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.     

Maintenance of immune tolerance by Foxp3+ regulatory T cells requires CD69 expression

Although FoxP3⁺ regulatory T cells are key players in the maintenance of immune tolerance and autoimmunity, the lack of specific markers constitute an obstacle to their use for immunotherapy protocols. In this study, we have investigated the role of the C-type lectin receptor CD69 in the suppressor function of Tregs and maintenance of immune tolerance towards harmless inhaled antigens. We identified a novel FoxP3⁺CD69⁺ Treg subset capable to maintain immune tolerance and protect to developing inflammation. Although CD69⁺ and CD69⁻FoxP3⁺ Tregs exist in homeostasis, only CD69-expressing Tregs express high levels of CTLA-4, ICOS, CD38 and GITR suppression-associated markers, secrete high amounts of TGFβ and have potent suppressor activity. This activity is regulated by STAT5 and ERK signaling pathways and is impaired by antibody-mediated down-regulation of CD69 expression. Moreover, immunotherapy with FoxP3⁺CD69⁺ Tregs restores the homeostasis in Cd69^−/− mice, that fail to induce tolerance, and is also highly proficient in the prevention of inflammation. The identification of the FoxP3⁺CD69⁺ Treg subset paves the way toward the development of new therapeutic strategies to control immune homeostasis and autoimmunity.     

Regulatory T‐cells in acute dengue viral infection

Although regulatory T‐cells (T_regs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of T_regs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4⁺ CD25⁺T‐cells (FoxP3⁺ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3⁺ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3⁺ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3⁺ cells of patients with acute dengue were predominantly CD45RA⁺ FoxP3^low, followed by CD45RA‐FoxP3^low, with only a small proportion of FoxP3⁺ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector T_reg population. Therefore, although FoxP3⁺ cells are expanded in acute dengue, they predominantly consist of naive T_regs, with poor suppressive capacity.     

Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses

An adequate effector response against pathogens and its subsequent inactivation after pathogen clearance are critical for the maintenance of immune homeostasis. This process involves an initial phase of T‐cell effector (Teff) activation followed by the expansion of regulatory T cells (Tregs), a unique cell population that limits Teff functions. However, significant questions remain unanswered about the mechanisms that regulate the balance between these cell populations. Using an in vitro system to mimic T‐cell activation in human peripheral blood mononuclear cells (PBMC), we analysed the patterns of Treg and Teff activation, with special attention to the role of type I interferon (IFN‐I). Interestingly, we found that IFN‐α, either exogenously added or endogenously induced, suppressed the generation of CD4⁺ FoxP3^HI IFN‐γ^Neg activated Tregs (aTregs) while simultaneously promoting propagation of CD4⁺ FoxP3^Low/Neg IFN‐γ^Pos activated Teffs (aTeffs). We also showed that IFN‐α‐mediated inhibition of interleukin (IL)‐2 production may play an essential role in IFN‐α‐induced suppression of aTregs. In order to test our findings in a disease state with chronically elevated IFN‐α, we investigated systemic lupus erythematosus (SLE). Plasma from patients with SLE was found to contain IFN‐I activity that suppressed aTreg generation. Furthermore, anti‐CD3 activated SLE PBMCs exhibited preferential expansion of aTeffs with a very limited increase in aTreg numbers. Together, these observations support a model whereby a transient production of IFN‐α (such as is seen in an early antiviral response) may promote CD4 effector functions by delaying aTreg generation, but a chronic elevation of IFN‐α may tip the aTeff:aTreg balance towards aTeffs and autoimmunity.     

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25^+/highCD127^Ø/lowFoxP3⁺, and effector T cells were defined as CD25⁺CD127⁺FoxP3^Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4⁺TREG and CD28⁺TREG cells and an increased frequency of CD40L⁺TREG cells. There was a decrease in the TREG/effector-T ratio for GITR⁺, HLA-DR⁺, OX40⁺, and CD45RO⁺ cells, and an increased ratio of TREG/effector-T CD40L⁺ cells in patients with SLE. In addition, CD40L⁺TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.     

