In vitro assessment of cord blood–derived proinsulin-specific regulatory T cells for cellular therapy in type 1 diabetes

Background

Antigen-specific regulatory T cells (Tregs) have proven to be effective in reversing established autoimmunity in type 1 diabetes (T1D). Cord blood (CB) can serve as an efficient and safe source for Tregs for antigen-specific immunomodulation in T1D, a strategy that is yet to be explored. Therefore, we assessed the potential of CB in generation of proinsulin (PI)-specific Tregs by using HLA class II tetramers.

Natural but not inducible regulatory T cells require TNF-alpha signaling for in vivo function

TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti-TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2(-/-) nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2(-/-) nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-β did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti-TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.     

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4⁺CD25^- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25⁺ cells prior to immunization, allowing the in vivo formation of Tregs from CD4⁺CD25^- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4⁺CD25⁺ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4⁺CD25^- T cells.     

Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta,Retinoic Acid,Rapamycin and Butyrate

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-β in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-β, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases.     

Natural regulatory T cells and de novo-induced regulatory T cells contribute independently to tumor-specific tolerance

Thymus-derived, naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) and Tregs induced in the periphery (iTregs) have both been implicated in regulating immune responses. However, the relationship between these populations in the same host, and their relative contribution to the overall Treg pool, has not been examined. Using a tumor-induced T cell tolerance model, we find that expansion of nTregs and de novo generation of iTregs both contribute to tumor-specific T cell tolerance. In this system in which the number of tumor-specific nTregs can be controlled, the efficiency of nTreg expansion significantly exceeds that of the induction of Tregs from uncommitted progenitors in the tumor-bearing host. However, pre-existing nTregs are neither required for the induction of Tregs nor measurably impact on the extent of their accumulation. Instead, induction of Ag-specific regulatory cells from naive cells is intrinsically influenced by the tumor microenvironment and the presence of tumor Ag.     

Identification of circulating human antigen-reactive CD4+ FOXP3+ natural regulatory T cells

Circulating human CD4(+)CD25(++)CD127(-)FOXP3(+) T cells with a persistent demethylated regulatory T cell (Treg)-specific demethylated region Foxp3 gene are considered natural Tregs (nTregs). We have shown that it is possible to identify functional Ag-reactive nTregs cells for a range of different common viral and vaccination Ags. The frequency of these Ag-reactive nTregs within the nTreg population is strikingly similar to the frequency of Ag-reactive T effector cells within the CD4(+) T cell population. The Ag-reactive nTregs could be recognized with great specificity by induction of CD154 expression. These CD154(+) Ag-reactive nTregs showed a memory phenotype and shared all phenotypical and functional characteristics of nTregs. The isolated CD154(+) nTregs could be most efficiently expanded by specific antigenic stimulation, while their Ag-reactive suppressive activity was maintained. After an in vivo booster Ag challenge, the ratio of Ag-reactive T cells to Ag-reactive Tregs increased substantially, which could be attributed to the rise in effector T cells but not Tregs. In conclusion, the nTreg population mirrors the effector T cell population in the frequency of Ag-reactive T cells. Isolation and expansion of functional Ag-reactive nTregs is possible and of potential benefit for specific therapeutic goals.     

Peroxisome proliferator-activated receptor gamma (PPARgamma) and immunoregulation: enhancement of regulatory T cells through PPARgamma-dependent and -independent mechanisms

Peroxisome proliferator-activated receptor (PPAR)gamma is a nuclear hormone receptor primarily characterized for its effect on insulin metabolism. PPARgamma ligands, used to treat human type 2 diabetes, also down-regulate most immune system cells including APCs and pathogenic T cells. These effects putatively underlie the efficacy of PPARgamma ligands in treating animal models of autoimmunity, leading to projections of therapeutic potential in human autoimmunity. However, the relationship between PPARgamma ligands and CD4+CD25+ regulatory T cells (Tregs) has not been examined. Specifically, no studies have examined the role of Tregs in mediating the in vivo immunoregulatory effects of PPARgamma ligands, and there have been no investigations of the use of PPARgamma ligands to treat autoimmunity in the absence of Tregs. We now characterize the novel relationship between ciglitazone, a thiazolidinedione class of PPARgamma ligand, and both murine natural Tregs (nTregs) and inducible Tregs (iTregs). In vitro, ciglitazone significantly enhances generation of iTregs in a PPARgamma-independent manner. Surprisingly, and contrary to the current paradigm, we find that, in a model of graft-vs-host disease, the immunotherapeutic effect of ciglitazone requires the presence of nTregs that express PPARgamma. Overall, our results indicate that, unlike its down-regulatory effect on other cells of the immune system, ciglitazone has an enhancing effect on both iTregs and nTregs, and this finding may have important implications for using PPARgamma ligands in treating human autoimmune disease.     

