Immunological Characteristics Of T Lymphocyte Subpopulations In Lymphocytic Pleural Effusion

Pleural cavity fluid is a thin layer of serous fluid normally exists in the closed cavity encompassed by the visceral and parietal pleura, and plays a critical role of lubrication during breathing exercise. Its generation and absorption are at the status of relatively constant homeostasis. In some pathological circumstance such as pleural malignancy or infection, vascular permeability is increased, and/or obstruction of lymphatic vessels in parietal pleura, accumulation of exudate which is rich in protein and cells exists in pleural cavity formed pleural effusion. Lymphocytic pleural effusion, which mainly (>90%) results from pleural tuberculosis or metastatic malignancy, accounted for a large proportion of pleural effusions, with an accumulation of lymphocyte take up to>50%proportions of total WBC in pleural fluid. Malignant pleural effusion is mainly caused by advanced cancer, especially lung cancer, with a poor median survival time of4-6M but lack of effective treatment. While tuberculous pleurisy is one type of tuberculosis infection, though curable, it is still a frequently-occurring disease which heavily burdens medicine and society, as the increases in immune-related diseases and multi-drug resistant Mycobacterium tuberculosis infection in prevalence. Although pleural effusion is a common disease or complication, it attracts little attention from academia, and to date, its immunopathological mechanisms and issues about clinical administration remain to be elucidated.T lymphocytes, of which CD4+helper T cells is the major component, are predominant in lymphocytic pleural effusion. Helper T cell is the bridge connecting innate immune and acquired immune, and plays a critical role in initiation and maintenance of immune response. Since identification of Thl and Th2more than tow decades ago, several helper T subsets were added to this group, such as Treg, Th17, Th22, and Th9cells. They are differentiated from naive CD4+T cells stimulated in different milieu, each of them shows unique biological features, mediating diverse specific immune response. Only little studies focus on the phenotypic characteristics and immunological activity of these brand new Th subpopulations in lymphocytic pleural effusion.Hence, present series of studies aimed at the international heat point that analyzed the biological features and specific immune responses of these T subsets, as well as the mechanisms about how they contribute to the development of pleural disease. These studies not only deepened the understanding of these important T subsets and elucidated pathogenesis of pleural disease from a brand new point of view, but also offered new intervention measures and theoretical basis for better clinical administration of malignancy and infection. The main research about T Lymphocyte Subpopulations in Lymphocytic Pleural Effusion was carried out as following6parts:Part I Generation and Differentiation of IL-17-Producing CD4+T Cells in Malignant Pleural EffusionIL-17-producing CD4+T (Th17) cells have been found to be increased in some human cancers; however, the possible implication of Th17cells in regulating antitumor responses in malignant pleural effusion (MPE) remains to be elucidated. In the current study, distribution and phenotypic features of Th17cells 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 Th17cell generation and differentiation were explored. The chemoattractant activity of chemokines CCL20and CCL22for Th17cells in vitro was also observed. It was found that the increased Thl7cells 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 Th17cell generation and differentiation from naive CD4+T cells. MPE was chemotactic for Th17cells, and this activity was partly blocked by anti-CCL20and/or CCL22Abs. Our data also showed that the accumulation of Th17cells in MPE predicted improved patient survival. It could be concluded that the overrepresentation of Thl7cells in MPE might be due to Th17cell differentiation and expansion stimulated by pleural proin-flammatory cytokines and to recruitment of Thl7cells from peripheral blood induced by pleural chemokines CCL20and CCL22. Furthermore, the accumulation of Th17cells 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. Part II CD39+Regulatory T cells Suppress Generation and Differentiation of Th17Cells in Human Malignant Pleural Effusion via a LAP-dependent MechanismBoth regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17cells in malignant pleural effusion remains to be elucidated. The numbers of both CD39+Tregs and Th17cells in malignant pleural effusion and peripheral blood from patients with lung cancer were deter mined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17cells were explored. Both CD39+Tregs and Th17cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39+Tregs were correlated negatively with those of Th17cells. It was also noted that high levels of IL-1β,IL-6, and TGF-β1could be observed in malignant pleural effusion when compared with the corresponding serum, and that pleural CD39+Tregs could express latency-associated peptide on their surface. When naive CD4+T cells were cocultured with CD39+Tregs, Th17cell numbers decreased as CD39+Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reversed the inhibitory effect exerted by CD39+Tregs. Therefore, the above results indicate that CD39+Tregs inhibit generation and differentiation of Th17cells via a