Interleukin‐10‐secreting type 1 regulatory T cells in rodents and humans

Summary: Interleukin‐10 (IL‐10)‐secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL‐10 and transforming growth factor‐β(TGF‐β), and by their ability to suppress antigen‐specific effector T‐cell responses via a cytokine‐dependent mechanism. In contrast to the naturally occurring CD4⁺CD25⁺ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL‐10‐dependent process in vitro and in vivo. Specialized IL‐10‐producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL‐10‐producing T‐cell populations that are induced by IL‐10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4⁺CD25⁺ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance.     

Human adipose‐tissue derived mesenchymal stem cells induce functional de‐novo regulatory T cells with methylated FOXP3 gene DNA

Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft‐versus‐host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC‐induced CD4⁺CD25⁺CD127^–forkhead box P3 (FoxP3)⁺ T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nT_reg). Perirenal adipose‐tissue derived MSC (ASC) obtained from kidney donors induced a 2·1‐fold increase in the percentage of CD25⁺CD127^–FoxP3⁺ cells within the CD4⁺ T cell population from allostimulated CD25^–/dim cells. Interleukin (IL)‐2 receptor blocking prevented this induction. The ASC‐induced T cells (iT_reg) inhibited effector cell proliferation as effectively as nT_reg. The vast majority of cells within the iTreg fraction had a methylated FOXP3 gene Treg‐specific demethylated region (TSDR) indicating that they were not of nT_reg origin. In conclusion, ASC induce T_reg from effector T cells. These iT_reg have immunosuppressive capacities comparable to those of nT_reg. Their induction is IL‐2 pathway‐dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de‐novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.     

Regulatory T-cell-derived interleukin-15 shapes cytotoxic T cell memory

It is well known that regulatory T-cells (Tregs) are required to prevent autoimmunity, but they may also have some less-well understood immune-stimulatory effects. In particular, in CD8⁺ T-cell responses Tregs select high-affinity clones upon priming and promote memory by inhibiting inflammation-dependent generation of short-lived effector cells. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 2149400 ], Madi et al. report the surprising finding that human and murine FOXP3⁺ Tregs are a physiologically relevant source of IL-15, a homeostatic cytokine that promotes antigen-independent maintenance of CD8⁺ memory T-cells. In mice that lack IL-15 selectively in FOXP3⁺Tregs the authors show that the composition of the CD8⁺ T-cell memory pool is altered in the absence of Treg-derived IL-15, since a subset of terminally effector memory cells is drastically reduced. Otherwise Treg-derived IL-15 is dispensable for antiviral immune responses and the generation of anti-viral CD8⁺ memory T-cells. These findings add to our understanding of the multifaceted role of Tregs in immune responses, and how IL-15 derived from different cellular sources maintains anti-viral T-cell memory.     

Two separate effects contribute to regulatory T cell defect in systemic lupus erythematosus patients and their unaffected relatives

Forkhead box P3 (FoxP3)⁺ regulatory T cells (T_regs) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)‐2 receptor alpha chain]. Low‐dose IL‐2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE T_reg phenotype, we studied its role through developmentally defined regulatory T cell (T_reg) subsets in 45 SLE patients, 103 SLE‐unaffected first‐degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25‐encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to T_reg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4⁺FoxP3⁺CD45RO^–CD31⁺ recent thymic emigrant T_regs. This first component effect influenced the proportions of circulating CD4⁺FoxP3^highCD45RO⁺ activated T_regs. (2) In contrast, patients and unaffected relatives differed sharply in their activated T_reg CD25 state: while relatives as control subjects up‐regulated CD25 strongly in these cells during differentiation from naive T_regs, SLE patients specifically failed to do so. This CD25 up‐regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated T_regs, but not to their circulating numbers. Both effects were found related to T cell IL‐2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early T_regs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up‐regulation upon peripheral T_reg activation that is selectively deficient in patients. We expect that T_reg‐directed therapies can be monitored more effectively when taking this distinction into account.     

