Transforming growth factor beta 1 plays an important role in inducing CD4+CD25+forhead box P3+ regulatory T cells by mast cells

The role of mast cells (MCs) in the generation of adaptive immune responses especially in the transplant immune responses is far from being resolved. It is reported that mast cells are essential intermediaries in regulatory T cell (T_reg) transplant tolerance, but the mechanism has not been clarified. To investigate whether bone marrow‐derived mast cells (BMMCs) can induce T_regs by expressing transforming growth factor beta 1 (TGF‐β1) in vitro, bone marrow cells obtained from C57BL/6 (H‐2^b) mice were cultured with interleukin (IL)‐3 (10 ng/ml) and stem cell factor (SCF) (10 ng/ml) for 4 weeks. The purity of BMMCs was measured by flow cytometry. The BMMCs were then co‐cultured with C57BL/6 T cells at ratios of 1:2, 1:1 and 2:1. Anti‐CD3, anti‐CD28 and IL‐2 were administered into the co‐culture system with (experiment groups) or without (control groups) TGF‐β1 neutralizing antibody. The percentages of CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ T_regs in the co‐cultured system were analysed by flow cytometry on day 5. The T_reg percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF‐β1 neutralizing antibody into the co‐culture system. Our results indicated that the CD4⁺ T cells can be induced into CD4⁺CD25⁺FoxP3⁺ T cells by BMMCs via TGF‐β1.     

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications

Although CD4⁺/CD25⁺ T regulatory cells (T_regs) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell‐separation strategy complying with good manufacturing practice guidelines. We isolated T_regs from standard leukapheresis products using double‐negative selection (anti‐CD8 and anti‐CD19 monoclonal antibodies) followed by positive selection (anti‐CD25 monoclonal antibody). The final cell fraction (CD4⁺/CD25⁺) showed a mean purity of 93·6% ± 1·1. Recovery efficiency was 81·52% ± 7·4. The CD4⁺/CD25^+bright cells were 28·4% ± 6·8. The CD4⁺/CD25⁺ fraction contained a mean of 51·9% ± 15·1 FoxP3 cells and a mean of 18·9% ± 11·5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4⁺CD25⁺FoxP3⁺ cells were in line with flow cytometric results. In Vβ spectratyping the complexity scores of CD4⁺/CD25⁺ cells and CD4⁺/CD25^‐ cells were not significantly different, indicating that T_regs had a broad T cell receptor repertoire. The inhibition assay showed that CD4⁺/CD25⁺ cells inhibited CD4⁺/CD25^‐ cells in a dose‐dependent manner (mean inhibition percentages: 72·4 ± 8·9 [ratio of T responder (T_resp) to T_regs, 1:2]; 60·8% ± 20·5 (ratio of T_resp to T_regs, 1:1); 25·6 ± 19·6 (ratio of T_resp to T_regs, 1:0·1)). Our study shows that negative/positive T_reg selection, performed using the CliniMACS device and reagents, enriches significantly CD4⁺CD25⁺FoxP3⁺ cells endowed with immunosuppressive capacities. The CD4⁺CD25⁺FoxP3⁺ population is a source of natural T_reg cells that are depleted of CD8⁺ and CD4⁺/CD25^‐ reacting clones which are potentially responsible for triggering graft‐versus‐host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft‐versus‐tumour effect.     

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.     

