Weishan Huang

Absence of Tec Family Kinases Interleukin-2 Inducible T cell Kinase (Itk) and Bruton's Tyrosine Kinase (Btk) Severely Impairs FcϵRI-dependent Mast Cell Responses

Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcϵRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcϵRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcϵRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcϵRI-induced cytokine secretion.

The signaling symphony: T cell receptor tunes cytokine-mediated T cell differentiation

T cell development, differentiation, and maintenance are orchestrated by 2 key signaling axes: the antigen‐specific TCR and cytokine‐mediated signals. The TCR signals the recognition of self‐ and foreign antigens to control T cell homeostasis for immune tolerance and immunity, which is regulated by a variety of cytokines to determine T cell subset homeostasis and differentiation. TCR signaling can synergize with or antagonize cytokine‐mediated signaling to fine tune T cell fate; however, the latter is less investigated. Murine models with attenuated TCR signaling strength have revealed that TCR signaling can function as regulatory feedback machinery for T cell homeostasis and differentiation in differential cytokine milieus, such as IL‐2‐mediated Treg development; IL‐7‐mediated, nai¨ve CD8+ T cell homeostasis; and IL‐4‐induced innate memory CD8+ T cell development. In this review, we discuss the symphonic cross‐talk between TCR and cytokine‐mediated responses that differentially control T cell behavior, with a focus on the negative tuning by TCR activation on the cytokine effects.

Cutting Edge: Innate Memory CD8+ T Cells Are Distinct from Homeostatic Expanded CD8+ T Cells and Rapidly Respond to Primary Antigenic Stimuli

Innate memory phenotype (IMP) CD8+ T cells are nonconventional αβ T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8+ T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8+ T cells in MHCI−/− recipients, resembling those in Itk−/− mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI−/− bone marrow does not lead to IMP CD8+ T cells in wild-type recipients. OTI IMP CD8+ T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific “naive” CD8+ IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.

Signal Transduction via the T cell Antigen Receptor in naïve and effector/memory T cells

T cells play an indispensable role in immune defense against infectious agents, but can also be pathogenic. These T cells develop in the thymus, are exported into the periphery as naïve cells and participate in immune responses. Upon recognition of antigen, they are activated and differentiate into effector and memory T cells. While effector T cells carry out the function of the immune response, memory T cells can last up to the life time of the individual, and are activated by subsequent antigenic exposure. Throughout this life cycle, the T cell uses the same receptor for antigen, the T cell Receptor, a complex multi-subunit receptor. Recognition of antigen presented by peptide/MHC complexes on antigen presenting cells unleashes signaling pathways that control T cell activation at each stage. In this review, we discuss the signals regulated by the T cell receptor in naïve and effector/memory T cells.

Self-Assembly Protein Nanogels for Safer Cancer Immunotherapy.

Soluble antigen-based cancer vaccines have poor retention in tissues along with suboptimal antigen processing by dendritic cells. Multiple booster doses are often needed, leading to dose-limiting systemic toxicity. A versatile, immunomodulatory, self-assembly protein nanogel vaccine is reported that induces robust immune cell response at lower antigen doses than soluble antigens, an important step towards biomaterials-based safer immunotherapy approaches.

IL-2–Inducible T Cell Kinase Tunes T Regulatory Cell Development and Is Required for Suppressive Function

IL-2–inducible T cell kinase (ITK) is a key signaling mediator downstream of TCR, mediating T cell positive selection, as well as innate T cell and CD4+ Th2/Th17 differentiation. In this article, we show that ITK also negatively tunes IL-2–induced expansion of conventional Foxp3-expressing regulatory T cells (Tregs). In vivo, Treg abundance is inversely correlated with ITK expression, and inducible Treg development is inversely dependent on ITK kinase activity. While Treg development normally requires both hematopoietic and thymic MHC class 2 (MHC2) expression, the absence of ITK allows Treg development with MHC2 expression in either compartment, with preference for selection by thymic MHC2, suggesting a gatekeeper role for ITK in ensuring that only Tregs selected by both thymic and hematopoietic MHC2 survive selection. Although ITK suppresses Treg development and is not required for maintenance of neuropilin-1–positive natural Tregs in the periphery, it is indispensable for Treg functional suppression of naive CD4+ T cell–induced colitis in Rag−/− recipients. ITK thus regulates the development and function of Tregs.

