Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection.

Intraepithelial T cells (IETs) are in close contact with intestinal epithelial cells and the underlying basement membrane, and they detect invasive pathogens. How intestinal epithelial cells and basement membrane influence IET survival and function, at steady state or after infection, is unclear. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is constitutively expressed by intestinal epithelial cells and is important for protection from pathogenic bacteria. Here, we showed that at steady-state LIGHT, an HVEM ligand, binding to epithelial HVEM promoted the survival of small intestine IETs. RNA-seq and addition of HVEM ligands to epithelial organoids indicated that HVEM increased epithelial synthesis of basement membrane proteins, including collagen IV, which bound to ß1 integrins expressed by IETs. Therefore, we proposed that IET survival depended on ß1 integrin binding to collagen IV and showed that ß1 integrin-collagen IV interactions supported IET survival in vitro. Moreover, the absence of ß1 integrin expression by T lymphocytes decreased TCR αß+ IETs in vivo. Intravital microscopy showed that the patrolling movement of IETs was reduced without epithelial HVEM. As likely consequences of decreased number and movement, protective responses to Salmonella enterica were reduced in mice lacking either epithelial HVEM, HVEM ligands, or ß1 integrins. Therefore, IETs, at steady state and after infection, depended on HVEM expressed by epithelial cells for the synthesis of collagen IV by epithelial cells. Collagen IV engaged ß1 integrins on IETs that were important for their maintenance and for their protective function in mucosal immunity.

Patient-Derived Organoid Serves as a Platform for Personalized Chemotherapy in Advanced Colorectal Cancer Patients.

Background: Addition of oxaliplatin to adjuvant 5-FU has significantly improved the disease-free survival and served as the first line adjuvant chemotherapy in advanced colorectal cancer (CRC) patients. However, a fraction of patients remains refractory to oxaliplatin-based treatment. It is urgent to establish a preclinical platform to predict the responsiveness toward oxaliplatin in CRC patients as well as to improve the efficacy in the resistant patients. Methods: A living biobank of organoid lines were established from advanced CRC patients. Oxaliplatin sensitivity was assessed in patient-derived tumor organoids (PDOs) in vitro and in PDO-xenografted tumors in mice. Based on in vitro oxaliplatin IC50 values, PDOs were classified into either oxaliplatin-resistant (OR) or oxaliplatin-sensitive (OS) PDOs. The outcomes of patients undergone oxaliplatin-based treatment was followed. RNA-sequencing and bioinformatics tools were performed for molecular profiling of OR and OS PDOs. Oxaliplatin response signatures were submitted to Connectivity Map algorithm to identify perturbagens that may antagonize oxaliplatin resistance. Results: Oxaliplatin sensitivity in PDOs was shown to correlate to oxaliplatin-mediated inhibition on PDO xenograft tumors in mice, and parallelled clinical outcomes of CRC patients who received FOLFOX treatment. Molecular profiling of transcriptomes revealed oxaliplatin-resistant and -sensitive PDOs as two separate entities, each being characterized with distinct hallmarks and gene sets. Using Leave-One-Out Cross Validation algorithm and Logistic Regression model, 18 gene signatures were identified as predictive biomarkers for oxaliplatin response. Candidate drugs identified by oxaliplatin response signature-based strategies, including inhibitors targeting c-ABL and Notch pathway, DNA/RNA synthesis inhibitors, and HDAC inhibitors, were demonstrated to potently and effectively increase oxaliplatin sensitivity in the resistant PDOs. Conclusions: PDOs are useful in informing decision-making on oxaliplatin-based chemotherapy and in designing personalized chemotherapy in CRC patients.

Btla signaling in conventional and regulatory lymphocytes coordinately tempers humoral immunity in the intestinal mucosa.

The Btla inhibitory receptor limits innate and adaptive immune responses, both preventing the development of autoimmune disease and restraining anti-viral and anti-tumor responses. It remains unclear how the functions of Btla in diverse lymphocytes contribute to immunoregulation. Here, we show that Btla inhibits activation of genes regulating metabolism and cytokine signaling, including Il6 and Hif1a, indicating a regulatory role in humoral immunity. Within mucosal Peyer's patches, we find T-cell-expressed Btla-regulated Tfh cells, while Btla in T or B cells regulates GC B cell numbers. Treg-expressed Btla is required for cell-intrinsic Treg homeostasis that subsequently controls GC B cells. Loss of Btla in lymphocytes results in increased IgA bound to intestinal bacteria, correlating with altered microbial homeostasis and elevations in commensal and pathogenic bacteria. Together our studies provide important insights into how Btla functions as a checkpoint in diverse conventional and regulatory lymphocyte subsets to influence systemic immune responses.

IL-22 initiates an IL-18-dependent epithelial response circuit to enforce intestinal host defence.

