Reversal of Experimental Autoimmune Diabetes With an sCD39/Anti-CD3 Treatment.

Extracellular (e)ATP, a potent proinflammatory molecule, is released by dying/damaged cells at the site of inflammation and is degraded by the membrane ectonucleotidases CD39 and CD73. In this study, we sought to unveil the role of eATP degradation in autoimmune diabetes. We then assessed the effect of soluble CD39 (sCD39) administration in prevention and reversal studies in NOD mice as well as in mechanistic studies. Our data showed that eATP levels were increased in hyperglycemic NOD mice compared with prediabetic NOD mice. CD39 and CD73 were found expressed by both α- and ß-cells and by different subsets of T cells. Importantly, prediabetic NOD mice displayed increased frequencies of CD3+CD73+CD39+ cells within their pancreata, pancreatic lymph nodes, and spleens. The administration of sCD39 into prediabetic NOD mice reduced their eATP levels, abrogated the proliferation of CD4+- and CD8+-autoreactive T cells, and increased the frequency of regulatory T cells, while delaying the onset of T1D. Notably, concomitant administration of sCD39 and anti-CD3 showed a strong synergism in restoring normoglycemia in newly hyperglycemic NOD mice compared with monotherapy with anti-CD3 or with sCD39. The eATP/CD39 pathway plays an important role in the onset of T1D, and its targeting might represent a potential therapeutic strategy in T1D.

Insulin-like Growth Factor-1 Synergizes with IL-2 to Induce Homeostatic Proliferation of Regulatory T Cells.

IL-2 has been proposed to restore tolerance via regulatory T cell (Treg) expansion in autoimmunity, yet off-target effects necessitate identification of a combinatorial approach allowing for lower IL-2 dosing. We recently reported reduced levels of immunoregulatory insulin-like growth factor-1 (IGF1) during type 1 diabetes progression. Thus, we hypothesized that IGF1 would synergize with IL-2 to expand Tregs. We observed IGF1 receptor was elevated on murine memory and human naive Treg subsets. IL-2 and IGF1 promoted PI3K/Akt signaling in Tregs, inducing thymically-derived Treg expansion beyond either agent alone in NOD mice. Increased populations of murine Tregs of naive or memory, as well as CD5lo polyclonal or CD5hi likely self-reactive, status were also observed. Expansion was attributed to increased IL-2Rγ subunit expression on murine Tregs exposed to IL-2 and IGF1 as compared with IL-2 or IGF1 alone. Assessing translational capacity, incubation of naive human CD4+ T cells with IL-2 and IGF1 enhanced thymically-derived Treg proliferation in vitro, without the need for TCR ligation. We then demonstrated that IGF1 and IL-2 or IL-7, which is also IL-2Rγ-chain dependent, can be used to induce proliferation of genetically engineered naive human Tregs or T conventional cells, respectively. These data support the potential use of IGF1 in combination with common γ-chain cytokines to drive homeostatic T cell expansion, both in vitro and in vivo, for cellular therapeutics and ex vivo gene editing.

CCL21 and beta-cell antigen releasing hydrogels as tolerance-inducing therapy in Type I diabetes.

