CD8+ T cells specific for β cells encounter their cognate antigens in the islets of NOD mice

CD8⁺ T cells play a key role in the initiation of insulitis. However, the site(s) where naive CD8⁺ T cells encounter β‐cell antigens and the mechanism(s) by which β‐cell autoimmunity is initiated remain to be determined. In the current study, an adoptive transfer model was employed assessing the initial site of priming and the nature of antigen recognition by naive β‐cell‐specific CD8⁺ T cells. Temporal analysis demonstrated that unlike CD4⁺ T cells that are primed in the draining pancreatic lymph nodes, initial proliferation of transferred CD8⁺ T cells was detected in the islets. These results indicate that in our model, naive β‐cell‐specific CD8⁺ T cells encounter β‐cell antigens in the islets. Furthermore, ectopic expression of CD80 by β cells accelerated the onset of insulitis mediated by β‐cell‐specific CD8⁺ T cells, but had no effect on CD4⁺ T‐cell‐mediated diabetes, suggesting an antigenic interaction between β cells and naive CD8⁺ T cells. However, it remains to be determined whether the initiation of insulitis in spontaneous diabetes is the result of a cognate interaction between naive CD8⁺ T cells and islet β cells.     

IL-12 and IL-23 modulate plasticity of FoxP3+ regulatory T cells in human Leprosy

Leprosy is a bacterial disease caused by M. leprae. Its clinical spectrum reflects the host’s immune response to the M. leprae and provide an ideal model to investigate the host pathogen interaction and immunological dysregulation. Tregs are high in leprosy patients and responsible for immune suppression of the host by producing IL-10 and TGF-β cytokines. In leprosy, plasticity of Tregs remain unstudied. This is the first study describing the conversion of Tregs into Th1-like and Th17-like cells using in vitro cytokine therapy in leprosy patients. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA), rIL-12 and rIL-23 for 48 h. Expression of FoxP3 in CD4⁺CD25⁺ Tregs, intracellular cytokines IFN-γ, TGF-β, IL-10 and IL-17 in Tregs cells were evaluated by flow cytometry (FACS) after stimulation. rIL-12 treatment increases the levels of pStat4 in Tregs and IFN-γ production. In the presence of rIL-23, pStat3⁺ and IL-17A⁺ cells increase. rIL-12 and r—IL-23 treatment downregulated the FoxP3 expression, IL-10 and TGF-β production by Tregs and enhances the expression of co-stimulatory molecules (CD80, CD86). In conclusion rIL-12 converts Tregs into IFN-γ producing cells through STAT-4 signaling while rIL-23 converts Tregs into IL-17 producing cells through STAT-3 signaling in leprosy patients. This study may helpful to provide a new avenue to overcome the immunosuprression in leprosy patients using in vitro cytokine.     

Lower proportions of CD4+CD25high and CD4+FoxP3, but not CD4+CD25+CD127low FoxP3+T cell levels in children with autoimmune thyroid diseases

The essence of autoimmune thyroid disease (AITD) is loss of tolerance of own tissues caused by malfunction of T lymphocytes, which affects the production of antibodies reacting with particular cell structures and tissues. Foxp3⁺ regulatory T cells (Tregs) take part in the regulation of immune response and play a leading role in developing immune tolerance through active suppression. The aim of the study was to estimate the expression of CD4+CD25^high, CD4+CD25+CD127^lowFoxP3⁺ and CD4+ FoxP3 T cells in patients with Graves' disease (GD) (n = 24, median age 15.5 years), in patients with Hashimoto's thyroiditis (HT) (n = 30, median age 15 years) in comparison with sex- and age-matched healthy control subjects (n = 30, median age 15 years). Polychromatic flow cytometry using a FACSCalibur (BD Biosciences) cytometer was applied to delineate T regulatory cell populations. In untreated patients with Graves' disease and HT we observed a significant decrease in CD4+FoxP3 (p < 0.001, p < 0.01) and CD4+CD25^high (p < 0.016, p < 0.048) T lymphocytes as compared to the healthy control subjects. After 6–12 months of L-thyroxine therapy in HT cases these phenotypes of Tregs were normalized, yet no such changes were observed during GD therapy. The analysis of CD4+CD25+CD127^lowFoxP3⁺T cells in the peripheral blood revealed comparable percentages of these cells in patients with thyroid autoimmune diseases to the controls. We conclude that the reduction number of Tregs with CD4+CD25^high and CD4+FoxP3 phenotype suggests their role in initiation and development of autoimmune process in thyroid disorders.     

