their ability to suppress AIG induced by fully differentiated autoantigen-specific Th17 lines. We demonstrate that neither polyclonal nTreg nor TGFβ-induced polyclonal iTreg could prevent disease induced by Th17 cells. In contrast, cotransfer of TGFβ-induced Ag-specific iTreg completely prevented tissue destruction in the gastric mucosa and reduced the numbers of transgenic effector T cells in the gastric lymph node (gLN). Furthermore, in a therapeutic approach, TxA23 transgenic iTreg suppressed Th17-mediated AIG when injected one week after transfer of the Th17 effectors. Thus, TGFβ-induced Ag-specific iTreg are capable of both preventing and treating AIG induced by fully differentiated Th17 effector cells.
Materials and Methods
Mice and reagents
Female BALB/c Thy1.1+ and BALB/c nu/nu (4–8 wk old) mice were purchased from the National Cancer Institute animal facility (Frederick, MD) and housed under specific pathogen-free conditions. TxA23 TCR transgenic mice have been described previously (11). All mice were maintained in our animal facility and cared for in accordance with institutional guidelines.
T cell purification and differentiation
Autoantigen-specific Th17 cell lines were generated from FACS-sorted CD4+CD8−CD25− thymocytes from TxA23 Thy1.1+/1.2+ mice as previously described (5, 8). nTreg were isolated from single cell suspensions of spleen cells from BALB/c Thy1.1+ mice as previously described (5). Optimal differentiation of Th17 cells was verified after 4–7 days of differentiation in vitro by intracellular staining for IL-17 expression. Polyclonal wild-type (WT) iTreg were generated from the CD4+CD25− T cells from BALB/c Thy1.1+ mice as previously described (10). Ag-specific iTreg were generated from naive CD4+CD8−CD25− thymocytes from TxA23 Thy1.1+ mice and were isolated and differentiated as previously described (8). On days 4–7 of culture the expression of FoxP3 was measured by flow cytometry and only populations with 80–99% FoxP3+ T cells were used for injection.
Cell transfer experiments
The TxA23 Th17 effector T cells and Treg were washed twice with PBS and diluted into PBS such that an i.v. injection of 0.25 ml/mouse resulted in the transfer of 50 × 103 TxA23 Thy1.1+/Thy1.2+ Th17 cells and 1 × 106 Thy1.1+ Treg, respectively.
Abs and flow cytometry
Cell surface staining was performed using Abs against CD25 (PC61), Thy-1.1, and Thy-1.2 directly conjugated to FITC or PE (all from BD Pharmingen). Intracellular cytokine staining was performed using mAbs for intracellular stainings (anti-IL-4, anti-IL-10, anti-IL-17, anti-IFN-γ, anti-TNFα) conjugated to PE or allophycocyanin (all from BD Pharmingen) as described previously (5). Allophycocyanin-conjugated mAb to FoxP3 (FJK-16s) was purchased from eBioscience. All flow cytometry was performed on a FACScan or FACSCalibur (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).
All group results are expressed as mean ± SD if not stated otherwise. Student’s t test was used for the comparison of group values and discriminatory parameters where appropriate. For comparing group values that did not follow Gaussian distribution, Mann-Whitney U test (two-tailed) was used. A value of p < 0.05 was considered significant.
Results and Discussion
Naive CD4+CD8−CD25− TxA23 thymocytes were stimulated in vitro for 5 days under Th17-polarizing conditions. After restimulation with PMA/ionomycin, the majority of Th17 cells produce IL-17 (Fig. 1,A, left panel). Because transfer of naive TxA23 CD4+ T cells induces a Th1 disease with IFN-γ as the main cytokine produced, we also measured IFN-γ in the in vitro differentiated Th17 cells. No IFN-γ was detectable by intracellular staining of different preparations of Th17 cells (Fig. 1,A, right panel). The in vitro differentiated Th17 cells also did not produce Th2 cytokines (IL-4, IL-5, IL-10, or TNFα) as measured by intracellular staining (data not shown). Stimulation of conventional CD4+CD25− T cells via the TCR in the presence of TGFβ and IL-2 induces FoxP3 expression and a regulatory phenotype (10, 12). We differentiated TGFβ-induced iTreg from naive Ag-specific TxA23 T cells and from naive WT CD4+CD25− T cells. On day 5 of the in vitro culture, the cells were stained for FoxP3 expression. Both populations of TGFβ-induced iTreg expressed high levels of FoxP3 comparable to freshly isolated nTreg (Fig. 1 B).
