CD8+ regulatory T cells are responsible for GAD-IgG gene-transferred tolerance induction in NOD mice

Our previous studies demonstrated that lipopolysaccharide (LPS)-stimulated splenocytes, retrovirally transduced with a glutamate decarboxylate 65 (GAD) and immunoglobulin G (IgG) fusion construct, can protect non-obese diabetic (NOD) mice from diabetes by inducing GAD-specific tolerance, and also that there are increased numbers of CD4(+) regulatory T cells (Tregs) in GAD-IgG-treated NOD mice. However, little is known about the role of CD8(+) Tregs in GAD-IgG gene-transferred tolerance induction in NOD mice. Here, we found that GAD-IgG-transduced splenocytes induced an increase in the number of CD8(+) Foxp3(+) Tregs in vitro. Using a T-cell depletion assay, we found that, compared with undepleted groups, NOD recipients transfused with CD8(-) or CD8(-) CD25(-) GAD-IgG-transduced splenocytes showed a decrease in the percentage of CD8(+) Foxp3(+) T cells, a high incidence of diabetes, serious insulitis, GAD-specific hyperresponsiveness at both the cellular and humoral levels, and changes in cytokine expression. These results indicate that CD8(+) Tregs, which were induced in vitro by GAD-IgG-transduced splenocytes, were also responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.     

CD8+ Foxp3+ T cells share developmental and phenotypic features with classical CD4+ Foxp3+ regulatory T cells but lack potent suppressive activity

"Suppressor T cells" were historically defined within the CD8(+) T-cell compartment and recent studies have highlighted several naturally occurring CD8(+) Foxp3(-) Treg populations. However, the relevance of CD8(+) Foxp3(+) T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8(+) Foxp3(-) T cells is counter-regulated by DC-mediated co-stimulation via CD80/CD86. CD8(+) Foxp3(+) T cells fail to develop in TCR-transgenic mice with Rag1(-/-) background, similar to classical CD4(+) Foxp3(+) Tregs. Notably, both naturally occurring and induced CD8(+) Foxp3(+) T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN-γ production upon restimulation when compared with their CD8(+) Foxp3(-) counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3(+) cells by eGFP reporter expression, we demonstrate that induced CD8(+) Foxp3(+) T cells similar to activated CD8(+) Foxp3(-) T cells only mildly suppress T-cell proliferation and IFN-γ production. We therefore categorize CD8(+) Foxp3(+) T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4(+) Foxp3(+) Tregs, but lacking potent suppressive activity.     

Diabetes is not prevented by Foxp3-transduced CD4(+)T cells under the IL-12Rbeta2 promoter control

Our previous studies have shown that Foxp3 under the control of IFN-gamma promoter (IgammaP-Foxp3) converts pathogenic CD4(+)Th1 cells into regulatory T cells (Tregs), which control diabetes in non-obese diabetic (NOD) mice. Here, we tested the other hypothesis that transient expression of Foxp3 as controlled by the transient expression of IL-12Rbeta2 during Th1 cell derivation is sufficient to convert cells to Tregs. Foxp3, under the control of IL-12Rbeta2 promoter (Ibeta2P), was lentivirally transduced into na?ve CD4(+)T cells from NOD mice. Ibeta2P-Foxp3-transduced CD4(+)T cells could not effectively suppress the incidence of diabetes when transferred into NOD mice. Furthermore, we found that Ibeta2P-Foxp3-transduced CD4(+)T cells, stimulated by a high dose of autoantigen, did not suppress CD4(+)T cell activation, produce CD4(+)Foxp3(+)Tregs, and up-regulate CTLA4 expression. These results suggest that Ibeta2P cannot mediate Foxp3 to convert pathogenic CD4(+)Th1 cells into Tregs which control diabetes in NOD mice.     