Ex vivo expanded regulatory T cells CD4+CD25+FoxP3+CD127Low develop strong immunosuppressive activity in patients with remitting-relapsing multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease characterized by defect in regulatory function of CD4⁺CD25⁺ T cells. We demonstrated difference in proportion of regulatory T cells CD4⁺CD25⁺FoxP3⁺CD127^low (Tregs) within the same patients’ relapse and remission. Proportion of peripheral Tregs (pTregs) dropped almost two times in the relapse compare to remission. Levels of pTregs in patients’ remission were lower than in healthy donors. Suppressive ability of pTregs was decreased in MS patients compared to healthy donors. Injections of expanded ex vivo autologous Tregs (eTregs) could be helpful in bringing up the level of Tregs in patients’ blood. We developed a simple method for ex vivo expansion of autologous Tregs within a short period of time. The final pool of cells consisted of 90-95% eTregs. When we started the culture with 10-20?×?10⁶ CD4⁺ T cells, we yield 300-400?×?10⁶ eTregs in a week. Expression of FoxP3 and Helios was calculated by two methods. Expanded ex vivo patients’ and donors’ Tregs were characterized by increased from three to five times expression of FoxP3, as well as almost doubled Helios expression. Peripheral Tregs in MS patients have decreased demethylation of FoxP3 gene promoter in comparison with donors. On the contrary, eTregs showed stable up-regulated demethylation without difference between MS patients and donors. MS patients’ and donors’ eTregs have much more suppressive ability than pTregs. Our data showed that eTregs can be applied as immunotherapy for MS patients and other autoimmune diseases if further investigated.     

Inhibition of phosphoantigen-mediated gammadelta T-cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells

Tumour growth promotes the expansion of CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) which suppress various arms of immune responses and might therefore contribute to tumour immunosurveillance. In this study, we found an inverse correlation between circulating Treg frequencies and phosphoantigen-induced gammadelta T-cell proliferation in cancer patients, which prompted us to address the role of Tregs in controlling the gammadelta T-cell arm of innate immune responses. In vitro, human Treg-peripheral blood mononuclear cell (PBMC) co-cultures strongly inhibited phosphoantigen-induced proliferation of gammadelta T cells and depletion of Tregs restored the impaired phosphoantigen-induced gammadelta T-cell proliferation of cancer patients. Tregs did not suppress other effector functions of gammadelta T cells such as cytokine production or cytotoxicity. Our experiments indicate that Tregs do not mediate their suppressive activity via a cell-cell contact-dependent mechanism, but rather secrete a soluble non-proteinaceous factor, which is independent of known soluble factors interacting with amino acid depletion (e.g. arginase-diminished arginine and indolamine 2,3-dioxygenase-diminished tryptophan) or nitric oxide (NO) production. However, the proliferative activity of alphabeta T cells was not affected by this cell-cell contact-independent suppressive activity induced by Tregs. In conclusion, these findings indicate a potential new mechanism by which Tregs can specifically suppress gammadelta T cells and highlight the strategy of combining Treg inhibition with subsequent gammadelta T-cell activation to enhance gammadelta T cell-mediated immunotherapy.     

Aberrant NKG2D expression with IL-17 production of CD4+ T subsets in patients with type 2 diabetes

Type 2 diabetes (T2D) is a systemic inflammatory disease. Although the natural killer group 2, member D (NKG2D) receptor, was not expressed normally on CD4+ T cells, the aberrant expression was found in pathological conditions such as in auto-immune diseases. However, the involvement of NKG2D in pathogenesis of T2D is unclear. We hypothesize that there is an inflammatory CD4+ T cell subpopulation expressing NKG2D and producing interleukin (IL)-17 in T2D. NKG2D expression on CD4+ T cells and their subsets were analyzed by multi-color staining using flow cytometry. Lymphocytes were activated by phorbol-12-myristate-13-acetate (PMA) and ionomycin, and were stained for intracellular IL-17. To investigate the mechanism of IL-17 production, patients’ lymphocytes were stimulated using specific anti-T cell receptor (TCR) alone, anti-NKG2D alone or a combination of the two antibodies. CD4+ T cells and particularly, CD4 + CD28^null T subset of T2D patients were highly expressed NKG2D and more prevalent compared to non-diabetic individuals (ND) (P = 0.039 and P = 0.022, respectively). Significantly higher percentages of CD4 + CD28^nullNKG2D + T cells of patients produced IL-17 when compared to those of ND (P = 0.024) and were positively correlated with the level of glycated hemoglobin A1c (HbA1c) (R² = 0.386, P = 0.041). Additionally, this cell population could be stimulated by specific monoclonal anti-NKG2D to produce IL-17. In conclusion, CD4 + CD28^nullNKG2D+ T cells were expanded in T2D, especially in patients with poor glycemic control. NKG2D may be one of the surrogate co-stimulatory receptors leading to irregular inflammatory function producing IL-17. An IL-17 producing CD4 + CD28^nullNKG2D + T cells may potentially be involved in pathogenesis and drive severity of the disease with the glycemic dependence. This particular cell type could be targeted for prognostic or therapeutic purposes.     

