Sifei Yu

Increased IL‐21 expression in chronic rhinosinusitis with nasalpolyps

IL‐21 is a key cytokine for regulating B cell immunity, which is involved in several inflammatory conditions. This study sought to define a role for IL‐21 in activated B lymphocytes and enhanced tissue eosinophilia in NP tissues during the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP).

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+TCRvβ11+ 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 CD45ROhighCD62LlowCCR7low. 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+TCRvβ11+ NKT cells. Sorted CD3+TCRvβ11+ NKT cells from PFMCs produced IFN-γ and IL-21 after stimulation, which expressed CD40L. Importantly, CD3+TCRvβ11+ NKT cells provided help to B cells for the production of IgG and IgA. Taken together, our data demonstrate that CD3+TCRvβ11+ 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.

Tuberculosis antigen-induced expression of IFN-α in tuberculosis patients inhibits production of IL-1β

The mechanism by which IFN‐α regulates the host response to Mycobacterium tuberculosis (M.tb) infection in humans is poorly understood. In the present study, we found that freshly isolated pleural fluid mononuclear cells (PFMCs) from tuberculous pleural effusion but not peripheral blood mononuclear cells (PBMCs) spontaneously expressed IFN‐α and IL‐1β in vivo. In addition, exogenous IFN‐α significantly inhibited production of IL‐1β in PFMCs after stimulation with Bacillus Calmette‐Guérin (BCG). To further evaluate the effect of endogenous IFN‐α on BCG‐induced IL‐1β production, a neutralizing antibody to IFN‐α was added to the cultures of BCG‐stimulated PFMCs. As expected, neutralization of IFN‐α by antibody significantly enhanced the production of IL‐1β. Notably, we showed that IFN‐α inhibited production of IL‐1β through 2 distinct mechanisms: IFN‐α signaling, via the STAT1 transcription factor, suppressed caspase‐1‐dependent IL‐1β maturation, and IFN‐α induced the production of IL‐10 in a STAT1‐dependent manner in which IL‐10 reduced the abundance of IL‐1β. In contrast, we found that IFN‐α enhanced the production of IFN‐γ, and IFN‐γ also suppressed IL‐1β production in the PFMCs during BCG stimulation. Our findings demonstrate that IFN‐α employs distinct pathways for regulating IL‐1β production and reveal that in the case of M.tb infection, the induction of IFN‐α and IFN‐γ might be associated with M.tb immune escape and disease progression in infected humans.—Ma, J., Yang, B., Yu, S., Zhang, Y., Zhang, X., Lao, S., Chen, X., Li, B., Wu, C. Tuberculosis antigen‐induced expression of IFN‐α in tuberculosis patients inhibits production of IL‐1β. FASEB J. 28, 3238–3248 (2014). www.fasebj.org

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-12Rβ1 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.

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.

IL-27 suppresses the production of IL-22 in human CD4(+) T cells by inducing the expression of SOCS1.

IL-27, a member of IL-6/IL-12 cytokine family, plays pro- and anti-inflammatory functions in immune responses. It can promote inflammation by inducing Th1 differentiation and exert the inhibitory effects on Th2 and Th17 mediated immune responses. Moreover, IL-27 suppresses CD28-mediated IL-2 production from mouse naive CD4(+) T cells. In the present study, we demonstrate that IL-27 inhibits the production of IL-22 and induces the expression of IFN-γ in CD4(+) T cells from human umbilical cord blood mononuclear cells (CBMCs) stimulated with anti-CD3 and anti-CD28 in dose-dependent manner. In addition, the suppression of IL-22 is not dependent on the production of IFN-γ and IL-10. Importantly, IL-27 promotes the expression of SOCS1, which could be inhibited by a Jak2/STAT inhibitor, AG490. Importantly, the expression of IL-22 could not be inhibited under the circumstances with the lower expression of SOCS1. Moreover, IL-27 inhibits the production of IL-22 in CD4(+)CD45RA(+) and CD4(+)CD45RO(+) T cells from PBMCs. These data identify that IL-27 may suppress the production of IL-22 by inducing the expression of SOCS1 in human CD4(+) T cells. Furthermore, it demonstrates that IL-27 may be a therapeutic approach in the treatment of IL-22-mediated diseases.

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+CD62LlowCCR7lowCD40LhighICOShigh 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.

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-exprssing 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 CD8+ T Cells from TB Pleurisy Respond to Four Immunodominant Epitopes in Mtb CFP10 Restricted by HLA-B Alleles

CD8+ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8+ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8+ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13–15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8+ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8+ T cells. The epitope-specific CD8+ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8+ T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8+ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8+ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.

Human Memory, but Not Naive, CD4+ T Cells Expressing Transcription Factor T-bet Might Drive Rapid Cytokine Production

Background: Human memory CD4+ T cells mediate adaptive immune responses via rapidly producing effector cytokines. Results: Memory CD4+ T cells highly express T-bet, which is closely correlated with IFN-γ production. Conclusion: Pre-existence and nuclear mobilization of T-bet in memory CD4+ T cells contribute to regulating the rapid production of IFN-γ. Significance: We declared a possible transcriptional mechanism for rapid production of cytokines by memory CD4+ T cells. 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 CD45RAlowCD62Llow CCR7low. 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.