Changyou Wu

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

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

IL-27 induces the differentiation of Tr1-like cells from human naive CD4+ T cells via the phosphorylation of STAT1 and STAT3

IL-27, a member of IL-6/IL-12 cytokine family, is mainly produced by activated antigen presenting cells (APC). It has been demonstrated that IL-27 has the pro- and anti-inflammatory properties during immune responses. However, the signaling pathways that contribute to the cytokine generation are still unclear in humans. In the present study, we showed that IL-27 induced IL-10 and IFN-γ, but had no effect on IL-2, TNF-α and IL-4 production from human umbilical cord blood mononuclear cells (CBMCs). For purified naive CD4(+) T cells, IL-27 elicited the differentiation of Tr1-like cells with expression of IL-10 and IFN-γ. Importantly, this induction was dependent on the signal transducer and activator of transcription (STAT) 1 and 3 factors. In addition, all of the phosphorylated STAT3 positive cells were also positive for phosphorylated STAT1, both of which could be inhibited by a JAK2/STAT inhibitor, AG490. However, there was no phosphorylation of STAT4, STAT5 and STAT6 in IL-27-stimulating conditions. Moreover, the biological function of IL-10 that was induced by IL-27 mainly enhanced the expression of CD16 without influencing CD14 expression on human monocytes. These data identify the function of IL-27 on the differentiation of human naive CD4(+) T cells and demonstrate that the STATs signaling pathways may play an important role in mediating immune responses in humans.

ESAT‐6‐ and CFP‐10‐Specific Th1, Th22 and Th17 Cells in Tuberculous Pleurisy May Contribute to the Local Immune Response Against Mycobacterium Tuberculosis Infection

Th1 cell‐mediated adaptive immune response is very important but may not be sufficient to control Mycobacterium tuberculosis (M. tuberculosis) infection. The roles of the various T cell subsets and cytokines in the inflammatory processes are not clearly elucidated. We investigated whether Th1, Th22 and Th17 cells mediated cellular immunity at the local site of M. tuberculosis infection in patients with tuberculous pleurisy (TBP). The results showed that the cytokines IFN‐γ and IL‐22 but not IL‐17 were elevated in tubercular pleural fluid. Following stimulation with immune‐dominant peptides of early secreted antigenic target‐6 (ESAT‐6), culture filtrate protein‐10 (CFP‐10) or Bacille Calmette–Guerin, pleural fluid mononuclear cells expressed high levels of cytokines IFN‐γ, IL‐22 and IL‐17 as revealed by mRNA and protein measurements. In addition, we showed that cytokines IFN‐γ, IL‐22 and IL‐17 were produced in M. tuberculosis‐specific immune response by distinct subsets of CD4+ T cells with the phenotype of CD45RA−CD62L−CCR7+CD27+. Our results demonstrated for the first time that ESAT‐6‐ and CFP‐10‐specific Th1, Th22 and Th17 cells existed in the patients with TBP and might play an essential role against M. tuberculosis infection. The findings of this study raised the possibility of unravelling the critical targets for therapeutic intervention in chronic inflammatory diseases such as TBP.

Polyunsaturated fatty acids promote the expansion of myeloid‐derived suppressor cells by activating the JAK/STAT3 pathway

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects that could prove beneficial in clinical therapies for certain autoimmune and inflammatory disorders. However, the mechanism of PUFA‐mediated immunosuppression is far from understood. Here, we provide evidence that PUFAs enhance the accumulation of myeloid‐derived suppressor cells (MDSCs), a negative immune regulator. PUFA‐induced MDSCs have a more potent suppressive effect on T‐cell responses than do control MDSCs. These observations were found both in cultured mouse bone marrow cells in vitro and in vivo in mice fed diets enriched in PUFAs. The enhanced suppressive activity of MDSCs by PUFAs administration was coupled with a dramatic induction of nicotinamide adenine dinucleo‐ tide phosphate oxidase subunit p47phox and was dependent on reactive oxygen species (ROS) production. Mechanistic studies revealed that PUFAs mediate its effects through JAK‐STAT3 signaling. Inhibition of STAT3 phosphorylation by JAK inhibitor JSI‐124 almost completely abrogated the effects of PUFAs on MDSCs. Moreover, the effects of PUFAs on MDSCs and the underlying mechanisms were confirmed in tumor‐bearing mice. In summary, this study sheds new light on the immune modulatory role of PUFAs, and demonstrates that MDSCs expansion may mediate the effects of PUFAs on the immune system.

