Association between discordant immunological response to highly active anti‐retroviral therapy,regulatory T cell percentage,immune cell activation and very low‐level viraemia in HIV‐infected patients

The mechanisms sustaining the absence of complete immune recovery in HIV‐infected patients upon long‐term effective highly active anti‐retroviral therapy (HAART) remain elusive. Immune activation, regulatory T cells (T_regs) or very low‐level viraemia (VLLV) have been alternatively suspected, but rarely investigated simultaneously. We performed a cross‐sectional study in HIV‐infected aviraemic subjects (mean duration of HAART: 12 years) to concomitantly assess parameters associated independently with inadequate immunological response. Patients were classified as complete immunological responders (cIR, n = 48) and inadequate immunological responders (iIR, n = 39), depending on the CD4⁺ T cell count (> or < 500/mm³). Clinical and virological data (including very low‐level viraemia) were collected. In parallel, immunophenotyping of CD4⁺ lymphocytes, including T_reg subsets, and CD8⁺ T cells was performed. Percentages of activated CD4⁺ T cells, T_regs, effector T_regs and terminal effector T_regs were found to be significantly elevated in iIR. Neither the percentage of activated CD8⁺ T cells nor VLLV were found to be associated with iIR. In the multivariate analysis, nadir of CD4⁺ T cell count and percentage of T_regs were the only two parameters associated independently with iIR [odds ratio (OR) = 2·339, P = 0·001, and OR = 0·803, P = 0·041]. We present here the largest study investigating simultaneously the immune response to long‐term HAART, activation of CD4⁺ and CD8⁺ T cells, T_reg percentages and very low‐level viraemia. Causative interactions between T_regs and CD4⁺ T cells should now be explored prospectively in a large patients cohort.     

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.     

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.     

CD4+HLA-G+ regulatory T cells: Molecular signature and pathophysiological relevance

The regulation of potentially harmful immune responses by regulatory T (T_reg) cells is essential for maintaining peripheral immune tolerance and homeostasis. Especially CD4⁺ T_reg cells have been regarded as pivotal regulators of autoreactive and inflammatory responses as well as inducers of immune tolerance by using a variety of immune suppressive mechanisms.Besides the well-known classical CD4⁺CD25⁺FoxP3⁺ T_reg cells, CD4⁺ T cells expressing the immune tolerizing molecule human leukocyte antigen G (HLA-G) have been recently described as another potent thymus-derived T_reg (tT_reg) cell subset. Albeit both tT_reg subsets share common molecular characteristics, the mechanisms of their immunosuppressive function differ fundamentally. Dysfunction and numerical abnormalities of classical CD4⁺ tT_reg cells have been implicated in the pathogenesis of several immune-mediated diseases such as multiple sclerosis (MS). Clearly, a deeper understanding of the various CD4⁺ tT_reg subsets and also the underlying mechanisms of impaired immune tolerance in these disorders are essential for the development of potential therapeutic strategies.This review focuses on the current knowledge on defining features and functioning of HLA-G⁺CD4⁺ tT_reg cells as well as their emerging role in various pathologies with special emphasis on the pathogenesis of MS. Furthermore, future research possibilities together with potential therapeutic applications are discussed.     

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.     

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.     

Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions

Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed‐type hypersensitivity (DTH) responses? To what extent can s.l.‐induced tolerance be explained by the generation of Foxp3⁺CD25⁺CD4⁺ regulatory T cells (T_reg)? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40‐μg doses of OVA/CTB conjugate suppressed T‐cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag‐specific T_reg in CLN, MLN and spleen and also transforming growth factor‐β (TGF‐β) levels in serum. The CLN and splenic cells from OVA/CTB‐treated BALB/c mice efficiently suppressed OVA‐specific T‐cell receptor (TCR) transgenic (DO11.10) CD25^?CD4⁺ effector T‐cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA‐specific DTH responses in vivo and T‐cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA‐specific (KJ1‐26⁺) CD4⁺ T cells from OVA/CTB‐treated mice. Thus, s.l. administration of CTB‐conjugated Ag can efficiently induce peripheral T‐cell tolerance associated with strong increases in serum TGF‐β levels and in Ag‐specific Foxp3⁺CD25⁺CD4⁺ T_reg cells.     

