Estrogen enhanced the expression of IL-17 by tissue-resident memory γδT cells from uterus via interferon regulatory factor 4.

Tissue-resident memory γδT cells at mucosal and epithelial sites play an important role for pathogen clearance, immunosurveillance, and participating in physiological processes. Different from other barrier sites, the immune cells in uterus face the protection against infections and tolerate an allogeneic fetus during a successful pregnancy. In the previous study, we found that tissue-resident memory γδT cells were enriched both in human and murine uterus and highly expressed IL-17 that promoted the invasion of trophocytes in vitro. In the current study, we found that γδT cells in uterus but not in blood or spleens expressed higher levels of estrogen receptors. The injection of estrogen into mice increased the proportion of γδT cells in uterus but not in spleens in vivo via CXCR3-CXCL10 chemokine axis. In addition, we found that estrogen enhanced the production of IL-17 but not IFN-γ in vivo and in vitro via interferon regulatory factor 4 but not RORγt and pSTAT3 at mRNA and protein levels. The analysis of cell transcriptome sequence further identified multiple differentially expressed genes between estrogen and control γδT cells. Our study demonstrated that estrogen directly act on γδT cells in uterus to enhance the production of IL-17 that might promote the invasion of trophocytes. Furthermore, our study might provide a new idea that estrogen increased the prevalence of autoimmune diseases in women by enhancing γδT cell-derived IL-17 production in uterus and uncover the critical pathological roles for estrogen in the development of autoimmune diseases.

Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69-CD8+ T cells expressed higher levels of CD45RO than CD103-CD69+CD8+ T cells did; CD103+CD69-CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103-CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69- memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-ß extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

Antigen-Specific Tissue-Resident Memory T Cells in the Respiratory System Were Generated following Intranasal Vaccination of Mice with BCG.

Tissue-resident memory T cells (TRM) are different from effector memory T cells (TEM) and central memory T cells (TCM) and contribute to the protective immunity against local challenges. Currently, we found that CD4+ and CD8+ TRM cells in the nasal mucosa, trachea, lungs, and lavage fluids were heterogeneous on the expression of CD69 and CD103 as well as the production of cytokines including IFN-γ, IL-2, and TNF-α. After intranasal vaccination of mice with BCG, respiratory tissues expressed higher levels of the chemokine CXCL16 and TRM cells expressed CXCR6 to CXCL16. In addition, antigen-specific CD4+ and CD8+ TRM cells expressed cytokines following the stimulation with BCG and persisted in the nasal mucosa, trachea, and lungs for more than a hundred days. At the same time, mice were infected intranasally with live BCG and the results showed that vaccinated mice cleared up live BCG faster than nonvaccinated mice in the respiratory system. Taken together, our data demonstrated that intranasal vaccination of mice with BCG could induce antigen-specific CD4+ and CD8+ TRM cells in the respiratory system and have the ability to provide protection against pulmonary reinfection.

The phenotypic and functional study of tissue B cells in respiratory system provided important information for diseases and development of vaccines.

The field of tissue-resident B cells has received increasing attention, yet the feature of tissue B cells in respiratory system is unclear. Here, we first show that non-circulating B cells obtained from nasal, trachea and lung tissues are numerically and phenotypically distinct from their circulating counterparts. Analysis of single cell transcriptome sequence identified multiple differentially expressed genes between non-circulating B cells and circulating B cells, which illustrated their heterogeneity. Furthermore, we found high expression of CXCR3 on non-circulating B cells, and the chemokine CXCL11 was also up-regulated in the respiratory tissues, suggesting that CXCR3-CXCL11 axis might accelerate the local resident of non-circulating B cells in respiratory tract. Interestingly, intranasal immunization with BCG in mice elicited a sustained humoral immune response via induction of IgA and IgG Abs, which revealed the role of B cells. Meanwhile, tissue-resident B cells, IgA+ and IgG+ memory B cells (MBCs) in respiratory tissues, as well as plasma cells in bone marrow, were expanded and maintained, and these subsets probably developed into antibody-producing cells to participate in the local humoral immunity. Our data illustrate the phenotype and function of tissue B cells in the upper and lower airways, provide references for the prospective development of vaccines.

Tissue Resident Memory γδT Cells in Murine Uterus Expressed High Levels of IL-17 Promoting the Invasion of Trophocytes.

