Interleukin-2 in the development and control of inflammatory disease

Summary: Interleukin-2 (IL-2) has multiple, sometimes opposing, functions during an inflammatory response. It is a potent inducer of T-cell proliferation and T-helper 1 (Th1) and Th2 effector T-cell differentiation and provides T cells with a long-lasting competitive advantage resulting in the optimal survival and function of memory cells. In a regulatory role, IL-2 is important for the development, survival, and function of regulatory T cells, it enhances Fas-mediated activation-induced cell death, and it inhibits the development of inflammatory Th17 cells. Thus, in its dual and contrasting functions, IL-2 contributes to both the induction and the termination of inflammatory immune responses.     

T cell responses induced by allergen-specific immunotherapy

Allergen‐specific immunotherapy is recognized as a highly effective practice in the treatment of patients with severe allergic rhinitis and/or asthma and is recommended by World Health Organization as an integrated part of allergy management strategy. Several studies have shown that allergen‐specific immunotherapy, based on the administration of increasing doses of allergen, achieves a hyposensitization and reduces both early and late responses occurring during the natural exposure to the allergen itself. This is the unique antigen‐specific immunomodulatory treatment in current use for human diseases. Successful immunotherapy is associated with reductions in symptoms and medication scores and improved quality of life. After interruption it usually confers long‐term remission of symptoms and prevents the onset of new sensitizations in children up to a number of years. Subcutaneous immunotherapy usually suppresses the allergen‐induced late response in target organs, likely due to the reduction of the infiltration of T cells, eosinophils, basophils, mast cells and neutrophils. In addition to the reduction of cells of allergic inflammation, immunotherapy also decreases inflammatory mediators at the site of allergen exposure. This review provides an update on the immunological T cell responses induced by conventional subcutaneous and sublingual immunotherapy, and gives a unifying view to reconciling the old dualism between immunoredirecting and immunoregulating mechanisms.     

TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells

Transforming growth factor (TGF)-beta-activating kinase 1 (TAK1) is critical for Toll-like receptor- and tumor necrosis factor-mediated cellular responses. In B cells, TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs), but not nuclear factor-kappaB (NF-kappaB), in antigen receptor signaling. In this study, we generate T cell-specific TAK1-deficient (Lck(Cre/(+))Tak1(flox/flox)) mice and show that TAK1 is indispensable for the maintenance of peripheral CD4 and CD8 T cells. In thymocytes, TAK1 is essential for TCR-mediated activation of both NF-kappaB and MAPKs. Additionally, Lck(Cre/(+))Tak1(flox/flox) mice developed colitis as they aged. In these mice, accumulations of activated/memory T cells as well as B cells were observed. Development of regulatory T (Treg) cells in thymus was abrogated in Lck(Cre/(+))Tak1(flox/flox) mice, suggesting that the loss of Treg cells is the cause of the disease. Together, the results show that TAK1, by controlling the generation of central Treg cells, is important for preventing spontaneously developing colitis.     

N‐Ethyl‐N‐nitrosourea mutagenesis in the mouse provides strong genetic and in vivo evidence for the role of the Caspase Recruitment Domain (CARD) of CARD‐MAGUK1 in T regulatory cell development

Natural regulatory T (nTreg) cells generated in the thymus are essential throughout life for the maintenance of T‐cell homeostasis and the prevention of autoimmunity. T‐cell receptor (TCR)/CD28‐mediated activation of nuclear factor‐κB and (J)un (N)‐terminal kinase pathways is known to play a key role in nTreg cell development but many of the predicted molecular interactions are based on extrapolations from non‐Treg cell TCR stimulation with non‐physiological ligands. For the first time, we provide strong genetic evidence of a scaffold function for the Caspase Recruitment Domain (CARD) of the TCR signalling protein CARD‐MAGUK1 (CARMA1) in nTreg cell development in vivo. We report two, new, N‐ethyl‐N‐nitrosourea‐derived mutant mice, Vulpo and Zerda, with a profound block in the development of nTreg cells in the thymus as well as impaired inducible Treg cell differentiation in the periphery. Despite independent heritage, both mutants harbour different point mutations in the CARD of the CARMA1 protein. Mutations in vulpo and zerda do not affect expression levels of CARMA1 but still impair signalling through the TCR due to defective downstream Bcl‐10 recruitment by the mutated CARD of CARMA1. Phenotypic differences observed between Vulpo and Zerda mutants suggest a role for the CARD of CARMA1 independent of Bcl‐10 activation of downstream pathways. We conclude that our forward genetic approach demonstrates a critical role for the CARD function of CARMA1 in Treg cell development in vivo.     