Glucagon-reactive islet-infiltrating CD8 T cells in NOD mice

Type 1 diabetes is characterized by T-cell-mediated destruction of the insulin-producing β cells in pancreatic islets. A number of islet antigens recognized by CD8 T cells that contribute to disease pathogenesis in non-obese diabetic (NOD) mice have been identified; however, the antigenic specificities of the majority of the islet-infiltrating cells have yet to be determined. The primary goal of the current study was to identify candidate antigens based on the level and specificity of expression of their genes in mouse islets and in the mouse β cell line MIN6. Peptides derived from the candidates were selected based on their predicted ability to bind H-2K^d and were examined for recognition by islet-infiltrating T cells from NOD mice. Several proteins, including those encoded by Abcc8, Atp2a2, Pcsk2, Peg3 and Scg2, were validated as antigens in this way. Interestingly, islet-infiltrating T cells were also found to recognize peptides derived from proglucagon, whose expression in pancreatic islets is associated with α cells, which are not usually implicated in type 1 diabetes pathogenesis. However, type 1 diabetes patients have been reported to have serum autoantibodies to glucagon, and NOD mouse studies have shown a decrease in α cell mass during disease pathogenesis. Our finding of islet-infiltrating glucagon-specific T cells is consistent with these reports and suggests the possibility of α cell involvement in development and progression of disease.     

Impaired IL-4 production by CD8+ T cells in NOD mice is related to a defect of c-Maf binding to the IL-4 promoter

CD8(+) T cells play an important role in the induction of the autoimmune response in non-obese diabetic (NOD) mice. Here we describe abnormalities in the control of cytokine production by NOD CD8(+) T cells. NOD CD8(+) T cells had an increased propensity to produce IFN-gamma upon TCR activation, in both adult and 2-week-old mice. NOD CD8(+) T cells had a reduced capacity to produce IL-4 in type 2 conditions compared to CD8(+) T cells from the diabetes-resistant strains BALB/c and C57BL/6. Both GATA-3 and c-Maf, two positive transactivators for IL-4 gene expression, were expressed in type 2 conditions at comparable levels in NOD CD8(+) T cells. The GATA-3 was functional since normal levels of IL-5 were produced and the IL-4 promoter was hyperacetylated in NOD CD8(+) T cells. In contrast, c-Maf failed to bind to its responsive element as determined by chromatin immunoprecipitation (ChIP) assay. These results suggest that NOD CD8(+) T cells possess an increased propensity to produce IFN-gamma and impaired c-Maf-dependent DNA binding activities in vivo that lead to reduced IL-4 production following TCR activation. These defects may facilitate the development of the autoimmune response by inducing an overall type 1-biased immune response in NOD mice.     

Reduced CD4+ T-cell-specific gene expression in human type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) in humans is characterized by the T-cell-dependent destruction of the insulin producing pancreatic beta cells; however, the precise pathogenesis of the disease, especially the initiation of pathologic immune response, is still largely unknown. We hypothesized that the function of human CD4+ T cells is altered in T1DM and analyzed unstimulated human peripheral blood CD4+ T-cell gene expression. We used a novel three-way comparison of DNA microarray data of CD4+ T cells isolated from patients with new onset T1DM, patients with long-term Type 2 diabetes (T2DM), and from healthy control subjects in order to eliminate any possible influence of glucose homeostasis on our findings. We analyzed the T1DM specific gene-expression changes and their functional relevance to T1DM autoimmunity. Our genetic and functional data show that T1DM CD4+ T cells are down-regulated specifically affecting key immune functions and cell cycle. Histone deacetylase gene expression, a key regulator of epigenetic modification is also reduced. The CD4+ T cells showed impaired function, including an abnormal immune response, which may be a key element that leads to the breakdown of self-tolerance.     