γδT cells are non-conventional T cells and serve as the bridge for connecting the innate and adaptive immune systems. γδT cells form a substantial population at barrier sites and play an important role in the development of physiology, inflammation, autoimmune diseases and tumors. γδT cells not only distribute in the maternal-fetal interface during pregnancy but also in non-pregnant uterus. However, the phenotypes and functions of γδT cells in uterus were not clear. In the current study, we found that the percentages of γδT cells were significantly higher in uterus than peripheral blood and most of γδT cells in uterus were distributed in endometrium. Further studies indicated that the majority of γδT cells in uterus were memory cells with higher expression of CD44 and CD27 but lower expression of CD62L and CCR7 compared to those in blood. In addition, we found that γδT cells in uterus were tissue resident memory γδT cells expressing CD69, expressed high levels of CCR6, GranzymeB and CD107a. Moreover, γδT cells in uterus were activated and fully expressed transcription factor RORγt. After short time of activation, γδT cells in uterus significantly expressed high levels of IL-17 but not IFN-γ, which promotes the invasion of murine trophocytes. Taken together, our study will lay the foundation for future research on uterine γδT cells in pregnancy and autoimmune disease.

A Novel RAG1 Mutation in a Compound Heterozygous Status in a Child With Omenn Syndrome.

Omenn syndrome is a rare autosomal recessive disorder characterized by severe, combined immunodeficiency and autoimmune features. In this case study, we found Omenn syndrome in a 3-month-old boy with recurrent infection, erythroderma, axillary lymphadenopathy, and hepatosplenomegaly. The numbers of eosinophile granulocytes and the levels of immunoglobulin E in his blood were distinctly elevated. Circulating B cells were absent, and the numbers of activated T lymphocytes were present in his peripheral blood. The production of T cell cytokines was significantly higher in the patient compared to the control samples except for interferon gamma. Whole exome sequencing revealed that the patient carried compound heterozygous mutations in the RAG1 gene, which included a previously undescribed frameshift mutation (exon 2, 2491_2497del, p. K830fsX4) and a missense mutation (exon 2, 2923 C > T, p.R975W).

Poly-functional T helper cells in human tonsillar mononuclear cells.

Tonsils are important lymphoid organs in which B cells and T cells complete their maturation and identify cells that are infected by pathogens. However, the functions of T cells in human tonsils remain unclear, especially the characteristics of polyfunctional CD4+ T helper cells. In this study, we used multi-color flow cytometry to analyze the expression or co-expression of effector cytokines in CD4+ T cells from tonsillar tissues. We have demonstrated that tonsillar CD4+ T cell can express various Th effector cytokines after short-term polyclonal stimulation, and that cytokine-producing CD4+ T cells were CD45RO+ T cells. In addition, we analyzed the co-expression of two or more kinds of cytokines at the level of a single cell. The results showed that tonsillar CD4+ T cells exhibited polyfunctionality by co-expressing two to five kinds of cytokines in the same time. These data furnished a basic theory for further understanding the differentiation of polyfunctional Th cells in human tonsils and their functions in resisting invasive microorganisms.

A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins.

Recent studies indicated that CXCR5+CD8+ T cells in lymph nodes could eradicate virus-infected target cells. However, in the current study we found that a subset of CXCR5+CD8+ T cells in the germinal centers from human tonsils or lymph nodes are predominately memory cells that express CD45RO and CD27. The involvement of CXCR5+CD8+ T cells in humoral immune responses is suggested by their localization in B cell follicles and by the concomitant expression of costimulatory molecules, including CD40L and ICOS after activation. In addition, CXCR5+CD8+ memory T cells produced significantly higher levels of IL-21, IFN-γ, and IL-4 at mRNA and protein levels compared to CXCR5-CD8+ memory T cells, but IL-21-expressing CXCR5+CD8+ T cells did not express Granzyme B and perforin. When cocultured with sorted B cells, sorted CXCR5+CD8+ T cells promoted the production of antibodies compared to sorted CXCR5-CD8+ T cells. However, fixed CD8+ T cells failed to help B cells and the neutralyzing antibodies against IL-21 or CD40L inhibited the promoting effects of sorted CXCR5+CD8+ T cells on B cells for the production of antibodies. Finally, we found that in the germinal centers of lymph nodes from HIV-infected patients contained more CXCR5+CD8+ T cells compared to normal lymph nodes. Due to their versatile functional capacities, CXCR5+CD8+ T cells are promising candidate cells for immune therapies, particularly when CD4+ T cell help are limited.

[IL-12 contributes to reconstruction of cellular immune function of tumor patients and mice after chemotherapeutics].

Objective To investigate the recovery effects of IL-12 on the immune suppression induced by chemotherapeutic medicine in patients and mouse with malignant tumors. Methods Peripheral blood mononuclear cells (PBMCs) from tumor patients with or without chemotherapy and healthy donors were stimulated with or without anti-CD3 antibody plus anti-CD28 antibody in the presence or absent of IL-12. The levels of IFN-γ and TNF-α in culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). The expression of IFN-γ in different subsets of T cells was analyzed by fluorescence activated cell sorter (FACS). Finally, we established the cisplatin toxicity mouse model and measured the levels of IFN-γ and TNF-α by ELISA and FACS. Results PBMCs from the patients with malignant tumors produced significantly lower levels of IFN-γ and TNF-α than PBMCs from healthy donors. The production of IFN-γ and TNF-α was higher in pre-chemotherapeutic patients compared with post-chemotherapeutic patients, whereas IL-12 could remarkably recover the production of IFN-γ and TNF-α in the patients with malignant tumors. FACS showed that IL-12 recovered the expression of IFN-γ by CD4+ and CD8+ T cells in post-chemotherapeutic patients. Finally, the results from the animal studies in vitro and in vivo proved that IL-12 recovered the inhibitory effect of chemotherapeutic drugs on immune function. Conclusion Chemotherapeutics inhibits the immune responses in patients and animals, and IL-12 can recover the suppressive effects of chemotherapeutics on the production of cytokines. Our results indicated that IL-12 might play an important role in the reconstruction of immune function in cancer patients with chemotherapeutics.

IL-12 reverses inhibitory effect of cisplatin on immune function of human NK cells and its mechanism / 中国肿瘤生物治疗杂志

@# Objective: To investigate the reverse effect and mechanism of IL-12 on chemotherapeutic medicine suppressing the immune function of NK cells. Methods: Purified NK cells were stimulated with PMAplus Ionomycin in the presence or absent of Cisplatin (DDP) and IL-12. The levels of IFN-γ and TNF-α in culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA); The content of IFN-γ and TNF-α, TRAIL (TNF-related apoptosis inducing ligand) and transcription factors including T-bet and p-STAT-4 in NK cells were analyzed by Flow cytometry. The cytotoxicity of purified NK cells (pretreated with/without chemotherapeutics and IL-12 for 48 h) to Jurkat cells was measured by Flow cytometry. Results: Chemotherapeutics significantly inhibited the production of IFN-γ, TNF-α and the expression of TRAILin NK cells, which were significantly reversed by IL-12 (P<0.05 or P<0.01). Further study revealed that chemotherapeutics down-regulated while IL-12 reversed the expression of p-STAT4 to restore cytokine production. In addition, DDP also inhibited but IL-12 recovered the cytotoxicity of NK cells against tumor cells by inducing the expression of TRAIL (P<0.05 or P<0.01). Conclusion: Chemotherapeutics inhibited the cytotoxicity of NK cells and its secretion of cytokines (IFNγ and TNF-α), which were reversed by IL-12 via up-regulating TRAIL and p-STAT-4; this might provide experimental evidence for the clinical application of IL-12 for rebuild the immune function of tumor patients receiving chemotherapy.

The ligands of translocator protein inhibit human Th1 responses and the rejection of murine skin allografts.

The translocator protein (TSPO) ligands affected inflammatory and immune responses. However, the exact effects of TSPO ligands on Th1 responses in vitro and in vivo are still unclear. In the present study, we found that TSPO ligands, FGIN1-27 and Ro5-4864, suppressed the cytokine production in a dose-dependent manner by purified human CD4+ T-cells from peripheral blood mononuclear cells (PBMCs) after stimulation. TSPO ligands inhibited the production of interferon γ (IFN-γ) by memory CD4+ T-cells and the differentiation of naïve CD4+ T-cells into Th1 cells via suppressing the activity of the corresponding transcription factors as indicated by reduced expression of T-bet and down-regulation of STAT1, STAT4 and STAT5 phosphorylation. TSPO ligands suppressed cell proliferation and activation of CD4+ T-cells by the inhibition of TCR signal transduction including membrane proteins: Zap, Lck, Src; cytoplasm proteins: Plcγ1, Slp-76, ERK, JNK and the nucleoproteins: c-Jun and c-Fos. In addition, FGIN1-27 inhibited mixed lymphocyte reactions by human or murine cells. After the transplantation of allogeneic murine skin, injection of FGIN1-27 into mice prevented graft rejection by inhibition of cell infiltration and IFN-γ production. Taken together, our data suggest that TSPO ligands inhibit Th1 cell responses and might be novel therapeutic medicine for the treatment of autoimmune diseases and prevention of transplant rejection.

Phenotypic and functional characteristics of IL-21-expressing CD8(+) T cells in human nasal polyps.

Although CD4(+) T cells are recognized to play an important role in the inflammatory response of nasal polyps (NPs), the biological functions of CD8(+) T cells in polypogenesis remain unclear. In this study, we analyzed cell markers, cytokine expression and transcription factors in IL-21-expressing CD8(+) T cells in polyp tissues of NP patients. The results showed that the majority of IL-21-producing CD8(+) T cells were effector memory cells and they co-expressed IFN-γ. IL-21-expressing CD8(+) T cells in polyp tissues expressed higher CXCR5, PD-1, and ICOS levels than cells in control tissues and showed significantly higher T-bet and Bcl-6 expression levels compared with IL-21(-)CD8(+) T cells. Purified polyp CD8(+) T cells promoted IgG production from isolated polyp B cells in vitro, and recombinant IL-12 modulated the expression of IL-21, IFN-γ and CD40L in purified polyp CD8(+) T cells. Moreover, the percentage of IL-21(+)CD8(+) T cells in polyp tissues was positively correlated with endoscopic and CT scan scores in NP patients. These findings indicated that polyp CD8(+) T cells, by co-expressing IL-21 and IFN-γ and other markers, display a Tfh cell functionality, which is associated with the clinical severity of NP patients.

Memory-Like Antigen-Specific Human NK Cells from TB Pleural Fluids Produced IL-22 in Response to IL-15 or Mycobacterium tuberculosis Antigens.

Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO+ or CD45RO- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO+ but not CD45RO- NK cells produced significantly higher level of IL-22. Anti-IL-12Rß1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45ROhighNKG2Dhighgranzyme Bhigh. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO+ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb.

Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12.

In the current study of Mycobacterium tuberculosis (MTB)-specific T and B cells, we found that MTB-specific peptides from early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) induced the expression of IL-21 predominantly in CD4(+) T cells. A fraction of IL-21-expressing CD4(+) T cells simultaneously expressed Th1 cytokines but did not secrete Th2 or Th17 cytokines, suggesting that MTB-specific IL-21-expressing CD4(+) T cells were different from Th1, Th2 and Th17 subpopulations. The majority of MTB-specific IL-21-expressing CD4(+) T cells co-expressed IFN-γ and IL-21+IFN-γ(+)CD4(+) T cells exhibited obviously polyfunctionality. In addition, MTB-specific IL-21-expressing CD4(+) T cells displayed a CD45RO+CD62Ll(ow)CCR7(low)CD40L(high)ICOS(high) phenotype. Bcl-6-expression was significantly higher in IL-21-expressing CD4(+) T cells than IL-21-CD4(+) T cells. Moreover, IL-12 could up-regulate MTB-specific IL-21 expression, especially the frequency of IL-21(+)IFN-γ+CD4(+) T cells. Taken together, our results demonstrated that MTB-specific IL-21(+)IFN-γ(+)CD4(+) T cells from local sites of tuberculosis (TB) infection could be enhanced by IL-12, which have the features of both Tfh and Th1 cells and may have an important role in local immune responses against TB infection.

IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells.

Interleukine-12 is critical for the differentiation of Th1 cells and can improve the development of Th1 cells with Tfh cell features in mouse model. Human effector CD4(+) T cells also exhibit poly-functionality by co-expressing IL-21 and IFN-γ. However, the effects of IL-12 on regulating generation of human IL-21- and IFN-γ-expressing CD4(+) T cells are still incompletely understood. Our studies found that IL-12 but not IL-21 could induce the differentiation of human naive CD4(+) T cells into multi-cytokine expressing CD4(+) T cells in vitro, which co-expressed IL-21 and IFN-γ with or without IL-2 and TNF-α. At early stage of differentiation, addition of excess exogenous IFN-γ could increase the generation of IL-21- and IFN-γ-expressing CD4(+) T cells, furthermore, anti-IFN-γ depressed the percentage of poly-functional CD4(+) T cells. Phenotypically, IL-21(+)IFN-γ(+)CD4(+) T cells exhibited more characteristic features about both of Th1 and Tfh cells than IL-21 or IFN-γ single-expressing CD4(+) T cells. Mechamistically, IL-12 modulated the differentiation of IL-21(+)IFN-γ(+)CD4(+) T cells from naive CD4(+) T cells via the pathways of STAT-1/4, T-bet and BCL(-)6. Different from naive CD4(+) T cells, IL-12 increasing the generation of IL-21(+)IFN-γ(+)CD4(+) T cells from memory CD4(+) T cells was only involved in STAT-4 pathway but not STAT-1. Poly-functional CD4(+) T cells were contributed to generation and progress of varies diseases and our studies provide basic theoretics for the designs of vaccine and therapies of diseases.

[Effects of different Toll-like receptor agonists on the function of T helper cells in mice].

OBJECTIVE: To investigate the secretion of IL-12p40 and IL-6 by splenocytes, dendritic cells stimulated by different Toll-like receptor (TLR) agonists or in the sera of mice immunized with different TLR agonists, and evaluate the effects of different TLR agonists on the function of T helper (Th) cells, especially the differentiation of Th1 cells. METHODS: Supernatants of splenocytes and dendritic cells stimulated with different TLR agonists for 24 hours or sera of mice immunized with different TLR agonists at different time points were used to determine the levels of IL-12p40 and IL-6 by ELISA. CD4⁺ T cells isolated from the spleens of ovalbumin-T cell receptor (OVA-TCR) transgenic BALB/c (DO11.10) mice were co-cultured with antigen presenting cells (APCs) from congenic BALB/c mice at 1:3 ratio of T:APCs. Cultures were stimulated with OVA peptide or OVA peptide plus different doses of TLR agonists and the supernatants collected at different time points were assayed by ELISA for detecting IFN-γ. RESULTS: Pam3CSK4, R848 and CpG oligodeoxynucleotide (ODN) promoted the production of IL-12p40 and IL-6 by splenocytes and dendritic cells obviously, and induced the expression of IFN-γ in antigen specific CD4⁺ T cells in a time- and dose-dependent manner. Monophosphoryl lipid A from Salmonella minnesota R595 lipopolysaccharide (MPLA-SM) induced low levels of cytokines by splenocytes and couldn't promote the production of IFN-γ by antigen specific CD4⁺ T cells, but increased the expressions of IL-12p40 and IL-6 by DCs. All the sera of mice immunized with the four TLR agonists expressed high levels of IL-12p40 and IL-6. CONCLUSION: Splenocytes, DCs stimulated or sera of mice immunized with different TLR agonists produced different levels of cytokines, which could further affect the differentiation of Th1 cells.

Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins.

Natural killer T (NKT) cells from mouse and human play an important role in the immune responses against Mycobacterium tuberculosis. However, the function of CD3(+)TCRvß11(+) NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that after stimulation with M. tuberculosis antigens, NKT cells isolated from tuberculosis (TB) pleural fluid mononuclear cells (PFMCs) produced IL-21 and other cytokines including IFN-γ, TNF-α, IL-2 and IL-17. IL-21-expressing NKT cells in PFMCs displayed effector memory phenotype, expressing CD45RO(high)CD62L(low)CCR7(low). Moreover, NKT cells expressed high levels of CXCR5 and all of IL-21-expressing NKT cells co-expressed CXCR5. The frequency of BCL-6-expression was higher in IL-21-expressing but not in non-IL-21-expressing CD3(+)TCRvß11(+) NKT cells. Sorted CD3(+)TCRvß11(+) NKT cells from PFMCs produced IFN-γ and IL-21 after stimulation, which expressed CD40L. Importantly, CD3(+)TCRvß11(+) NKT cells provided help to B cells for the production of IgG and IgA. Taken together, our data demonstrate that CD3(+)TCRvß11(+) NKT cells from a local site of M. tuberculosis infection produce IL-21, express CXCR5 and CD40L, help B cells to secrete IgG and IgA, and may participate in local immune responses against M. tuberculosis infection.

Human IL-21+IFN-γ+CD4+ T cells in nasal polyps are regulated by IL-12.

In the previous study, we found that the levels of IL-21 in nasal polyps (NPs) were significantly increased and associated with polyp size and recurrence. However, it is unclear that the cell source of IL-21 and the regulation of IL-21 in NP tissues. In the present study, we isolated the lymphocytes from NP tissues, uncinate tissues and peripheral blood of patients with NPs. The cells were analyzed for cell surface markers, cytokines and transcriptional factors by flow cytometry. The results indicated that CD4(+) T cells were the major IL-21-expressing cells in NP tissues and the majority of IL-21 producing CD4(+) T cells co-expressed IFN-γ or IL-17A. IL-21(+)IFN-γ(+)CD4(+) T cells in NP tissues exhibited the features of both Tfh and Th1 cells which co-expressed significantly higher amount of CXCR5, ICOS, PD-1, Bcl-6 and T-bet than did IL-21(+)IFN-γ(-)CD4(+) T cells (p < 0.05). Treatment of the lymphocytes from NP tissues with IL-12 enhanced the production of IL-21 and IFN-γ, especially the frequency of IL-21(+)IFN(-)γ(+)CD4(+) T cells (p < 0.05). The blockade of IL-12 inhibited the production of IL-21 and IFN-γ (p < 0.05). These findings indicated that IL-12 positively enhanced the generation of IL-21(+)IFN-γ(+)CD4(+) T cells having the features of both Tfh and Th1 cells in NP tissues.

Human memory, but not naive, CD4+ T cells expressing transcription factor T-bet might drive rapid cytokine production.

We found that after stimulation for a few hours, memory but not naive CD4(+) T cells produced a large amount of IFN-γ; however, the mechanism of rapid response of memory CD4(+) T cells remains undefined. We compared the expression of transcription factors in resting or activated naive and memory CD4(+) T cells and found that T-bet, but not pSTAT-1 or pSTAT-4, was highly expressed in resting memory CD4(+) T cells and that phenotypic characteristics of T-bet(+)CD4(+) T cells were CD45RA(low)CD62L(low) CCR7(low). After short-term stimulation, purified memory CD4(+) T cells rapidly produced effector cytokines that were closely associated with the pre-existence of T-bet. By contrast, resting naive CD4(+) T cells did not express T-bet, and they produced cytokines only after sustained stimulation. Our further studies indicated that T-bet was expressed in the nuclei of resting memory CD4(+) T cells, which might have important implications for rapid IFN-γ production. Our results indicate that the pre-existence and nuclear mobilization of T-bet in resting memory CD4(+) T cells might be a possible transcriptional mechanism for rapid production of cytokines by human memory CD4(+) T cells.

Mycobacterium tuberculosis-specific memory NKT cells in patients with tuberculous pleurisy.

Natural killer T (NKT) cells from mouse and human play a protective role in the immune responses against the infection of Mycobacterium tuberculosis. However, the characteristic of CD3(+)TCRvß11(+) NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that the numbers of CD3(+)TCRvß11(+) NKT cells in pleural fluid mononuclear cells (PFMCs) were significantly lower than those in peripheral blood mononuclear cells (PBMCs). However, CD3(+)TCRvß11(+) NKT cells from PFMCs spontaneously expressed high levels of CD69 and CD25 and effector memory phenotypes of CD45RO(high)CD62L(low)CCR7(low). After stimulation with the antigens of M. tuberculosis, CD3(+)TCRvß11(+) NKT cells from PFMCs produced high levels of IFN-γ. Sorted CD3(+)TCRvß11(+) NKT cells from PFMCs cultured with antigen presenting cells (APCs) produced IFN-γ protein and mRNA. The production of IFN-γ could be completely inhibited by AG490 and Wortmannin. In addition, CD3(+)TCRvß11(+) NKT cells from PFMCs expressed higher levels of Fas (CD95), FasL (CD178) and perforin but lower levels of granzyme B compared with those from PBMCs. Taken together, our data demonstrated for the first time that M. tuberculosis-specific CD3(+)TCRvß11(+) NKT cells participated in the local immune responses against M. tuberculosis through the production of IFN-γ and the secretion of cytolytic molecules.