Treg Maintenance Research Articles

Differentiating the roles of STAT5B and STAT5A in human CD4+ T cells

STAT5A and STAT5B are highly homologous proteins whose distinctive roles in human immunity remain unclear. However, STAT5A sufficiency cannot compensate for STAT5B defects, and human STAT5B deficiency, a rare autosomal recessive primary immunodeficiency, is characterized by chronic lung disease, growth failure and autoimmunity associated with regulatory T cell (Treg) reduction. We therefore hypothesized that STAT5A and STAT5B play unique roles in CD4(+) T cells. Upon knocking down STAT5A or STAT5B in human primary T cells, we found differentially regulated expression of FOXP3 and IL-2R in STAT5B knockdown T cells and down-regulated Bcl-X only in STAT5A knockdown T cells. Functional ex vivo studies in homozygous STAT5B-deficient patients showed reduced FOXP3 expression with impaired regulatory function of STAT5B-null Treg cells, also of increased memory phenotype. These results indicate that STAT5B and STAT5A act partly as non-redundant transcription factors and that STAT5B is more critical for Treg maintenance and function in humans.

TCR signaling by conventional T cells is required for peripheral maintenance of Foxp3+ regulatory T cells (P1155)

Abstract Regulatory T cells (Tregs) control inappropriate conventional T cell (Tconv) activation. Treg maintenance is highly dependent on their IL-2-mediated proliferation in the periphery. Since Tconvs but not Tregs produce IL-2, IL-2 could serve as a gauge for Tregs to adjust their numbers accordingly to the size of the Tconv pool in the periphery. As the production of IL-2 by Tconvs is dependent on TCR signaling, we hypothesized that TCR signaling by Tconvs in response to self MHC class II (MHC-II) molecules is required for Treg maintenance. Indeed, when TCR signaling was specifically ablated in Tconvs but not in Tregs, an abrupt drop in Treg numbers was observed. Moreover, Treg numbers could not be maintained when T cells were adoptively transferred into MHC-II-deficient bone marrow chimeric mice, where MHC-II molecules were missing on radiosensitive antigen presenting cells. The drop in Treg numbers was associated with Tconv activation and the development of severe dermatitis, gut pathology, and weight loss. The decrease in Treg numbers and the onset of this disease was delayed by the injection of IL-2. These results suggest that IL-2 produced through TCR signaling by Tconvs in response to self MHC-II molecules is critical for the maintenance of Tregs and for subsequent prevention of a widespread inflammatory syndrome. This points to a potential role of positive selection in producing Tconvs bearing TCRs with adequate affinity to self MHC-II complexes in Treg maintenance.

Blocking of PD-L1 on therapeutic dendritic cells modulate in vitro T cells proliferation and differentiation to Th1 and Th17 subpopulations (P4230)

Abstract DCs constitute a promising treatment against melanoma. Recently, we showed the effectiveness of an allogeneic melanoma lysate DC-based immunotherapy for improving long-term survival in melanoma patients. Moreover, an elevated proportion of regulatory T lymphocytes (Tregs) was observed in non-responder patients. PD-L1 is an important co-inhibitory molecule expressed on DCs binding to PD-1 on T cells. This interaction promotes the induction, conversion and maintenance of Tregs, suggesting a role in immunosuppression. Here, we studied if the blockade of PD-1-PD-L1 interaction reverses Tregs differentiation and increases T helper populations. Monocyte-DCs were generated in the presence of pro and anti-inflammatory factors, and the PD-L1/PD-1 signaling was blocked by anti PD-L1 mAbs. Allo-stimulatory capacity of DCs was impaired, and inversely correlated with PD-L1/CD86 ratio. However, blocking PD-L1 on DCs increased of Th1 and Th17 proliferation, concomitant with a Tregs reduction, independently of the DCs phenotype and functional characteristics. We demonstrated that the PD-L1/CD86 ratio play a key role in the functional capability of DCs. Therefore, blocking this molecule on DCs becomes an attractive strategy to improve the efficacy of DCs-based vaccines in melanoma patients.