PARP-1 inhibitor-AG14361 suppresses acute allograft rejection via stabilizing CD4+FoxP3+ regulatory T cells

Acute allograft rejection is the most common complication in organ transplantation leading to organ loss. Treg cells play an important role in preventing acute rejection, but they are unstable and easily lose function. Poly(ADP-ribose) polymerase 1(PARP-1) is involved in the differentiation stabilization of Treg cells, it has been suggested that PARP-1 inhibition could prevent acute rejection and prolong allograft survival. This study investigated AG14361 effects on acute allograft rejection. We used a fully MHC-mismatched murine heart transplantation model to compare the effect of PARP-1 inhibitor-AG14361 on alloimmunity to the control. Mice treated with PARP-1 inhibitors showed a longer median survival time of allografts (MS14 compared with the control group, MST was 8 days, and AG14361 was 6 days, P = 0.019). The combination of sirolimus and AG14361 significantly delayed allograft MST (AG14361 + sirolimus for 30 days, sirolimus for 16 days, P = 0.002). AG14361 markedly augmented the number of the CD25+FoxP3+ Treg cells in the graft and periphery. In addition, it could enhance the suppressive function of Treg cells by upregulating the level of CTLA-4, PD-1 and ICOS. In vivo, the Treg/Th17 ratio increased significantly in the AG14351 group compared to the control. In the combination with sirolimus treatment, AG14361 promoted the long-term allograft survival. Our results highlight novel effects of a PARP-1 inhibitor. PARP-1 inhibitor AG14361 may be a promising agent to attenuate acute allograft rejection as it can maintain the number and function of Treg cells in allografts.     

Reduced IL-2 and IL-4 mRNA expression in CD4+ T cells from bovine leukemia virus-infected cows with persistent lymphocytosis

Camelpox virus (CMLV), a member of the Orthopoxvirus genus in the Poxviridae, is the etiologic agent of a disease of camels. Here we report the CMLV genomic sequence with analysis. The 205,719-bp CMLV genome contains 211 putative genes and consists of a central region bound by identical inverted terminal repeats of approximately 7 kb. A high degree of similarity in gene order, gene content, and amino acid composition in the region located between CMLV017 and CMLV184 (average 96% amino acid identity to vaccinia virus (VACV)) indicates a close structural and functional relationship between CMLV and other known orthopoxviruses (OPVs). Notably, CMLV contains a unique region of approximately 3 kb, which encodes three ORFs (CMLV185, CMLV186, CMLV187) absent in other OPVs. These ORFs are most similar to B22R homologues found in other chordopoxvirus genera. Among OPVs, CMLV is the most closely related to variola virus (VARV), sharing all genes involved in basic replicative functions and the majority of genes involved in other host-related functions. Differences between CMLV and VARV include deletion and disruption of a large number of genes. Twenty-seven CMLV ORFs are absent in VARV, including seven full-length homologues of NMDA-like receptor, phospholipase D, Schlafen, MT-4 virulence, kelch, VACV C8L, and cowpox (CPXV) B21R proteins. Thirty-eight CMLV ORFs, some of which are fragments of larger genes, differ in size from corresponding VARV ORFs by more than 10% (amino acids). Genome structure and phylogenetic analysis of DNA sequences for all ORFs indicate that CMLV is clearly distinct from VARV and VACV and, as it has been suggested for VARV, it may have originated from a CPXV virus-like ancestor.     

Evidence for a preferential Vβ usage by the T cells which adoptively transfer diabetes in NOD mice

Non-obese diabetic (NOD) mice become spontaneously diabetic as a result of a genetically programmed autoimmune process mediated by autoreactive T lymphocytes and directed against β cell antigen(s). Studies dealing with T cell receptor (TcR) variable (V) gene usage by such autoreactive T lymphocytes have given contrasted results. Various reasons may explain these discrepancies: the multiplicity of antigenic epitopes putatively recognized by T cells, the ambiguity between specifically committed T cells and passenger lymphocytes homing randomly to the pancreas, the necessarily limited size of the T cell clone panels which have been analyzed for TcR rearrangements and, last but not least, the flexibility of T cell repertoires. To circumvent some of these difficulties, we have decided to concentrate upon the T cell population present in diseased animals and capable of transferring diabetes into young naive NOD recipients. This population, composed of CD4⁺ and CD8⁺ T cells, is presumably committed against the relevant β cell antigens and is the most likely to reveal a bias in V gene usage if such a bias does indeed exist. To find out whether certain V_β genes are more frequently used than others by such pathogenic T cells, T lymphocytes from diabetic donors have been depleted in vitro of defined V_β subsets before being reinoculated into permissive recipients. Out of four V_β families probed under such conditions, three (V_β8, V_β5 and V_β11 are neutral. Their absence neither increases nor reduces the final incidence of successful transfers, indicating that these gene segments are not preferentially used. In contrast, the depletion of V_β6-positiveT cells results in a severe reduction of transfers, suggesting that V_β6 gene is used with a relatively high frequency by diabetogenic CD4⁺ and/or CD8⁺ T cells. To define more precisely which subset uses V_β6 gene preferentially, we have performed mixing experiments with deleted and intact subsets. The results, based on disease transfer and insulitis severity, indicate that the V_β6 bias affects predominantly the CD4⁺ subset. Thus, at variance with several studies concluding that V gene usage in NOD mice is heterogeneous, our present data suggest that disease transferring T cells use a relatively restricted set of V_β genes.     

