Tim‐1+ B cells suppress T cell interferon‐gamma production and promote Foxp3 expression,but have impaired regulatory function in coronary artery disease

Atherosclerosis and its associated coronary artery disease (CAD) represent another chronic low‐grade inflammatory disorder. Regulatory B cells (Bregs) possess essential functions in maintaining peripheral tolerance and inhibiting pathogenic inflammation through IL‐10. Here, we investigated one subset of Bregs, Tim‐1⁺ B cell, and its role in atherosclerosis and CAD patients. In healthy individuals, IL‐10‐producing B cells were predominantly found in the Tim‐1⁺ B cells. Upon stimulation of the B cell receptor (BCR) and Toll‐like receptor 9 (TLR‐9) by anti‐BCR antibodies and CpG, respectively, the Tim‐1⁺ B cells could further upregulate IL‐10 expression. In contrast, the Tim‐1⁺ B cells were present at normal frequency in CAD patients, but showed impaired capacity to upregulate IL‐10 with or without BCR + CpG stimulation. The stimulated Tim‐1⁺ B cells from healthy individuals also suppressed expression of interferon gamma (IFN‐γ), an atherogenic cytokine in T cells, in an IL‐10‐dependent fashion, and strongly promoted the expression of Foxp3 in naive CD4⁺CD45RO^? T cells. In contrast, the Tim‐1⁺ B cells from CAD patients were unable to suppress IFN‐γ secretion, and only minimally increased the expression of Foxp3 in naive CD4⁺CD45RO^? T cells. Despite this, the frequency of Tim‐1⁺ B cells in the atherosclerotic lesions from CAD patients was inversely correlated with the frequency of IFN‐γ‐expressing T cells. Together, these results demonstrated that CAD patients presented an inflammatory disorder in regulatory B cells, which could be used as a therapeutic target.     

Membrane‐bound Dickkopf‐1 in Foxp3+ regulatory T cells suppresses T‐cell‐mediated autoimmune colitis

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3⁺ regulatory T (Treg) cells use Dickkopf‐1 (DKK‐1) to regulate T‐cell‐mediated tolerance in the T‐cell‐mediated autoimmune colitis model. Treg cells from DKK‐1 hypomorphic doubleridge mice failed to control CD4⁺ T‐cell proliferation, resulting in CD4 T‐cell‐mediated autoimmune colitis. Thymus‐derived Treg cells showed a robust expression of DKK‐1 but not in naive or effector CD4 T cells. DKK‐1 expression in Foxp3⁺ Treg cells was further increased upon T‐cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3⁺ Treg cells expressed DKK‐1 in the cell membrane and the functional inhibition of DKK‐1 using DKK‐1 monoclonal antibody abrogated the suppressor function of Foxp3⁺ Treg cells. DKK‐1 expression was dependent on de novo protein synthesis and regulated by the mitogen‐activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane‐bound DKK‐1 as a novel Treg‐derived mediator to maintain immunological tolerance in T‐cell‐mediated autoimmune colitis.     

Enhanced suppressor function of TIM‐3+FoxP3+ regulatory T cells

T‐cell immunoglobulin and mucin domain 3 (TIM‐3) is an Ig‐superfamily member expressed on IFN‐γ‐secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM‐3 is expressed by a subset of activated CD4⁺ T cells, and anti‐CD3/anti‐CD28 stimulation increases both the level of expression and the number of TIM‐3⁺ T cells. In mice, TIM‐3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4⁺ T‐cell differentiation. Here, we examined TIM‐3 expression on human Treg cells to determine its role in T‐cell suppression. In contrast to mice, TIM‐3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM‐3⁺ Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM‐3^? Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT‐3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM‐3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1‐ and Th17‐cell responses.     

Phenotypic characterization of regulatory T cells from antiretroviral‐naive HIV‐1‐infected people

Regulatory T (Treg) cells play a key role in dampening excessive immune activation. However, antiretroviral therapy (ART) ‐naive HIV‐1 infection maintains the immune system in a sustained state of activation that could alter both Treg cell surface markers and functions. As Treg cell surface markers are directly linked to their functions the overall objective of this study was to determine how ART‐naive HIV infection affects the phenotypic properties of Treg cells. Our data showed that in ART‐naive HIV‐1 infection, Treg cells are dominated by effector (CD45RA⁺ CD27⁻ CCR7⁻ CD62L⁻) and effector memory (CD45RA⁻ CD27⁻ CCR7⁻ CD62L⁻) cells. In contrast Treg cells from HIV‐negative individuals were mainly naive (CD45RA⁺ CD27⁺ CCR7⁺ CD62L⁺) and central memory (CD45RA⁻ CD27⁺ CCR7⁺ CD62L⁺) cells. Whereas effector and effector memory Treg cells showed enhanced expression of CD39 (P < 0·05), CD73 (P < 0·001), HLA‐DR and CD38 (P < 0·001); naive and central memory Treg cells showed a significant reduction in the expression of these markers. Overall Treg cell frequencies within total CD4⁺ T cells correlated positively with plasmatic HIV‐1 viral load. As increased viral load is associated with augmented CD4⁺ T‐cell destruction; this could suggest a resistance of peripheral Treg cells to HIV‐1 destruction. Hence the modulation of Treg cell phenotype and frequencies could be considered in designing immunotherapeutic strategies targeting immune system restoration during HIV‐1 infection.     