Identification of Mycobacterium tuberculosis-Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4+ T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4+CD40L+ but not CD4+CD40L− T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4+CD40L+ T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA−CD45RO+CCR7−CD62L−ICOS−). To determine the specificity of CD4+CD40L+ T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4+CD40L+ and CD4+CD40L− T cells by flow cytometry. We further demonstrated that sorted CD4+CD40L+, but not CD4+CD40L− fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4+ T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.

Interleukin‐21 induces the differentiation of human Tc22 cells via phosphorylation of signal transducers and activators of transcription

Interleukin‐21 (IL‐21) exerts critical functions in T helper type 17 (Th17) cell development. However, the effect of IL‐21 on the differentiation of IL‐22‐producing T cells is not clear. Here we showed that IL‐21 induced the differentiation of human naive CD8+ T cells into Tc22 cells without the expression of IL‐17. The addition of transforming growth factor‐β inhibited the production of IL‐22 but induced the production of IL‐17. Both IL‐15 and IL‐2 induced interferon‐γ production but did not induce differentiation of Tc22, which suggests that common γ‐chain signals are not specific to promote IL‐22 synthesis. The IL‐21 induced naive CD8+ T cells to produce IL‐22 in greater amounts than memory CD8+ T cells. In addition, we demonstrated that IL‐21 promoted the proliferation and increased the expression of IL‐21 receptors on activated naive CD8+ T cells. Furthermore, IL‐21 increased the expression of granzyme B molecules. Analysis of molecular mechanisms indicated that IL‐21 induced phosphorylation of signal transducers and activators of transcription 1, 3 and 5 in CD8+ T cells. Overall, our data indicated that IL‐21, an effector cytokine produced by CD4+ T cells, might mediate the cross‐talk between CD4+ and CD8+ T cells through the production of IL‐22.

The immune responses of central and effector memory BCG-specific CD4+ T cells in BCG-vaccinated PPD+ donors were modulated by Treg cells.

Most of the studies evaluating the function of tuberculosis (TB)-specific T cells were only based on the ability to produce cytokines, which may not fully reflect the function of T cells. In the present study, we confirmed that Bacille Calmette Guerin (BCG) could significantly induce cytokine production by CD4(+) T cells from BCG-vaccinated PPD(+) donors. In addition, CD4(+) T cells were activated, divided and proliferated in response to BCG stimulation. Phenotypic analysis showed that IFN-γ(+)CD4(+) T cells displayed CD45RA(-)CCR7(+/-)CD62L(-), indicating that these CD4(+) T cells were central and effector memory cells. The analysis of cytokine profiles demonstrated that most of BCG-specific BrdU(+)CD4(+) T cells produced Th1 cytokines in response to polyclonal stimulation. In addition, we found that regulatory T cells (Treg) suppressed BCG-induced proliferation and IFN-γ production by memory CD4(+) T cells. The suppressive effects of Treg on BCG-specific responses of CD4(+) T cells could be partially reversed by blocking the production of IL-10. Taken together, our results demonstrated that functional central and effector memory BCG-specific CD4(+) T cells could be detected based on the activation, proliferation and division of these cells, and modulated by Treg in PBMCs from BCG-vaccinated PPD(+) donors.

