Depletion of CD4+CD25+ regulatory T cells enhances natural killer T cell‐mediated anti‐tumour immunity in a murine mammary breast cancer model

Both invariant natural killer T (NK T) cells and CD4⁺CD25⁺ T regulatory cells (T_regs) regulate the immune system to maintain homeostasis. In a tumour setting, NK T cells activated by α‐galactosylceramide (α‐GalCer) execute anti‐tumour activity by secreting cytokines. By contrast, T_regs intrinsically suppress antigen‐specific immune responses and are often found to be elevated in tumour patients. In this study, we have shown that T_regs regulate NK T cell function negatively in vitro, suggesting a direct interaction between these cell types. In a murine mammary tumour model, we demonstrated that administration of either α‐GalCer or anti‐CD25 antibody alone markedly suppressed tumour formation and pulmonary metastasis, and resulted in an increase in the survival rate up to 44% (from a baseline of 0%). When treatments were combined, depletion of T_regs boosted the anti‐tumour effect of α‐GalCer, and the survival rate jumped to 85%. Our results imply a potential application of combining T_reg cell depletion with α‐GalCer to stimulate NK T cells for cancer therapy.     

Th17, synchronically increased with Tregs and Bregs,promoted by tumour cells via cell‐contact in primary hepatic carcinoma

Documented reports about T helper type 17 (Th17) cells have revealed that Th17 plays a critical role in inflammation and autoimmunity diseases. However, the role of Th17 in cancer remains contradictory. The interplay between Th17 and tumour cells in the tumour microenvironment of primary hepatic carcinoma (PHC) needs to be explored further and the relationship between Th17, regulatory T cells (T_regs) and regulatory B cells (B_regs) has not been defined completely. In this study, numerous experiments were undertaken to elucidate the interaction of Th17 and T_reg/B_reg cells involved in PHC. Our work demonstrated that an increased Th17 was detected in the peripheral circulation and in tumour tissues in PHC patients. In addition, increases in peripheral blood Th17 corresponded with tumour–node–metastasis (TNM) stage progression. Also, further studies indicated that Th17 cells were promoted by tumour cells in the PHC tumour microenvironment through both contact‐dependent and ‐independent mechanisms, but cell‐contact played the major important role in promoting the production and proliferation of Th17. When isolated CD4⁺CD25⁺CD127^low T_regs and CD4⁺CD25^–CD127⁺ non‐T_regs were cultured with autologous tumour cells, it implied that the phenotype of Th17 and T_regs was modified by tumour cells in the tumour microenvironment. As well as this, Th17 cells were also found to correlate positively with CD4⁺forkhead box protein 3⁺ T_regs and CD19⁺CD5⁺CD1d^hi B_regs in PHC. Notably, Th17 increased synchronically with T_regs and B_regs in PHC. These findings may provide new clues to reveal the mechanisms of immune escape in PHC.     

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus‐infected patients after 5 years of highly active anti‐retroviral therapy may be due to increased thymic production of naive Tregs

This study determines levels of regulatory T cells (T_regs), naive T_regs, immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)‐infected patients receiving prolonged highly active anti‐retroviral therapy (HAART) who have known thymic output, and explores if naive T_regs may represent recent thymic emigrant T_regs. HIV‐infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T_regs (CD3⁺CD4⁺CD25⁺CD127^low), naive T_regs (CD3⁺CD4⁺CD25⁺CD45RA⁺) and activation markers (CD38⁺human leucocyte antigen D‐related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T_REC) content in CD4⁺ cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T_regs were significantly higher in HIV‐infected patients compared with controls, both after 1 and 5 years of HAART (P < 0·001), despite fully suppressed HIV‐RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T_regs were elevated significantly in HIV‐infected patients (P < 0·001) and were associated with thymic output measured as the T_REC frequency in CD4⁺ cells (P = 0·038). In summary, T_reg levels in HIV‐infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T_regs may contribute to higher T_reg levels in HIV‐infection.     

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.     

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.     