Type-I Diabetes (T1D) is caused by defective immunotolerance mechanisms enabling autoreactive T cells to escape regulation in lymphoid organs and destroy insulin-producing ß-cells in the pancreas, leading to insulin dependence. Strategies to promote ß-cell tolerance could arrest T1D. We previously showed that secretion of secondary lymphoid chemokine CCL21 by CCL21 transgenic ß-cells induced tolerance and protected non-obese diabetic (NOD) mice from T1D. T1D protection was associated with formation of lymph node-like stromal networks containing tolerogenic fibroblastic reticular cells (FRCs). Here, we developed a polyethylene glycol (PEG) hydrogel platform with hydrolytically degradable PEG-diester dithiol crosslinkers to provide controlled and sustained delivery of CCL21 and ß-cell antigens for at least 28 days in vitro and recapitulate properties associated with the tolerogenic environment of CCL21 transgenic ß-cells in our previous studies. CCL21 and MHC-II restricted antigens were tethered to gels via simple click-chemistry while MHC-I restricted antigens were loaded in PEG-based polymeric nanovesicles and incorporated in the gel networks. CCL21 and antigen release kinetics depended on the PEG gel tethering strategy and the linkers. Importantly, in vitro functionality, chemotaxis, and activation of antigen-specific T cells were preserved. Implantation of CCL21 and ß-cell antigen gels under the kidney capsule of pre-diabetic NOD mice led to enrichment of adoptively transferred antigen-specific T cells, formation of gp38 + FRC-like stromal cell networks, and increased regulation of specific T cells with reduced accumulation within pancreatic islets. Thus, our platform for sustained release of ß-cell antigens and CCL21 immunomodulatory molecule could enable the development of antigen-specific tolerance therapies for T1D.

Immunosuppressive PLGA TGF-ß1 Microparticles Induce Polyclonal and Antigen-Specific Regulatory T Cells for Local Immunomodulation of Allogeneic Islet Transplants.

Allogeneic islet transplantation is a promising cell-based therapy for Type 1 Diabetes (T1D). The long-term efficacy of this approach, however, is impaired by allorejection. Current clinical practice relies on long-term systemic immunosuppression, leading to severe adverse events. To avoid these detrimental effects, poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were engineered for the localized and controlled release of immunomodulatory TGF-ß1. The in vitro co-incubation of TGF-ß1 releasing PLGA MPs with naïve CD4+ T cells resulted in the efficient generation of both polyclonal and antigen-specific induced regulatory T cells (iTregs) with robust immunosuppressive function. The co-transplantation of TGF-ß1 releasing PLGA MPs and Balb/c mouse islets within the extrahepatic epididymal fat pad (EFP) of diabetic C57BL/6J mice resulted in the prompt engraftment of the allogenic implants, supporting the compatibility of PLGA MPs and local TGF-ß1 release. The presence of the TGF-ß1-PLGA MPs, however, did not confer significant graft protection when compared to untreated controls, despite measurement of preserved insulin expression, reduced intra-islet CD3+ cells invasion, and elevated CD3+Foxp3+ T cells at the peri-transplantation site in long-term functioning grafts. Examination of the broader impacts of TGF-ß1/PLGA MPs on the host immune system implicated a localized nature of the immunomodulation with no observed systemic impacts. In summary, this approach establishes the feasibility of a local and modular microparticle delivery system for the immunomodulation of an extrahepatic implant site. This approach can be easily adapted to deliver larger doses or other agents, as well as multi-drug approaches, within the local graft microenvironment to prevent transplant rejection.

In vitro platform establishes antigen-specific CD8+ T cell cytotoxicity to encapsulated cells via indirect antigen recognition.

The curative potential of non-autologous cellular therapy is hindered by the requirement of anti-rejection therapy. Cellular encapsulation within nondegradable biomaterials has the potential to inhibit immune rejection, but the efficacy of this approach in robust preclinical and clinical models remains poor. While the responses of innate immune cells to the encapsulating material have been characterized, little attention has been paid to the contributions of adaptive immunity in encapsulated graft destabilization. Avoiding the limitations of animal models, we established an efficient, antigen-specific in vitro platform capable of delineating direct and indirect host T cell recognition to microencapsulated cellular grafts and evaluated their consequential impacts. Using ovalbumin (OVA) as a model antigen, we determined that alginate microencapsulation abrogates direct CD8+ T cell activation by interrupting donor-host interaction; however, indirect T cell activation, mediated by host antigen presenting cells (APCs) primed with shed donor antigens, still occurs. These activated T cells imparted cytotoxicity on the encapsulated cells, likely via diffusion of cytotoxic solutes. Overall, this platform delivers unique mechanistic insight into the impacts of hydrogel encapsulation on host adaptive immune responses, comprehensively addressing a long-standing hypothesis of the field. Furthermore, it provides an efficient benchtop screening tool for the investigation of new encapsulation methods and/or synergistic immunomodulatory agents.