A Unique Role for ITK in Survival of Invariant NKT Cells Associated with the p53-Dependent Pathway in Mice

Invariant NKT (iNKT) cells play important roles in the immune response. ITK and TXK/RLK are Tec family kinases that are expressed in iNKT cells; the expression level of ITK is ∼7-fold higher than that of TXK. Itk−/− mice have reduced iNKT cell frequency and numbers, with defects in development and cytokine secretion that are exacerbated in Itk/Txk double-knockout mice. In contrast, there is no iNKT cell defect in Txk−/− mice. To determine whether ITK and TXK play distinct roles in iNKT cell development and function, we examined mice that overexpress TXK in T cells at levels similar to Itk. Overexpression of TXK rescues the maturation and cytokine secretion of Itk−/− iNKT cells, as well as altered expression of transcription factors T-bet, eomesodermin, and PLZF. In contrast, the increased apoptosis observed in Itk−/− splenic iNKT cells is not affected by TXK overexpression, likely due to the lack of effect on the elevated expression of p53 regulated proapoptotic pathways Fas, Bax, and Bad in those cells. Supporting this idea, p53−/− and Bax−/− mice have increased splenic iNKT cells. Our results suggest that TXK plays an overlapping role with ITK in iNKT cell development and function but that ITK also has a unique function in the survival of iNKT cells, likely via a p53-dependent pathway.

Plasmin-Mediated Activation of Pandemic H1N1 Influenza Virus Hemagglutinin Is Independent of the Viral Neuraminidase

ABSTRACT Influenza virus is well recognized to modulate host tropism and pathogenesis based on mutations in the proteolytic cleavage site of the viral hemagglutinin (HA), which activates HA and exposes the fusion peptide for membrane fusion. Instead of the conventional trypsin-mediated cleavage event, modification of the cleavage site allows extended use of host cell proteases and enhanced spread in vivo. For H1N1 influenza viruses, the mouse-adapted A/WSN/33 strain is known to replicate in the brain based on recruitment of plasminogen by the viral neuraminidase (NA), as well as a Ser-Tyr substitution at the P2 position of the HA cleavage site. Here, we show that an equivalent Ser-Tyr substitution has occurred in the HA of naturally occurring human H1N1 influenza viruses. We characterize one of these viruses (A/Beijing/718/2009), as well as the prototype A/California/04/2009 with a Ser-Tyr substitution in the cleavage site, and show that these HAs are preferentially cleaved by plasmin. Importantly, cleavage activation by plasmin/plasminogen was independent of the viral NA, suggesting a novel mechanism for HA cleavage activation. We show that the viral HA itself can recruit plasminogen for HA cleavage. We further show that cellular factors, as well as streptokinase from bacteria commonly coinfecting the respiratory tract of influenza patients, can be a source of activated plasminogen for plasmin-mediated cleavage of influenza virus HAs that contain a Ser-Tyr substitution in the cleavage site.

ITK tunes IL-4-induced development of innate memory CD8+ T cells in a γδ T and invariant NKT cell-independent manner

True memory CD8+ T cells develop post antigenic exposure and can provide life‐long immune protection. More recently, other types of memory CD8+ T cells have been described, such as the memory‐like CD8+ T cells (IMP; CD44hiCD122+) that arise spontaneously in Itk−/− mice, which are suggested to develop as a result of IL‐4 secreted by NKT‐like γδ T or PLZF+ NKT cells found in Itk−/− mice. However, we report here that whereas IMP CD8+ T cell development in Itk−/− mice is dependent on IL‐4/STAT6 signaling, it is not dependent on any γδ T or iNKT cells. Our experiments suggest that the IMP develops as a result of tuning of the CD8+ T cell response to exogenous IL‐4 and TCR triggering by ITK and challenge the current model of IMP CD8+ T cell development as a result of NKT‐like γδ T or iNKT cells. These findings suggest that some naive CD8+ T cells may be preprogrammed by weak homeostatic TCR signals in the presence of IL‐4 to become memory phenotype cells with the ability to elaborate effector function rapidly. The role of ITK in this process suggests a mechanism by which IMP CD8+ T cells can be generated rapidly in response to infection.

Cutting Edge: STAT6 Signaling in Eosinophils Is Necessary for Development of Allergic Airway Inflammation

Eosinophils are critical cellular mediators in allergic asthma and inflammation; however, the signals that regulate their functions are unclear. The transcription factor STAT6 regulates Th2 cytokine responses, acting downstream of IL-4 and IL-13. We showed previously that eosinophil-derived IL-13 plays an important role in the recruitment of T cells to the lung and the subsequent development of allergic asthma. However, whether eosinophils respond to Th2 signals to control allergic airway inflammation is unclear. In this report, we show that STAT6−/− eosinophils are unable to induce the development of allergic lung inflammation, including recruitment of CD4+ T cells, mucus production, and development of airways hyperresponsiveness. This is likely due to the reduced migration of STAT6−/− eosinophils to the lung and in response to eotaxin. These data indicate that, like Th cells, eosinophils need to respond to Th2 cytokines via STAT6 during the development of allergic airway inflammation.