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.     

Comparative study of GAD65-specific CD4+ T cells in healthy and type 1 diabetic subjects

Glutamic acid decarboxylase 65 (GAD65) is a putative autoantigen associated with the pathogenesis of type 1 diabetes (T1D). The prevalence of autoreactive CD4+ T cells towards the immunodominant GAD65(555-567) epitope in DR4 healthy and T1D subjects was investigated with class II tetramers. A slightly higher percentage of diabetic subjects had GAD65(555-567) tetramer-positive T cells upon GAD65(555-567) peptide stimulation on the total CD4+ T-cell populations compared to healthy subjects. In contrast, three quarters of subjects in both groups had tetramer-positive T cells resulting from stimulation of the CD4+CD25+ regulatory T-cell depleted CD4+ T cells. The frequencies and TCR Vbeta gene usages of GAD65(555-567) T cells were also similar in both groups. Experiments demonstrated that GAD65(555-567)-reactive T cells in healthy and diabetic subjects had different CD45RA phenotypes. For the healthy group, GAD65(555-567)-reactive T cells were generally found in the CD45RA+ na?ve T-cell pool while GAD65(555-567)-reactive T cells from T1D subjects were present in both CD45RA+ na?ve and CD45RA- memory T-cell pools. These findings suggested that there is no difference in thymic selection of DR4 restricted GAD-reactive T cells amongst healthy and T1D individuals but GAD65(555-567)-reactive T cells have been preferentially activated in diabetic patients.     

类风湿性关节炎患者CD4+CD25+Foxp3+调节性T细胞检测及意义

目的:研究CD4 CD25 FoxP3 调节性T细胞在类风湿关节炎患者(RA)外周血中的比例改变,并探讨其在疾病进程中的意义.方法:选取活动期及稳定期RA患者,采用细胞内染色的流式细胞术及定量PCR的方法,分别在蛋白质和mRNA水平检测FoxP3表达,并与正常人进行比较.结果:RA患者CD4和CD25双阳性细胞所占比例与对照组没有明显差异,而活动期患者外周血CD4 CD25high和CD4 CD25 FoxP3 细胞明显低于稳定期和对照组(P＜0.05).FoxP3 mRNA表达水平与蛋白质表达水平变化相一致.结论:类风湿性关节炎活动期时CD4 CD25 FoxP3 调节性T细胞明显减少,这群调节性T细胞可能参与了类风湿性关节炎的病理进程.     

IL-2抑制小鼠CD4~+ CD62L~+ T细胞分化为Th17细胞

目的研究IL-2对小鼠脾脏CD4+CD62L+T细胞在体外向Th17细胞分化的作用。方法免疫磁珠法分选C57BL/6小鼠脾脏CD4+CD62L+T细胞,于抗体包被的培养板中培养3 d,实验分为对照组和IL-2处理组。对照组为经典Th17诱导分化培养基,IL-2处理组在对照组基础上于培养体系中添加IL-2。CFSE染色检测细胞增殖,Annexin V-PI法检测细胞凋亡,ELISA检测培养上清中IL-17A的浓度,荧光定量PCR检测Rorγt mRNA的表达,流式细胞术检测CD4~+IL-17~+Th17的生成比例以及Rorγt的表达。结果磁珠分选的小鼠脾脏CD4+CD62L+nave T细胞纯度高于95%。与对照组相比,IL-2处理组细胞数目明显增多,增殖能力明显增强(P0.05),细胞凋亡比例降低(P0.05);IL-2处理组培养上清中IL-17A的浓度明显降低(P0.05),且CD4+IL-17+细胞比例下降,其特异性转录因子Rorγt的表达水平也显著降低(P0.05)。结论 IL-2在CD4+CD62L+T细胞分化为Th17的过程中,能够促进T细胞的增殖并且抑制Th17的分化。     

Flt3 ligand expands CD4+FoxP3+ regulatory T cells in human subjects

CD4⁺CD25⁺FoxP3⁺ naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8⁺ T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg‐cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L‐induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L‐expanded pool of CD1c⁺ myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.