Protection of Tregs, Suppression of Th1 and Th17 Cells, and Amelioration of Experimental Allergic Encephalomyelitis by a Physically-Modified Saline

In multiple sclerosis (MS) and other autoimmune diseases, the autoreactive T cells overcome the resistance provided by the regulatory T cells (Tregs) due to a decrease in the number of Foxp3-expressing Tregs. Therefore, upregulation and/or maintenance of Tregs during an autoimmune insult may have therapeutic efficacy in autoimmune diseases. Although several immunomodulatory drugs and molecules are available, most present significant side effects over long-term use. Here we have undertaken an innovative approach to upregulate Tregs and achieve immunomodulation. RNS60 is a 0.9% saline solution generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) and PNS60 (saline containing excess oxygen without TCP modification), was found to upregulate Foxp3 and enrich Tregs in MBP-primed T cells. Moreover, RNS60, but not NS, RNS10.3 and PNS60, inhibited the production of nitric oxide (NO) and the expression of iNOS in MBP-primed splenocytes. Incubation of the cells with an NO donor abrogated the RNS60-mediated upregulation of Foxp3. These results suggest that RNS60 boosts Tregs via suppression of NO production. Consistent to the suppressive activity of Tregs towards autoreactive T cells, RNS60, but not NS, RNS10.3, or PNS60, suppressed the differentiation of Th17 and Th1 cells and shifted the balance towards a Th2 response. Finally, RNS60 treatment exhibited immunomodulation and ameliorated adoptive transfer of experimental allergic encephalomyelitis, an animal model of MS, via Tregs. These results describe a novel immunomodulatory property of RNS60 and suggest its exploration for therapeutic intervention in MS and other autoimmune disorders.

IL-10-independent regulatory role for B cells in suppressing autoimmunity through GITRL-mediated maintenance of Tregs (176.18)

Abstract A regulatory role for B cells has been shown in animal models of autoimmunity and several studies suggested an IL-10-dependent mechanism. In our model of experimental autoimmune encephalomyelitis (EAE), WT mice exhibited a monophasic disease and recovered, but B cell deficiency, either genetic (µMT) or anti-CD20-mediated, led to unresolved EAE. Although B cells regulated EAE, we did not detect IL-10-producing B cells. Hence, we investigated IL-10-independent roles for B cells in immune regulation. B cell deficiency resulted in reduced numbers of CD4+Foxp3+ T regulatory cells (Tregs) in the periphery, suggesting involvement of B cells in their peripheral homeostasis. Interestingly, B cell depletion therapy in humans has been associated with the onset of autoimmunity. Likewise, reduction of Tregs post anti-CD20-mediated depletion of B cells resulted in spontaneous EAE and accelerated colitis in genetically susceptible mice. To study B cell-Treg interactions, we transferred B cells into µMT mice, which resulted in the proliferation and partial restoration of Treg numbers, sufficient for the resolution of EAE. Mechanistically, expression of glucocorticoid-induced tumor necrosis factor receptor-ligand (GITRL) by B cells was required, but IL-10 and B7 molecules were dispensable. Our results demonstrate a novel IL-10-independent role for B cells in regulating autoimmunity by maintenance of Tregs via GITRL.

A Novel IL-10–Independent Regulatory Role for B Cells in Suppressing Autoimmunity by Maintenance of Regulatory T Cells via GITR Ligand

B cells are important for the regulation of autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), B cells are required for spontaneous recovery in acute models. Production of IL-10 by regulatory B cells has been shown to modulate the severity EAE and other autoimmune diseases. Previously, we suggested that B cells regulated the number of CD4(+)Foxp3(+) T regulatory cells (Treg) in the CNS during EAE. Because Treg suppress autoimmune responses, we asked whether B cells control autoimmunity by maintenance of Treg numbers. B cell deficiency achieved either genetically (μMT) or by depletion with anti-CD20 resulted in a significant reduction in the number of peripheral but not thymic Treg. Adoptive transfer of WT B cells into μMT mice restored both Treg numbers and recovery from EAE. When we investigated the mechanism whereby B cells induce the proliferation of Treg and EAE recovery, we found that glucocorticoid-induced TNF ligand, but not IL-10, expression by B cells was required. Of clinical significance is the finding that anti-CD20 depletion of B cells accelerated spontaneous EAE and colitis. Our results demonstrate that B cells play a major role in immune tolerance required for the prevention of autoimmunity by maintenance of Treg via their expression of glucocorticoid-induced TNFR ligand.