Memory IL‐22‐producing CD4+ T cells specific for Candida albicans are present in humans

Co‐expression of IL‐22 and IL‐17 has been identified and demonstrated to be involved in the immunopathogenesis of some autoimmune diseases as well as the defense against pathogenic infections in animal studies. However, the properties of IL‐22‐producing cells in humans remain largely unclear. In the present study, we showed that IL‐22 could be induced from human PBMC following various polyclonal stimulations. The majority of IL‐22‐producing cells in PBMC were CD4⁺ T cells with a memory cell phenotype. In addition, we found that a subset of IL‐22⁺ T cells produced IL‐22 alone, whereas other IL‐22⁺ T cells co‐expressed cytokines typical of Th1, Th2 and Th17 cells. Importantly, stimulation of PBMC from healthy adults with heat‐inactivated Candida albicans (C. albicans) yeast or hyphae resulted in IL‐22 production by central and effector memory CD4⁺ T cells. Moreover, CD4⁺CCR6⁺ but not CD4⁺CCR6^? T cells produced IL‐22 when stimulated with either C. albicans or PMA and ionomycin. In addition, PBMC from the individuals infected with C. albicans produced a significantly higher amount of IL‐22 compared with healthy controls following stimulation with C. albicans. These data demonstrate that IL‐22‐producing T cells in humans may play an important role in the defense against fungal infections such as C. albicans.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

A unique DNA methylation signature defines a population of IFN‐γ/IL‐4 double‐positive T cells during helminth infection

Th1 and Th2 cell fates are traditionally viewed as mutually exclusive, but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4⁺ T cells from mice infected with the parasitic helminth Schistosoma mansoni, we observed a defined population of IFN‐γ/IL‐4 double‐positive cells. These IFN‐γ⁺IL‐4⁺ cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN‐γ⁺IL‐4^? (Th1) and IFN‐γ^?IL‐4⁺ (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN‐γ⁺IL‐4⁺ cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind the co‐existence of Th1 and Th2 characteristics.     

HCV‐infected hepatocytes drive CD4+CD25+Foxp3+ regulatory T‐cell development through the Tim‐3/Gal‐9 pathway

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T‐cell Ig and mucin domain protein‐3 (Tim‐3) and galectin‐9 (Gal‐9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim‐3/Gal‐9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim‐3/Gal‐9 interactions regulate HCV‐mediated Treg‐cell development, here we provide pilot data showing that HCV‐infected human hepatocytes express higher levels of Gal‐9 and TGF‐β, and upregulate Tim‐3 expression and regulatory cytokines TGF‐β/IL‐10 in co‐cultured human CD4⁺ T cells, driving conventional CD4⁺ T cells into CD25⁺Foxp3⁺ Treg cells. Additionally, recombinant Gal‐9 protein can transform TCR‐activated CD4⁺ T cells into Foxp3⁺ Treg cells in a dose‐dependent manner. Importantly, blocking Tim‐3/Gal‐9 ligations abrogates HCV‐mediated Treg‐cell induction by HCV‐infected hepatocytes, suggesting that Tim‐3/Gal‐9 interactions may regulate human Foxp3⁺ Treg‐cell development and function during HCV infection.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

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.     

Expansion of Foxp3+ T‐cell populations by Candida albicans enhances both Th17‐cell responses and fungal dissemination after intravenous challenge

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3⁺ regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3⁺ Treg‐cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4⁺Foxp3⁺ population that correlated positively with fungal burden. Depletion from Foxp3^hCD2 reporter mice in vivo confirmed that Foxp3⁺ cells exacerbated fungal burden and inflammatory renal disease. The CD4⁺Foxp3⁺ population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg‐cell subset, together with conversion of Foxp3^? cells to the induced Treg‐cell form, and to a cell type sharing effector Th17‐cell characteristics, expressing ROR‐γt, and secreting IL‐17A. The expanded Foxp3⁺ T cells inhibited Th1 and Th2 responses, but enhanced Th17‐cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17‐cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3⁺ T cells promotes Th17‐cell responses that drive pathology.     

CD26‐mediated co‐stimulation in human CD8+ T cells provokes effector function via pro‐inflammatory cytokine production

CD26 is an activation marker of human CD4⁺ T cells, and is associated with T‐cell signal transduction processes as a co‐stimulatory molecule. We have previously demonstrated that high CD26 cell surface expression on CD4⁺ T cells is correlated with the production of T helper type 1 cytokines, whereas CD26⁺ T helper cells stimulate antibody synthesis in B cells. Although the cellular and molecular mechanisms involved in CD26‐mediated CD4⁺ T‐cell activation have been extensively evaluated by our group and others, the role of CD26 in CD8⁺ T cells has not been clearly elucidated. In the present study, we examine the effector function of CD8⁺ T cells via CD26‐mediated co‐stimulation in comparison with CD28‐mediated co‐stimulation. We found that CD26^high CD8⁺ T cells belong to the early effector memory T‐cell subset, and that CD26‐mediated co‐stimulation of CD8⁺ T cells exerts a cytotoxic effect preferentially via granzyme B, tumour necrosis factor‐α, interferon‐γ and Fas ligand. The effector function associated with CD26‐mediated co‐stimulation is enhanced compared with that obtained through CD28‐mediated co‐stimulation, suggesting that the CD26 co‐stimulation pathway in CD8⁺ T cells is distinct from the CD28 co‐stimulation pathway. Targeting CD26 in CD8⁺ T cells therefore has the potential to be useful in studies of immune responses to new vaccine candidates as well as innovative therapy for immune‐mediated diseases.     

Mycobacterium tuberculosis PstS1 amplifies IFN‐γ and induces IL‐17/IL‐22 responses by unrelated memory CD4+ T cells via dendritic cell activation

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4⁺ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4⁺ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4⁺ and CD8⁺ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α^? subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.