Peripheral CD4 loss of regulatory T cells is associated with persistent viraemia in chronic HIV infection

Chronic HIV infection is associated with T cell abnormalities and altered effector function. Regulatory T cells (Treg) are CD4+ T cells that play a critical role in regulating the immune system. The impact of regulatory T cells on HIV infection and disease progression may be highly significant. We hypothesize that chronic antigenic stimulation from a persistent, high viraemic state may promote a population of Treg that contributes to HIV-associated immune dysfunction. We evaluated the pattern of Treg in chronically infected, HIV-positive individuals over a course of 6 months. Treg are depleted at a distinct rate from that of absolute CD4 cells and loss of Treg is slower in the presence of viral suppression. In vitro depletion of CD25+ CD4+ cells resulted in increased Gag-specific CD4 and CD8 responses. A significant correlation between ex vivo measurement of Treg and Gag-specific CD4 T cell responses was observed (r=-0 x 41, P=0 x 018) with a trend observed with Gag-specific CD8 T cell responses (P=0 x 07). The impact of HIV infection on the Treg population directly complicates the measured effect of Treg on the immune dysfunction although our data support the important role of Treg on modulating the effector T cell response in chronic infection.     

Differential T‐cell receptor signals for T helper cell programming

Upon encounter with their cognate antigen, naive CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen‐presenting cells, cytokines and co‐stimulatory molecules. The strength of the T‐cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength.     

Regulatory T cell-derived interleukin-15 promotes the diversity of immunological memory

While the immunosuppressive function of regulatory T (Treg) cells has been extensively studied, their immune-supportive roles have been less well investigated. Using a lymphocytic choriomeningitis virus (LCMV) Armstrong infection mouse model, we found that Treg cell-derived interleukin (IL)-15 is required for long-term maintenance of the KLRG1⁺IL-7Rα⁻CD62L⁻ terminal effector memory CD8⁺ T (tTEM) cell subset, but dispensable for the suppressive function of Treg cells themselves. In contrast, deletion of Il15 from other sources, including myeloid cells and muscles, did not affect the composition of the memory CD8⁺ T cell pool. Our findings identify Treg cells as an essential IL-15 source maintaining tTEM cells and suggest that Treg cells promote the diversity of immunological memory.     

Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro

Cross-talk has been shown to occur between the immune system and bone metabolism pathways. In the present study, we investigated the impact of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells on osteoclastogenesis and bone resorption. Treg cells that were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy adults inhibited both the differentiation of osteoclasts (OCs) from human embryo bone marrow cells (BMCs) and the pit formation in a dose-dependent manner. In cell cocultures, the production levels of both interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1) were proportionally upregulated as the ratio of Treg cells to BMCs was increased, and the inhibition of OC differentiation and bone resorption by Treg cells was completely reversed by anti-IL-10 and anti-TGF-β1 antibodies. Treatment of BMC and Treg cell cocultures with 17β-estradiol (E2) at concentrations between 10⁻⁷ and 10⁻⁹ mol/l suppressed OC differentiation and bone resorption more efficiently than it did in cultures of BMCs alone; this enhanced suppression occurred via the stimulation of Treg cell IL-10 and TGF-β1 expression. These data suggest that Treg cells suppress OC differentiation and bone resorption by secreting IL-10 and TGF-β1. E2 enhances the suppressive effects of Treg cells on OC differentiation and bone resorption by stimulating IL-10 and TGF-β1 secretion from these cells. Therefore, Treg cell-derived IL-10 and TGF-β1 are likely involved in the regulation of E2 on bone metabolism and represent potential therapeutic targets for the treatment of postmenopausal osteoporosis (PMO).     

Oestrogen modulates experimental autoimmune encephalomyelitis and interleukin-17 production via programmed death 1

The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17β-oestradiol (E₂) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4⁺ FoxP3⁺ regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E₂ at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E₂ treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E₂ treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E₂ failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E₂-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.     

Mechanisms of autoimmunity in the non‐obese diabetic mouse: effector/regulatory cell equilibrium during peak inflammation

Immune imbalance in autoimmune disorders such as type 1 diabetes may originate from aberrant activities of effector cells or dysfunction of suppressor cells. All possible defective mechanisms have been proposed for diabetes‐prone species: (i) quantitative dominance of diabetogenic cells and decreased numbers of regulatory T cells, (ii) excessive aggression of effectors and defective function of suppressors, (iii) perturbed interaction between effector and suppressor cells, and (iv) variations in sensitivity to negative regulation. The experimental evidence available to date presents conflicting information on these mechanisms, with identification of perturbed equilibrium on the one hand and negation of critical role of each mechanism in propagation of diabetic autoimmunity on the other hand. In our analysis, there is no evidence that inherent abnormalities in numbers and function of effector and suppressor T cells are responsible for the immune imbalance responsible for propagation of type 1 diabetes as a chronic inflammatory process. Possibly, the experimental tools for investigation of these features of immune activity are still underdeveloped and lack sufficient resolution, in the presence of the extensive biological viability and functional versatility of effector and suppressor elements.     