Suppressive effect of diazepam on IFN-γ production by human T cells

Many studies showed that benzodiazepines could modulate immune responses through interaction with peripheral benzodiazepine receptors (PBRs) in immune cells but most of the studies were focused on monocytes and macrophages. In the present study, we revealed that diazepam, a mixed-type benzodiazepine, inhibited IFN-gamma production by human peripheral blood mononuclear cells (PBMCs) induced by anti-CD3 in dose-dependent manner. Flow cytometry analysis demonstrated that diazepam could inhibit the frequency of IFN-gamma-producing CD4(+) and CD8(+) T cells. The inhibitory effect of diazepam on IFN-gamma production is similar to that of R(0)5-4864, a selective PBRs ligand. However, D8555, a selective ligand for PBRs in microglia in the central nervous system, is a much weak inhibitor compared with R(0)5-4864 or diazepam. The inhibitory effect of R(0)5-4864 could be antagonized by PK11195, which is recognized as selective PBRs antagonist, and suppressive effect of diazepam on T cells is partially antagonized by PK11195. Collectively, these results suggested that diazepam suppressed human T cell function through PBRs.

B lymphocytes that migrate to tuberculous pleural fluid via the SDF-1/CXCR4 axis actively respond to antigens specific for Mycobacterium tuberculosis.

B‐cell biology has been largely uncharacterized in the field of tuberculosis (TB). In this study, we investigated the immunophenotypical and functional characteristics of B cells obtained from the pleural fluid (PF) and peripheral blood of patients with tuberculous pleuritis (TP). Our results indicated that the total numbers of B cells, CD27+ memory B cells and plasmablasts were clearly lower in the PF than in peripheral blood. Furthermore, we found significantly higher expression of CXCR4 on B cells in the PF, and a chemotaxis assay showed that B cells in the PF were more responsive to stromal cell‐derived factor‐1 (SDF‐1) than B cells from peripheral blood. In addition, SDF‐1 levels in PF were remarkably high compared with SDF‐1 levels in plasma, suggesting that the SDF‐1/CXCR4 axis might facilitate the migration of circulating B cells into tuberculous pleural space. Importantly, we observed that significantly more antibodies were produced by B cells in the PF following stimulation with BCG, early secretory antigenic target (ESAT‐6)/culture filtrate protein‐10 (CFP‐10) or ESAT‐6 protein. Collectively, these data demonstrate that Mycobacterium tuberculosis‐specific B cells exist at local sites of infection in TP patients and this localization might influence the immune response to M. tuberculosis.

Identification of M.tuberculosis-Specific Th1 Cells Expressing CD69 Generated in vivo in Pleural Fluid Cells from Patients with Tuberculous Pleurisy

Th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of M.tuberculosis (MTB) and for the spontaneous resolution of patients with tuberculous pleurisy (TBP). In the present study, we found that without any stimulation, CD4+ T cells in pleural fluid cells (PFCs) from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with tuberculosis (TB) or healthy donors. CD4+CD69+ T cells expressed T-bet and IL-12Rβ2. After stimulation with MTB-specific antigens, CD4+CD69+ T cells expressed significantly higher levels of IFN-γ, IL-2 and TNF-α than CD4+CD69− T cells, demonstrating that CD4+CD69+ T cells were MTB-specific Th1 cells. In addition, CD4+CD69+ T cells were mostly polyfunctional Th1 cells that simultaneously produced IFN-γ, IL-2, TNF-α and displayed an effector or effector memory phenotype (CD45RA−CCR7−CD62L−CD27−). Moreover, the percentages of CD4+CD69+ T cells were significantly and positively correlated with polyfunctional T cells. Interestingly, sorted CD4+CD69+ but not CD4+CD69− fractions by flow cytometry produced IFN-γ, IL-2 and TNF-α that were significantly regulated by CD4+CD25+ Treg cells. Taken together, based on the expression of CD69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated MTB-specific polyfunctional CD4+ T cells in PFCs from patients with TBP. This method can be used for the potential diagnosis and enrichment or isolation of MTB-specific Th1 cells in the investigations.