Regulatory T cells in the periphery

Summary: Recognition of a systemic antigen by CD4⁺ T cells in a lymphopenic host leads to the sequential generation of pathogenic effector cells and protective CD25⁺ forkhead box protein (Foxp3⁺) regulatory T cells (Tregs) in the periphery. Such an experimental model is potentially valuable for defining the stimuli that determine the balance of effector and regulatory T cells. Our studies have shown that interleukin‐2 (IL‐2) enhances the development of effector cells and is essential for the peripheral generation of regulatory cells. Other models of peripheral Treg generation suggest that the concentration of antigen, the nature of the antigen‐presenting cells, and cytokines such as transforming growth factor‐β and IL‐10 may all influence the peripheral generation of Tregs.     

HLA DRB1*1503 allelic haplotype predominance and associated immunodysregulation in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, relapsing, and remitting disease affecting primarily African American females of child bearing age. Familial aggregation of this disease suggests that at least part of the susceptibility for this disease is genetic, although environmental and hormonal influences are also likely to play a role. Early studies of genetic susceptibility to SLE revealed several of the major histocompatibility complex molecules, namely HLA DR, to be linked to SLE. Meta-analysis of genome scans has yielded loci significant for lupus patients, one of which includes the MHC region.Regulatory T cells are immunoregulatory cells that modulate activated immune cells. These cells play a large role in homeostasis of the immune responses and maintenance of immunologic tolerance, i.e., prevention of autoimmunity. Decreased numbers of regulatory T cells have been described in many autoimmune diseases, including systemic lupus erythematosus.Autoantibody production in systemic lupus erythematosus and the resulting immune complex formation and complex deposition into tissues are arguably the central core of immune dysregulation leading to disease manifestations and symptoms. Inability of the immune system to recognize and inhibit autoreactive immune cells in this particular autoimmune disease may be the result of inappropriate numbers and function of regulatory T cells.This study aims to characterize the immune cell population in patients from our community suffering from systemic lupus erythematosus and to prove that these patients exhibit a unique cellular profile compared to healthy age, race and gender matched control subjects. Surprisingly, our findings demonstrate that patients from the local Mississippi area exhibit increased proportions of CD25⁺ FoxP3⁺ regulatory T cells and CD25⁺ FoxP3⁻ T cells (of CD45⁺ CD3⁺ CD4⁺ helper T cells) as compared to healthy controls.HLA tissue-typing of these lupus patients revealed a prominent subgroup (~ 30%) of patients possessing the HLA DRB1*1503 allele. The investigation of this subgroup demonstrated regulatory T cell composition similar to that of the total lupus group and to that of the non-HLA DRB1*1503 subgroup.Genetic analysis for molecular gene expression levels of various lupus-associated genes by real-time PCR demonstrated a unique profile as compared to healthy controls. Increased gene expression of FoxP3 together with decreased gene expression levels of GATA3, TNFAIP3, and TNFSF4 suggest that variations in gene products compared to healthy controls may be playing a role in the immune cell dysregulation and disproportionate CD25⁺ FoxP3⁺ regulatory T cells.     

Trafficking of FoxP3<Superscript>+</Superscript> regulatory T cells: myths and facts

Fork head box P3 (FoxP3⁺) regulatory T cells (Tregs) are specialized T cells for prevention of hyperimmune responses and autoimmunity. Tumors and pathogens can hijack FoxP3⁺ Tregs to evade host immune responses. There is an increasing body of evidence that trafficking of FoxP3⁺ Tregs is important for their effective suppression of target cells. Because of their distinctive functions and gene expression phenotype, the migratory behavior of FoxP3⁺ Tregs has been somewhat mystified. The myths are that they have unique trafficking receptors and migratory behaviors that are different from those of conventional T cells. Another related myth is that FoxP3⁺ regulatory T cell subsets have a fixed trafficking behavior from the time they are generated in the thymus. The recent progress in trafficking receptors and migratory behavior of FoxP3⁺ Tregs is reviewed here and the validity of these myths is examined.     