Tremelimumab (anti-CTLA4) mediates immune responses mainly by direct activation of T effector cells rather than by affecting T regulatory cells

Cytotoxic T Lymphocyte Antigen 4 (CTLA4) blockade has shown antitumor activity against common cancers. However, the exact mechanism of immune mediation by anti-CTLA4 remains to be elucidated. Further understanding of how CTLA4 blockade with tremelimumab mediates immune responses may allow a more effective selection of responsive patients. Our results show that tremelimumab enhanced the proliferative response of T effector cells (Teff) upon TCR stimulation, and abrogated Treg suppressive ability. In the presence of tremelimumab, frequencies of IL-2-secreting CD4(+) T cells and IFN-γ-secreting CD4(+) and CD8(+) T cells were increased in response to polyclonal activation and tumor antigens. Importantly, Treg frequency was not reduced in the presence of tremelimumab, and expanded Tregs in cancer patients treated with tremelimumab expressed FoxP3 with no IL-2 release, confirming them as bona fide Tregs. Taken together, this data indicates that tremelimumab induces immune responses mainly by direct activation of Teff rather than by affecting Tregs.     

Interaction between natural killer cells and regulatory T cells: perspectives for immunotherapy

Regulatory T (Treg) cells and natural killer (NK) cells are key players in the immune system. The interaction between these two cell types has been reported to be beneficial in healthy conditions such as pregnancy. However, in the case of certain pathologies such as autoimmune diseases and cancer this interaction can become detrimental, as Treg cells have been described to suppress NK cells and in particular to impair NK cell effector functions. This review aims to discuss the recent information on the interaction between Treg cells and NK cells under healthy and pathologic conditions, to describe the specific conditions in which this interaction takes place, the effect of Treg cells on hematopoietic stem cell differentiation and the consequences of this interaction on the optimization of immunotherapeutic protocols.     

Direct Engagement of TLR9 Ligand with T Helper Cells Leads to Cell Proliferation & Up-regulation of Cytokines

Purpose: Toll like receptor (TLR) engagement is primarily a function of the innate immune cells. The purpose of the study was to assess direct uptake of ODN 2216 in T helper cells and effects on cell proliferation and cytokine expression.

Methods: We isolated CD4+ CD25- T helper cells by magnetic sorting and studied the uptake of ODN 2216 using flow cytometry and confocal microscopy. We then studied the effect of ODN 2216 engagement on cell proliferation and cytokine expression using flow cytometry and gene expression of TLR9 signaling genes using real time RT-PCR.

Results: We made a chance observation that purified T helper cells from healthy individuals consistently bind to the TLR9 ligand ODN 2216. In PBMCs, on the other hand, 98% of monocytes preferentially bound to ODN 2216 FITC, indicating that they competed with the lymphocytes. We confirmed intracellular localization of ODN 2216 FITC as well as intracellular expression of TLR9 in Thelper cells. Furthermore, ODN 2216 FITC was also co-localized with the lysosomal membrane associated protein 1. The uptake of TLR9 ligand culminated in cellular proliferation, up-regulation of cytokines and increased mRNA expression of TLR9 and IRF7 in T helper cells, in the absence of antigen presenting cells. ODN 2216 uptake was inhibited by promethazine as well as by TLR9 antagonist.

Conclusions: Our results show a direct engagement of TLR9 ligand in T helper cells and suggest involvement of TLR9 signalling in CD4+T cells, which may envisage novel targets for TLR inhibitors.     

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.     

Role of interleukin-2 and interleukin-6 in the mitogen responsiveness of T cells from patients with ''common-variable'' hypogammaglobulinaemia.

We have assessed the ability of interleukin-2 (IL-2) and interleukin-6 (IL-6) to augment the proliferative response of T lymphocytes from 'common-variable' hypogammaglobulinaemia (CVH) patients and from normal controls, to the mitogens phytohaemagglutinin (PHA) and OKT3. We show that with cells from the control group and from those patients whose T cells respond to PHA within the control range, both IL-2 and IL-6 will significantly augment the response to OKT3. However, in those patients with a T cell defect in which the PHA response is below the control range, neither IL-2 nor IL-6 could restore the PHA or OKT3 response to normal. Responses to IL-2 or IL-6 alone were always in or above the control range.     