Revival of CD8+ Treg-mediated suppression

Despite their first recognition almost 40 years ago, CD8(+) 'suppressor' T cells remain poorly characterized. Recent studies of these lymphocytes, now popularly referred to as regulatory CD8(+) T cells (CD8(+) Tregs), have helped clarify their important role in the regulation of autoimmune disease. Here, we review progress related to the identification, phenotype and function of CD8(+) Tregs. We also focus on a newly described subset, CD8alphaalpha(+)TCRalphabeta(+) Tregs, which in mice recognize a T-cell receptor-derived peptide in the context of the class Ib major histocompatibility complex molecule Qa-1. These Tregs target only activated T cells and complement the suppression provided by CD4(+)Foxp3(+) Tregs. Investigations leading to the detailed identification, expansion, maintenance and function of CD8alphaalpha(+) Tregs should result in new therapeutic strategies for human inflammatory diseases.     

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice

Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3⁺ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3⁺CD103⁺CD38^? stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3⁺CD103^?CD38⁺ labile-phenotype Tregs in the thymus and increased autoreactive CD4⁺ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4⁺ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.     

Diabetes in non-obese diabetic mice is not associated with quantitative changes in CD4+ CD25+ Foxp3+ regulatory T cells

The role of regulatory T cells (Tregs) in maintaining self tolerance has been intensively researched and there is a growing consensus that a decline in Treg function is an important step towards the development of autoimmune diseases, including diabetes. Although we show here that CD25+ cells delay diabetes onset in non-obese diabetic (NOD) mice, we found, in contrast to previous reports, neither an age-related decline nor a decline following onset of diabetes in the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. Furthermore, we demonstrate that CD4+ CD25+ cells from both the spleen and pancreatic draining lymph nodes of diabetic and non-diabetic NOD mice are able to suppress the proliferation of CD4+ CD25- cells to a similar extent in vitro. We also found that pretreatment of NOD mice with anti-CD25 antibody allowed T cells with a known reactivity to islet antigen to proliferate more in the pancreatic draining lymph nodes of NOD mice, regardless of age. In addition, we demonstrated that onset of diabetes in NOD.scid mice is faster when recipients are co-administered splenocytes from diabetic NOD donors and anti-CD25. Finally, we found that although diabetic CD4+ CD25+ T cells are not as suppressive in cotransfers with effectors into NOD.scid recipients, this may not indicate a decline in Treg function in diabetic mice because over 10% of CD4+ CD25+ T cells are non-Foxp3 and the phenotype of the CD25- contaminating population significantly differs in non-diabetic and diabetic mice. This work questions whether onset of diabetes in NOD mice is associated with a decline in Treg function.     

Reduced IL-2 expression in NOD mice leads to a temporal increase in CD62Llo FoxP3+ CD4+ T cells with limited suppressor activity

IL-2 plays a critical role in the induction and maintenance of FoxP3-expressing regulatory T cells (FoxP3(+) Tregs). Reduced expression of IL-2 is linked to T-cell-mediated autoimmune diseases such as type 1 diabetes (T1D), in which an imbalance between FoxP3(+) Tregs and pathogenic T effectors exists. We investigated the contribution of IL-2 to dysregulation of FoxP3(+) Tregs by comparing wildtype NOD mice with animals congenic for a C57BL/6-derived disease-resistant Il2 allele and in which T-cell secretion of IL-2 is increased (NOD.B6Idd3). Although NOD mice exhibited a progressive decline in the frequency of CD62L(hi) FoxP3(+) Tregs due to an increase in CD62L(lo) FoxP3(+) Tregs, CD62L(hi) FoxP3(+) Tregs were maintained in the pancreatic lymph nodes and islets of NOD.B6Idd3 mice. Notably, the frequency of proliferating CD62L(hi) FoxP3(+) Tregs was elevated in the islets of NOD.B6Idd3 versus NOD mice. Increasing levels of IL-2 in vivo also resulted in larger numbers of CD62L(hi) FoxP3(+) Tregs in NOD mice. These results demonstrate that IL-2 influences the suppressor activity of the FoxP3(+) Tregs pool by regulating the balance between CD62L(lo) and CD62L(hi) FoxP3(+) Tregs. In NOD mice, reduced IL-2 expression leads to an increase in nonsuppressive CD62L(lo) FoxP3(+) Tregs, which in turn correlates with a pool of CD62L(hi) FoxP3(+) Tregs with limited proliferation.     

IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.     

Alterations of peripheral CD4+CD25+Foxp3+ T regulatory cells in mice with STZ-induced diabetes

Complications arising from abnormal immune responses are the major causes of mortality and morbidity in diabetic patients. CD4+CD25+T regulatory cells (Tregs) play pivotal roles in controlling immune homeostasis, immunity and tolerance. The effect of hyperglycemia on CD4+CD25+Tregs has not yet been addressed. Here we used streptozotocin (STZ)-induced diabetic mice to study the effects of long-term hyperglycemia on CD4+CD25+Tregs in vivo. Four months after the onset of diabetes, the frequency of CD4+CD25+Foxp3+ T regulatory cells was significantly elevated in the spleen, peripheral blood lymphocytes (PBLs), peripheral lymph nodes (pLNs) and mesenteric LNs (mLNs). CD4+CD25+Tregs obtained from mice with diabetes displayed defective immunosuppressive functions and an activated/memory phenotype. Insulin administration rescued these changes in the CD4+CD25+ Tregs of diabetic mice. The percentage of thymic CD4+CD25+ naturally occurring Tregs (nTregs) and peripheral CD4+Helios+Foxp3+ nTregs were markedly enhanced in diabetic mice, indicating that thymic output contributed to the increased frequency of peripheral CD4+CD25+Tregs in diabetic mice. In an in vitro assay in which Tregs were induced from CD4+CD25- T cells by transforming growth factor (TGF)-β, high glucose enhanced the efficiency of CD4+CD25+Foxp3+ inducible Tregs (iTregs) induction. In addition, CD4+CD25- T cells from diabetic mice were more susceptible to CD4+CD25+Foxp3+ iTreg differentiation than those cells from control mice. These data, together with the enhanced frequency of CD4+Helios-Foxp3+ iTregs in the periphery of mice with diabetes, indicate that enhanced CD4+CD25+Foxp3+ iTreg induction also contributes to a peripheral increase iCD4+CD25+Tregs in diabetic mice. Our data show that hyperglycemia may alter the frequency of CD4+CD25+Foxp3+ Tregs in mice, which may result in late-state immune dysfunction in patients with diabetes.     

The different effects of indirubin on effector and CD4+CD25+ regulatory T cells in mice: potential implication for the treatment of autoimmune diseases

CD4(+)CD25(+) regulatory T (Treg) cells play an essential role in the induction and maintenance of peripheral self-tolerance. Indirubin, a traditional Chinese medicine, was clinically used in the treatment of chronic myelocytic leukemia as well as some autoimmune diseases, including Alzheimer's disease, diabetes, and so on. The effects of indirubin on CD4(+)CD25(+)Treg cells, which play a critical role in controlling autoimmunity, have not been addressed. In the present study, we observed the cell levels, phenotypes, and immunoregulatory function of CD4(+)CD25(+)Treg cells in indirubin-treated mice. Treatment with indirubin significantly enhanced the ratios of CD4(+)CD25(+)Treg cells or CD4(+)CD25(+)Foxp3(+)Treg cells to CD4(+)T cells in peripheral blood, lymph nodes, and spleens (P < 0.01 compared with control mice). CD4(+)CD25(+)Foxp3(+)Treg cells to CD4 single positive cells in the thymi of indirubin-treated mice were significantly higher than those in control mice. Furthermore, splenic CD4(+)CD25(+)Treg cells in indirubin-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4(+)CD25(+)Treg cells in vitro. The present studies indicate that CD4(+)CD25(+)Treg cells are more resistant to indirubin than effector T cells in vivo. The selectively enhanced CD4(+)CD25(+)Treg cell levels by indirubin made host to be more favorable for immune tolerance induction, which opened one possibility for indirubin to treat autoimmune diseases.     