Immunomodulation Followed by Antigen-Specific Treg Infusion Controls Islet Autoimmunity.

Optimal immune-based therapies for type 1 diabetes (T1D) should restore self-tolerance without inducing chronic immunosuppression. CD4+Foxp3+ regulatory T cells (Tregs) are a key cell population capable of facilitating durable immune tolerance. However, clinical trials with expanded Tregs in T1D and solid-organ transplant recipients are limited by poor Treg engraftment without host manipulation. We showed that Treg engraftment and therapeutic benefit in nonautoimmune models required ablative host conditioning. Here, we evaluated Treg engraftment and therapeutic efficacy in the nonobese diabetic (NOD) mouse model of autoimmune diabetes using nonablative, combinatorial regimens involving the anti-CD3 (αCD3), cyclophosphamide (CyP), and IAC (IL-2/JES6-1) antibody complex. We demonstrate that αCD3 alone induced substantial T-cell depletion, impacting both conventional T cells (Tconv) and Tregs, subsequently followed by more rapid rebound of Tregs Despite robust depletion of host Tconv and host Tregs, donor Tregs failed to engraft even with interleukin-2 (IL-2) support. A single dose of CyP after αCD3 depleted rebounding host Tregs and resulted in a 43-fold increase in donor Treg engraftment, yet polyclonal donor Tregs failed to reverse diabetes. However, infusion of autoantigen-specific Tregs after αCD3 alone resulted in robust Treg engraftment within the islets and induced remission in all mice. This novel combinatorial therapy promotes engraftment of autoantigen-specific donor Tregs and controls islet autoimmunity without long-term immunosuppression.

CCL21 Expression in ß-Cells Induces Antigen-Expressing Stromal Cell Networks in the Pancreas and Prevents Autoimmune Diabetes in Mice.

Tumors induce tolerance toward their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, nonobese diabetic (NOD) mice express CCL21 in pancreatic ß-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes. Ins2-CCL21 mice develop TLOs by 4 weeks of age, consisting of naive CD4+ T cells compartmentalized within networks of CD45-gp38+CD31- fibroblastic reticular cell (FRC)-like cells. Importantly, 12-week-old Ins2-CCL21 TLOs contained FRC-like cells with higher contractility, regulatory, and anti-inflammatory properties and enhanced expression of ß-cell autoantigens compared with nontransgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and a higher proportion of regulatory T cells in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for the regulation of autoimmunity, and although the effect is systemic, the induction is mediated locally likely by lymphocyte trafficking through TLOs. Overall, our findings suggest that CCL21 promotes TLOs that differ from inflammatory TLOs found in type 1 diabetic islets in that they resemble lymph nodes, contain FRC-like cells expressing ß-cell autoantigens, and are able to induce systemic and antigen-specific tolerance leading to diabetes prevention.

Immunoisolation of murine islet allografts in vascularized sites through conformal coating with polyethylene glycol.

Islet encapsulation may allow transplantation without immunosuppression, but thus far islets in large microcapsules transplanted in the peritoneal cavity have failed to reverse diabetes in humans. We showed that islet transplantation in confined well-vascularized sites like the epididymal fat pad (EFP) improved graft outcomes, but only conformal coated (CC) islets can be implanted in these sites in curative doses. Here, we showed that CC using polyethylene glycol (PEG) and alginate (ALG) was not immunoisolating because of its high permselectivity and strong allogeneic T cell responses. We refined the CC composition and explored PEG and islet-like extracellular matrix (Matrigel; MG) islet encapsulation (PEG MG) to improve capsule immunoisolation by decreasing its permselectivity and immunogenicity while allowing physiological islet function. Although the efficiency of diabetes reversal of allogeneic but not syngeneic CC islets was lower than that of naked islets, we showed that CC (PEG MG) islets from fully MHC-mismatched Balb/c mice supported long-term (>100 days) survival after transplantation into diabetic C57BL/6 recipients in the EFP site (750-1000 islet equivalents/mouse) in the absence of immunosuppression. Lack of immune cell penetration and T cell allogeneic priming was observed. These studies support the use of CC (PEG MG) for islet encapsulation and transplantation in clinically relevant sites without chronic immunosuppression.

Altered homeostasis and development of regulatory T cell subsets represent an IL-2R-dependent risk for diabetes in NOD mice.

The cytokine interleukin-2 (IL-2) is critical for the functions of regulatory T cells (Tregs). The contribution of polymorphisms in the gene encoding the IL-2 receptor α subunit (IL2RA), which are associated with type 1 diabetes, is difficult to determine because autoimmunity depends on variations in multiple genes, where the contribution of any one gene product is small. We investigated the mechanisms whereby a modest reduction in IL-2R signaling selectively in T lymphocytes influenced the development of diabetes in the NOD mouse model. The sensitivity of IL-2R signaling was reduced by about two- to threefold in Tregs from mice that coexpressed wild-type IL-2Rß and a mutant subunit (IL-2RßY3) with reduced signaling (designated NOD-Y3). Male and female NOD-Y3 mice exhibited accelerated diabetes onset due to intrinsic effects on multiple activities in Tregs Bone marrow chimera and adoptive transfer experiments demonstrated that IL-2RßY3 Tregs resulted in impaired homeostasis of lymphoid-residing central Tregs and inefficient development of highly activated effector Tregs and that they were less suppressive. Pancreatic IL-2RßY3 Tregs showed impaired development into IL-10-secreting effector Tregs The pancreatic lymph nodes and pancreases of NOD-Y3 mice had increased numbers of antigen-experienced CD4+ effector T cells, which was largely due to impaired Tregs, because adoptively transferred pancreatic autoantigen-specific CD4+ Foxp3- T cells from NOD-Y3 mice did not accelerate diabetes in NOD.SCID recipients. Our study indicates that the primary defect associated with chronic, mildly reduced IL-2R signaling is due to impaired Tregs that cannot effectively produce and maintain highly functional tissue-seeking effector Treg subsets.

The Folate Cycle As a Cause of Natural Killer Cell Dysfunction and Viral Etiology in Type 1 Diabetes.

The folate pathway is critical to proper cellular function and metabolism. It is responsible for multiple functions, including energy (ATP) production, methylation reactions for DNA and protein synthesis and the production of immunomodulatory molecules, inosine and adenosine. These play an important role in immune signaling and cytotoxicity. Herein, we hypothesize that defects in the folate pathway in genetically susceptible individuals could lead to immune dysfunction, permissive environments for chronic cyclical latent/lytic viral infection, and, ultimately, the development of unchecked autoimmune responses to infected tissue, in this case islet beta cells. In the context of type 1 diabetes (T1D), there has been a recent increase in newly diagnosed cases of T1D in the past 20 years that has exceeded previous epidemiological predictions with yet unidentified factor(s). This speaks to a potential environmental trigger that adversely affects immune responses. Most research into the immune dysfunction of T1D has focused on downstream adaptive responses of T and B cells neglecting the role of the upstream innate players such as natural killer (NK) cells. Constantly, surveilling the blood and tissues for pathogens, NK cells remove threats through direct cytotoxic responses and recruitment of adaptive responses using cytokines, such as IL-1ß and IFN-γ. One long-standing hypothesis suggests viral infection as a potential trigger for the autoimmune response in T1D. Recent data suggest multiple viruses as potential causal agents. Intertwined with this is an observed reduced NK cell enumeration, cytotoxicity, and cytokine signaling in T1D patients. Many of the viruses implicated in T1D are chronic latent/lysogenic infections with demonstrated capacity to reduce NK cell response and number through mechanisms that resemble those of pregnancy tolerance. Defects in the folate pathway in T1D patients could result in decreased immune response to viral infection or viral reactivation. Dampened NK responses to infections result in improper signaling, improper antigen presentation, and amplified CD8+ lymphocyte proliferation and cytotoxicity, a hallmark of beta cell infiltrates in patients with T1D onset. This would suggest a critical role for NK cells in T1D development linked to viral infection and the importance of the folate pathway in maintaining proper NK response.