Follicular bronchiolitis as phenotype associated with CD25 deficiency

Regulatory T cells [T_regs; CD4⁺CD25⁺ forkhead box protein 3 (FoxP3⁺)] are subsets of T cells involved in the maintenance of peripheral self‐tolerance by actively suppressing the activation and expansion of autoreactive T cells. Signalling through the interleukin‐2 receptor (IL‐2R) contributes to T cell tolerance by controlling three important aspects of regulatory T cell (T_reg) biology. CD25 is the α‐chain of the IL‐2R that, in concert with the β‐chain and γ‐chain, constitutes the complete IL‐2R. CD25 contributes only to IL‐2 binding affinity but not to the recruitment of signalling molecules. However, its importance in the development of a normal immune response is emphasized by the finding that a truncation mutant of CD25 results in an immunodeficiency in humans characterized by an increased susceptibility to viral, bacterial and fungal infections. In 1997, Sharfe et al. described an infant with severe bacterial, viral and fungal infections. Counts of autologous T lymphocytes were moderately low, T cells displayed a weak proliferative response to mitogens in vitro and the patient displayed no rejection of an allogeneic skin graft. However, unlike children with severe combined immunodeficiency (SCID), besides not having circulating T cells, the patient also developed peripheral lymphocytic proliferation and autoimmune primary biliary cirrhosis. We present the first female Argentine patient with mutation in CD25 associated with chronic and severe inflammatory lung disease (follicular bronchiolitis with lymphocyte hyperplasia), eczema and infections. She has no expression of CD25 on CD4⁺ T cells and an extremely low amount of T_regs. The molecular study confirmed homozygous missense mutation in the alpha subunit of the IL‐2 receptor (CD25αR) (c. 122 a > c; p. Y41S).     

CD4+CD45RA−FoxP3high activated regulatory T cells are functionally impaired and related to residual insulin‐secreting capacity in patients with type 1 diabetes

Accumulating lines of evidence have suggested that regulatory T cells (T_regs) play a central role in T cell-mediated immune response and the development of type 1A and fulminant type 1 diabetes. CD4⁺forkhead box protein 3 (FoxP3)⁺ T cells are composed of three phenotypically and functionally distinct subpopulations; CD45RA⁺FoxP3^low resting T_regs (r-T_regs), CD45RA⁻FoxP3^high activated T_regs (a-T_regs) and CD45RA⁻FoxP3^low non-suppressive T cells (non-T_regs). We aimed to clarify the frequency of these three subpopulations in CD4⁺FoxP3⁺ T cells and the function of a-T_regs with reference to subtypes of type 1 diabetes. We examined 20 patients with type 1A diabetes, 15 patients with fulminant type 1 diabetes, 20 patients with type 2 diabetes and 30 healthy control subjects. A flow cytometric analysis in the peripheral blood was performed for the frequency analysis. The suppressive function of a-T_regs was assessed by their ability to suppress the proliferation of responder cells in a 1/2:1 co-culture. A flow cytometric analysis in the peripheral blood demonstrated that the frequency of a-T_regs was significantly higher in type 1A diabetes, but not in fulminant type 1 diabetes, than the controls. Further, the proportion of a-T_regs among CD4⁺FoxP3⁺ T cells was significantly higher in patients with type 1A diabetes with detectable C-peptide but not in patients with type 1A diabetes without it and with fulminant type 1 diabetes. A proliferation suppression assay showed that a-T_regs were functionally impaired both in fulminant type 1 diabetes and in type 1A diabetes. In conclusion, a-T_regs were functionally impaired, related to residual insulin-secreting capacity and may be associated with the development of type 1 diabetes.     

Significant augmentation of regulatory T cell numbers occurs during the early neonatal period

Regulatory T cells (T_regs) control immune responses by suppressing various inflammatory cells. T_regs in newborn babies may play an important role in preventing excessive immune responses during their environmental change. We examined the number and phenotype of T_regs during the neonatal period in 49 newborn babies. T_regs were characterized by flow cytometry using cord blood (CB) and peripheral blood (PB) from the early (7–8 days after birth) and late (2–4 weeks after birth) neonatal periods. CD4⁺forkhead box protein 3 (FoxP3⁺) T cells were classified into resting T_regs (CD45RA⁺FoxP3^low), activated T_regs (CD45RA^– FoxP3^high) and newly activated T cells (CD45RA^– FoxP3^low). Compared with CB and PB during the late neonatal period, the percentage of T_regs and all T_reg subpopulations in the CD4⁺ lymphocyte population were increased significantly during the early neonatal period. Furthermore, the proportion and absolute number of activated T_regs were increased markedly compared with other T_reg subpopulations, such as resting T_regs and newly activated T cells (non‐T_regs), in the early neonatal period. Increased T_regs concomitantly expressed the suppressive molecule cytotoxic T lymphocyte antigen‐4 (CTLA‐4). The up‐regulated expression of chemokine receptor 4 (CCR4) and down‐regulated expression of CCR7 were also observed in expanded T_regs. When cord blood cells were cultured in vitro with CD3 monoclonal antibodies (mAb) for 5 days, CD4⁺CD45RA^–FoxP3^high cells were increased significantly during the culture. Thus, the presence of increased activated T_regs in early neonates may play an important role in immunological regulation by suppressing excessive T cell activation caused by the immediate exposure to ubiquitous antigens after birth.     

