Virus-specific interleukin-17-producing CD4+ T cells are detectable in early human immunodeficiency virus type 1 infection

T(H)-17 cells have been shown to play a role in bacterial defense, acute inflammation, and autoimmunity. We examined the role of interleukin 17 (IL-17) production in human immunodeficiency virus type 1 (HIV-1) infection. Both HIV-1- and cytomegalovirus (CMV)-specific IL-17-producing CD4(+) T cells were detectable in early HIV-1 infection but were reduced to nondetectable levels in chronic and nonprogressive HIV-1 infection. IL-17-producing CMV-specific cells were not detected in blood from HIV-1-uninfected normal volunteers. Virus-specific T(H)-17 cells could coexpress other cytokines and could express CCR4 or CXCR3. Although the etiology of these cells has yet to be established, we propose that microbial translocation may induce them.     

Interleukin-9 stimulates the production of interleukin-5 in CD4+ T cells

We recently showed that interleukin-9 (IL-9), a Th2 cytokine, promotes IL-5-mediated rejection of allografts in mice. This observation led us to investigate the functional link between IL-9 and IL-5 production during alloreactive T cell responses in vitro and in vivo. Firstly, we found that IL-9 was produced by alloreactive Th2 cells, and IL-9 mRNA was detected in skin allograft during Th2-type rejection. We then established that IL-5 production was impaired in alloreactive Th2 cells isolated from IL-9-deficient mice and that optimal IL-5 production after allogeneic stimulation requires a functional IL-9 receptor (IL-9R) on the responding cells. Finally, the production of IL-5 by anti-CD3-stimulated CD4+ T cells was abolished by neutralization of IL-9. Despite the fact that IL-9 promotes IL-5 production by alloreactive T cells, IL-9-deficient recipients of skin allografts still developed eosinophilic graft infiltrates and neither IL-9 nor IL-9R deficiency modified Th2-type allograft rejection.     

Generation and differentiation of IL-17-producing CD4+ T cells in malignant pleural effusion

IL-17-producing CD4(+) T (Th17) cells have been found to be increased in some human cancers; however, the possible implication of Th17 cells in regulating antitumor responses in malignant pleural effusion (MPE) remains to be elucidated. In the current study, distribution and phenotypic features of Th17 cells in both MPE and peripheral blood from patients with lung cancer were determined by flow cytometry or double immunofluorescence staining. The impacts of cytokines on Th17 cell generation and differentiation were explored. The chemoattractant activity of chemokines CCL20 and CCL22 for Th17 cells in vitro was also observed. It was found that the increased Th17 cells could be found in MPE compared with blood. The in vitro experiments showed that IL-1β, IL-6, IL-23, or their various combinations could promote Th17 cell generation and differentiation from naive CD4(+) T cells. MPE was chemotactic for Th17 cells, and this activity was partly blocked by anti-CCL20 and/or CCL22 Abs. Our data also showed that the accumulation of Th17 cells in MPE predicted improved patient survival. It could be concluded that the overrepresentation of Th17 cells in MPE might be due to Th17 cell differentiation and expansion stimulated by pleural proinflammatory cytokines and to recruitment of Th17 cells from peripheral blood induced by pleural chemokines CCL20 and CCL22. Furthermore, the accumulation of Th17 cells in MPE predicted improved patient survival. These data provide the basis for developing immune-boosting strategies based on ridding the cancer patient of this cell population.     

Lectin-mediated induction of IL-4-producing CD4+ T cells.