Characterization of CD4+CD25+ natural regulatory T cells in the inflammatory infiltrate of human chronic periodontitis

Periodontitis is an infectious disease, where putative periodontopathogens trigger chronic inflammatory and immune responses against periodontal structures, in which an unbalanced host response is also determinant to the disease outcome. It is reasonable to assume that patient susceptibility to periodontal tissue destruction could be determined by the balance between the response against periodontopathogens and regulatory mechanisms of these events mediated by suppressive T cells. In the present study, we identified and characterized natural regulatory T cells (Tregs) in the inflammatory infiltrate of human chronic periodontitis (CP) with emphasis on phenotypic analyses that were carried out to address the participation of Tregs in CP. Results showed that patients with CP presented increased frequency of T lymphocytes and CD4+CD25+ T cells in the inflammatory infiltrate of gingival tissues. These cells exhibited the phenotypic markers of Tregs such as forkhead box p3 (Foxp3), CTLA-4, glucocorticoid-inducible TNFR, CD103, and CD45RO and seemed to be attracted to the inflammation site by the chemokines CCL17 and CCL22, as their expression and its receptor CCR4 were increased in CP patients. Moreover, besides the increased detection of Foxp3 mRNA, diseased tissues presented high expression of the regulatory cytokines IL-10 and TGF-beta. In addition, the inflammatory infiltrate in CP biopsies was composed of CD25+Foxp3+ and CD25+TGF-beta+ cells, thus corroborating the hypothesis of the involvement of Tregs in the pathogenesis of CP. Finally, these results indicate that Tregs are found in the chronic lesions and must be involved in the modulation of local immune response in CP patients.     

Essential roles of TGF-beta in anti-CD3 antibody therapy: reversal of diabetes in nonobese diabetic mice independent of Foxp3+CD4+ regulatory T cells

Anti-CD3 mAb have potentials to treat overt autoimmunity as reported recently. However, the underlying mechanisms remain unclear. In this report, using an animal model of type 1 diabetes, we found that TGF-beta1, an important immunoregulatory cytokine, plays a critical role in anti-CD3-mediated diabetes reversion and immune tolerance. Anti-CD3 treatment increased the TGF-beta1 production, lasting for a long period of time, which contributed to maintaining peripheral tolerance by controlling pathogenic cells. Furthermore, we found that anti-CD3 treatment did not increase the forkhead box p3+ (Foxp3+)CD4+ regulatory T cells (Tregs). When fractionated from anti-CD3-treated, remitting mice and cotransferred with splenic cells from diabetic NOD mice, these Tregs failed to inhibit diabetes development in NOD.scid mice. Moreover, we found that the depletion of these Tregs did not affect an anti-CD3-mediated, therapeutic effect and the level of TGF-beta1 production, which suggested that an increased level of TGF-beta1 may not derive from these Tregs. Thus, our data showed a dispensable role of Foxp3+CD4+ Tregs in anti-CD3 antibody-reversed diabetes in NOD mice. These findings may have an important implication for understanding the involved mechanisms responsible for immunomodulatory function of anti-CD3 antibody on autoimmune diseases.     

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.     

Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes

Although regulatory T cells (Tregs) are well described, identifying autoaggressive effector T cells has proven more difficult. However, we identified CD4loCD40+ (Th40) cells as being necessary and sufficient for diabetes in the NOD mouse model. Importantly, these cells are present in pancreata of prediabetic and diabetic NOD mice, and Th40 cells but not CD4+CD40(-) T cells transfer progressive insulitis and diabetes to NOD.scid recipients. Nonobese-resistant (NOR) mice have the identical T cell developmental background as NOD mice, yet they are diabetes-resistant. The seminal issue is how NOR mice remain tolerant to diabetogenic self-antigens. We show here that autoaggressive T cells develop in NOR mice and are confined to the Th40 subset. However, NOR mice maintain Treg numbers equivalent to their Th40 numbers. NOD mice have statistically equal numbers of CD4+CD25+forkhead box P3+intrinsic Tregs compared with NOR or nonautoimmune BALB/c mice, and NOD Tregs are equally as suppressive as NOR Tregs. A critical difference is that NOD mice develop expanded numbers of Th40 cells. We suggest that a determinant factor for autoimmunity includes the Th40:Treg ratio. Mechanistically, NOD Th40 cells have low susceptibility to Fas-induced cell death and unlike cells from NOR and BALB/c mice, have predominantly low Fas expression. CD40 engagement of Th40 cells induces Fas expression but further confers resistance to Fas-mediated cell death in NOD mice. A second fundamental difference is that NOD Th40 cells undergo much more rapid homeostatic expansion than Th40 cells from NOR mice.     

A dual altered peptide ligand inhibits myasthenia gravis associated responses by inducing phosphorylated extracellular-regulated kinase 1,2 that upregulates CD4+CD25+Foxp3+ cells

Myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG), are T-cell dependent, antibody-mediated autoimmune disorders. A dual altered peptide ligand (APL) composed of the tandemly arranged two single amino acids analogs of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to downregulate, in vitro and in vivo, MG-associated autoimmune responses. Upregulation of regulatory CD4(+)CD25(+) cells plays a key role in the mechanism of action of the dual APL. The objectives of the present study were to address the involvement of extracellular-regulated kinase (ERK)1,2 in the mechanisms by which the dual APL-induced CD4(+)CD25(+) cells suppress MG-associated autoimmune responses. We demonstrate here that administration of the dual APL increased activated ERK1,2 in the CD4(+)CD25(+)-enriched population. Further, inhibition of ERK1,2 by its inhibitor, U0126, in dual APL-induced CD4(+)CD25(+) cells, abrogated their ability to suppress interferon (IFN)-gamma secretion by lymph node (LN) cells of mice that were immunized with the myasthenogenic peptide. Moreover, inhibition of ERK1,2 in the dual APL-induced regulatory CD4(+)CD25(+) cells, resulted in downregulation of the forkhead box p3 (Foxp3) gene and protein expression levels, as well as in the downregulation of CD4(+)CD25(+) development, suggesting that the active suppression exerted by the dual APL via CD4(+)CD25(+) cells depends on ERK1,2 activity.     

Detectable expression of IL-35 in CD4+ T cells from peripheral blood of chronic hepatitis B patients

Epstein-Barr virus-induced gene 3 (Ebi3) and the p35 subunit of IL-12 have been reported to form a heterodimeric cytokine, named IL-35, in human and mouse. In mice, IL-35 has been shown to be constitutively expressed by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) and suggested to contribute to their suppressive activity. However, human CD4(+)CD25(+)Foxp3(+) Tregs do not constitutively express detectable amounts of IL-35 in both mRNA and protein levels. Circulating CD4(+)CD25(+) Treg frequency of chronic Hepatitis B patients significantly correlates with serum viral load. In this study, we investigated whether IL-35 expression could be detected in CD4(+) T cells from peripheral blood of chronic Hepatitis B patients. Using both RT-PCR and immunoprecipitation plus Western blot analysis, we demonstrated that IL-35 expression could be detected in the CD4(+) T cells from peripheral blood of Chronic Hepatitis B patients.     