A Role for IL-27 in Limiting T Regulatory Cell Populations

IL-27 is a cytokine that regulates Th function during autoimmune and pathogen-induced immune responses. Although previous studies have shown that regulatory T cells (Tregs) express the IL-27R, and that IL-27 inhibits forkhead box P3 upregulation in vitro, little is known about how IL-27 influences Tregs in vivo. The studies presented in this article show that mice that overexpress IL-27 had decreased Treg frequencies and developed spontaneous inflammation. Although IL-27 did not cause mature Tregs to downregulate forkhead box P3, transgenic overexpression in vivo limited the size of a differentiating Treg population in a bone marrow chimera model, which correlated with reduced production of IL-2, a vital cytokine for Treg maintenance. These data identify an indirect role for IL-27 in shaping the Treg pool.

A role of TGFβ in p53-induced CD28-dependent T cell apoptosis (168.18)

Abstract Naturally arising CD4+CD25+ Tregs (nTregs) are essential for maintaining immune homeostasis and peripheral tolerance. The balance between nTregs and conventional T cells appears to play a significant role in immune regulation. The majority of nTregs are considered to recognize antigen peptides derived from self tissues and are predicted to receive continuous antigen stimulation, but regulation of Treg growth and survival against activation-induced cell death (AICD) in vivo is not well-understood. Recently, we reported a novel form of apoptosis (p53-dependent CD28- induced T cell apoptosis: PICA) which is distinguishable from classical AICD. We demonstrated that conventional T cells undergo massive p53- induced apoptosis when stimulated by anti-CD3 and anti-CD28 antibodies coated on flat surface. Most importantly, nTregs survive and expand effectively under the same culture conditions, showing that there is a different survival/ apoptosis mechanism between nTregs and non-Tregs in regards to PICA. Here, we report that transforming growth factor β (TGFβ) signaling is required for survival of Tregs against PICA and the presence of the exogenous active form of TGFβ renders conventional T cells resistant to PICA. TGFβ-mediated survival of nTregs is in part mediated by downmodulation of a pro-apoptotic protein, Bim, by a posttranscriptional mechanism. Together, the data show that TGFβ plays a critical role in PICA and may contribute to the maintenance of Tregs: non-Tregs balance.

A Major Role for Bim in Regulatory T Cell Homeostasis

We have previously shown that regulatory T cells (Treg) accumulate dramatically in aged animals and negatively impact the ability to control persistent infection. However, the mechanisms underlying the age-dependent accrual of Treg remain unclear. In this study, we show that Treg accumulation with age is progressive and likely not the result of increased thymic output, increased peripheral proliferation, or from enhanced peripheral conversion. Instead, we found that Treg from aged mice are more resistant to apoptosis than Treg from young mice. Although Treg from aged mice had increased expression of functional IL-7Rα, we found that IL-7R signaling was not required for maintenance of Treg in vivo. Notably, aged Treg exhibit decreased expression of the proapoptotic molecule Bim compared with Treg from young mice. Furthermore, in the absence of Bim, Treg accumulate rapidly, accounting for >25% of the CD4(+) T cell compartment by 6 mo of age. Additionally, accumulation of Treg in Bim-deficient mice occurred after the cells left the transitional recent thymic emigrant compartment. Mechanistically, we show that IL-2 drives preferential proliferation and accumulation of Bim(lo) Treg. Collectively, our data suggest that chronic stimulation by IL-2 leads to preferential expansion of Treg having low expression of Bim, which favors their survival and accumulation in aged hosts.

Hepatitis C Virus Induces Regulatory T Cells by Naturally Occurring Viral Variants to Suppress T Cell Responses

Regulatory T cell markers are increased in chronically infected individuals with the hepatitis C virus (HCV), but to date, the induction and maintenance of Tregs in HCV infection has not been clearly defined. In this paper, we demonstrate that naturally occurring viral variants suppress T cell responses to cognate NS3358-375 in an antigen-specific manner. Of four archetypal variants, S370P induced regulatory T cell markers in comparison to NS3358-375-stimulated CD4 T cells. Further, the addition of variant-specific CD4 T cells back into a polyclonal culture in a dose-dependent manner inhibited the T cell response. These results suggest that HCV is able to induce antigen-specific regulatory T cells to suppress the antiviral T cell response in an antigen-specific manner, thus contributing to a niche within the host that could be conducive to HCV persistence.

