This paper aimed to compare the phenotypes of Tfh cells as well as evaluate the relationship of Tfh cells with Th1,Th17 and Th22 cell subsets in human peripheral blood and tonsils.We isolated PBMCs and tonsillar mononuclear cells,then detected the production of cytokines by ELISA and FACS after stimulation with anti-CD3 plus anti-CD28 or PMA plus ionomycin.Finally,the relationship of Tfh cells with Th1,Th17 and Th22 cell subsets was identified.Different from what observed in PBMCS,higher expression of CXCR5 and CD45RO and lower expression of CCR7 and CD62L were demonstrated in tonsillar CD4+ T cells.Tonsillar CD4+ T cells produced higher levels of IL-21 and IL-17 but lower level of IFN-γ compared with CD4+ T cells in PBMCs.While the level of IL-22 was similar in PBMC and tonsillar CD4+ T cells.A few of IL-21+ IL-17+,IL-21+ IL-22+ and IL-21+ IFN-γ+ cells were existed both in PBMC and tonsillar CD4+ T cells.Furthermore,the frequencies of IL-21 single positive cells were higher in tonsillar CD4+ T cells than in CD4+ T cells from PBMCs.Besides co-expression with IL-21,some proportion of CD4+ CXCR5+ cells in PBMCs and tonsils are IL-17-positve,IL-22-positive or IFN-γ-positive.We concluded that there are higher amount of Tfh cells in tonsils than in PBMCs,and the majority of Tfh cells belong to a distinct subset from Th1,Th17,or Th22 cells.

Immunosuppressive mediators in tuberculosis pleurisy (pleural fluid (PF)) are associated with the course of disease, but they remain poorly defined. To study the local immune status of patients with tuberculosis pleurisy, we examined the effect of PF on the functions of T cells and the differentiation of Th1 cells. PF could inhibit the ability of T cells to produce cytokines. However, tumor-necrosis factor (TNF)-α derived from non-T cells was not impaired. Further analysis indicated that cell activation and cell cycle progression were also suppressed. Moreover, PF could inhibit Th1 cell differentiation. Importantly, we found that inhibitors of indoleamine 2,3-dioxygenase (IDO) and adenosine and neutralizing antibodies against IL-10 and transforming growth factor (TGF)-β could reverse cytokine production, suggesting that IDO, adenosine, IL-10 and Transforming growth factor–β1 in PF might take part in impairing T-cell functions. Taken together, our data demonstrate for the first time that several immunopathological factors participate in the downregulation of T-cell functions in local PF.

This paper aimed to compare the characteristics of IL-22-producing T cells in human normal membrane of intestinal tract and peripheral blood mononuclear cells(PBMC).We isolated the mononuclear cells from the membrane of intestinal tract and peripheral blood,respectively.The production of IL-22 by T cells and the correlation of IL-22 with IFN-γ and IL-17 were detected by FACS after stimulation with anti-CD3 plus anti-CD28.Finally,the surface markers CD45RO,CD62L,CCR7,CCR6,CCR10 and CCR4 were analyzed.Compared with the lower secretion of IL-22 by CD4+ and CD8+ T cells from PBMCs(0.6%;0.57%),3.15% of CD4+ IL-22+ and 4% CD8+ IL-22+ could be detected in membrane of intestinal tract after stimulation with anti-CD3 plus anti-CD28.A certain subset of CD4+ IL-22+ and CD8+ IL-22+ T cells was existed and they were distinct from Th1,Th17,Tc1,Tc17 cells.IL-22+ T cells in the membrane of intestinal tract expressed higher levels of CD45RO,part of these cells expressed CCR7 and fewer expressed CD62L.Furthermore,CD4+IL-22+ and CD8+IL-22+ T cells expressed higher levels of CCR10(55.3%;73.9%) and part of CCR6 and CCR4 in the membrane of intestinal tract.In conclusion,IL-22 is mainly produced by the effector or central memory T cells from the membrane of intestinal tract.In addition,a certain subset of IL-22-producing T cells is distinct from Th1,Th17,Tc1,Tc17 cells.