The role of physiological self-antigen in the acquisition and maintenance of regulatory T-cell function

Summary: The CD4⁺CD25⁺ regulatory T cells (Tregs) are efficient regulators of autoimmunity, but the mechanism remains elusive. We summarize recent data for the conclusion that disease‐specific Tregs respond to tissue antigens to maintain physiological tolerance and prevent autoimmunity. First, polyclonal Tregs from antigen‐positive donors suppress autoimmune ovarian disease (AOD) or experimental autoimmune prostatitis in day 3 thymectomized (d3tx) mice more efficiently than Tregs from antigen‐negative donors. Second, Tregs of antigen‐negative adult mice respond to cognate antigen in vivo and rapidly gain disease‐specific Treg function. Third, in d3tx female recipients devoid of neonatal ovarian antigens, only female Tregs suppressed AOD; the male Tregs gain AOD‐suppressing function by responding to the ovarian antigen in the recipients and mask the supremacy of female Tregs in AOD suppression. Fourth, when Tregs completely suppress AOD, the ovary‐draining lymph node is the only location with evidence of profound and persistent (but reversible) host T‐cell suppression. Fifth, from these nodes, highly potent AOD‐suppressing Tregs are retrievable. We conclude that self‐tolerance involves the continuous priming of Tregs by autoantigens, and in autoimmune disease suppression, the effector T‐cell response is continuously negated by potent disease‐specific Tregs that accumulate at the site of autoantigen presentation.     

Dendritic cells expand antigen-specific Foxp3+CD25+CD4+ regulatory T cells including suppressors of alloreactivity

Summary: Thymic derived naturally occurring CD25⁺CD4⁺ T regulatory cells (Tregs) suppress immune responses, including transplantation. Here we discuss the capacity of dendritic cells (DCs) to expand antigen‐specific Tregs, particularly polyclonal Tregs directed to alloantigens. Initial studies have shown that mature DCs are specialized antigen‐presenting cells (APCs) for expanding antigen‐specific CD25⁺ CD4⁺ Tregs from TCR transgenic mice. When triggered by specific antigen, these Tregs act back on immature DCs to block the upregulation of CD80 and CD86 costimulatory molecules. More recently, DCs have been used to expand alloantigen‐specific CD25⁺CD4⁺ Tregs from the polyclonal repertoire in the presence of interleukin‐2 (IL‐2). Allogeneic DCs are much more effective than allogeneic spleen cells for expanding CD25⁺CD4⁺ Tregs. The DC‐expanded Tregs continue to express high levels of Foxp3, even without supplemental IL‐2, whereas spleen cells poorly sustain Foxp3 expression. When suppressive activity is tested, relatively small numbers of DC‐expanded CD25⁺CD4⁺ Tregs exert antigen‐specific suppression in the mixed leukocyte reaction (MLR), blocking immune responses to the original stimulating strain 10 times more effectively than to third party stimulating cells. DC‐expanded Tregs also retard graft versus host disease (GVHD) across full major histocompatibility complex (MHC) barriers. In vitro and in vivo, the alloantigen‐specific CD25⁺CD4⁺ Tregs are much more effective suppressors of transplantation reactions than polyclonal populations. We suggest that the expansion of Tregs from a polyclonal repertoire via antigen‐presenting DCs will provide a means for antigen‐specific control of unwanted immune reactions.     