CD25 Appears Non Essential for Human Peripheral Treg Maintenance In Vivo

BackgroundIL-2 has been reported to be critical for peripheral Treg survival in mouse models. Here, we examined Treg maintenance in a series of paediatric liver transplant recipients who received basiliximab, a therapeutic anti-CD25 monoclonal antibody.Methodology/Principal FindingsFoxP3+ CD4 T cells were analyzed by flow cytometry before liver grafting and more than 9 months later. We found that in vivo CD25 blockade did not lead to Treg depletion: the proportion of FoxP3+ cells among CD4 T cells and the level of FoxP3 expression were both unchanged. IL-2Rβ expression was enhanced in FoxP3+ cells both before and after basiliximab treatment, while the level of IL-2Rγ expression was similar in Tregs and non-Tregs. No significant change in the weak or absent expression of IL-7Rα and IL-15Rα expression on FoxP3+ cells was observed. Although the proportion of FoxP3+ cells among CD4 T cells did not vary, food allergies occurred more rapidly after liver grafting in patients who received basiliximab, raising questions as to Treg functionality in vivo in the absence of functional CD25.ConclusionsCD25 appears non essential for human Treg peripheral maintenance in vivo. However, our results raise questions as to Treg functionality after therapeutic CD25 targeting.

IFN-γ expression by regulatory T cells in the CNS of mice persistently infected with neurotropic coronavirus (137.27)

Abstract Mice infected with mouse hepatitis virus, strain JHM develop a chronic infection in the CNS characterized by low levels of viral antigen persistence and retention of virus-specific CD4 and CD8 T cells at the site of infection. We have previously demonstrated that T cell maintenance in the infected CNS is a dynamic process, requiring replenishment from peripheral antigen- experienced and naïve T cell pools. Since mice continued to show clinical improvement, these results raised the possibility that Tregs contributed to a quiescent state in the CNS. Here we show a preferential influx of Tregs to the CNS at very early stage of the infection. They are not converted from Foxp3- cells. Intriguingly, not only IL-10 but also IFN-γ producing Tregs are found in the CNS as early as d 5 p.i. While there are much less (<1%) Tregs making IL-10 during chronic infection, a proportion (>15%) of them continue to express IFN-γ. IFN-γ expression is correlated with T-bet upregulation in Tregs. IFN-γ producing Tregs are induced locally in the CNS from both virus-specific and nonspecific Treg pools. While the frequencies of total Tregs decrease dramatically by d 7 p.i. and remain constant thereafter, two thirds of Tregs lose their Foxp3 expression between d 7 and d 28 p.i. in adoptive transfer experiments. We propose that Tregs acquire effector cell phenotype during type 1 inflammation and the maintenance of Tregs in the persistently infected CNS is also dynamic, requiring recruitment of new Tregs.

Infestation of sheep withPsoroptes ovis, the sheep scab mite, results in recruitment of Foxp3+T cells into the dermis

Regulatory T cells (Tregs) play a central role in maintenance of immune homeostasis by controlling harmful immune responses to inappropriate antigens and are thought to play a key role in modulating hypersensitivity reactions. Infestation of sheep with Psoroptes ovis results in a pronounced cutaneous hypersensitivity-type response, which appears to be crucial for mite survival. We hypothesize that (i) Tregs are involved in sheep scab lesions and (ii) Treg responses may crucially affect lesion development and subsequent mite survival. Foxp3 is a key transcription factor required for generation and maintenance of Tregs in rodents and humans, and is the most widely used marker for Tregs in these species. In this study, we sequence ovine foxp3 and show that it exhibits a high degree of homology with foxp3 from other species. Using a validated immunohistochemical staining technique, we demonstrate that infestation of sheep with P. ovis results in an influx of Foxp3(+) T cells into the skin. Future work will investigate the regulatory function of ovine Foxp3(+) T cells and determine whether the quality of the Treg response to P. ovis plays a role in individual susceptibility to the mite.