The Expanding Role of Natural Killer Cells in Type 1 Diabetes and Immunotherapy.

Treatments for autoimmune diseases including type 1 diabetes (T1D) are aimed at resetting the immune system, especially its adaptive arm. The innate immune system is often ignored in the design of novel immune-based therapies. There is increasing evidence for multiple natural killer (NK) subpopulations, but their role is poorly understood in autoimmunity and likely is contributing to the controversial role reported for NKs. In this review, we will summarize NK subsets and their roles in tolerance, autoimmune diabetes, and immunotherapy.

Adoptive T Regulatory Cell Therapy for Tolerance Induction.

There is a clear need to develop strategies to induce tolerance without the need of chronic immunosuppression in transplant recipient and in patients with autoimmunity. Adoptive T regulatory cell (Treg) therapy offers the potential of long-lasting protection. However, based on results of clinical trials so far with ex vivo expanded autologous Tregs in type 1 diabetic (T1D) patients, it seems unlikely that single immunotherapy with Treg infusion without immunomodulation regimens that promote stable donor Treg engraftment and persistence would afford truly significant clinical benefit. Combination therapies could provide improved outcomes with consideration of the fundamental factors required for Treg generation, homeostasis, and function to promote long-term donor Treg persistence to provoke beneficial therapeutic outcomes.

The IL-2/IL-2R system: from basic science to therapeutic applications to enhance immune regulation.

IL-2 plays a critical role in the normal function of the immune system. A trophic factor for lymphocytes, IL-2 is required for mounting and sustaining adaptive T cell responses; however, IL-2 is also critical for immune regulation via its effects on regulatory T cells (Treg cells). Over the years, we have contributed to the understanding of the biology of IL-2 and its signaling through the IL-2 receptor and helped define the key role played by IL-2 in Treg development and function. Our data show that Treg cells have a heightened sensitivity to IL-2, which may create a therapeutic window to promote immune regulation by selective stimulation of Treg cells. We are now developing new efforts to translate this knowledge to the clinical arena, through our focused interest in Type 1 diabetes as a prototypic autoimmune disease. Specifically, we aim at developing IL-2-based therapeutic regimens and incorporate means to enhance antigen-specific Treg responses, for improved and more selective regulation of islet autoimmunity. In parallel, we are pursuing studies in preclinical models of autoimmunity and transplantation to define critical factors for successful adoptive Treg therapy and develop clinically applicable therapeutic protocols.

Prevention of autoimmune diabetes and induction of ß-cell proliferation in NOD mice by hyperbaric oxygen therapy.

We evaluated the effects of hyperbaric oxygen therapy (HOT) on autoimmune diabetes development in nonobese diabetic (NOD) mice. Animals received no treatment or daily 60-min HOT 100% oxygen (HOT-100%) at 2.0 atmospheres absolute and were monitored for diabetes onset, insulitis, infiltrating cells, immune cell function, and ß-cell apoptosis and proliferation. Cyclophosphamide-induced diabetes onset was reduced from 85.3% in controls to 48% after HOT-100% (P < 0.005) and paralleled by lower insulitis. Spontaneous diabetes incidence reduced from 85% in controls to 65% in HOT-100% (P = 0.01). Prediabetic mice receiving HOT-100% showed lower insulitis scores, reduced T-cell proliferation upon stimulation in vitro (P < 0.03), increased CD62L expression in T cells (P < 0.04), reduced costimulation markers (CD40, DC80, and CD86), and reduced major histocompatibility complex class II expression in dendritic cells (DCs) (P < 0.025), compared with controls. After autoimmunity was established, HOT was less effective. HOT-100% yielded reduced apoptosis (transferase-mediated dUTP nick-end labeling-positive insulin-positive cells; P < 0.01) and increased proliferation (bromodeoxyuridine incorporation; P < 0.001) of insulin-positive cells compared with controls. HOT reduces autoimmune diabetes incidence in NOD mice via increased resting T cells and reduced activation of DCs with preservation of ß-cell mass resulting from decreased apoptosis and increased proliferation. The safety profile and noninvasiveness makes HOT an appealing adjuvant therapy for diabetes prevention and intervention trials.

Expansion of a restricted residual host T reg-cell repertoire is dependent on IL-2 following experimental autologous hematopoietic stem transplantation.

We previously identified a population of residual T(reg) cells following autologous hematopoietic stem transplantation (HSCT), that rapidly undergoes significant expansion in lymphopenic transplant recipients prior to repopulation by donor de novo derived T(reg) cells. These CD4(+) Foxp3(+) T cells provide protection from the development of autoimmune disease. Although ablative conditioning results in excess IL-7 and IL-15, IL-2 is typically not found at high levels following autologous HSCT. We therefore examined the role of these three STAT-5 signaling cytokines in the expansion of residual T(reg) cells after autologous HSCT. The present study found that the residual T(reg) cell population included surviving peripheral host Foxp3(+) CD4(+) T cells whose expansion was critically dependent on IL-2, which could be solely provided by surviving host cells. IL-7 was found to contribute to T(reg) cell homeostasis, however, not as a growth factor but rather for their persistence. In conjunction with this expansion, TCR spectratype analyses revealed that the residual host T(reg) -cell compartment differed from that present in non-conditioned healthy mice since the residual host Treg cells exhibit a limited TCR diversity. Collectively, these data indicate that the proliferation of T(reg) and T effector (T(eff) ) cells post-HSCT utilize separate pools of cytokines which has important implications regarding the development of clinical strategies to elicit the desired immune responses in patients post-transplant.

High-resolution, noninvasive longitudinal live imaging of immune responses.

Intravital imaging emerged as an indispensible tool in biological research, and a variety of imaging techniques have been developed to noninvasively monitor tissues in vivo. However, most of the current techniques lack the resolution to study events at the single-cell level. Although intravital multiphoton microscopy has addressed this limitation, the need for repeated noninvasive access to the same tissue in longitudinal in vivo studies remains largely unmet. We now report on a previously unexplored approach to study immune responses after transplantation of pancreatic islets into the anterior chamber of the mouse eye. This approach enabled (i) longitudinal, noninvasive imaging of transplanted tissues in vivo; (ii) in vivo cytolabeling to assess cellular phenotype and viability in situ; (iii) local intervention by topical application or intraocular injection; and (iv) real-time tracking of infiltrating immune cells in the target tissue.

Host CD4+CD25+ T cells can expand and comprise a major component of the Treg compartment after experimental HCT.

Reconstitution of the recipient lymphoid compartment following hematopoietic cell transplantation (HCT) is typically delayed. The present studies investigated the residual host CD4(+)CD25(+)Foxp3(+) (Treg) compartment after several conditioning regimens, including T cell-depleted and T cell-replete HCT and observed (1) a small number of recipient Treg cells survived aggressive conditioning; (2) the surviving, that is, residual Tregs underwent marked expansion; and (3) recipient CD4(+)FoxP3(+) cells composed the majority of the Treg compartment for several months post-syngeneic HCT. Notably, residual Tregs also dominated the compartment post-HCT with T cell-depleted (TCD) major histocompatibility complex-matched allogeneic bone marrow but not following T cell-replete transplantations. The residual Treg cell compartment was functionally competent as assessed by in vitro lymphoid suppression and in vivo autoimmune disease transfer assay. These observations support the notion that functional host Tregs initially occupy a niche in lymphopenic transplantation recipients, undergo significant expansion, and contribute to the compartment for an extended period before donor-derived CD4(+)FoxP3(+) T cells eventually compose the majority of the compartment. In total, the findings suggest that the presence of host Tregs may be important to consider regarding elicitation of immune (eg, antitumor, vaccine) responses in recipients during the early post-transplant period involving autologous and certain allogeneic HCT regimens.

IL-2 family of cytokines in T regulatory cell development and homeostasis.

INTRODUCTION: Interleukin 2 (IL-2) induces an essential signal for T regulatory (Treg) cells. Without a functional IL-2R, only immature CD4(+) Foxp3(low) CD25(neg) T cells develop, and these cells fail to suppress autoreactive T cells in the periphery. DISCUSSION: IL-2 functions during Treg cell development by upregulating Foxp3 and CD25 and by increasing the number of thymic Treg cells. Upon exiting the thymus during neonatal life, IL-2 is responsible for rapid amplification of the number of Treg cells in peripheral lymph nodes to insure suppression of autoreactive T cells that escape negative selection, thereby maintaining tolerance. The homeostasis of Treg cells in mature immunocompetent mice also depends on IL-2. However, there is an alternative mechanism for Treg cells homeostasis that may represent a minor IL-2-independent pathway or the consequence of weak and very transient IL-2R signaling. CONCLUSION: Thus, IL-2 provides importance signals for Treg cell development and for their homeostasis in peripheral immune tissues.

A function for IL-7R for CD4+CD25+Foxp3+ T regulatory cells.

The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-deficient mice are not completely devoid of Foxp3+ cells, but rather lack population of mature CD4+CD25+Foxp3high Treg cells and contain few immature CD4+CD25-Foxp3low T cells. Interestingly, common gamma chain (gammac) knockout mice have been shown to have a near complete absence of Foxp3+ Treg cells, including the immature CD25-Foxp3low subset. Therefore, other gammac-cytokine(s) must be critically important during thymic development of CD4+CD25+Foxp3+ Treg cells apart from the IL-2. The present study was undertaken to determine whether the gammac-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Treg cell production. These studies revealed that mice double deficient in IL-2Rbeta and IL-7Ralpha contained a striking lack in the CD4+Foxp3+ population and the Treg cell defect recapitulated the gammac knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production of CD4+CD25+Foxp3+ Treg cells, indicating that normal thymic Treg cell production likely depends on signaling through both IL-2 and IL-7 receptors. Selective thymic reconstitution of IL-2Rbeta in mice double deficient in IL-2Rbeta and IL-7Ralpha established that IL-2Rbeta is dominant and sufficient to restore production of Treg cells. Furthermore, the survival of peripheral CD4+Foxp3low cells in IL-2Rbeta-/- mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Treg cell development and peripheral homeostasis.

Function of the IL-2R for thymic and peripheral CD4+CD25+ Foxp3+ T regulatory cells.

IL-2 contributes to the production, function, and homeostasis of CD4+CD25+ T(reg) cells. However, it remains uncertain whether IL-2 is essential for the development of T(reg) cells in the thymus, their homeostasis in the periphery, or both. The present study was undertaken to investigate the contribution of IL-2 during thymic T(reg) cell development and its maintenance in peripheral immune tissue. Relying on genetic mouse models where IL-2R signaling was either completely blocked or selectively inhibited in peripheral CD4+CD25+ T(reg) cells, we show that the IL-2/IL-2R interaction is active in the thymus at the earliest stage of the development of T(reg) cells to promote their expansion and to up-regulate Foxp3 and CD25 to normal levels. Furthermore, CD4+CD25+Foxp3+ T(reg) cells with impaired IL-2-induced signaling persist in the periphery and control autoimmunity without constant thymic output. These peripheral T(reg) cells with poor responsiveness to IL-2 exhibited slower growth and extended survival in vivo, somewhat lower suppressive activity, and poor IL-2-dependent survival in vitro. Mixed thymic and bone marrow chimeric mice showed that wild-type-derived T(reg) cells were substantially more effective in populating peripheral immune tissue than T(reg) cells with impaired IL-2 signaling. Collectively, these data support the notion that normally IL-2 is a dominant mechanism controlling the number of thymic and peripheral T(reg) cells.