Optimal Isolation of Functional Foxp3(+) Induced Regulatory T Cells Using DEREG Mice

Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells) mice have greatly helped in exploring the biology of Foxp3(+) Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC)-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3(+) Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs) are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3(+) iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4(+)Foxp3(+) iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP(+)Foxp3(-) cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP(-) cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP(+)Foxp3(+) iTregs exhibited potent suppressive activity comparable to that of natural eGFP(+)Foxp3(+) Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP(+)Foxp3(+) iTregs by use of DEREG mice. Isolation of functional Foxp3(+) iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP expression are driven from distinct Foxp3 loci and because this dichotomy preferentially occurs only under defined in vitro conditions.     

Antigen-specific TGF-β-induced regulatory T cells secrete chemokines, regulate T cell trafficking, and suppress ongoing autoimmunity

The ability to regulate ongoing inflammation using regulatory T cells (Tregs) is under intense investigation. Strategies to induce and expand Ag-specific Tregs are being developed, and whether various types of Tregs are suppressive in the inflammatory conditions associated with ongoing disease needs to be determined. In this study, we report that TGF-β-induced Tregs (iTregs) and expanded Tregs specific for a major self-Ag in autoimmune gastritis suppress inflammation and associated pathology when administered late in the process of ongoing disease. Transferred iTregs localized to the stomach, maintained Foxp3 and suppressor functions, and engaged several distinct mechanisms to alleviate disease progression. In addition to suppressing the production of inflammatory cytokines in the stomach and preventing the destruction of parietal cells, we show that iTregs secrete numerous chemokines and regulate both iTreg and effector T cell trafficking into the stomach. These data support efforts to use iTregs in therapies to treat autoimmunity and inflammatory diseases and provide novel insight into the biological mechanisms of iTreg-mediated immune suppression.     

The type of responder T-cell has a significant impact in a human in vitro suppression assay

Background

In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4⁺CD25^+high, or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4⁺CD25^low T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease.

Methods/Principal Findings

We investigated human CD4⁺CD25^low T cells and compared them to CD4⁺CD25^- T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4⁺CD25^low T cells divided more rapidly than CD4⁺CD25^- T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25^low compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25^low T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively).

Conclusions/Significance

The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases.     

Induced Treg Cells Augment the Th17-Mediated Intestinal Inflammatory Response in a CTLA4-Dependent Manner

Th17 cells and Foxp3⁺ regulatory T cells (Tregs) are thought to promote and suppress inflammatory responses, respectively. However, whether they counteract each other or synergize in regulating immune reactions remains controversial. To determine their interactions, we describe the results of experiments employing mouse models of intestinal inflammation by transferring antigen-specific Th cells (Th1, Th2, and Th17) differentiated in vitro followed by the administration of the cognate antigen via enema. We show that cotransfer of induced Tregs (iTregs) suppressed Th1- and Th2-mediated colon inflammation. In contrast, colon inflammation induced by transfer of Th17 cells, was augmented by the cotransfer of iTregs. Furthermore, oral delivery of antigen potentiated Th17-mediated colon inflammation. Administration of a blocking antibody against cytotoxic T lymphocyte-associated antigen 4 (CTLA4) abrogated the effects of cotransfer of iTregs, while the injection of a soluble recombinant immunoglobulin (Ig) fusion protein, CTLA4-Ig substituted for the cotransfer of iTregs. These results suggest that antigen-specific activation of iTregs in a local environment stimulates the Th17-mediated inflammatory response in a CTLA4-dependent manner.     

Autoantigen-specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T cells

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+ Foxp3- T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFbeta. TGFbeta-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.     