Paradoxical Functions of B7: CD28 Costimulation in a MHC Class II-Mismatched Cardiac Transplant Model

Blockade of the B7: CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, this pathway has also been demonstrated to be important for the generation and maintenance of regulatory T cells. In this study, we investigated the role of the B7: CD28 pathway in the 'bm12 into B6' MHC class II-mismatched vascularized cardiac transplant model of chronic rejection. Allograft rejection was remarkably accelerated in B6 background B7DKO and CD28KO recipients compared with B6 wild-type (WT) recipients. Allograft rejection was associated with a significantly enhanced Th1/Th2 alloreactivity and marked reduction in the ratio of regulatory T cells to CD4(+) effector/memory cells. We noted that administration of anti-B7-1 and anti-B7-2 mAb prior to transplantation also accelerated allograft rejection. Furthermore, depleting CD25(+) cells in B6 WT recipients of bm12 hearts prior to transplant also precipitated rejection at a similar rate. Neither B7/CD28 deficiency nor CD25 depletion affected graft survival in single MHC class I-mismatched (bm1 into B6) recipients. This study highlights the paradoxical functions of B7: CD28 costimulation in a MHC class II-mismatched model, in which the B7: CD28 pathway is demonstrated to be important in preventing rejection through the generation and maintenance of Tregs.

Regulatory T Cells in Renal Transplantation and Modulation by Immunosuppression

The poor long-term graft survival rate counteracts the important advance that transplantation is for end-stage renal disease patients. This is mainly due to the employment of immunosuppression that inhibits nonspecifically the alloimmune response to avoid graft rejection, but, at the same time, brings a number of adverse effects leading to chronic rejection. Thus, the major goal in transplantation is to reach an absence of immune response towards donor alloantigens without the need of long-term immunosuppressant drugs. In recent years, regulatory T cells, mainly those with a CD4CD25FOXP3 phenotype (named as Tregs), have demonstrated an inhibitory effect on immune responses against donor alloantigens. As a consequence, they are a potential tool in the development of transplant tolerance in vivo. Most of the evidence comes from experimental models, although recent works address the role of Tregs in the clinical arena of transplantation. In such a setting, the coexistence of immunosuppression in almost 100% patients is an essential factor to consider. Recent findings show that different drugs favor the induction and maintenance of Tregs in renal transplant recipients. Among them, mammalian target of rapamycin inhibitors seem to better promote the development of Tregs at present. The present work reviews all the evidence published up to date about Tregs in human renal transplantation with a special focus on the effect of clinical protocols of immunosuppression.

Homeostasis of CD4+ memory and regulatory T cells occurs independently of NF-¿B (82.12)

Abstract Deletion of I kappa B kinase (IKK2) under the control of cre-recombinant drivers lck-Cre and CD4-Cre, both expressed before thymic selection, interferes with normal naïve T cell activation and consequently mice fail to generate regulatory and memory CD4 T cells. However, it is not known whether the requirement for IKK2 expression extends beyond the initial inductive events required for generation of these populations. With this premise, the current study asks whether IKK2 signals are necessary for survival and/or maintenance of Treg and CD4 memory. We achieved this by specifically deleting IKK2 in mature peripheral T cells using an OX40cre driver, specifically deleting NF-κB signalling in regulatory and memory T cells post activation. Surprisingly, we find that IKK2 signalling is dispensable for normal homeostatic maintenance of both memory and regulatory T cells as mice with OX40 regulated deletion of IKK2 have normal numbers of Treg and CD4 memory. However, studies are ongoing to elucidate the mechanism by which of IKK2 mediates memory and regulatory T cell survival.

Preservation of FoxP3+Regulatory T Cells in the Peripheral Blood of Human Immunodeficiency Virus Type 1-Infected Elite Suppressors Correlates with Low CD4+T-Cell Activation

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4(+) T-cell counts and control viremia to levels that are below the limit of detection of current assays. The mechanisms involved in long-term control of viremia have not been fully elucidated. CD4(+) CD25(+) regulatory T cells (Tregs) downmodulate chronic inflammation by suppressing the activation and proliferation of effector lymphocytes. We found that while Tregs were functional in ES and patients on highly active antiretroviral therapy (HAART), ES maintained high levels of Tregs in peripheral blood mononuclear cells whereas patients on HAART had evidence of Treg depletion. We also demonstrated that Tregs can serve as reservoirs for HIV-1 in vivo. These data suggest that both direct infection by HIV-1 and tissue redistribution are possible explanations for declining FoxP3(+) Tregs in progressive HIV-1 infection. Furthermore, the maintenance of Tregs may be one mechanism associated with the nonprogressive nature of HIV-1 infection in ES.

B7‐1 and B7‐2 contribute unequally to peripheral homeostasis of regulatory T cells (Tregs)

Tregs maintain immunological self tolerance, prevent autoimmunity and control immune responses to transplants. Observations of decreased numbers of Treg cells in CD28‐ or B7‐1/B7‐2‐deficient non‐obese diabetic (NOD) mice suggest that the CD28/B7 interaction is essential for the development and peripheral homeostasis of the Tregs. Although targeting the CD28/B7 interaction remains among the most promising approaches to treat autoimmune diseases and transplant rejection, little information exists regarding the relative role of B7‐1 and B7‐2, the only two known ligands of CD28, on the generation and maintenance of Tregs. To clarify whether B7‐1 and B7‐2 have identical or overlapping effects on Tregs development and homeostasis, in this study we measured the frequencies of CD4+CD25+Foxp3+ Tregs in the blood, thymus and peripheral lymphatic tissues of mice deficient in B7‐1, B7‐2 or both B7‐1/B7‐2 and compared with that in wild type mice. Our results suggest that the absence of both B7‐1 and B7‐2 results in a dramatic reduction in the frequencies of Tregs. Interestingly, we observed a significant but similar reduction in the frequency of Tregs in thymus of both mice deficient for either ligands, however, with more impact in Treg peripheral homeostasis seen in mice deficient in B7‐2 rather B7‐1. Consistent with these results we found that B7‐2 deficient DCs were less efficient in promoting the expansion of Tregs in vitro. Our data provides the first direct comparison of B7‐1 and B7‐2 roles in thymic development and peripheral homeostasis of Tregs. TAMUSHSC‐OVPRGS Award.

Enhanced thymic selection of FoxP3+ regulatory T cells in the NOD mouse model of autoimmune diabetes.

FoxP3(+)CD4(+) regulatory T cells (Tregs) play a key role in the maintenance of peripheral self-tolerance, and it has been suggested that diabetes-susceptible nonobese diabetic (NOD) mice are defective in the generation and numbers of Tregs. We found thymic selection of Tregs to be under genetic control. Fetal thymic organ cultures on the NOD background required 3- to 10-fold more antigen than corresponding cultures on the B6 background for optimal induction of Tregs, but once the threshold for induction was reached the NOD background yielded close to 10-fold more Tregs. This increased selection of Tregs was also found in nontransgenic NOD mice in fetal through adult stages. This trait did not map to the MHC, idd3, or the chromosome 3 (Chr3) regions that control clonal deletion, but mainly to two regions on Chr1 and Chr11. Thus, NOD mice do not have a global defect in the generation or maintenance of Tregs; if anything, they show the opposite.

IL‐2Rβ links IL‐2R signaling with Foxp3 expression

Immunological tolerance to self antigens is a tightly regulated process. Recent work has demonstrated that the forkhead family member Foxp3 is a critical element in the differentiation and function of mouse CD4(+)CD25(+) regulatory T cells (Treg). Recent work has suggested an important role for IL-2 in the development and maintenance of Treg. To directly assess the effect of IL-2 signaling on Treg development and function, we analyzed mice that were genetically deficient in components of the IL-2 receptor (IL-2R). Mice lacking CD25 (IL-2Ralpha) displayed a slight decrease in Treg within the thymus, while peripheral numbers are unchanged. In contrast, we found that mice deficient in CD122 (IL-2Rbeta) had a profound reduction in both thymic and peripheral Treg, coinciding with more rapid development of a fatal lymphoproliferative disease. Expression of a Foxp3 transgene restored Treg and protected against the onset of autoimmunity. Thus, a signal mediated by IL-2Rbeta is essential for the development and homeostasis of Foxp3(+) Treg in vivo.