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

FTY720-induced conversion of conventional Foxp3- CD4+ T cells to Foxp3+ regulatory T cells in NOD mice

Citation Sun Y, Wang W, Shan B, Di J, Chen L, Ren L, Li W, Li D‐J, Lin Y. FTY720‐induced conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in NOD mice. Am J Reprod Immunol 2011; 66: 349–362 Problem FTY720 is known as an agonist of sphingosine‐1‐phosphate (S1P) receptor, but little is known about the possibility that FTY720 induces the conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in non‐obese diabetic (NOD) mice. Method of study FTY720 treatment was performed using Foxp3^?CD4⁺ T cells purified from NOD mice. Results FTY720 caused an increase in Foxp3⁺ Treg cells in lymphoid organs in NOD mice. FTY720 effectively induced Foxp3 expression in Foxp3^?CD4⁺ T cells both in vitro and in vivo, an effect that was inhibited by a TGF‐β‐neutralizing antibody or the proinflammatory cytokine IL‐6. T‐cell‐mediated embryo rejection in NOD mice was prevented upon FTY720 treatment. Conclusions The use of FTY720 along with Ag administration may represent a useful therapeutic strategy to selectively expand Ag‐specific Foxp3⁺ Tregs to intervene autoimmune and infectious diseases.     

Relations of regulatory T cells with hepatitis markers in chronic hepatitis B virus infection

To assess regulatory T cells (Treg) in chronic hepatitis B (CHB) infected patients and to evaluate the presence of a possible relation between them and hepatitis B markers, flow cytometry analysis was carried out to calculate the percentages of Tregs, Tregs secreting IL-10 and CD4(+) T cells secreting interferon-γ (IFN-γ) and enzyme-linked immunosorbent assay was used to detect hepatitis B virus (HBV) markers in 59 patients and 32 healthy controls. CD4(+)CD25(+), CD4(+)CD25(+)Foxp3(+), CD4(+)D25(high), CD4(+)CD25(high)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) T cells and Treg cells secreting IL-10 were higher in CHB patients than in healthy controls. CD4(+)CD25(+), CD4(+)CD25(-), and total CD4(+)T cells secreting IFN-γ were generally lower in CHB patients than in healthy controls. Fair correlations were observed between CD4(+)CD25(+)Foxp3(+) T cells and alanine aminotransferase (ALT) levels and between HBsAb and both CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(high)Foxp3(+) T cells. CD4(+)CD25(+) T cells were significantly higher in CHB virus infected patients positive for HBeAg than in those negative for HBeAg and a good correlation was observed between CD4(+)CD25(+) T cells and HBeAg. Fair negative correlations were observed between CD4(+)CD25(high) T cells and both HBeAb and HBcAb. These data suggest that Tregs contribute to viral persistence. It was not possible to say that Tregs were the cause of immune suppression in this group of patients.     

Changes of CD4+CD25+Foxp3+ regulatory T cells in aged Balb/c mice

A progressive decline in the integrity of the immune system is one of the physiologic changes during aging. The frequency of autoimmune diseases or immune disorders increases in the aging population, but the state of regulatory T (Treg) cells in aged individuals has not been well determined. In the present study, we investigated the levels, phenotypes, and function of CD4(+)CD25(+) Treg cells in Balb/c mice, which were older than 20 months. Significantly enhanced percentages of CD4(+)CD25(+) Treg cells in the periphery (blood, spleen, and lymph nodes) of the aged mice were observed. These Treg cells showed modified Vbeta family distribution, reduced levels of CD45 receptor B and CD62 ligand molecules, as well as normal levels of forkhead box p3. However, when the inhibiting function of Treg cells was assayed in the in vitro assays and in a delayed-type hypersensitivity (DTH) model, CD4(+)CD25(+) Treg cells of aged mice displayed significantly lower inhibiting ability on alloantigen-induced DTH reaction or cytokine productions (IL-2 and IFN-gamma) but not cell proliferation of effector T cells, as compared with CD4(+)CD25(+) Treg cells of young mice. In addition, the percentages of CD4(+)CD8(-)CD25(+) Treg cells in the thymi of aged mice increased significantly, but their total cell numbers decreased markedly in these mice. Our present studies indicated collectively that the percentages, phenotypes, the size of TCR repertoire, and function of CD4(+)CD25(+) Treg cells were altered significantly with aging in mice. The functional defects of CD4(+)CD25(+) Treg cells may shed light on the role of CD4(+)CD25(+) Treg cells in the increased sensitivity to autoimmune diseases of aged populations.