Phosphoinositide 3-kinase δ is a regulatory T-cell target in cancer immunotherapy

Tumour infiltration by regulatory T (Treg) cells contributes to suppression of the anti-tumour immune response, which limits the efficacy of immune-mediated cancer therapies. The phosphoinositide 3-kinase (PI3K) pathway has key roles in mediating the function of many immune cell subsets, including Treg cells. Treg function is context-dependent and depends on input from different cell surface receptors, many of which can activate the PI3K pathway. In this review, we explore how PI3Kδ contributes to signalling through several major immune cell receptors, including the T-cell receptor and co-stimulatory receptors such as CD28 and ICOS, but is antagonized by the immune checkpoint receptors CTLA-4 and PD-1. Understanding how PI3Kδ inhibition affects Treg signalling events will help to inform how best to use PI3Kδ inhibitors in clinical cancer treatment.     

吸入糖皮质激素对哮喘儿童外周血Foxp3~+调节性T细胞及IL-2、IL-6的影响及其机制研究

研究吸入丙酸氟替卡松(FP)对哮喘患儿外周血单个核细胞(PBMC)中CD4+Foxp3+调节T细胞、细胞因子IL-2、IL-6以及转录因子STAT5的影响。以30例确诊为支气管哮喘的患儿为研究对象,随机分为未治疗哮喘组(15例)、吸入FP缓解组(15例),10例同期正常儿童为对照组。流式细胞仪检测外周血PBMC中的CD4+Foxp3+调节T细胞比率,ELISA检测血浆或培养上清中IL-2、IL-6细胞因子水平,Western blot检测PBMC中磷酸化及非磷酸化STAT5的水平。结果1.未治疗哮喘组PBMC中CD4+Foxp3+T细胞百分率在PHA刺激培养前后均明显低于正常对照组,吸入FP缓解组明显升高,与正常对照组没有差异;各组刺激后CD4+Foxp3+T细胞百分率均有升高,吸入FP缓解组、正常对照组分别升高约1.89、2.01倍,而未治疗哮喘组升高仅1.56倍;2.未治疗哮喘组血浆中IL-6水平明显高于正常组及吸入FP缓解组,而IL-2水平没有明显差异;3.PHA刺激24 h后未治疗哮喘组磷酸化STAT5(p-STAT)表达水平明显低于吸入FP组及正常对照组,而各组STAT5表达水平没有明显差异,结论吸入FP能增加哮喘患儿外周血PBMC中CD4+Foxp3+调节T细胞数量,其机制可能与降低血浆IL-6,上调STAT5磷酸化水平有关。     

Alpha1beta1 integrin and interleukin-7 receptor up-regulate the expression of RANKL in human T cells and enhance their osteoclastogenic function

Activated T cells, through the production of the receptor activator of NF-kappaB ligand (RANKL) cytokine, have been implicated in the osteoclast development and bone loss that are associated with autoimmune diseases such as rheumatoid arthritis. However, the cellular pathways that regulate the expression of RANKL and the induction of osteoclasts are still unclear. In this study, we show that, in human effector CD4(+) T cells, activation of alpha1beta1 integrin and interleukin (IL)-7 receptor (IL-7R) up-regulates the expression and production of RANKL but has no effect on the production of interferon-gamma, an inhibitor of T-cell-mediated osteoclastogenesis. Thus, both alpha1beta1 integrin and IL-7R enhance the ability of these cells to induce the formation of osteoclasts from human monocytes. Furthermore, we found that simultaneous activation of effector CD4(+) T cells via alpha1beta1 integrin and IL-7R synergistically increases the production of RANKL and enhances their osteoclastogenic function. We also show that, although alpha1beta1 integrin does not protect human effector CD4(+) T cells from IL-2-withdrawal-induced apoptosis, it does enhance the pro-survival effect of IL-7, further emphasizing the importance of the alpha1beta1/IL-7R synergistic effect. Together our results identify a new function of alpha1beta1 integrin in T cells and suggest that activation of effector CD4(+) T cells through alpha1beta1 integrin and IL-7R is an important regulatory pathway in T-cell-dependent osteoclastogenesis. Further understanding of the mechanisms by which IL-7R and alpha1beta1 integrin promote T-cell-mediated osteoclastogenesis will lead to new insights into the regulatory pathways of T-cell-dependent bone resorption associated with autoimmune diseases.