Binyan Yang

Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hiCD16−, CD56loCD16−, CD56+CD16+ and CD56−CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)‐2 stimulation, interferon‐γ was preferentially produced by CD56+CD16− NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16− and CD56+/−CD16+ subsets could be augmented in response to IL‐2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.

Phenotypic and Functional Heterogeneity of Natural Killer Cells from Umbilical Cord Blood Mononuclear Cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-γ and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56hiCD16− NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-γ expression by CD56hiCD16− and CD56loCD16− subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.

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).

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

Human memory-like NK cells migrating to tuberculous pleural fluid via IP-10/CXCR3 and SDF-1/CXCR4 axis produce IFN-γ in response to Bacille Calmette Guerin.

We have previously shown that human memory-like NK cells were persistent in tuberculous pleurisy but it was unclear how NK cells migrated into the pleural fluids. At present, we found that NK cells from TB pleural fluid cells (PFCs) expressed significantly higher levels of CXCR3 and CXCR4 than NK cells from PBMCs. Migration assay demonstrated that IP-10 and SDF-1 induced more migration of NK cells from PFCs than PBMCs. CD45RO(+) or CD45RO(-) NK cells from PFCs were co-cultured with autologous monocytes and stimulated with BCG. The results showed CD45RO(+) but not CD45RO(-) NK cells produced significantly higher levels of IFN-γ, which was IL-12-dependent since anti-IL-12Rβ1 mAbs could significantly inhibit the IFN-γ by NK cells. Collectively, our data demonstrated that human Mycobacterium tuberculosis-specific NK cells were migrated into the local site of TB infection mainly via IP-10/CXCR3 and SDF-1/CXCR4 axis, memory-like NK cells might display an important role against M. tuberculosis infection.

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.

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+TCRvβ11+ NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that the numbers of CD3+TCRvβ11+ NKT cells in pleural fluid mononuclear cells (PFMCs) were significantly lower than those in peripheral blood mononuclear cells (PBMCs). However, CD3+TCRvβ11+ NKT cells from PFMCs spontaneously expressed high levels of CD69 and CD25 and effector memory phenotypes of CD45ROhighCD62LlowCCR7low. After stimulation with the antigens of M. tuberculosis, CD3+TCRvβ11+ NKT cells from PFMCs produced high levels of IFN-γ. Sorted CD3+TCRvβ11+ 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+TCRvβ11+ 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+TCRvβ11+ NKT cells participated in the local immune responses against M. tuberculosis through the production of IFN-γ and the secretion of cytolytic molecules.

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.