Jr-Wen Shui

Transcriptional reprogramming of mature CD4 + helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes

TCRαβ thymocytes differentiate into either CD8αβ+ cytotoxic T lymphocytes or CD4+ helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II–restricted CD4+ thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4+ T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4+ T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II–restricted CD4+ cytotoxic T lymphocytes.

Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell–mediated immune responses

HPK1 is a Ste20-related serine-threonine kinase that inducibly associates with the adaptors SLP-76 and Gads after T cell receptor (TCR) signaling. Here, HPK1 deficiency resulted in enhanced TCR-induced phosphorylation of SLP-76, phospholipase C-γ1 and the kinase Erk, more-persistent calcium flux, and increased production of cytokines and antigen-specific antibodies. Furthermore, HPK1-deficient mice were more susceptible to experimental autoimmune encephalomyelitis. Although the interaction between SLP-76 and Gads was unaffected, the inducible association of SLP-76 with 14-3-3τ (a phosphorylated serine–binding protein and negative regulator of TCR signaling) was reduced in HPK1-deficient T cells after TCR stimulation. HPK1 phosphorylated SLP-76 and induced the interaction of SLP-76 with 14-3-3τ. Our results indicate that HPK1 negatively regulates TCR signaling and T cell–mediated immune responses.

HVEM signalling at mucosal barriers provides host defence against pathogenic bacteria

The herpes virus entry mediator (HVEM), a member of the tumour-necrosis factor receptor family, has diverse functions, augmenting or inhibiting the immune response. HVEM was recently reported as a colitis risk locus in patients, and in a mouse model of colitis we demonstrated an anti-inflammatory role for HVEM, but its mechanism of action in the mucosal immune system was unknown. Here we report an important role for epithelial HVEM in innate mucosal defence against pathogenic bacteria. HVEM enhances immune responses by NF-κB-inducing kinase-dependent Stat3 activation, which promotes the epithelial expression of genes important for immunity. During intestinal Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli infection, Hvem−/− mice showed decreased Stat3 activation, impaired responses in the colon, higher bacterial burdens and increased mortality. We identified the immunoglobulin superfamily molecule CD160 (refs 7 and 8), expressed predominantly by innate-like intraepithelial lymphocytes, as the ligand engaging epithelial HVEM for host protection. Likewise, in pulmonary Streptococcus pneumoniae infection, HVEM is also required for host defence. Our results pinpoint HVEM as an important orchestrator of mucosal immunity, integrating signals from innate lymphocytes to induce optimal epithelial Stat3 activation, which indicates that targeting HVEM with agonists could improve host defence.

HIP-55 is important for T-cell proliferation, cytokine production, and immune responses

ABSTRACT Engagement of the T-cell receptor (TCR) triggers a series of signaling events that lead to the activation of T cells. HIP-55 (SH3P7 or mAbp1), an actin-binding adaptor protein, interacts with and is tyrosine phosphorylated by ZAP-70, which is a crucial proximal protein tyrosine kinase for TCR signaling. HIP-55 is important for JNK and HPK1 activation induced by TCR signaling. In this study, we report the generation and characterization of HIP-55 knockout mice. We found that HIP-55 knockout mice were viable and fertile but showed decreased body weight and increased occurrence of death within the first 4 weeks after birth. The lymphoid organs in HIP-55 knockout mice showed cellularity and T-cell development comparable to that of the wild-type mice. HIP-55 knockout T cells displayed defective T-cell proliferation, decreased cytokine production, and decreased up-regulation of the activation markers induced by TCR stimulation. TCR internalization was slightly increased in HIP-55 knockout T cells. These phenotypes were accompanied by reduced immune responses, including antigen-specific antibody production and T-cell proliferation in HIP-55 knockout mice. The TCR-induced signaling events, including LAT/phospholipase Cγ1 phosphorylation and HPK1/JNK activation, were partially defective in HIP-55 knockout T cells. These results demonstrate the importance of HIP-55 as an adaptor protein in the TCR signaling and immune system.

The SH3 Domain-containing Adaptor HIP-55 Mediates c-Jun N-terminal Kinase Activation in T Cell Receptor Signaling

HIP-55 (hematopoietic progenitor kinase 1 (HPK1)-interacting protein of 55 kDa, also called SH3P7 and mAbp1) is a novel SH3 domain-containing protein. HIP-55 binds to actin filaments both in vitro and in vivo. HIP-55 activates HPK1 and c-Jun N-terminal kinase (JNK), which are two important lymphocyte signaling molecules. Until now, the regulation and function of HIP-55 in T cell receptor (TCR) signaling were unknown. We found that HIP-55 was recruited to glycolipid-enriched microdomains upon TCR stimulation, which indicates that HIP-55 is regulated by TCR signaling. HIP-55 interacted with ZAP-70, a critical protein-tyrosine kinase in TCR signaling, and this interaction was induced by TCR signaling. ZAP-70 phosphorylated HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo, and the HIP-55 mutant (Y334F/Y344F) was not tyrosine-phosphorylated in stimulated T cells. To study its function in T cell activation, HIP-55-deficient Jurkat T cells were established using the RNA interference approach. In the HIP-55-deficient cells, TCR (but not UV)-stimulated JNK activation was decreased. Furthermore, the activation of HPK1, a known JNK upstream activator and HIP-55-interacting protein, was also decreased in the HIP-55-deficient cells. Our data reveal the regulation of HIP-55 during TCR signaling, and using a genetic approach, we demonstrate for the first time that HIP-55 plays a functional role in TCR signaling.