Suppression,subversion and escape: the role of regulatory T cells in cancer progression

Regulatory T cells (T_regs) are crucial in mediating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. However, in the context of cancer their role is more complex, and they are thought to contribute to the progress of many tumours. As cancer cells express both self‐ and tumour‐associated antigens, T_regs are key to dampening effector cell responses, and therefore represent one of the main obstacles to effective anti‐tumour responses. Suppression mechanisms employed by T_regs are thought to contribute significantly to the failure of current therapies that rely on induction or potentiation of anti‐tumour responses. This review will focus on the current evidence supporting the central role of T_regs in establishing tumour‐specific tolerance and promoting cancer escape. We outline the mechanisms underlying their suppressive function and discuss the potential routes of T_regs accumulation within the tumour, including enhanced recruitment, in‐situ or local proliferation, and de‐novo differentiation. In addition, we review some of the cancer treatment strategies that act, at least in part, to eliminate or interfere with the function of T_regs. The role of T_regs is being recognized increasingly in cancer, and controlling the function of these suppressive cells in the tumour microenvironment without compromising peripheral tolerance represents a significant challenge for cancer therapies.     

Pulmonary sarcoidosis is associated with high‐level inducible co‐stimulator (ICOS) expression on lung regulatory T cells – possible implications for the ICOS/ICOS‐ligand axis in disease course and resolution

Sarcoidosis is a granulomatous inflammatory disorder of unknown aetiology. The increased frequency of activated lung CD4⁺ T cells with a T helper type 1 (Th1) cytokine profile in sarcoidosis patients is accompanied by a reduced proportion and/or impaired function of regulatory T cells (T_regs). Here we evaluated the expression of the inducible co‐stimulator (ICOS) on lung and blood CD4⁺ T cell subsets in sarcoidosis patients with different prognosis, by flow cytometry. Samples from the deep airways were obtained by bronchoalveolar lavage (BAL). We show that T_regs from the inflamed lung of sarcoidosis patients were characterized by a unique ICOS^high phenotype. High‐level ICOS expression was restricted to T_regs from the inflamed lung and was absent in blood T_regs of sarcoidosis patients as well as in lung and blood T_regs of healthy volunteers. In addition, lung T_regs exhibited increased ICOS expression compared to sarcoid‐specific lung effector T cells. Strikingly, ICOS expression on T_regs was in particularly high in the lungs of Löfgren's syndrome (LS) patients who present with acute disease which often resolves spontaneously. Moreover, blood monocytes from LS patients revealed increased ICOS‐L levels compared to healthy donors. Sarcoidosis was associated with a shift towards a non‐classical monocyte phenotype and the ICOS‐L^high phenotype was restricted to this particular monocyte subset. We propose a potential implication of the ICOS/ICOS‐L immune‐regulatory axis in disease activity and resolution and suggest to evaluate further the suitability of ICOS as biomarker for the prognosis of sarcoidosis.     

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.     

Immunological monitoring of extracorporeal photopheresis after heart transplantation

Extracorporeal photopheresis (ECP) has been used as a prophylactic and therapeutic option to avoid and treat rejection after heart transplantation (HTx). Tolerance‐inducing effects of ECP such as up‐regulation of regulatory T cells (T_regs) are known, but specific effects of ECP on regulatory T cell (T_reg) subsets and dendritic cells (DCs) are lacking. We analysed different subsets of T_regs and DCs as well as the immune balance status during ECP treatment after HTx. Blood samples were collected from HTx patients treated with ECP for prophylaxis (n = 9) or from patients with histologically proven acute cellular rejection (ACR) of grade ≥ 1B (n = 9), as well as from control HTx patients without ECP (HTxC; n = 7). Subsets of T_regs and DCs as well as different cytokine levels were analysed. Almost 80% of the HTx patients showed an effect to ECP treatment with an increase of T_regs and plasmacytoid DCs (pDCs). The percentage of pDCs before ECP treatment was significantly higher in patients with no ECP effect (26·3% ± 5·6%) compared to patients who showed an effect to ECP (9·8% ± 10·2%; P = 0·011). Analysis of functional subsets of CD4⁺CD25^highCD127^low T_regs showed that CD62L‐, CD120b‐ and CD147‐positive T_regs did not differ between the groups. CD39‐positive T_regs increased during ECP treatment compared to HTxC. ECP‐treated patients showed higher levels for T helper type 1 (Th1), Th2 and Th17 cytokines. Cytokine levels were higher in HTx patients with rejection before ECP treatment compared to patients with prophylactic ECP treatment. We recommend a monitoring strategy that includes the quantification and analysis of T_regs, pDCs and the immune balance status before and up to 12 months after starting ECP.     

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.     

