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Although diverse functions of different toll-like receptors (TLR) on human natural regulatory T cells have been demonstrated recently, the role of TLR-related signals on human induced regulatory T cells remain elusive. Previously our group developed an ex vivo high-efficient system in generating human alloantigen-specific CD4hiCD25+ regulatory T cells from naïve CD4+CD25− T cells using allogeneic CD40-activated B cells as stimulators. In this study, we investigated the role of TLR5-related signals on the generation and function of these novel CD4hiCD25+ regulatory T cells. It was found that induced CD4hiCD25+ regulatory T cells expressed an up-regulated level of TLR5 compared to their precursors. The blockade of TLR5 using anti-TLR5 antibodies during the co-culture decreased CD4hiCD25+ regulatory T cells proliferation by induction of S phase arrest. The S phase arrest was associated with reduced ERK1/2 phosphorylation. However, TLR5 blockade did not decrease the CTLA-4, GITR and FOXP3 expressions, and the suppressive function of CD4hiCD25+ regulatory T cells. In conclusion, we discovered a novel function of TLR5-related signaling in enhancing the proliferation of CD4hiCD25+ regulatory T cells by promoting S phase progress but not involved in the suppressive function of human CD40-activated B cell-induced CD4hiCD25+ regulatory T cells, suggesting a novel role of TLR5-related signals in the generation of induced regulatory T cells. [ABSTRACT FROM AUTHOR]

Background. Mannose-binding lectin (MBL) is a pattern-recognition molecule, which functions as a first line of host defense. Pandemic H1N1 (pdmH1N1) influenza A virus caused massive infection in 2009 and currently circulates worldwide. Avian influenza A H9N2 (H9N2/G1) virus has infected humans and has the potential to be the next pandemic virus. Antiviral function and immunomodulatory role of MBL in pdmH1N1 and H9N2/G1 virus infection have not been investigated. Methods. In this study, MBL wild-type (WT) and MBL knockout (KO) murine models were used to examine the role of MBL in pdmH1N1 and H9N2/G1 virus infection. Results. Our study demonstrated that in vitro, MBL binds to pdmH1N1 and H9N2/G1 viruses, likely via the carbohydrate recognition domain of MBL. Wild-type mice developed more severe disease, as evidenced by a greater weight loss than MBL KO mice during influenza virus infection. Furthermore, MBL WT mice had enhanced production of proinflammatory cytokines and chemokines compared with MBL KO mice, suggesting that MBL could upregulate inflammatory responses that may potentially worsen pdmH1N1 and H9N2/G1 virus infections. Conclusions. Our study provided the first in vivo evidence that MBL may be a risk factor during pdmH1N1 and H9N2/G1 infection by upregulating proinflammatory response. [ABSTRACT FROM AUTHOR]

Murine Foxp3 [ABSTRACT FROM AUTHOR]

Inducible co-stimulator (ICOS) is a member of CD28/Cytotoxic T-lymphocyte Antigen-4 (CTLA-4) family and broadly expressed in activated CD4+ T cells and induced regulatory CD4+ T cells (CD4+ iTreg). ICOS-related signal pathway could be activated by the interaction between ICOS and its ligand (ICOSL). In our previous work, we established a cost-effective system to generate a novel human allo-antigen specific CD4hi Treg by co-culturing their naïve precursors with allogeneic CD40-activated B cells in vitro. Here we investigate the role of ICOS in the generation and function of CD4hi Treg by interrupting ICOS-ICOSL interaction with ICOS-Ig. It is found that blockade of ICOS-ICOSL interaction impairs the induction and expansion of CD4hi Treg induced by allogeneic CD40-activated B cells. More importantly, CD4hi Treg induced with the addition of ICOS-Ig exhibits decreased suppressive capacity on alloantigen-specific responses. Dysfunction of CD4hi Treg induced with ICOS-Ig is accompanied with its decreased exocytosis and surface CTLA-4 expression. Through inhibiting endocytosis with E64 and pepstatin A, surface CTLA-4 expression and suppressive functions of induced CD4hi Treg could be partly reversed. Conclusively, our results demonstrate the beneficial role of ICOS-ICOSL signal pathway in the generation and function of CD4hi Treg and uncover a novel relationship between ICOS and CTLA-4. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are indispensable components of both the innate and adaptive immune response. However, their precise roles in the cross-talk between innate and adaptive immunity during influenza virus infection remain controversial. By comparing NK cell dynamics and activity under a sub-lethal dose and high dose of influenza virus infection, we showed that influenza virus PR8 directly infected NK cells during natural infection, which was consistent with our previous findings obtained from an in vitro investigation of human NK cells. The impairments in cytotoxicity and IFN-γ production by spleen NK cells following high-dose infection were accompanied by decreased virus-specific killing mediated by cytotoxic T lymphocytes (CTLs). Importantly, the weakened CTL activity could be reversed by adoptive transfer of spleen NK cells harvested from low-dose-infected mice but not healthy donors. Taken together, our data provide direct evidence supporting the contribution of NK cells to antiviral T-cell responses. This study also indicates that a novel NK-targeted immune evasion strategy is used by influenza virus to shrink both innate and adaptive immune responses.

Graft-versus-host disease (GVHD) is a lethal complication of allogeneic bone marrow transplantation (BMT). Immunosuppressive agents are currently used to control GVHD but may cause general immune suppression and limit the effectiveness of BMT. Adoptive transfer of regulatory T cells (T(regs)) can prevent GVHD in rodents, suggesting a therapeutic potential of T(regs) for GVHD in humans. However, the clinical application of T(reg)-based therapy is hampered by the low frequency of human T(regs) and the lack of a reliable model to test their therapeutic effects in vivo. Recently, we successfully generated human alloantigen-specific CD8(hi) T(regs) in a large scale from antigenically naïve precursors ex vivo using allogeneic CD40-activated B cells as stimulators. We report a human allogeneic GVHD model established in humanized mice to mimic GVHD after BMT in humans. We demonstrate that ex vivo-induced CD8(hi) T(regs) controlled GVHD in an allospecific manner by reducing alloreactive T cell proliferation as well as decreasing inflammatory cytokine and chemokine secretion within target organs through a CTLA-4-dependent mechanism in humanized mice. These CD8(hi) T(regs) induced long-term tolerance effectively without compromising general immunity and graft-versus-tumor activity. Our results support testing of human CD8(hi) T(regs) in GVHD in clinical trials.

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus-infected cells and inhibit viral replication in vitro. In Rag2(-/-)γc(-/-) immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus-infected Rag2(-/-)γc(-/-) mice reconstituted with Vγ9Vδ2 T cell-depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Paget's disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza.