In‐vitro effect of pembrolizumab on different T regulatory cell subsets

Programmed death‐1 (PD‐1) and interactions with PD‐ligand 1 (PD‐L1) play critical roles in the tumour evasion of immune responses through different mechanisms, including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumour‐infiltrating T cells and regulatory T cell (T_reg) expansion. Effective blockade of immune checkpoints can therefore potentially eliminate these detrimental effects. The aim of this study was to investigate the effect of anti‐PD‐1 antibody, pembrolizumab, on various T_reg subpopulations. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and primary breast cancer patients (PBC) were treated in vitro with pembrolizumab, which effectively reduced PD‐1 expression in both cohorts. We found that PD‐1 was expressed mainly on CD4⁺CD25⁺ T cells and pembrolizumab had a greater effect on PD‐1 expression in CD4⁺CD25^? T cells, compared to CD4⁺CD25⁺ cells. In addition, pembrolizumab did not affect the expression levels of T_reg‐related markers, including cytotoxic T lymphocyte antigen‐4 (CTLA‐4), CD15s, latency‐associated peptide (LAP) and Ki‐67. Moreover, we report that CD15s is expressed mainly on forkhead box P3 (FoxP3)^?Helios⁺ T_reg in HD, but it is expressed on FoxP3⁺Helios^? T_reg subset in addition to FoxP3^?Helios⁺ T_reg in PBC. Pembrolizumab did not affect the levels of FoxP3^+/?Helios^+/? T_reg subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect T_reg or change their phenotype or function but rather blocks signalling via the PD‐1/PD‐L1 axis in activated T cells.     

The protein tyrosine phosphatase PTPN22 controls forkhead box protein 3 T regulatory cell induction but is dispensable for T helper type 1 cell polarization

Protein tyrosine phosphatases (PTPs) regulate T cell receptor (TCR) signalling and thus have a role in T cell differentiation. Here we tested whether the autoimmune predisposing gene PTPN22 encoding for a PTP that inhibits TCR signalling affects the generation of forkhead box protein 3 (FoxP3)⁺ T regulatory (T_reg) cells and T helper type 1 (Th1) cells. Murine CD4⁺ T cells isolated from Ptpn22 knock‐out (Ptpn22^KO) mice cultured in T_reg cell polarizing conditions showed increased sensitivity to TCR activation compared to wild‐type (WT) cells, and subsequently reduced FoxP3 expression at optimal‐to‐high levels of activation. However, at lower levels of TCR activation, Ptpn22^KO CD4⁺ T cells showed enhanced expression of FoxP3. Similar experiments in humans revealed that at optimal levels of TCR activation PTPN22 knock‐down by specific oligonucleotides compromises the differentiation of naive CD4⁺ T cells into T_reg cells. Notably, in vivo T_reg cell conversion experiments in mice showed delayed kinetic but overall increased frequency and number of T_reg cells in the absence of Ptpn22. In contrast, the in vitro and in vivo generation of Th1 cells was comparable between WT and Ptpn22^KO mice, thus suggesting PTPN22 as a FoxP3‐specific regulating factor. Together, these results propose PTPN22 as a key factor in setting the proper threshold for FoxP3⁺ T_reg cell differentiation.     

Plasmacytoid dendritic cells and type 1 interferon promote peripheral expansion of forkhead box protein 3+ regulatory T cells specific for the ubiquitous RNA‐binding nuclear antigen La/Sjögren's syndrome (SS)‐B

RNA‐binding nuclear antigens are a major class of self‐antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)‐B (La), is controlled by CD4⁺ T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa‐specific CD4⁺ T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte‐replete recipient mice expressing hLa as a neo‐self‐antigen. After initial antigen‐specific cell division, hLa‐specific donor CD4⁺ T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)?10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3‐negative donor cells demonstrated that accumulation of hLa‐specific regulatory T cells (T_reg) was due primarily to expansion of small numbers of donor T_reg. Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3⁺ donor T_reg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co‐stimulatory and co‐inhibitory molecules than B cells. Adoptive transfer of hLa peptide‐loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa‐specific T_reg. Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa‐specific T cells impaired FoxP3⁺ donor T cell accumulation. Therefore, peripheral expansion of T_reg specific for an RNA‐binding nuclear antigen is mediated by antigen‐presenting pDC in a type 1 IFN‐dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA‐binding nuclear antigens.     

Antigen‐specific over‐expression of human cartilage glycoprotein 39 on CD4+CD25+ forkhead box protein 3+ regulatory T cells in the generation of glucose‐6‐phosphate isomerase‐induced arthritis

Human cartilage gp‐39 (HC gp‐39) is a well‐known autoantigen in rheumatoid arthritis (RA). However, the exact localization, fluctuation and function of HC gp‐39 in RA are unknown. Therefore, using a glucose‐6‐phosphate isomerase (GPI)‐induced model of arthritis, we investigated these aspects of HC gp‐39 in arthritis. The rise in serum HC gp‐39 levels was detected on the early phase of GPI‐induced arthritis (day 7) and the HC gp‐39 mRNA was increased significantly on splenic CD4⁺T cells on day7, but not on CD11b⁺cells. Moreover, to identify the characterization of HC gp‐39⁺CD4⁺T cells, we assessed the analysis of T helper (Th) subsets. As a result, HC gp‐39 was expressed dominantly in CD4⁺CD25⁺ forkhead box protein 3 (FoxP3)⁺ refulatory T cells (T_reg), but not in Th1, Th2 or Th17 cells. Furthermore, to investigate the effect of HC gp‐39 to CD4⁺T cells, T cell proliferation assay and cytokine production from CD4⁺T cells using recombinant HC gp‐39 was assessed. We found that GPI‐specific T cell proliferation and interferon (IFN)‐γ or interleukin (IL)‐17 production were clearly suppressed by addition of recombinant HC gp‐39. Antigen‐specific over‐expression of HC gp‐39 in splenic CD4⁺CD25⁺ FoxP3⁺ T_reg cells occurs in the induction phase of GPI‐induced arthritis, and addition of recombinant HC gp‐39 suppresses antigen‐specific T‐cell proliferation and cytokine production, suggesting that HC gp‐39 in CD4⁺ T cells might play a regulatory role in arthritis.     

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‐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.     

Critical roles of regulatory B and T cells in helminth parasite-induced protection against allergic airway inflammation

The prevalence of allergic asthma and incidences of helminth infections in humans are inversely correlated. Although experimental studies have established the causal relation between parasite infection and allergic asthma, the mechanism of the parasite-associated immunomodulation is not fully elucidated. Using a murine model of asthma and nematode parasite Heligmosomoides polygyrus, we investigated the roles of regulatory B cells (B_reg) and T cells (T_reg) in mediation of the protection against allergic asthma by parasite. H. polygyrus infection significantly suppressed ovalbumin (OVA)-induced allergic airway inflammation (AAI) evidenced by alleviated lung histopathology and reduced numbers of bronchoalveolar inflammatory cell infiltration, and induced significant responses of interleukin (IL)-10⁺ B_reg, IL-10⁺ T_reg and forkhead box protein 3 (FoxP3)⁺ T_reg in mesenteric lymph node and spleen of the mice. Adoptive transfer of IL-10⁺ B_reg and IL-10⁺ T_reg cell prevented the lung immunopathology in AAI mice. Depletion of FoxP3⁺ T_reg cells in FoxP3-diphtheria toxin (DT) receptor transgenic mice by diphtheria toxin (DT) treatment exacerbated airway inflammation in parasite-free AAI mice and partially abrogated the parasite-induced protection against AAI. IL-10⁺ B_reg cells were able to promote IL-10⁺ T_reg expansion and maintain FoxP3⁺ T_reg cell population. These two types of T_regs failed to induce CD19⁺ B cells to transform into IL-10⁺ B_reg cells. These results demonstrate that B_reg, IL-10⁺ T_reg and FoxP3⁺ T_reg cells contribute in A discrepant manner to the protection against allergic airway immunopathology by parasiteS. B_reg cell might be a key upstream regulatory cell that induces IL-10⁺ T_reg response and supports FoxP3⁺ T_reg cell population which, in turn, mediate the parasite-imposed immunosuppression of allergic airway inflammation. These results provide insight into the immunological relationship between parasite infection and allergic asthma.     

Harnessing the power of regulatory T‐cells to control autoimmune diabetes: overview and perspective

Type 1 diabetes (T1D) is a T‐cell‐mediated autoimmune disease resulting in islet β‐cell destruction, hypoinsulinaemia and severely altered glucose homeostasis. Although the mechanisms that initiate T1D still remain elusive, a breakdown of immune tolerance between effector T‐cells (T_eff) and regulatory T‐cells (T_reg) is considered to be the crucial component leading to autoimmunity. As such, strategies have been developed to boost the number and/or function of T_reg in the hope of specifically hampering the pathogenic T_eff activity. In this review, we will summarize the current understanding of biomarkers and functions of both forkhead box protein 3 (FoxP3)⁺ T_reg and type 1 regulatory T (Tr1) cells in health and in T1D, examine the outcome of experimental therapies in both animal models and humans via manipulation of T_reg responses and also provide an outlook on the potential of T_reg‐based immunotherapies in the prevention and treatment of this disease. Discussed immunotherapies include adoptive transfer of ex‐vivo expanded FoxP3⁺ T_reg, manipulation of T_reg cells via the interleukin (IL)‐2/IL‐2R pathway and induction of T_reg by tolerogenic peptides, tolerogenic dendritic cells or altered gut microbiota.     

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.     

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.     

An increased CD25‐positive intestinal regulatory T lymphocyte population is dependent upon Cox‐2 activity in the Apcmin/+ model

Only mismatch repair (MMR)‐deficient colorectal cancer (CRC) appears to respond well to programmed death (PD)‐1 inhibition at the present time. Emerging evidence suggests a role for micro‐environmental factors such as CD25⁺ cells modulating response to PD‐1 inhibition. In the Apc^Min/+ model of familial adenomatous polyposis (MMR‐proficient CRC), increased Cyclooxygenase‐2 (Cox‐2) expression by cells which include alternatively activated mononuclear phagocytes promotes intestinal tumorigenesis by mechanisms which may include immune suppression. To gain insight into this, we compared regulatory T cell (T_reg) populations between Apc^Min/+ and wild‐type mice prior to and after the phase of increased intestinal Cox‐2‐dependent prostaglandin E₂ (PGE₂) production. There was no difference in systemic T_reg function or numbers between Apc^Min/+ and wild‐type mice. However, increased numbers of small intestinal CD25⁺ T_regs were observed with increased Cox‐2 activity in the absence of any difference in the expression of Tgf‐β or Tslp between Apc^Min/+ and wild‐type mice. Cox‐2 inhibitor therapy (Celecoxib) reversed the increase in Apc^Min/+ intestinal CD25⁺ T_reg numbers, without decreasing numbers of CD25⁺ systemic T_regs. Forkhead box protein 3 (FoxP3⁺) and Cox‐2⁺ cells were co‐localized to the interstitium of adenomas of Apc^min/+ mice. These results suggest selective dependence of an ‘activated T_reg’ phenotype on paracrine Cox‐2 activity in Apc^Min/+ small intestine. For therapeutic potential, further studies are required to evaluate the relevance of these findings to human cancer as well as the functional significance of CD25⁺ intestinal T_regs in cancer.     

KLRG1 expression identifies short-lived Foxp3+ Treg effector cells with functional plasticity in islets of NOD mice

A progressive waning in Foxp3⁺ regulatory T (T_reg) cell function provokes autoimmunity in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), a cellular defect rescued by prophylactic IL-2 therapy. We showed that most islet-infiltrating T_reg cells express inducible T-cell co-stimulator (ICOS) in pre-diabetic NOD mice, and that ICOS⁺ T_reg cells display enhanced fitness and suppressive function in situ. Moreover, T1D progression is associated with decreased expansion and suppressive activity of ICOS⁺Foxp3⁺ T_reg cells, in islets, an observation consistent with the exacerbated T1D seen in NOD.BDC2.5 mice in which the ICOS pathway is abrogated. Here, we show that a large proportion of islet-resident T_reg cells express the KLRG1 marker of terminally differentiation, in contrast to islet-infiltrating ICOS^? T_reg or T_eff cells. We hypothesized that KLRG1 expression designates a subpopulation of ICOS⁺ T_reg cells in islets that progressively loses function, and contributes to the immune dysregulation observed at T1D onset. Indeed, KLRG1-expressing ICOS⁺ T_reg cells are prone to apoptosis, and have an impaired proliferative capacity and suppressive function in vitro and in vivo. T1D protective low-dose IL-2 treatment in vivo could not rescue the loss of KLRG1-expressing T_reg cells in situ. While the global pool of Foxp3⁺ T_reg cells displays some degree of functional plasticity in vivo, the KLRG1⁺ ICOS⁺ T_reg cell subset is particularly susceptible to lose Foxp3 expression and reprogram into Th1- or Th17-like effector T (T_eff) cells in the pancreas microenvironment. Overall, KLRG1 expression delineates a subpopulation of dysfunctional T_reg cells during T1D progression in autoantigen-specific TCR transgenic NOD mice.     

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.     

Proinflammatory cytokine interferon‐γ and microbiome‐derived metabolites dictate epigenetic switch between forkhead box protein 3 isoforms in coeliac disease

Coeliac disease (CD) is an autoimmune enteropathy triggered by gluten and characterized by a strong T helper type 1 (Th1)/Th17 immune response in the small intestine. Regulatory T cells (T_reg) are CD4⁺CD25⁺⁺forkhead box protein 3 (FoxP3⁺) cells that regulate the immune response. Conversely to its counterpart, FoxP3 full length (FL), the alternatively spliced isoform FoxP3 Δ2, cannot properly down‐regulate the Th17‐driven immune response. As the active state of CD has been associated with impairments in T_reg cell function, we aimed at determining whether imbalances between FoxP3 isoforms may be associated with the disease. Intestinal biopsies from patients with active CD showed increased expression of FOXP3 Δ2 isoform over FL, while both isoforms were expressed similarly in non‐coeliac control subjects (HC). Conversely to what we saw in the intestine, peripheral blood mononuclear cells (PBMC) from HC subjects did not show the same balance between isoforms. We therefore hypothesized that the intestinal microenvironment may play a role in modulating alternative splicing. The proinflammatory intestinal microenvironment of active patients has been reported to be enriched in butyrate‐producing bacteria, while high concentrations of lactate have been shown to characterize the preclinical stage of the disease. We show that the combination of interferon (IFN)‐γ and butyrate triggers the balance between FoxP3 isoforms in HC subjects, while the same does not occur in CD patients. Furthermore, we report that lactate increases both isoforms in CD patients. Collectively, these findings highlight the importance of the ratio between FoxP3 isoforms in CD and, for the first time, associate the alternative splicing process mechanistically with microbial‐derived metabolites.