CD8+ Foxp3+ regulatory T cells are induced during graft-versus-host disease and mitigate disease severity

Regulatory T cells (Tregs), in particular CD4(+) Foxp3(+) T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8(+) Foxp3(+) T cells that are induced early during graft-versus-host disease (GVHD), constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4(+) Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4(+)-induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8(+) and CD4(+) iTregs had accelerated GVHD mortality compared with animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4(+) and CD8(+) T cells that secreted IFN-γ and IL-17. The presence of CD8(+) iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4(+) Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8(+) iTregs could compensate for CD4(+) iTregs. These studies define a novel population of CD8(+) Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.     

Natural naive CD4+CD25+CD127low regulatory T cell (Treg) development and function are disturbed in multiple sclerosis patients: recovery of memory Treg homeostasis during disease progression

Patients with relapsing-remitting multiple sclerosis (RR-MS) show a suboptimal CD4(+)CD25(+) regulatory T cell (Treg) function, whereas no Treg alterations are observed in secondary progressive MS (SP-MS) patients. To clarify the difference in Treg activity between early and chronic disease stages in MS, we analyzed the functional capacity and homeostatic parameters of naive CD4(+)CD25(+)CD127(low)CD45RA(+) Tregs (nTregs) and their memory counterparts CD4(+)CD25(+)CD127(low)CD45RO(+) Tregs (mTregs) in untreated MS patients and healthy controls. Interestingly, whereas the suppressive capacity of FACS-sorted nTregs was impaired in both early and chronic MS patients, only the latter group showed a restored mTreg function. Consistent with this observation, chronic MS patients had increased numbers of mTregs as compared with age-matched early MS patients, whereas nTreg frequencies did not differ significantly. TCR excision circle numbers were reduced in nTregs of early MS patients, suggestive of a diminished nTreg thymic output. Moreover, a decreased number of CD31(+) mTregs were observed in early vs chronic MS patients, indicating that inflammatory processes drive the homeostatic turnover of mTregs during the early disease stage. Additionally, early MS patients showed a more restricted nTreg and mTreg TCR BV gene profile as compared with healthy controls and chronic MS patients. Finally, analysis of IFN-beta and glatiramer acetate-treated MS patients showed that these immunomodulatory drugs modify nTreg homeostasis. Taken together, this study provides strong evidence for a disturbed thymic nTreg development and function in MS patients. Moreover, memory Treg but not naive Treg homeostasis recovers during disease progression.     

Pilus backbone protein PitB of Streptococcus pneumoniae contains stabilizing intramolecular isopeptide bonds

Naturally occurring CD4⁺CD25⁺ regulatory T cells (nTregs) play a pivotal role in the maintenance of self-tolerance and immune homeostasis. To gain insight into the mechanism of action of nTregs in pathological and physiological immune responses, it is important to analyze bioactive molecules that modulate the maintenance and function of nTregs. From a library of bioactive lipids, we obtained lysophosphatidylcholine (LPC) as a molecule that enhanced the Foxp3 expression and suppressive function of human nTregs significantly in comparison with those of DMSO-treated nTregs (control). The expression levels of TGF-β1 mRNA and protein in LPC-treated nTregs were significantly higher than those in control nTregs. After treatment with anti-TGF-β1 antibody, the increases in Foxp3 expression and the suppressive properties of LPC-treated nTregs returned to the levels observed in control nTregs. These findings indicate that LPC enhances Foxp3 expression and the suppressive function of nTregs through TGF-β1 produced by nTregs themselves. Experimental knockdown of G2A and GPR4 showed that this LPC-induced TGF-β1 expression in nTregs was due to G2A signaling, and did not involve GPR4. Moreover, JNK was a major contributor to LPC-induced TGF-β1 expression in nTregs, although LPC activated MAPKs including ERK1/2, p38 MAPK, and JNK via G2A. LPC is a bioactive lysolipid highly abundant in the circulation. Therefore, LPC may contribute to the maintenance and function of human nTregs in vivo.     

Interaction between Treg apoptosis pathways, Treg function and HLA risk evolves during type 1 diabetes pathogenesis