Cultured murine CD4+ T cells have been shown to differentiate into IL-2 or IL-4-producing subsets. The factors responsible for the development of CD4+ T cells which produce IL-2 but not IL-4 and cells capable of producing IL-4 but not IL-2 are unknown. Here we describe a system that allows the controlled induction of IL-2- or IL-4-producing T cells after one single round of activation. Freshly isolated CD8-depleted T cells were activated with various polyclonal T cell activators for 48 h, washed, and then expanded under different conditions. IL-2 and IL-4 production were induced by restimulation of T cells and were measured with CTLL cells that respond to both cytokines and mAb to IL-2 and IL-4. T cells produced mainly IL-2 and small amounts of IL-4 when restimulated after expansion culture for 12 days with rIL-2 alone. However, after expansion for 12 days in the presence of rIL-2 plus Con A, we observed a 30- to 100-fold up-regulation of IL-4 activity and a 100-fold down-regulation of IL-2 when assessed by responses of CTLL cells incubated with the supernatant of restimulated T cells and by responses of CTLL cells cocultured with restimulated cells. An increase of IL-4 and decrease of IL-2 was also observed when the results were based on the cell numbers at the beginning of the expansion culture. The induction of IL-4 and the down-regulation of IL-2 1) were not reproduced with alpha-methyl-mannoside-treated supernatant of Con A-stimulated spleen cells, 2) were not dependent on the presence of large numbers of APC, 3) did not result from differential consumption of lymphokines after restimulation, 4) were not due to a difference in the time course of IL-2 or IL-4 release in either T cell population, and 5) were obtained regardless of the agents used to activate or to restimulate the T cells. Because Con A remained detectable on the T cell surface and because expansion of activated T cells with IL-2 plus Con A for several days was necessary, our results indicate that mainly IL-4-producing CD4+ T cells can be induced by prolonged engagement of T cell surface molecules.     

Effector activity of decidual CD8+ T lymphocytes in early human pregnancy

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.     

CD8+ IL-17-producing T cells are important in effector functions for the elicitation of contact hypersensitivity responses

Allergen-induced contact hypersensitivity (CHS) is a T cell-mediated delayed-type immune response which has been considered to be primarily mediated by CD8+ T cytotoxic type I (Tc1) cells. IFN-gamma, the prototype Tc1 (Th1) cytokine, has been implicated as the primary inflammatory cytokine for CHS. In this study, we demonstrate that neutralization of IL-17 rather than IFN-gamma suppresses the elicitation of CHS. The suppression does not result from inhibition of the proliferation of allergen-activated T cells. Allergen sensitization induces the development of distinct CD8+ T cell subpopulations that produce IFN-gamma or IL-17. Although CD8+ IL-17-producing cells are stimulated by IL-23, they are inhibited by IL-12, a prototypical stimulator of IFN-gamma-producing Tc1 cells. This indicates that CD8+ IL-17-producing cells are distinct from Tc1 cells and are important in effector functions at the elicitation of CHS. These studies provide insights into a novel mechanism for CHS.     

Functions of metallothionein generating interleukin-10-producing regulatory CD4+ T cells potentiate suppression of collagen-induced arthritis

Metallothionein, a cysteine-rich stress response protein that is naturally induced by a variety of immunologic stressors, has been shown to suppress autoimmune disorders through mechanisms not yet fully defined. In the present study, we examined the underlying mechanisms by which metallothionein might mediate such regulation of autoimmunity. Na?ve CD4+ T cells from metallothionein-deficient mice differentiated to produce significantly less IL-10, TGF-gamma, and repressor of GATA, but more IFN-gamma and T-bet, when compared with those from wild-type mice. The levels of IL-4 and GATA-3 production were not different between the two groups of mice. Conversely, treatment with exogenous metallothionein during the priming phase drove na?ve wild-type CD4+ T cells to differentiate into cells producing more IL-10 and TGF-beta, but less IFN-gamma than untreated cells. Metallothionein-primed cells were hyporesponsive to restimulation, and suppressive to T cell proliferation in an IL-10-dependent manner. Lymphocytes from metallothionein-deficient mice displayed significantly elevated levels of AP-1 and JNK activities in response to stimulation compared with those from wild-type controls. Importantly, transgenic mice overexpressing metallothionein exhibited significantly reduced susceptibility to collagen-induced arthritis and enhanced IL-10 level in the serum, relative to their nontransgenic littermates. Taken together, these data suggest that metallothionein is able to promote the generation of IL-10- and TGF-beta-producing type 1 regulatory T-like cells by downregulating JNK-dependent AP-1 activity. Thus, metallothionein may play an important role in the regulation of Th1-dependent autoimmune arthritis, and may represent both a potential target for therapeutic manipulation and a critical element in the diagnostic assessment of disease potential.     