Phenotypical characterization of regulatory T cells in humans and rodents

Regulatory T cells (T_regs) constitute a fascinating subpopulation of CD4⁺ T cells due to their ability to limit the immune response against self and non‐self antigens. Murine models and antibodies directed against surface and intracellular molecules have allowed elucidation of the mechanisms that govern their development and function. However, these markers used to their classification lack of specificity, as they can be expressed by activated T cells. Similarly, there are slight differences between animal models, in steady state and pathological conditions, anatomical localization and strategy of analysis by flow cytometry. Here, we revised the most common markers utilized for T_reg typification by flow cytometry such as CD25, forkhead box protein 3 (FoxP3) and CD127, along with our data obtained in different body compartments of humans, mice and rats. Furthermore, we revised and determined the expression of other molecules important for the phenotypical characterization of T_reg cells. We draw attention to the drawbacks of those markers used in chronic states of inflammation. However, until a specific marker for the identification of T_regs is discovered, the best combination of markers will depend upon the tissue or the degree of inflammation from which T_regs derive.     

Heparin affects the induction of regulatory T cells independent of anti-coagulant activity and suppresses allogeneic immune responses

Heparin is a widely used anti-coagulant that enhances anti-thrombin (AT) activity. However, heparin also suppresses immune and inflammatory responses in various rodent models and clinical trials, respectively. The mechanism by which heparin suppresses immune responses is unclear. The effect of heparin on regulatory T cells (T_regs) in allogeneic immune responses was analysed using an acute graft-versus-host disease (aGVHD) mouse model and mixed lymphocyte reactions (MLRs). In-vitro culture systems were utilized to study the effects of heparin on T_regs. Heparin administration reduced mortality rates and increased the proportion of T_regs in the early post-transplantation period of aGVHD mice. In both murine and human MLRs, heparin increased T_regs and inhibited responder T cell proliferation. Heparin promoted functional CD4⁺CD25⁺forkhead box protein 3 (FoxP3)⁺ T_reg generation from naive CD4⁺ T cells, increased interleukin (IL)-2 production and enhanced the activation of pre-existing T_regs with IL-2. Heparin-induced T_reg increases were not associated with anti-coagulant activity through AT, but required negatively charged sulphation of heparin. Importantly, N-acetyl heparin, a chemically modified heparin without anti-coagulant activity, induced T_regs and decreased mortality in aGVHD mice. Our results indicate that heparin contributes to T_reg-mediated immunosuppression through IL-2 production and suggest that heparin derivatives may be useful for immunopathological control by efficient T_reg induction.     

Patients treated with high‐dose intravenous immunoglobulin show selective activation of regulatory T cells

Intravenous immunoglobulin (IVIg) is used to treat autoimmune and systemic inflammatory diseases caused by derailment of humoral and cellular immunity. In this study we investigated whether IVIg treatment can modulate regulatory T cells (T_regs) in humans in vivo. Blood was collected from IVIg-treated patients with immunodeficiency or autoimmune disease who were treated with low-dose (n = 12) or high-dose (n = 15) IVIg before, immediately after and at 7 days after treatment. Percentages and activation status of circulating CD4⁺CD25⁺forkhead box protein 3 (FoxP3⁺) T_regs and of conventional CD4⁺FoxP3⁻ T-helper cells (T_conv) were measured. The suppressive capacity of T_regs purified from blood collected at the time-points indicated was determined in an ex-vivo assay. High-dose, but not low-dose, IVIg treatment enhanced the activation status of circulating T_regs, as shown by increased FoxP3 and human leucocyte antigen D-related (HLA-DR) expression, while numbers of circulating T_regs remained unchanged. The enhanced activation was sustained for at least 7 days after infusion, and the suppressive capacity of purified T_regs was increased from 41 to 70% at day 7 after IVIg treatment. The activation status of T_conv was not affected by IVIg. We conclude that high-dose IVIg treatment activates T_regs selectively and enhances their suppressive function in humans in vivo. This effect may be one of the mechanisms by which IVIg restores imbalanced immune homeostasis in patients with autoimmune and systemic inflammatory disorders.     

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.     

The influence of bone marrow‐ and synovium‐derived mesenchymal stromal cells from osteoarthritis patients on regulatory T cells in co‐culture