IL-18 is emerging as an IL-22-induced and epithelium-derived cytokine which contributes to host defence against intestinal infection and inflammation. In contrast to its known role in Goblet cells, regulation of barrier function at the molecular level by IL-18 is much less explored. Here we show that IL-18 is a bona fide IL-22-regulated gate keeper for intestinal epithelial barrier. IL-22 promotes crypt immunity both via induction of phospho-Stat3 binding to the Il-18 gene promoter and via Il-18 independent mechanisms. In organoid culture, while IL-22 primarily increases organoid size and inhibits expression of stem cell genes, IL-18 preferentially promotes organoid budding and induces signature genes of Lgr5+ stem cells via Akt-Tcf4 signalling. During adherent-invasive E. coli (AIEC) infection, systemic administration of IL-18 corrects compromised T-cell IFNγ production and restores Lysozyme+ Paneth cells in Il-22-/- mice, but IL-22 administration fails to restore these parameters in Il-18-/- mice, thereby placing IL-22-Stat3 signalling upstream of the IL-18-mediated barrier defence function. IL-18 in return regulates Stat3-mediated anti-microbial response in Paneth cells, Akt-Tcf4-triggered expansion of Lgr5+ stem cells to facilitate tissue repair, and AIEC clearance by promoting IFNγ+ T cells.

The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis.

The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.

ATF3 Sustains IL-22-Induced STAT3 Phosphorylation to Maintain Mucosal Immunity Through Inhibiting Phosphatases.

In gut epithelium, IL-22 transmits signals through STAT3 phosphorylation (pSTAT3) which provides intestinal immunity. Many components in the IL-22-pSTAT3 pathway have been identified as risk factors for inflammatory bowel disease (IBD) and some of them are considered as promising therapeutic targets. However, new perspectives are still needed to understand IL-22-pSTAT3 signaling for effective clinical interventions in IBD patients. Here, we revealed activating transcription factor 3 (ATF3), recently identified to be upregulated in patients with active IBD, as a crucial player in the epithelial IL-22-pSTAT3 signaling cascade. We found ATF3 is central to intestinal homeostasis and provides protection during colitis. Loss of ATF3 led to decreased crypt numbers, more shortened colon length, impaired ileal fucosylation at the steady state, and lethal disease activity during DSS-induced colitis which can be effectively ameliorated by rectal transplantation of wild-type colonic organoids. Epithelial stem cells and Paneth cells form a niche to orchestrate epithelial regeneration and host-microbe interactions, and IL-22-pSTAT3 signaling is a key guardian for this niche. We found ATF3 is critical for niche maintenance as ATF3 deficiency caused compromised stem cell growth and regeneration, as well as Paneth cell degeneration and loss of anti-microbial peptide (AMP)-producing granules, indicative of malfunction of Paneth/stem cell network. Mechanistically, we found IL-22 upregulates ATF3, which is required to relay IL-22 signaling leading to STAT3 phosphorylation and subsequent AMP induction. Intriguingly, ATF3 itself does not act on STAT3 directly, instead ATF3 regulates pSTAT3 by negatively targeting protein tyrosine phosphatases (PTPs) including SHP2 and PTP-Meg2. Furthermore, we identified ATF3 is also involved in IL-6-mediated STAT3 activation in T cells and loss of ATF3 leads to reduced capacity of Th17 cells to produce their signature cytokine IL-22 and IL-17A. Collectively, our results suggest that via IL-22-pSTAT3 signaling in the epithelium and IL-6-pSTAT3 signaling in Th17 cells, ATF3 mediates a cross-regulation in the barrier to maintain mucosal homeostasis and immunity.

LIGHT-HVEM Signaling in Innate Lymphoid Cell Subsets Protects Against Enteric Bacterial Infection.

Innate lymphoid cells (ILCs) are important regulators of early infection at mucosal barriers. ILCs are divided into three groups based on expression profiles, and are activated by cytokines and neuropeptides. Yet, it remains unknown if ILCs integrate other signals in providing protection. We show that signaling through herpes virus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor superfamily, in ILC3 is important for host defense against oral infection with the bacterial pathogen Yersinia enterocolitica. HVEM stimulates protective interferon-γ (IFN-γ) secretion from ILCs, and mice with HVEM-deficient ILC3 exhibit reduced IFN-γ production, higher bacterial burdens and increased mortality. In addition, IFN-γ production is critical as adoptive transfer of wild-type but not IFN-γ-deficient ILC3 can restore protection to mice lacking ILCs. We identify the TNF superfamily member, LIGHT, as the ligand inducing HVEM signals in ILCs. Thus HVEM signaling mediated by LIGHT plays a critical role in regulating ILC3-derived IFN-γ production for protection following infection. VIDEO ABSTRACT.

LIGHT-HVEM signaling in keratinocytes controls development of dermatitis.