Regulatory CD4+ T cells are crucial for preventing CD8+ T cell-mediated autoimmunity

In vivo studies have shown that regulatory CD4(+) T cells regulate conventional CD4(+) T cell responses to self- and environmental Ags. However, it remains unclear whether regulatory CD4(+) T cells control CD8(+) T cell responses to self, directly, or indirectly by decreasing available CD4(+) T cell help. We have developed an experimental mouse model in which suppressive and helper T cells cannot mediate their functions. The mouse chimeras generated were not viable and rapidly developed multiple organ autoimmunity. These features were correlated with strong CD8(+) T cell activation and accumulation in both lymphoid and nonlymphoid organs. In vivo Ab treatment and secondary transfer experiments demonstrated that regulatory CD4(+) T cells play an important direct role in the prevention of peripheral CD8(+) T cell-mediated autoimmunity.     

Interleukin-21-producing HIV-1-specific CD8 T cells are preferentially seen in elite controllers

A hallmark of human immunodeficiency virus type 1 (HIV-1) pathogenesis is the rapid loss of CD4 T cells leading to generalized immune dysfunction, including an exhausted CD8 T cell phenotype. Understanding the necessary factors that govern the functional quality and protective potential of antiviral T cell responses would facilitate rational vaccine design and improve therapeutic strategies to combat persistent infections. Mouse models of chronic viral infection demonstrate that interleukin-21 (IL-21), produced primarily by CD4 T cells, is required for the generation and maintenance of functionally competent CD8 T cells and viral containment. We reasoned that preserved IL-21 production during HIV-1 infection would be associated with enhanced CD8 T cell function, allowing improved viral control. Here we analyzed the ability of CD4 and CD8 T cells to produce several cytokines in addition to IL-21 ex vivo following stimulation with overlapping HIV-1 peptides. Both CD4 and CD8 T cells were able to produce IL-21 in response to HIV-1 infection, with the latter cell type more closely associated with viral control. Furthermore, IL-21-producing HIV-1-specific CD4 T cells (compared to those producing other cytokines) were the best indicator of functional CD8 T cells. Our results demonstrate that HIV-1-specific IL-21-producing CD8 T cells are induced following primary infection and enriched in elite controllers, suggesting a critical role for these cells in the maintenance of viremia control.     

Increased IL-17-producing CD4(+) T cells in patients with esophageal cancer

Increased interleukin-17 (IL-17)-producing Th (Th17) cells have been described in a variety of human carcinoma cases, however, the mechanism of Th17 cells' accumulation in a tumor microenvironment remains elusive. This study was designed to investigate whether Th17 cells were involved in the development of esophageal cancer. We found that the proportion of Th17 cells increased within the peripheral blood and tumor tissues of esophageal cancer patients. Furthermore, the proportion of circulating Th17 cells was higher in advanced esophageal cancer patients than that in early esophageal cancer patients. In addition, the Th17 cells differentiation-related cytokines (IL-23, IL-1β, and IL-6) and accumulation-related chemokines (CCL22 and CCL20) were present in a tumor microenvironment. Therefore, the findings may partly explain the cause for the increased proportion of Th17 cells and indicate a potential prognostic marker of Th17 cells in esophageal cancer.     

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression,but little interleukin-22 and interleukin-23R expression

Introduction  

Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.     

Tim-3+ decidual Mφs induced Th2 and Treg bias in decidual CD4+T cells and promoted pregnancy maintenance via CD132

T-cell immunoglobulin mucin-3 (Tim-3) plays roles in the functional regulation of both adaptive and innate immune cells and is greatly involved in many diseases. However, the precise roles of Tim-3 on macrophages (Mφs) in pregnancy remain unstated. In the current study, we found the higher frequency of Tim-3⁺ decidual Mφs (dMφs) in response to trophoblasts. The reduced abundance of Tim-3 on Mφs was accompanied by disordered anti- and pro-inflammatory cytokine profiles in miscarriage. Adoptive transfer of Tim-3⁺Mφs, but not Tim-3⁻Mφs, relieved murine embryo absorption induced by Mφ depletion. Our flow cytometry results and the extensive microarray analysis confirmed that Tim-3⁺ and Tim-3⁻dMφs were neither precisely pro-inflammatory (M1) nor anti-inflammatory (M2) Mφs. However, with higher CD132 expression, Tim-3⁺dMφs subset induced Th2 and Treg bias in decidual CD4⁺T cells and promoted pregnancy maintenance. Blockade of Tim-3 or CD132 pathways leaded to the dysfunction of maternal-fetal tolerance and increased fetal loss. These findings underscored the important roles of Tim-3 in regulating dMφ function and maintaining normal pregnancy, and suggested that Tim-3 on Mφs is a potential biomarker for diagnosis of miscarriage. Our study also emphasized the importance of careful consideration of reproductive safety when choosing immune checkpoint blockade therapies in real world clinical care. Though IL-4 treated Tim-3⁻Mφs could rescue the fetal resorption induced by Mφ depletion, whether IL-4 represent novel therapeutic strategy to prevent pregnancy loss induced by checkpoint inhibition still needs further research.Subject terms: Infertility, Translational immunology, Cell death and immune response     