There is increasing evidence that inflammation in the synovium plays a major role in the progression of osteoarthritis (OA). However, the immunogenic properties of mesenchymal stromal cells (MSCs), which are considered to regulate immunity in various diseases, remain largely unknown in OA. The purpose of this study was to determine the influence of MSCs from OA patients on regulatory T cells (T_regs) in an allogeneic co‐culture model. Bone marrow (BM) and synovial membrane (SM) were harvested from hip joints of OA patients and co‐cultured with lymphocytes enriched in CD4⁺CD25⁺CD127^– regulatory T cells (T_reg⁺LC) from healthy donors. T_reg proportions and MSC markers were assessed by flow cytometry. Cytokine levels were assessed after 2 and 5 days of co‐cultivation. Additionally, T_reg⁺LC cultures were analysed in the presence of interleukin (IL)‐6 and MSC‐supernatant complemented medium. B‐MSCs and S‐MSCs were able to retain the T_reg proportion compared to lymphocyte monocultures. T cell–MSC co‐cultures showed a significant increase of IL‐6 compared to MSC cultures. S‐MSCs produced higher amounts of IL‐6 compared to B‐MSCs, both in single and T cell co‐cultures. The effect of retaining the T_reg percentage could be reproduced partially by IL‐6 addition to the medium, but could only be observed fully when using MSC culture supernatants. Our data demonstrate that retaining the T_reg phenotype in MSC–T cell co‐cultures can be mediated by MSC derived from OA patients. IL‐6 plays an important role in mediating these processes. To our knowledge, this study is the first describing the interaction of MSCs from OA patients and T_regs in an allogeneic co‐culture model.     

Serum inflammatory factors and circulating immunosuppressive cells are predictive markers for efficacy of radiofrequency ablation in non‐small‐cell lung cancer

In recent years, percutaneous radiofrequency ablation (RFA) has been developed as a new tool in the treatment of non‐small‐cell lung cancer (NSCLC) in non‐surgical patients. There is growing evidence that RFA‐mediated necrosis can modulate host immune responses. Here we analysed serum inflammatory factors as well as immunosuppressive cells in the peripheral blood to discover possible prognostic indicators. Peripheral blood and serum samples were collected before RFA and within 3 months after the treatment in a total of 12 patients. Inflammatory cytokines and growth factors were measured in serum by the Bio‐Plex assay. Myeloid‐derived suppressor cells (MDSCs) and regulatory T cells (T_regs) were evaluated in the peripheral blood via flow cytometry. In patients developing local or lymphogenic tumour relapse (n = 4), we found an early significant increase in the concentration of tumour necrosis factor (TNF)‐α as well as chemokine (C‐C motif) ligand (CCL)‐2 and CCL‐4 compared to patients without relapse (n = 4) and healthy donors (n = 5). These changes were associated with an elevated activity of circulating MDSC indicated by an increased nitric oxide (NO) production in these cells. Elevated serum levels of TNF‐α, CCL‐2 and CCL‐4 associated with an increased NO production in circulating MDSCs might be an early indicator of the incomplete RFA and subsequently a potential tumour relapse in NSCLC.     

Immunosuppressive drugs affect interferon (IFN)‐γ and programmed cell death 1 (PD‐1) kinetics in patients with newly diagnosed autoimmune hepatitis

Autoimmune hepatitis (AIH) is characterized by overwhelming effector immune responses associated with defective regulatory T cells (T_regs). Several lines of evidence indicate CD4 as the main effectors involved in autoimmune liver damage. Herein we investigate the in‐vitro effects of prednisolone, 6‐mercaptopurine, cyclosporin, tacrolimus, mycophenolic acid (MPA) and rapamycin, immunosuppressive drugs (ISDs) used in AIH treatment, on the expression of proinflammatory cytokines, co‐inhibitory molecules and ability to proliferate of CD4⁺CD25^– cells, isolated from the peripheral blood of treatment‐naive patients with AIH. We note that in healthy subjects (HS) following polyclonal stimulation and in the absence of ISDs, the expression of interferon (IFN)‐γ, interleukin (IL)‐17 and tumour necrosis factor (TNF)‐α by CD4 effectors peaks at 48 h and decreases at 96 h to reach baseline levels. In contrast, in AIH the expression of all these proinflammatory cytokines continue rising between 48 and 96 h. Levels of programmed cell death‐1 (PD‐1), T cell immunoglobulin and mucin domain‐containing molecule‐3 (TIM‐3) and cytotoxic T lymphocyte antigen‐4 (CTLA‐4) increase over 96‐h culture both in HS and AIH, although with faster kinetics in the latter. Exposure to ISDs contains IFN‐γ and PD‐1 expression in AIH, where control over CD4⁺CD25^– cell proliferation is also noted upon exposure to MPA. Treatment with tacrolimus and cyclosporin render CD4⁺CD25^– cells more susceptible to T_reg control. Collectively, our data indicate that in treatment‐naive patients with AIH, all ISDs restrain T helper type 1 (Th1) cells and modulate PD‐1 expression. Furthermore, they suggest that tacrolimus and cyclosporin may ameliorate effector cell responsiveness to T_regs.