Objective:To study the synergic effect of flagellin and interleukine-12(IL-12) on the production of IFN-γ by human NK cells and its mechanism.Methods:PBMCs isolated from normal donors were cultured with medium,flagellin,IL-12 or flagellin plus IL-12.Culture supernatants were collected at day 3 and the level of IFN-γ was measured by ELISA.Cells under primary culture conditions were collected,washed,restimulated,and the production of IFN-γ was detected after 3 days.The expression of IL-12 receptor β1 and toll-like receptor 5 on NK cell subsets was detected and analyzed by flow cytometry.Results:In the primary culture conditions,application of suboptimal dose of flagellin or IL-12 could induce low levels of IFN-γ.Stimulation of PBMCs with flagellin plus IL-12 could synergistically induce IFN-γ production.Under the secondary culture conditions,cells that pre-treated with flagellin were restimulated with different concentrations of IL-12,which could induce high levels of IFN-γ in a dose-dependent manner.Cells pre-stimulated with IL-12 could,in a dose-dependent manner,enhance the production of IFN-γ in the presence of different concentrations of flagellin.Flow cytometry analysis indicated that the expression of IL-12 receptorβ1 on NK cell subsets was upregulated when stimulated with flagellin and IL-12 could enhance the expression of toll-like receptor 5 on NK cell subsets.Conclusion:Up-regulation of IL-12 receptorβ1 by flagellin and enhanced expression of the toll-like receptor 5 by IL-12 contribute to the synergistic effects of IFN-γ production by NK cell subsets.

The local milieu at the site of Mycobacterium tuberculosis infection that modulates T-cell functions is the main battleground for the host to build counter-M. tuberculosis immune responses. CD4+T cells are enriched predominantly in tuberculosis pleurisy and their roles are of considerable importance, but their nature and functional profiles linked with local condition remain elusive. Here we evaluated the functions of M. tuberculosis-specific CD4+T cells from the major three profiles: cytokines production, cell activation and division. Results showed that pleural fluid (PF) from tuberculosis patients in a dose dependent manner inhibited the production of IFN-γ, IL-2 and TNF-α by M. tuberculosis-specific peptides or BCG activated CD4+T cells from pleural fluid mononuclear cells (PFMCs). Surface staining for activation molecules indicated that PF could also blunt cell activation process. CFSE labeling showed that antigen-specific CD4+T cell division ceased following co-incubation with PF. Pre- or post-treatment with PF could disturb subsequent cell activities. The strong inhibitory effect mediated by PF on CD4+T cells was functional predominance. Moreover, application of inhibitors of IDO, adenosine, neutralizing Abs to IL-10 and TGF-β could partially reverse IFN-γ production. Our current research provided novel information that the functions of antigen-specific CD4+T cells coincubated with PF were apparently impaired, which were distinct from cells that cultured in fresh culture medium. We concluded that CD4+T cell mediated antigen-specific cellular immune response that occurred locally might be impaired by PF.

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 .

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.

Natural killer (NK) cells are known as innate immune lymphocytes that respond rapidly when challenged by pathogens but little is known about adaptive immune features including memory related to NK cells from human beings. In the present study, we demonstrate for the first time that human NK cells expressing the memory-associated marker CD45RO were persistent in pleural fluid cells (PFCs) from tuberculous patients. CD45RO(+) NK cells produced significantly more interferon-gamma and were more cytotoxic compared with CD45RO(-) NK cells from PFCs when stimulated with interleukin-12 (IL-12). Consistently, IL-12 enhanced the expression of granzyme B, CD69, CD25, NKG2D, IL-12 receptors beta 1 and beta 2 on CD45RO(+) NK cells from PFCs. Our experiments contribute to a better understanding of the NK cells from PFCs and indicate that human CD45RO(+) NK cells from PFCs expressing a 'memory-like' phenotype may have an important role in defending against infection by Mycobacterium tuberculosis.

This paper is aimed to study the effects of R848 on the cytotoxicity of human natural killer cells and its mechanism.We isolated peripheral blood mononuclear cell(PBMC) and purified NK cells from normal human peripheral blood,and then cultured them with R848.The cultured cells were harvested at different time points (0,2,6,24 and 48 hours) for follow tests.The expression of activating molecules such as CD69 and CD25 were detected by flow cytometry;the cytolytic activity and mechanism of R848-activated NK cells were evaluated by detecting the expression of granzymes,perforin,TRAIL,and CD107a/b.The results indicated that the percentage and MFI of the expression of CD69 and CD25 molecules on R848-activated human NK cells reached to peak levels after culturing for 24 hours and then decreased slightly;the cytotoxicity of R848 activated NK cells were enhanced significantly.After coincubation with the K562 target cells the degranulation of granzymes and perforin of R848 activated NK cells were more significant than those of the unstimulated cells.Therefore,the expression of CD107a/b and TRAIL was increased (P 0.05).This study provides valuable information that R848 can directly promote the cytotoxic activity of human NK cells by enhancing the degranulation of granzymes and perforin,and by up-regulating the expression of TRAIL.