Conditional knockout mice reveal an essential role of protein phosphatase 4 in thymocyte development and pre-T-cell receptor signaling∇

ABSTRACT Okadaic acid-sensitive serine/threonine phosphatases have been shown to regulate interleukin-2 transcription and T-cell activation. Okadaic acid inhibits protein phosphatase 4 (PP4), a novel PP2A-related serine/threonine phosphatase, at a 50% inhibitory concentration (IC50) comparable to that for PP2A. This raises the possibility that some cellular functions of PP2A, determined in T cells by using okadaic acid, may in fact be those of PP4. To investigate the in vivo roles of PP4 in T cells, we generated conventional and T-cell-specific PP4 conditional knockout mice. We found that the ablation of PP4 led to the embryonic lethality of mice. PP4 gene deletion in the T-cell lineage resulted in aberrant thymocyte development, including T-cell arrest at the double-negative 3 stage (CD4− CD8− CD25+ CD44−), abnormal thymocyte maturation, and lower efficacy of positive selection. PP4-deficient thymocytes showed decreased proliferation and enhanced apoptosis in vivo. Analysis of pre-T-cell receptor (pre-TCR) signaling further revealed impaired calcium flux and phospholipase C-γ1-extracellular signal-regulated kinase activation in the absence of PP4. Anti-CD3 injection in PP4-deficient mice led to enhanced thymocyte apoptosis, accompanied by increased proapoptotic Bim but decreased antiapoptotic Bcl-xL protein levels. In the periphery, antigen-specific T-cell proliferation and T-cell-mediated immune responses in PP4-deficient mice were dramatically compromised. Thus, our results indicate that PP4 is essential for thymocyte development and pre-TCR signaling.

IL-10-producing intestinal macrophages prevent excessive antibacterial innate immunity by limiting IL-23 synthesis.

Innate immune responses are regulated in the intestine to prevent excessive inflammation. Here we show that a subset of mouse colonic macrophages constitutively produce the anti-inflammatory cytokine IL-10. In mice infected with Citrobacter rodentium, a model for enteropathogenic Escherichia coli infection in humans, these macrophages are required to prevent intestinal pathology. IL-23 is significantly increased in infected mice with a myeloid cell-specific deletion of IL-10, and the addition of IL-10 reduces IL-23 production by intestinal macrophages. Furthermore, blockade of IL-23 leads to reduced mortality in the context of macrophage IL-10 deficiency. Transcriptome and other analyses indicate that IL-10-expressing macrophages receive an autocrine IL-10 signal. Interestingly, only transfer of the IL-10 positive macrophages could rescue IL-10-deficient infected mice. Therefore, these data indicate a pivotal role for intestinal macrophages that constitutively produce IL-10, in controlling excessive innate immune activation and preventing tissue damage after an acute bacterial infection.

Regulation of inflammation, autoimmunity, and infection immunity by HVEM-BTLA signaling

The HVEM, or TNFRSF14, is a membrane‐bound receptor known to activate the NF‐κB pathway, leading to the induction of proinflammatory and cell survival‐promoting genes. HVEM binds several ligands that are capable of mediating costimulatory pathways, predominantly through its interaction with LIGHT (TNFSF14). However, it can also mediate coinhibitory effects, predominantly by interacting with IGSF members, BTLA or CD160. Therefore, it can function like a “molecular switch” for various activating or inhibitory functions. Furthermore, recent studies suggest the existence of bidirectional signaling with HVEM acting as a ligand for signaling through BTLA, which may act as a ligand in other contexts. Bidirectional signaling, together with new information indicating signaling in cis by cells that coexpress HVEM and its ligands, makes signaling within a HVEM‐mediated network complicated, although potentially rich in biology. Accumulating in vivo evidence has shown that HVEM‐mediated, coinhibitory signaling may be dominant over HVEM‐mediated costimulatory signaling. In several disease models the absence of HVEM‐BTLA signaling predominantly resulted in severe mucosal inflammation in the gut and lung, autoimmune‐like disease, and impaired immunity during bacterial infection. Here, we will summarize the current view about how HVEM‐BTLA signaling is involved in the regulation of mucosal inflammation, autoimmunity, and infection immunity.

Regulating the mucosal immune system: the contrasting roles of LIGHT, HVEM, and their various partners.

LIGHT and herpes virus entry mediator (HVEM) comprise a ligand–receptor pair in the tumor necrosis factor superfamily. These molecules play an important role in regulating immunity, particularly in the intestinal mucosa. LIGHT also binds the lymphotoxin β receptor, and HVEM can act as a ligand for immunoglobulin family molecules, including B- and T-lymphocyte attenuator, which suppresses immune responses. Complexity in this pivotal system arises from several factors, including the non-monogamous pairing of ligands and receptors, and reverse signaling or the ability of some ligands to serve as receptors. As a result, recognition events in this fascinating network of interacting molecules can have pro- or anti-inflammatory consequences. Despite complexity, experiments we and others are carrying out are establishing rules for understanding when and in what cell types these molecules contribute to intestinal inflammation.

Genomic structure of the mouse PP4 gene: a developmentally regulated protein phosphatase.

Protein phosphatases play important roles in the control of various cellular processes. Here, we report the cloning and characterization of the murine cDNA and genomic DNA encoding the serine/threonine protein phosphatase 4 (PP4), also called PPX. While the nucleotide sequences of murine and human PP4 are distinct, their amino acid sequences are identical. We have analyzed the protein, cDNA and genomic PP4 sequences to provide insight into the structure, function and potential regulation of PP4. Genomic Southern blots demonstrated the conservation of PP4 across species. Using Northern blotting and in situ hybridization, we have examined the expression of PP4 in murine embryos and adult tissues. In adult tissues, PP4 was expressed at high levels in the testis, kidney, liver, and lung, and at lower levels in virtually all tissues. PP4 was differentially expressed in murine embryos at different developmental stages, suggesting that PP4 is a developmentally regulated protein phosphatase.