n‐Butyrate Anergized Effector CD4+ T Cells Independent of Regulatory T cell Generation or Activity

CD4⁺ T cell anergy reflects the inability of CD4⁺ T cells to respond functionally to antigenic stimulation through proliferation or IL‐2 secretion. Histone deacetylase (HDAC) inhibitors have been shown to induce anergy in antigen‐activated CD4⁺ T cells. However, questions remain if HDAC inhibitors mediate anergy through direct action upon activated CD4⁺ T cells or through the generation and/or enhancement of regulatory T (T_reg) cells. To assess if HDAC inhibitor n‐butyrate induces anergy independent of the generation or expansion of FoxP3⁺ T_reg cells in vitro, we examine n‐butyrate‐treated murine CD4⁺ T cells for anergy induction and FoxP3⁺ T_reg activity. Whereas n‐butyrate decreases CD4⁺ T cell proliferation and IL‐2 secretion, n‐butyrate did not augment FoxP3 protein production or confer a suppressive phenotype upon CD4⁺ T cells. Collectively, these data suggest that HDAC inhibitors can facilitate CD4⁺ T cell functional unresponsiveness directly and independently of T_reg cell involvement.     

Curcumin induces maturation-arrested dendritic cells that expand regulatory T cells in vitro and in vivo

Dendritic cells (DC) and regulatory T cells (T_regs) are vital to the development of transplant tolerance. Curcumin is a novel biological agent extracted from Curcuma longa (turmeric), with anti‐inflammatory and anti‐oxidant activity mediated via nuclear factor (NF)‐κB inhibition. We investigated the immunomodulatory effects of curcumin on human monocyte‐derived and murine DC. Human monocyte‐derived DC (hu‐Mo‐DC) were generated in the presence (CurcDC) or absence (matDC) of 25 µM curcumin, and matured using lipopolysaccharide (1 µg/ml). DC phenotype and allostimulatory capacity was assessed. CD11c⁺ DC were isolated from C57BL/6 mice, pretreated with curcumin and injected into BALB/c mice, followed by evaluation of in vivo T cell populations and alloproliferative response. Curcumin induced DC differentiation towards maturation‐arrest. CurcDC demonstrated minimal CD83 expression (<2%), down‐regulation of CD80 and CD86 (50% and 30%, respectively) and reduction (10%) in both major histocompatibility complex (MHC) class II and CD40 expression compared to matDC. CurcDC also displayed decreased RelB and interleukin (IL)‐12 mRNA and protein expression. Functionally, CurcDC allostimulatory capacity was decreased by up to 60% (P < 0·001) and intracellular interferon (IFN‐γ) expression in the responding T cell population were reduced by 50% (P < 0·05). T cell hyporesponsiveness was due to generation of CD4⁺CD25^hiCD127^loforkhead box P3 (FoxP3)⁺ T_regs that exerted suppressive functions on naïve syngeneic T cells, although the effect was not antigen‐specific. In mice, in vivo infusion of allogeneic CurcDC promoted development of FoxP3⁺ T_regs and reduced subsequent alloproliferative capacity. Curcumin arrests maturation of DC and induces a tolerogenic phenotype that subsequently promotes functional FoxP3⁺ T_regsin vitro and in vivo.     

