Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation.

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell-mediated airway inflammation. Using STAP-1 knockout mice and STAP-1-overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK-LCK and ITK-phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell-mediated immune disorders.

The Majority of the Serine/Threonine Phosphorylation Sites in Bcl11b Protein Are Dispensable for the Differentiation of T Cells.

Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets-Th1, Th2, Th17, and regulatory T-was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation.

Influenza virus infection expands the breadth of antibody responses through IL-4 signalling in B cells.

Influenza viruses are a major public health problem. Vaccines are the best available countermeasure to induce effective immunity against infection with seasonal influenza viruses; however, the breadth of antibody responses in infection versus vaccination is quite different. Here, we show that nasal infection controls two sequential processes to induce neutralizing IgG antibodies recognizing the hemagglutinin (HA) of heterotypic strains. The first is viral replication in the lung, which facilitates exposure of shared epitopes that are otherwise hidden from the immune system. The second process is the germinal center (GC) response, in particular, IL-4 derived from follicular helper T cells has an essential role in the expansion of rare GC-B cells recognizing the shared epitopes. Therefore, the combination of exposure of the shared epitopes and efficient proliferation of GC-B cells is critical for generating broadly-protective antibodies. These observations provide insight into mechanisms promoting broad protection from virus infection.

Protective neutralizing influenza antibody response in the absence of T follicular helper cells.

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

Syk-dependent signaling pathways in neutrophils and macrophages are indispensable in the pathogenesis of anti-collagen antibody-induced arthritis.

Spleen tyrosine kinase (Syk) is associated with Fcγ receptors (FcγRs) and transmits activation signals through FcγRs in myeloid cells. Thus, application of drugs to inhibit Syk activity can affect the development of immune diseases mediated by autoantibodies, while unexpected systemic effects by the inhibition may be concerned because Syk has multiple physiological functions. We used tamoxifen-inducible systemic conditional Syk knockout (KO) mice to evaluate the role of Syk in the pathogenesis of autoimmune arthritis and to investigate the systemic effects of Syk deletion. In a collagen antibody-induced arthritis model, Syk KO mice were almost completely protected from disease induction and showed significantly attenuated accumulation of neutrophils and macrophages in the joints. Syk-deleted macrophages showed less IL-6 and MCP-1 production upon FcγR ligation and exhibited reduced FcγR-mediated phagocytosis in vitro. Syk-deleted macrophages produce more RANTES upon FcγR ligation, indicating a Syk-independent signaling through the FcγR. We further found that both wild-type and Syk-deleted macrophages induced neutrophil chemotaxis upon FcγR ligation in vitro, and air-pouch model demonstrated that Syk-deleted neutrophils have a potential to infiltrate into local tissues in response to collagen and anti-collagen antibodies. However, Syk-deleted neutrophils exhibited greatly decreased neutrophil extracellular traps formation and FcγR-mediated phagocytosis. Our results indicated that Syk deficiency rendered mice completely unresponsive to immune activation by anti-collagen antibodies with disabling one pathway of FcγR-mediated signaling that was crucial for arthritis induction.

The 3' enhancer CNS2 is a critical regulator of interleukin-4-mediated humoral immunity in follicular helper T cells.

A main role for interleukin-4 (IL-4) is in humoral immunity, and follicular helper CD4(+) T (Tfh) cells may be an intrinsic IL-4 source. Here we demonstrate that conserved noncoding sequence 2 (CNS2) is an essential enhancer element for IL-4 expression in Tfh cells but not in Th2 cells. Mice with a CNS2 deletion had a reduction in IgG1 and IgE production and in IL-4 expression in Tfh cells. Tracking of CNS2 activity via a GFP reporter mouse demonstrated that CNS2-active cells expressed several markers of Tfh cells: CXCR5, PD-1, and ICOS; the transcriptional master regulator Bcl6; and the cytokines IL-21 and IL-4. These CNS2-active cells were mainly localized in B cell follicles and germinal centers. The GFP(+) Tfh cells were derived from GFP(-) naive T cells after in vivo systemic immunization. These results indicate that CNS2 is an essential enhancer element required for IL-4 expression in Tfh cells controlling humoral immunity.

Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness.

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.

Transcription factors Foxo3a and Foxo1 couple the E3 ligase Cbl-b to the induction of Foxp3 expression in induced regulatory T cells.

The transcription factor Foxp3 is essential for optimal regulatory T (T reg) cell development and function. Here, we show that CD4(+) T cells from Cbl-b RING finger mutant knockin or Cbl-b-deficient mice show impaired TGF-beta-induced Foxp3 expression. These T cells display augmented Foxo3a phosphorylation, but normal TGF-beta signaling. Expression of Foxo3a rescues Foxp3 expression in Cbl-b-deficient T cells, and Foxo3a deficiency results in defective TGF-beta-driven Foxp3 induction. A Foxo3a-binding motif is present in a proximal region of the Foxp3 promoter, and is required for Foxo3a association. Foxo1 exerts similar effects as Foxo3a on Foxp3 expression. This study reveals that Foxo factors promote transcription of the Foxp3 gene in induced T reg cells, and thus provides new mechanistic insight into Foxo-mediated T cell regulation.