We have previously reported increased apoptosis of regulatory T cells (Tregs) in recent-onset Type 1 Diabetes subjects (RO T1D) in the honeymoon phase and in multiple autoantibody-positive (Ab+) subjects, some of which are developing T1D. We have also reported that increased Treg apoptosis was associated with High HLA risk and that it subsided with cessation of honeymoon period. In this report, we present results generated using genetics, genomics, functional cell-based assays and flow cytometry to assess cellular changes at the T-cell level during T1D pathogenesis. We measured ex vivo Treg apoptosis and Treg function, surface markers expression, expression of HLA class II genes, the influence of HLA risk on Treg apoptosis and function, and evaluated contribution of genes reported to be involved in the apoptosis process. This integrated comprehensive approach uncovered important information that can serve as a basis for future studies aimed to modulate Treg cell responsiveness to apoptotic signals in autoimmunity. For example, T1D will progress in those subjects where increased Treg apoptosis is accompanied with decreased Treg function. Furthermore, Tregs from High HLA risk healthy controls had increased Treg apoptosis levels and overexpressed FADD but not Fas/FasL. Tregs from RO T1D subjects in the honeymoon phase were primarily dying through withdrawal of growth hormones with contribution of oxidative stress, mitochondrial apoptotic pathways, and employment of TNF-receptor family members. Ab+ subjects, however, expressed high inflammation level, which probably contributed to Treg apoptosis, although other apoptotic pathways were also activated: withdrawal of growth hormones, oxidative stress, mitochondrial apoptosis and Fas/FasL apoptotic pathways. The value of these results lie in potentially different preventive treatment subjects would receive depending on disease progression stage when treated.     

Analysis of circulating regulatory T cells in patients with metastatic prostate cancer pre- versus post-vaccination

We have previously shown that the suppressive function of regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) is enhanced in patients with prostate cancer when compared with healthy individuals. Two phase II studies using the PSA-TRICOM vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC) showed evidence of patient benefit in terms of enhanced survival. The Halabi nomogram has been used to predict survival (HPS) of patients with mCRPC treated with conventional chemotherapy or second-line hormonal therapy. Tregs from PBMCs of patients (n = 23) with mCRPC were obtained pre- and post-three monthly vaccinations, and analyzed for number, phenotype, and suppressive function. Changes post- versus pre-vaccination in these parameters were compared with 3-year survival and HPS. No differences in Treg numbers were observed post- versus pre-vaccination. Trends (P = 0.029) were observed between overall survival (OS) and a decrease in Treg suppressive function post- versus pre-vaccination. Trends were also observed in analyzing effector:Treg (CD4⁺CD25⁺CD127⁻FoxP3⁺CTLA4⁺) ratio post- versus pre-vaccination with OS versus HPS. These data provide preliminary evidence for a possible association between improved OS and a decrease in Treg function when PBMCs are analyzed after three monthly vaccinations. Patients with an OS > HPS were more likely to have decreased Treg function following vaccine. Larger studies to confirm and extend these findings are warranted.     

Myelin-reactive, TGF-β-induced regulatory T cells can be programmed to develop Th1-like effector function but remain less proinflammatory than myelin-reactive Th1 effectors and can suppress pathogenic T cell clonal expansion in vivo

Interest in the use of regulatory T cells (Tregs) as cellular therapeutics has been tempered by reports of naturally occurring Tregs losing Foxp3 expression and producing IL-17, raising concerns over a switch to pathogenic function under inflammatory conditions in vivo. TGF-β-induced Tregs (inducible Tregs [iTregs]), generated in large numbers in response to disease-relevant Ags, represent the most amenable source of therapeutic Tregs. Using Foxp3-reporter T cells recognizing myelin basic protein (MBP), we investigated the capacity of iTregs to produce effector-associated cytokines under proinflammatory cytokine conditions in vitro and whether this translated into proinflammatory function in vivo. In contrast with naturally occurring Tregs, iTregs resisted conversion to an IL-17-producing phenotype but were able to express T-bet and to produce IFN-γ. iTregs initiated their T-bet expression during their in vitro induction, and this was dependent on exposure to IFN-γ. IL-12 reignited iTreg expression of T-bet and further promoted iTreg production of IFN-γ upon secondary stimulation. Despite losing Foxp3 expression and expressing both T-bet and IFN-γ, MBP-responsive IL-12-conditioned iTregs induced only mild CNS inflammation and only when given in high numbers. Furthermore, iTregs retained an ability to suppress naive T cell clonal expansion in vivo and protected against the development of experimental autoimmune encephalomyelitis. Therefore, despite bearing predictive hallmarks of pathogenic effector function, previously Foxp3(+) iTregs have much lower proinflammatory potential than that of MBP-responsive Th1 cells. Our results demonstrate that autoprotective versus autoaggressive functions in iTregs are not simply a binary relationship to be determined by their relative expression of Foxp3 versus T-bet and IFN-γ.