To compare the suppressive capacity of the different types of Treg, we cotransferred Th17 effector T cells with Treg at a fixed ratio of 1:20 (50 × 103 Th17 effectors and 1 × 106 Treg). Transfer of Th17 effector cells alone induced destructive AIG (score 5–6). Six wk after transfer, the majority of mice showed high-grade AIG as defined by severe or complete parietal cell loss with replacement by so-called “foamy cells,” epithelial hyperplasia, and massive inflammatory infiltrates (Fig. 2,A). Cotransfer of either nTreg or polyclonal iTreg did not prevent the development of destructive gastritis (Fig. 2,A). In contrast, cotransfer of TxA23 iTreg completely prevented development of disease, and destructive tissue pathology was not observed in any of the animals cotransferred with the Ag-specific iTreg (Fig. 2, A and B). The results of a large series of cotransfers are summarized in Fig. 2,B. Cotransfer of nTreg significantly reduced the percentage of animals with destructive gastritis from 85 to 66%, whereas only a slight reduction of the percentage of animals with destructive tissue pathology was seen when WT iTreg were cotransferred. In contrast, Ag-specific iTreg completely prevented development of destructive disease (Fig. 2 B). The enhanced suppressive capacity of the Ag-specific iTreg compared with the polyclonal nTreg was not secondary to their preactivation in vitro, as in vitro preactivated nTreg were no more efficient that freshly isolated nTreg in their capacity to prevent destructive AIG (data not shown).
One of the proposed mechanisms for the therapeutic effect of Ag-specific iTreg in the AIG model is the down-regulation of costimulatory molecules on APC, resulting in inhibition of the expansion of the effector T cells in the draining lymph node (8). The H/K ATPase is presented by dendritic cells in the gLN (13), and it is likely that the initial activation of the effector cells in AIG occurs in the node with subsequent migration to the gastric mucosa. Following transfer of the Th17 effector cells, marked enhancement of the cellularity of the gastric lymph node was observed (Fig. 3,A). Cotransfer of TxA23 iTreg significantly reduced the cellularity in the gLN (Fig. 3,A), whereas neither nTreg nor WT iTreg prevented the increase in cellularity. As the Th17 effector T cells were labeled with a congenic marker, we also calculated the absolute number of Th17 effector cells. Both the percentage of Th17 effectors (Fig. 3,B) and their absolute numbers (Fig. 3,C) were significantly reduced only in animals that received the TxA23 iTreg, but not the nTreg or the polyclonal iTreg. The higher percentages of Ag-specific iTreg in the gLN (Fig. 3 B) could either be due to their preferential homing or increased expansion in comparison to the other Treg populations. CFSE-labeling of all three Treg populations revealed a higher CFSE-dilution in the gLN of the Ag-specific iTreg compared with WT iTreg or nTreg. All three populations displayed similar CFSE profiles in the inguinal LN (data not shown). This result indicates that preferential expansion of the Ag-specific iTreg after Ag encounter in the draining node likely accounts for their higher numbers in the gLN and their higher suppressive capacity toward Th17 effector T cells as compared with WT iTreg or nTreg.
Because the Ag-specific iTreg had a strong suppressive effect on development of AIG in cotransfer experiments, we next asked whether these cells were also capable of suppressing disease in a therapeutic experimental setting. We used the same Treg:T effector ratio of 20:1 as in the previous experiments but injected the Ag-specific iTreg 6 days after transfer of the in vitro differentiated Th17 effector cells. When analyzed 6 wk after transfer of the Th17 effector T cells, marked inhibition of their expansion was seen in the animals that received the iTreg (Fig. 4,A). All animals in the group that received Th17 effector cells alone had developed destructive AIG, whereas cotransfer of Ag-specific iTreg completely prevented development of destructive disease (Fig. 4,B). Furthermore, the cotransferred TxA23 iTreg were also readily identifiable in cell suspensions prepared from the gastric mucosa, and their presence was associated with a reduced percentage of infiltrating Th17 effector T cells (Fig. 4 C).
Th17 cells play an important role in the pathogenesis of many autoimmune diseases, and some studies have suggested that Th17 cells may be resistant to suppression by Treg as they produce or induce the production of cytokines such as IL6 and TNFα that dampen Treg function. For example, even though high numbers of Treg could be detected in the CNS of animals with myelin oligodendrocyte glycoprotein (MOG)35–55-induced experimental autoimmune encephalomyelitis, they could not prevent the function of the pathogenic T effector cells, and T effector cells from the acutely inflamed CNS were resistant to suppression by Treg in vitro (7). Similarly, in our AIG model the disease induced by Th17 effectors was highly aggressive and resistant to suppression by polyclonal nTreg. It was therefore of interest to compare the capacity of different Foxp3+ Treg subpopulations to suppress the induction of AIG by fully differentiated Th17 cells. Consistent with our previous results, both polyclonal nTreg and polyclonal iTreg were ineffective in preventing the induction of AIG. In contrast, Ag-specific iTreg were potent inhibitors of disease induction and even exerted considerable therapeutic effects when administered 6 days after transfer of the Th17 at a time point when the inflammatory response in the target organ had already been initiated. Taken together, these results demonstrate that Th17-induced AIG is susceptible to suppression, but only by Ag-specific Treg. As Th17 cells may play an important role in many human autoimmune diseases, these findings have important implications regarding the use of Treg for cellular biotherapy.
We thank C. Henry and T. Moyer for cell sorting and the staff of the animal facilities for their work maintaining the mice.
The authors have no financial conflict of interest.
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.
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.
Abbreviations used in this paper: AIG, autoimmune gastritis; gLN, gastric lymph node; Treg, regulatory T cell; iTreg, TGFβ-induced Treg; nTreg, thymic-derived Treg; WT, wild type.