latency-associated peptide-dependent mechanism.Part Ⅲ Differentiation and Recruitment of IL-22-Producing Helper T Cells Stimulated by Pleural Mesothelial Cells in Tuberculous PleurisyIL-22-producing helper T cells (Th22cells) have been reported to be involved in tuberculosis infection. However, differentiation and immune regulation of Th22cells in tuberculous pleural effusion (TPE) remain unknown. Present work is to elucidate the mechanism by which Th22cells differentiate and recruit into the pleural space. The distribution and phenotypic features of Th22cells in both TPE and blood were determined. The impacts of proinflammatory cytokines and antigen presentation by pleural mesothelial cells (PMCs) on Th22cell differentiation were explored. The chemoattractant activity of chemokines produced by PMCs for Th22cells was observed. Th22cells were significantly higher in TPE than in blood. IL-1β,IL-6, and/or tumor necrosis factor-α promoted Th22cell differentiation from CD4+T cells. It was found that PMCs expressed CCL20, CCL22, and CCL27, and that TPE and PMC supernatants were chemotactic for Th22cells. This activity was partly blocked by anti-CCL20, anti-CCL22, and anti-CCL27antibodies. IL-22and IL-17significantly improved PMC wound healing. Moreover, PMCs were able to stimulate CD4+T cell proliferation and Th22cell differentiation by presenting tuberculosis-specific antigen. The overrepresentation of Th22cells in TPE may be due to pleural cytokines and to PMC-produced chemokines. Our data suggest a collaborative loop between PMCs and Th22cells in TPE. In particular, PMCs were able to function as antigen-presenting cells to stimulate CD4+T-cell proliferation and Th22cell differentiation.Part Ⅳ Interleukin22-producing CD4+T Cells in Malignant Pleural EffusionTh22cells have been reported to be involved in human cancers. However, differentiation and immune regulation of Th22.cells in malignant pleural effusion (MPE) remain unknown. We noted that Th22cell numbers were increased in MPE, and that IL-22substantially promoted the proliferation and migratory activity of A549cells. Moreover, IL-22could strongly facilitate intercellular adhesion of A549cells to pleural mesothelial cell monolayers. Present study revealed that the increase in Th22cells in MPE was due to pleural cytokines and chemokines, and that Th22exerted an important immune regulation on cancer cells in human pleural malignant environment.Part V Differentiation and Recruitment of Th9Cells Stimulated by Pleural Mesothelial Cells in Human Mycobacterium tuberculosis InfectionNewly discovered IL-9-producing CD4+helper T cells (Th9cells) have been reported to contribute to tissue inflammation and immune responses, however, differentiation and immune regulation of Th9cells in tuberculosis remain unknown. In the present study, our data showed that increased Th9cells with the phenotype of effector memory cells were found to be in tuberculous pleural effusion as compared with blood. TGF-β was essential for Th9cell differentiation from naive CD4+T cells stimulated with PMA and ionomycin in vitro for5h, and addition of IL-1β, IL-4or IL-6further augmented Th9cell differentiation. Tuberculous pleural effusion and supernatants of cultured pleural mesothelial cells were chemotactic for Th9cells, and this activity was partly blocked by anti-CCL20antibody. IL-9promoted the pleural mesothelial cell repairing and inhibited IFN-y-induced pleural mesothelial cell apoptosis. Moreover, pleural mesothelial cells promoted Th9cell differentiation by presenting antigen. Collectively, these data provide new information concerning Th9cells, in particular the collaborative immune regulation between Th9cells and pleural mesothelial cells in human M. tuberculosis infection. In particular, pleural mesothelial cells were able to function as antigen-presenting cells to stimulate Th9cell differentiation.Part VI Differentiation and Immune Regulation of IL-9-Producing CD4+T Cells in Malignant Pleural EffusionIL-9-producing CD4+T cells (Th9cells) have been reported to be involved in inflammation and immune diseases. However, the involvement of Th9cells in malignancy has not been investigated. This study is to elucidate the mechanism by which Th9cells differentiate in malignant pleural effusion (MPE), and to explore the immune regulation of Th9cells on lung cancer cells. Distribution of Th9cells in relation to Th17and Thl cells in both MPE and peripheral blood were determined by flow cytometry. The effects and mechanisms of proinflammatory cytokines and regulatory T cells on differentiation of Th9cells in vitro were explored. The impacts and signal transductions of IL-9, IL-17and IFN-γ on lung cell lines were also investigated. The numbers of Th9, Th17and Thl cells were all increased in MPE when compared with their compartments in blood. By activating STAT3signaling, both IL-9and IL-17substantially promoted the proliferation and migratory activity of lung cancer cells, whereas IFN-y which tended to activate STAT1signaling was noted to suppress lung cancer cell proliferation and migration; whereas IFN-y could induce lung cancer cell apoptosis. Moreover, IL-9and LFN-γ, but not by IL-17, could strongly facilitate intercellular adhesion of lung cancer cells to pleural mesothelial cell monolayers. These data revealed that the increase in Th9cells in MPE was due to the promotion by cytokines and regulatory T cells, and that Th9exerted an important immune regulation on lung cancer cells in human tumor environment.