Tolerance induction after specific immunotherapy with pollen allergoids adjuvanted by monophosphoryl lipid A in children

Specific immunotherapy (SIT) is a well‐established and clinically effective treatment for allergic diseases. A pollen allergoid formulated with the T helper type 1 (Th1)‐inducing adjuvant monophosphoryl lipid A (MPL) facilitates short‐term SIT. Little is known about mechanisms of tolerance induction in this setting. In a prospective study, 34 patients allergic to grass pollen (25 male, nine female, median age 10·2 years) received a total of 44 SIT courses (20 in the first, 24 in the second) with MPL‐adjuvanted pollen allergoids. Immunogenicity was measured by levels of specific immunoglobulin G (IgG_grass) and IgG4_grass by antibody blocking properties on basophil activation, and by induction of CD4⁺, CD25⁺ and forkhead box P3 (FoxP3⁺) regulatory T cells (T_reg). Specific IgG and IgG4 levels increased only slightly in the first year of SIT. In the second year these changes reached significance (P < 0·0001). In keeping with these findings, we were able to show an increase of T_reg cells and a decreased release of leukotrienes after the second year of treatment. In the first year of treatment we found little evidence for immunological changes. A significant antibody induction was seen only after the second course of SIT. Short‐course immunotherapy with pollen allergoids formulated with the Th1‐inducing adjuvant MPL needs at least two courses to establish tolerance.     

Down syndrome, autoimmunity and T regulatory cells

Autoimmune diseases are more represented in Down syndrome (DS) individuals compared to chromosomally normal people. Natural T regulatory cells (nT_reg) have been considered to be primary in the role of controlling the intensity and targets of the immune response. We have investigated the phenotypical and functional alteration of nT_reg in a group of DS people. The phenotypical characteristic of T_reg cells of 29 DS was analysed and compared with an age‐matched healthy control group. The inhibitory potential of CD4⁺CD25^highCD127^low T regulatory cells was evaluated on autologous CD4⁺CD25^‐ T cell proliferation in response to activation with a mytogenic pan‐stimulus (anti‐CD2, anti‐CD3 and anti‐CD28 antibodies). The CD4⁺CD25^high cells in the DS and control groups were 2·692 ± 0·3808%, n = 29 and 1·246 ± 0·119, n = 29%, respectively (P = 0.0007), with a percentage of forkhead box protein 3 (FoxP3)‐expressing cells of 79·21 ± 3·376%, n = 29 and 59·75 ± 4·496%, respectively (P = 0.0015). CD4⁺CD25⁺FoxP3⁺ cells were increased in peripheral blood from DS subjects (DS mean 5·231 ± 0·6065% n = 29, control mean 3·076 ± 0·3140% n = 29). The majority of CD4⁺CD25^high were CD127^low and expressed a high percentage of FoxP3 (natural T_reg phenotype). While the proliferative capacity of DS T cells was not altered significantly compared to normal individuals, a reduced inhibitory potential of T_reg compared to healthy controls was clearly observed (mean healthy control inhibition in T_eff : T_reg 1:1 co‐culture: 58·9% ± 4·157%, n = 10 versus mean DS inhibition in T_eff : T_reg 1:1 co‐culture: 39·8 ± 4·788%, n = 10, P = 0.0075; mean healthy control inhibition in T_eff : T_reg 1:0·5 co‐culture: 45·10 ± 5·858%, n = 10 versus DS inhibition in T_eff : T_reg 1:0·5 co‐culture: 24·10 ± 5·517%, n = 10, P = 0.0177). DS people present an over‐expressed peripheral nT_reg population with a defective inhibitory activity that may partially explain the increased frequency of autoimmune disease.     

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.     

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögren's syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4⁺CD25⁺ regulatory T cells (T_reg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of T_reg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4⁺CD25⁺ T_reg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4⁺CD25⁺ T_reg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4⁺CD25⁺ T_reg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4⁺CD25⁺ T_reg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.     

Level,phenotype and activation status of CD4+FoxP3+ regulatory T cells in patients chronically infected with human immunodeficiency virus and/or hepatitis C virus

CD4⁺ regulatory T (T_reg) cells have been involved in impaired immunity and persistence of viral infections. Herein, we report the level, phenotype and activation status of T_reg cells in patients chronically infected with human immunodeficiency virus (HIV) and/or hepatitis C virus (HCV). Expression of CD25, CD45RA, CD27, CD127 and CD38 was assessed on these cells using polychromatic flow cytometry in 20 healthy controls, 20 HIV‐monoinfected, 20 HCV‐monoinfected and 31 HIV/HCV‐co‐infected patients. T_reg cells were defined as CD4⁺forkhead box P3 (FoxP3)⁺. The percentage of T_reg cells was increased significantly in HIV patients compared with controls. Moreover, there was a significant inverse correlation between CD4 counts and T_reg cell levels. Fewer than 50% of T_reg cells expressed CD25, with differences in terms of CD127 expression between CD25⁺ and CD25(^–) T_reg cells. CD4⁺Foxp3⁺ T_reg cells displayed predominantly a central memory phenotype (CD45RA^–CD27⁺), without differences between patients and healthy controls. Activated T_reg cells were increased in HIV patients, particularly considering the central memory subset. In summary, HIV infection, but not HCV, induces an up‐regulation of highly activated T_reg cells, which increases in parallel with CD4 depletion. Hypothetically, this might contribute to the accelerated course of HCV‐related liver disease in HIV‐immunosuppressed patients.     

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.     

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.