The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins

Rabbit anti-thymocyte globulins (rATG) induce CD4⁺CD25⁺forkhead box P3 (FoxP3⁺) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4⁺CD25⁺FoxP3⁺CD127^−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25^neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25⁺ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4⁺CD25⁺FoxP3⁺CD127^−/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3⁺T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3⁺ T cells. The rATG tacrolimus-induced CD25⁺ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4⁺CD25⁺FoxP3⁺CD127^−/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25⁺ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-γ, perforin and granzyme B in contrast to natural CD25⁺ T cells (all P = 0·03), while FoxP3 was expressed at a lower level (P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4⁺CD25⁺ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.     

Imbalance of different types of CD4+forkhead box protein 3 (FoxP3)+ T cells in patients with new‐onset systemic lupus erythematosus

The aim of this study was to examine the numbers of CD4⁺CD25⁻forkhead box protein 3 (FoxP3)⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells in patients with new-onset systemic lupus erythematosus (SLE). The numbers of CD4⁺CD25⁻FoxP3⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and the concentrations of serum interleukin (IL)-10 in 23 patients and 20 healthy controls (HC) were measured. The potential correlations between CD4⁺FoxP3⁺ T cells, serum IL-10 and clinical measures in SLE patients were analysed. In comparison with that in the HC, significantly reduced numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells, but increased numbers of CD4⁺CD25⁻FoxP3⁺ T cells, were detected, accompanied by significantly lower levels of serum IL-10 in the patients. Stratification analysis indicated the numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and serum IL-10 levels in the patients with seropositive anti-dsDNA were significantly less than that in those with seronegative anti-dsDNA. Treatment with the anti-SLE therapy, particularly with prednisone, leflunomide and methotrexate, significantly improved the imbalance of these types of FoxP3⁺ T cells and increased the concentrations of serum IL-10 in the drug-responding patients. The numbers of CD4⁺CD25⁺FoxP3⁺ T cells were correlated negatively with the values of SLE disease activity index (SLEDAI), whereas the numbers of CD4⁺CD25⁻FoxP3⁺ T cells were correlated positively with the values of SLEDAI, erythrocyte sedimentation rate (ESR) and serum C3. In addition, the concentrations of serum IL-10 were correlated positively with the numbers of CD4⁺CD25⁺FoxP3⁺ T cells, but negatively with the values of SLEDAI, serum C3, CRP and ESR in these patients. Our data indicate that the imbalance of different types of FoxP3⁺CD4⁺ T cells may contribute to the development of SLE in Chinese patients.     

Upregulated IL-17A secretion and CCR6 co-expression in Treg subsets are related to the imbalance of Treg/Th17 cells in active UC patients

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disease, which is characterized with overactive immune response. It is well established that the imbalance between Tregs and Th17 cells plays a pivotal role in pathogenesis of UC. In this study, we investigated the impact of functional changes in Treg subsets on Treg/Th17 ratio and further explored their clinical significance in the activity of UC. Treg subsets were comprehensively analysed using flow cytometry and in vitro cultured in both active and remission UC patients, of which nine active UC patients were further followed up. The correlation analyses were performed to explore the potential associations between Treg subsets and clinical indicators, as well as the impact of serum cytokines, detected by ELISA, on IL-17A secretion and CCR6 co-expression of Treg subsets. In active UC patients, we found CD45RA^-FoxP3^hiTregs were obviously decreased and inversely correlated with disease activity, while CD45RA⁺FoxP3^loTregs were increased and positively correlated with disease activity. Meanwhile, IL-17A secretion and CCR6 co-expression levels in Tregs were significantly increased in active UC. Moreover, Tregs co-expressing CCR6 possesses higher level of IL-17A secretion. In nine followed up patients, we observed downregulated IL-17A secreting and CCR6 co-expression when achieving remission from active stage. In addition, IL-17A⁺FoxP3⁺ and IL-17A⁺FoxP3⁺CCR6⁺Tregs were positively correlated with serum IL-21 and disease activity, respectively. These findings suggested that upregulated IL-17A secretion and CCR6 co-expression in Treg subsets may be related to the imbalance between Tregs and Th17 cells and associated with the disease activity in UC patients.