Increased resistance to CD4+CD25hi regulatory T cell‐mediated suppression in patients with type 1 diabetes

Type I diabetes (T1D) is a T cell‐mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin‐producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4⁺CD25^hi naturally occurring regulatory T cells (T_regs), defects in which could contribute to loss of self‐tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4⁺CD25^hi T_regs of autologous CD4⁺CD25^‐ responder cells. Here we demonstrate that this defective regulation is also present in subjects with long‐standing T1D (> 3 years duration; P = 0·009). No difference was observed in forkhead box P3 or CD127 expression on CD4⁺CD25^hi T cells in patients with T1D that could account for this loss of suppression. Cross‐over co‐culture assays demonstrate a relative resistance to CD4⁺CD25^hi T_reg‐mediated suppression within the CD4⁺CD25^‐ T cells in all patients tested (P = 0·002), while there appears to be heterogeneity in the functional ability of CD4⁺CD25^hi T_regs from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.     

Selective depletion of CD11c+CD11b+ dendritic cells partially abrogates tolerogenic effects of intravenous MOG in murine EAE

Intravenous (i.v.) injection of a soluble myelin antigen can induce tolerance, which effectively ameliorates experimental autoimmune encephalomyelitis (EAE). We have previously shown that i.v. myelin oligodendrocyte glycoprotein (MOG) induces tolerance in EAE and expands a subpopulation of tolerogenic CD11c⁺CD11b⁺ dendritic cells (DCs) with an immature phenotype having low expression of IA and co‐stimulatory molecules CD40, CD86, and CD80. Here, we further investigate the role of tolerogenic DCs in i.v. tolerance by injecting clodronate‐loaded liposomes, which selectively deplete CD11c⁺CD11b⁺ and immature DCs, but not CD11c⁺CD8⁺ DCs and mature DCs. I.v. MOG‐induced suppression of EAE was partially, yet significantly, blocked by CD11c⁺CD11b⁺ DC depletion. While i.v. MOG inhibited IA, CD40, CD80, CD86 expression and induced TGF‐β, IL‐27, IL‐10 production in CD11c⁺CD11b⁺ DCs, these effects were abrogated after injection of clodronate‐loaded liposomes. Depletion of CD11c⁺CD11b⁺ DCs also precluded i.v. autoantigen‐induced T‐cell tolerance, such as decreased production of IL‐2, IFN‐γ, IL‐17 and numbers of IL‐2⁺, IFN‐γ⁺, and IL‐17⁺ CD4⁺ T cells, as well as an increased proportion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and CD4⁺IL‐10⁺Foxp3^? Tr1 cells. CD11c⁺CD11b⁺ DCs, through low expression of IA and costimulatory molecules as well as high expression of TGF‐β, IL‐27, and IL‐10, play an important role in i.v. tolerance‐induced EAE suppression.     

Alterations in regulatory T-cells: Rediscovered pathways in immunotoxicology

In addition to the effector T-cells subsets, T-cells can also differentiate into cells that play a suppressive or regulatory role in adaptive immune responses. The cell types currently identified as regulatory T-cells (T_regs) include natural or thymic-derived T_regs, T-cells which express Foxp3⁺CD25⁺CD4⁺ and can suppress immune responses to autoreactive T-cells, as well as inducible T_regs, that are generated from naïve T-cells in the periphery after interaction with antigens presented by dendritic cells. Inducible T_regs include T_H3 cells, T_r1 cells, and Foxp3⁺-inducible T_regs. T_regs have been shown to be critical in the maintenance of immune responses and T-cell homeostasis. These cells play an important role in suppressing responses to self-antigens and in controlling inappropriate responses to non-self-antigens, such as commensal bacteria or food in the gut. For example, depletion of CD4⁺CD25⁺ T_regs from mice resulted in the development of multi-organ autoimmune diseases. CD4⁺CD25⁺ T_regs and/or IL-10-producing T_r1 cells are capable of suppressing or attenuating T_H2 responses to allergens. Moreover, adoptive transfer of CD4⁺CD25⁺ T_regs from healthy to diseased animals resulted in the prevention or cure of certain autoimmune diseases, and was able to induce transplantation tolerance. Clinical improvement seen after allergen immunotherapy for allergic diseases such as rhinitis and asthma is associated with the induction of IL-10- and TGFβ-producing T_r1 cells as well as FoxP3-expressing IL-10 T-cells, with resulting suppression of the T_H2 cytokine milieu. Activation, expansion, or suppression of CD4⁺CD25⁺ T_regs in vivo by xenobiotics, including drugs, may therefore represent a relevant mechanism underlying immunotoxicity, including immunosuppression, allergic asthma, and autoimmune diseases.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