Expression and regulation of IL-22 in the IL-17-producing CD4＋ T lymphocytes

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4＋ helper T lymphocytes （TH） were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFI3 and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-l 7 signaling pathways in tissue inflammation and autoimmune diseases.     

CD4+ NKT cells,but not conventional CD4+ T cells,are required to generate efferent CD8+ T regulatory cells following antigen inoculation in an immune-privileged site

Following inoculation of Ag into the anterior chamber (a.c.), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8(+) T regulatory (Tr) cells. This model of tolerance is called a.c.-associated immune deviation. The generation of the efferent CD8(+) Tr cell in a.c.-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Valpha14(+) NKT (iNKT) cells. The iNKT cell subpopulations are either CD4(+) or CD4(-)CD8(-) double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II(-/-) (class II(-/-)) mice generate efferent Tr cells following a.c. inoculation, we conclude that conventional CD4(+) T cells are not needed for the development of efferent CD8(+) T cells. Furthermore, Ab depletion of CD4(+) cells in both wild-type mice (remove both conventional and CD4(+) NKT cells) and class II(-/-) mice (remove CD4(+) NKT cells) abrogated the generation of Tr cells. We conclude that CD4(+) NKT cells, but not the class II molecule or conventional CD4(+) T cells, are required for generation of efferent CD8(+) Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8(+) Tr cells may lead to novel immunotherapy for immune inflammatory diseases.     

CD4+CD25+Foxp3+ regulatory T cells are dispensable for controlling CD8+ T cell-mediated lung inflammation

Every person harbors a population of potentially self-reactive lymphocytes controlled by tightly balanced tolerance mechanisms. Failures in this balance evoke immune activation and autoimmunity. In this study, we investigated the contribution of self-reactive CD8(+) T lymphocytes to chronic pulmonary inflammation and a possible role for naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) in counterbalancing this process. Using a transgenic murine model for autoimmune-mediated lung disease, we demonstrated that despite pulmonary inflammation, lung-specific CD8(+) T cells can reside quiescently in close proximity to self-antigen. Whereas self-reactive CD8(+) T cells in the inflamed lung and lung-draining lymph nodes downregulated the expression of effector molecules, those located in the spleen appeared to be partly Ag-experienced and displayed a memory-like phenotype. Because ex vivo-reisolated self-reactive CD8(+) T cells were very well capable of responding to the Ag in vitro, we investigated a possible contribution of nTregs to the immune control over autoaggressive CD8(+) T cells in the lung. Notably, CD8(+) T cell tolerance established in the lung depends only partially on the function of nTregs, because self-reactive CD8(+) T cells underwent only biased activation and did not acquire effector function after nTreg depletion. However, although transient ablation of nTregs did not expand the population of self-reactive CD8(+) T cells or exacerbate the disease, it provoked rapid accumulation of activated CD103(+)CD62L(lo) Tregs in bronchial lymph nodes, a finding suggesting an adaptive phenotypic switch in the nTreg population that acts in concert with other yet-undefined mechanisms to prevent the detrimental activation of self-reactive CD8(+) T cells.     

CD40L-expressing CD4+ T cells prime adipose-derived stromal cells to produce inflammatory chemokines