AIM:To characterize IL-21-producing T cells in human PBMCs.METHODS:PBMCs from healthy individuals were stimulated with or without anti-CD3 (OKT3), OKT3 coupled with anti-CD28, or PMA coupled with ionomycin. The cell subsets of IL-21-producing T cells were determined by FACS. PBMCs, CD4+, CD4+CD45RA⁻, CD4+ CD45RA+ or CBMCs were stimulated with PMA coupled with ionomycin. The phenotype of CD4+ IL-21+ T cells and the correlation of IL-21-producing CD4+; T cells with Th1, Th2, Th17 and Th22 cell populations were analyzed by FACS.RESULTS:PMA and ionomycin induced the highest level of IL-21 production among the stimuli tested. CD4+ T cells but not CD8+ T cells mainly expressed IL-21. CD4+ IL-21+ T cells expressed CD45RO instead of CD45RA. Some of the CD4+ IL-21+ T cells expressed CCR6, CCR7 or CXCR5. CD4+ CD45RA⁻ T cells expressed much more IL-21 than CD4+ CD45RA+ T cells. Furthermore, CD4+ T cells from PBMCs but not CBMCs, expressed IL-21. Approximately 24% of CD4+ IL-21+ cells expressed IFN-γ. Less than 10% of CD4+ IL-21+ cells expressed IL-4, IL-17 or IL-22.CONCLUSION:IL-21 is induced from human PBMCs following various polyclonal stimulations. The majority of IL-21-producing cells in PBMCs are memory CD4+ T cells. In addition, some of the CD4+ IL-21+ T cells are distinct from Th1, Th2 and Th17 cells.

Immunological memory is the ability to make a second and more effective immune response to an antigen that encountered previously. Antigen is processed to short peptides and presented by DCs on MHC molecules to initiate the responses of T cells. A TCR signal with co-stimuli leads to clonal expansion of antigen-specific T cells and follows a phase of contraction, in which most short-lived antigen-specific effector T cells die. Some of these cells survive and form long-lived memory cells. Memory cells are heterogeneous in phenotype and function. Understanding the properties of memory T cells will help in the design of vaccines and the immunotherapy for diseases.

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.

目的:研究正常人外周血单个核细胞(PBMCs)在白色念珠菌刺激条件下,环孢素A(CsA)对IL-17产生的影响.方法:PBMCs用白色念珠菌+抗CD28单克隆抗体(mAb)刺激后,用ELISA法检测培养上清中IL-17及IFN-γ的水平.同时利用ELISPOT法检测IL-17产生细胞的频率.结果:CsA对白色念珠菌刺激条件下诱导的PBMCs产生的IL-17与IFN-γ均为剂量依赖性的抑制,且对IL-17的抑制率高于IFN-γ,而且CsA在培养的早期和晚期均能抑制IL-17与IFN-γ的产生.同时,与单独白色念珠菌刺激相比,加入CsA后,能抑制IL-17产生细胞的频率.结论:CsA剂量依赖性抑制白色念珠菌诱导产生IL-17,进一步证明了CsA用于治疗白色念珠菌感染的疗效.

To evaluate the effect of the piliated Pseudomonas aeruginosa(PPA) and interleukin-12(IL-12)on the secretion of IFN-γ from human PBMC and NK cells,PBMC or purified NK cells were co-cultivated with culture medium,PPA,IL-12 or PPA + IL-12,and the level of IFN-γ in the cell-free culture supernatants was measured by ELISA.The lymphocyte subsets producing IFN-γ were examined at single cell level by means of flow cytometry.It was demonstrated that application of suboptimal dose of PPA or IL-12 alone induced low or undetectable levels of IFN-γ by human PBMC or NK cells.However,use of PPA plus IL-12 showed a synergistic effect on the induction of high level of IFN-γ production from human PBMC in a dose and time-dependent manner.As demonstrated from the flow cytometry analysis,PPA plus IL-12 induced the production of IFN-γ from CD56+ NK cells,but not from CD4+ or CD8+ T cells.It is apparent that the synergistic effect of PPA and IL-12 on the production of IFN-γ from CD56+ NK cells reflects the fact that they can stimulate NK cells directly,so as to induce immune responses.