Ovarian cancer stem cells promote tumour immune privilege and invasion via CCL5 and regulatory T cells

Emerging evidence indicates a link between the increased proportion of regulatory T cells (T_regs) and reduced survival in patients who have been diagnosed with cancer. Cancer stem cells (CSCs) have been indicated to play a vital role in tumour initiation, drug resistance and recurrence. However, the relationship between T_regs and CSCs remains largely unknown. Here, we sorted out ovarian cancer stem‐like side population (SP) cells and CD133⁺ cells to investigate the influence of ovarian CSCs on T_regs. Among the various immune‐related molecules that we assessed, C‐C motif chemokine ligand 5 (CCL5) was the most elevated in ovarian CSCs relative to that in the non‐CSCs. The expression of its receptor, C‐C motif chemokine receptor 5 (CCR5), was also increased on the surface of T_regs in ovarian cancer patients. This receptor‐ligand expression profile indicated that ovarian CSCs recruit T_regs via CCL5–CCR5 interactions. We further assessed the expression of interleukin (IL)‐10 in T_regs cultured with different cancer cells. T_regs cultured in conditioned medium (CM) from ovarian CD133⁺ cells expressed a higher level of IL‐10 than T_regs cultured in CM from CD133^– cells, indicating that T_regs exert pronounced immune‐inhibitory functions in CSC‐rich environments. Furthermore, co‐culture with ovarian cancer cell lines induced the expression of matrix metalloproteinase‐9 (MMP9) in T_regs which, in turn, enhanced the degradation of the extracellular matrix and enabled the invasion of tumour cells, thereby facilitating tumour metastasis. For the first time, to our knowledge, our findings describe the relationship between ovarian CSCs and T_regs, and demonstrated that these two cell populations co‐operate to promote tumour immune tolerance and enhance tumour progression.     

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (T_reg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of T_reg (CD4⁺CD25^highforkhead box protein 3⁺), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4⁺CD25⁺, CD4⁺CD25^highFoxP3⁺ and CD8⁺CD25⁺ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4⁺CD25^low/negIL‐10⁺ in PB and CD4⁺CD25^low/negIFN‐γ⁺ in PF; meanwhile, CD25^high mainly expressed IL‐10 in both compartments. A high proportion of CD4⁺CD107⁺ and CD8⁺CD107⁺ cells was observed in PF. T_reg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4⁺ and CD8⁺ degranulation responses and decreased CD4⁺IL‐10⁺ cells in PF. Our results demonstrated that in TB pleurisy T_reg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4⁺ and CD8⁺ cells to degranulate in response to M. tuberculosis.     

T‐bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death‐1‐deficient mice

Programmed cell death‐1 (PD‐1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD‐1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3⁺) regulatory T cells (T_regs). PD‐1‐deficient T cell‐specific T‐bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T‐bet over‐expression, increased interferon (IFN)‐γ production by CD4⁺ T cells and significantly low FoxP3⁺ T_reg cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3⁺ T_reg count. The study identified a unique, previously undescribed role for PD‐1 in Th1 and T_reg differentiation, with potential implication in the development of Th1 cell‐targeted therapy.     

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.