The therapeutic potential of culture-adapted adipose-derived stromal cells (ASCs) is largely related to their production of immunosuppressive factors that are inducible in vitro by priming with inflammatory stimuli, in particular tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). In vivo, obesity is associated with chronic inflammation of white adipose tissue, including accumulation of neutrophils, infiltration by IFNγ/TNFα-producing immune cells, and ASC dysfunction. In the current study, we identified in obese patients a simultaneous upregulation of CD40Lin the adipose tissue stroma vascular fraction (AT-SVF), correlated with the Th1 gene signature, and an overexpression of CD40 by native ASCs. Moreover, activated CD4⁺ T cells upregulated CD40 on culture-expanded ASCs and triggered their production of IL-8 in a CD40L-dependent manner, leading to an increased capacity to recruit neutrophils. Finally, activation of ASCs by sCD40L or CD40L-expressing CD4⁺ T cells relies on both canonical and non-canonical NF-κB pathways, and IL-8 was found to be coregulated with NF-κB family members in AT-SVF. These data identify the CD40-CD40L axis as a priming mechanism of ASCs, able to modulate their cross talk with neutrophils in an inflammatory context, and their functional capacity for therapeutic applications.     

Interleukin-7 influences FOXP3+CD4+ regulatory T cells peripheral homeostasis

Mechanisms governing peripheral CD4+ FOXP3+ regulatory T cells (Treg) survival and homeostasis are multiple suggesting tight and complex regulation of regulatory T cells homeostasis. Some specific factors, such as TGF-β, interleukin-2 (IL-2) and B7 costimulatory molecules have been identified as essentials for maintenance of the peripheral Treg compartment. Conversely, Treg dependency upon classical T cell homeostatic factors such as IL-7 is still unclear. In this work, we formally investigated the role of IL-7 in Treg homeostasis in vivo in murine models. We demonstrated that IL-7 availability regulated the size of peripheral Treg cell pool and thus paralleled the impact of IL-7 on conventional T cell pool. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing thymic-independent Treg peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Our results definitively identify IL-7 as a central factor contributing to Treg peripheral homeostasis, thus reassembling Treg to other T cell subsets in respect of their need for IL-7 for their peripheral maintenance.     

HIV-1-specific memory CD4+ T cells are phenotypically less mature than cytomegalovirus-specific memory CD4+ T cells

HIV-1-specific CD4(+) T cells are qualitatively dysfunctional in the majority of HIV-1-infected individuals and are thus unable to effectively control viral replication. The current study extensively details the maturational phenotype of memory CD4(+) T cells directed against HIV-1 and CMV. We find that HIV-1-specific CD4(+) T cells are skewed to an early central memory phenotype, whereas CMV-specific CD4(+) T cells generally display a late effector memory phenotype. These differences hold true for both IFN-gamma- and IL-2-producing virus-specific CD4(+) T cells, are present during all disease stages, and persist even after highly active antiretroviral therapy (HAART). In addition, after HAART, HIV-1-specific CD4(+) T cells are enriched for CD27(+)CD28(-)-expressing cells, a rare phenotype, reflecting an early intermediate stage of differentiation. We found no correlation between differentiation phenotype of HIV-1-specific CD4(+) T cells and HIV-1 plasma viral load or HIV-1 disease progression. Surprisingly, HIV-1 viral load affected the maturational phenotype of CMV-specific CD4(+) T cells toward an earlier, less-differentiated state. In summary, our data indicate that the maturational state of HIV-1-specific CD4(+) T cells cannot be a sole explanation for loss of containment of HIV-1. However, HIV-1 replication can affect the phenotype of CD4(+) T cells of other specificities, which might adversely affect their ability to control those pathogens. The role for HIV-1-specific CD4(+) T cells expressing CD27(+)CD28(-) after HAART remains to be determined.     

Naive human CD4+ T cells are a major source of lymphotoxin alpha

It is generally accepted that immunologically naive T cells display a very restricted cytokine production profile consisting mainly of IL-2, which is used as an autocrine growth factor. Here we report that activated naive CD4+ T cells, of neonatal or adult origin, express very high levels of soluble lymphotoxin (LT) alpha (LTalpha3), as determined by ELISA, RNase protection assay, and intracytoplasmic staining. Besides LTalpha3 and IL-2, these cells also produce high levels of TNF-alpha together with significant amounts of IFN-gamma and IL-13. Naive cells also express LTbeta mRNA and the membrane form of LTalpha (LTalphabeta). On average, naive CD4+ T cells secrete four times more LTalpha3 than Th1-like cells, twice more than naive CD8+ T cells, and ten times more than B cells. Thus, naive T cells express a large spectrum of cytokines, mainly of the Th1 type, and the very high levels of LTalpha3/TNF-alpha that they release may play an hitherto unsuspected role in the early stage of T cell-dependent immune responses.