Dermatitis is often associated with an allergic reaction characterized by excessive type 2 responses leading to epidermal acanthosis, hyperkeratosis, and dermal inflammation. Although factors like IL-4, IL-13, and thymic stromal lymphopoietin (TSLP) are thought to be instrumental for the development of this type of skin disorder, other cytokines may be critical. Here, we show that the tumor necrosis factor (TNF) superfamily protein LIGHT (homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes) is required for experimental atopic dermatitis, and LIGHT directly controls keratinocyte hyperplasia, and production of periostin, a matricellular protein that contributes to the clinical features of atopic dermatitis as well as other skin diseases such as scleroderma. Mice with a conditional deletion of the LIGHT receptor HVEM (herpesvirus entry mediator) in keratinocytes phenocopied LIGHT-deficient mice in exhibiting reduced epidermal thickening and dermal collagen deposition in a model of atopic dermatitis driven by house dust mite allergen. LIGHT signaling through HVEM in human epidermal keratinocytes directly induced proliferation and periostin expression, and both keratinocyte-specific deletion of HVEM or antibody blocking of LIGHT-HVEM interactions after disease onset prevented expression of periostin and limited atopic dermatitis symptoms. Developing reagents that neutralize LIGHT-HVEM signaling might be useful for therapeutic intervention in skin diseases where periostin is a central feature.

Murine Corneal Inflammation and Nerve Damage After Infection With HSV-1 Are Promoted by HVEM and Ameliorated by Immune-Modifying Nanoparticle Therapy.

Purpose: To determine cellular and temporal expression patterns of herpes virus entry mediator (HVEM, Tnfrsf14) in the murine cornea during the course of herpes simplex virus 1 (HSV-1) infection, the impact of this expression on pathogenesis, and whether alterations in HVEM or downstream HVEM-mediated effects ameliorate corneal disease. Methods: Corneal HVEM levels were assessed in C57BL/6 mice after infection with HSV-1(17). Leukocytic infiltrates and corneal sensitivity loss were measured in the presence, global absence (HVEM knockout [KO] mice; Tnfrsf14-/-), or partial absence of HVEM (HVEM conditional KO). Effects of immune-modifying nanoparticles (IMPs) on viral replication, corneal sensitivity, and corneal infiltrates were measured. Results: Corneal HVEM+ populations, particularly monocytes/macrophages during acute infection (3 days post infection [dpi]) and polymorphonuclear neutrophils (PMN) during the chronic inflammatory phase (14 dpi), increased after HSV-1 infection. Herpes virus entry mediator increased leukocytes in the cornea and corneal sensitivity loss. Ablation of HVEM from CD45+ cells, or intravenous IMP therapy, reduced infiltrates in the chronic phase and maintained corneal sensitivity. Conclusions: Herpes virus entry mediator was expressed on two key populations: corneal monocytes/macrophages and PMNs. Herpes virus entry mediator promoted the recruitment of myeloid cells to the cornea in the chronic phase. Herpes virus entry mediator-associated corneal sensitivity loss preceded leukocytic infiltration, suggesting it may play an active role in recruitment. We propose that HVEM on resident corneal macrophages increases nerve damage and immune cell invasion, and we showed that prevention of late-phase infiltration of PMN and CD4+ T cells by IMP therapy improved clinical symptoms and mortality and reduced corneal sensitivity loss caused by HSV-1.

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.

HVEM is a TNF Receptor with Multiple Regulatory Roles in the Mucosal Immune System.

The herpes virus entry mediator (HVEM) is a member of the tumor necrosis factor receptor superfamily (TNFRSF), and therefore it is also known as TNFRSF14 or CD270 (1,2). In recent years, we have focused on understanding HVEM function in the mucosa of the intestine, particularly on the role of HVEM in colitis pathogenesis, host defense and regulation of the microbiota (2,3,4). HVEM is an unusual TNF receptor because of its high expression levels in the gut epithelium, its capacity to bind ligands that are not members of the TNF super family, including immunoglobulin (Ig) superfamily members BTLA and CD160, and its bi-directional functionality, acting as a signaling receptor or as a ligand for the receptor BTLA. Clinically, Hvem recently was reported as an inflammatory bowel disease (IBD) risk gene as a result of genome wide association studies (5,6). This suggests HVEM could have a regulatory role influencing the regulation of epithelial barrier, host defense and the microbiota. Consistent with this, using mouse models, we have revealed how HVEM is involved in colitis pathogenesis, mucosal host defense and epithelial immunity (3,7). Although further studies are needed, our results provide the fundamental basis for understanding why Hvem is an IBD risk gene, and they confirm that HVEM is a mucosal gatekeeper with multiple regulatory functions in the mucosa.

HVEM: An unusual TNF receptor family member important for mucosal innate immune responses to microbes.

The herpes virus entry mediator (HVEM or CD270) is a member of the tumor necrosis factor receptor superfamily (TNFRSF), and therefore it is also known as TNFRSF14. We have recently provided evidence showing a novel signaling pathway downstream of HVEM leading to signal transducer and activator of transcription 3 (STAT3) activation in epithelial cells. As STAT3 regulates the expression of genes important for host defense in epithelial cells, as well as the differentiation of retinoid-related orphan receptor (ROR)γt+ Th17 and innate lymphoid cells (ILC), our finding that HVEM activates STAT3 has revealed fresh insights into the potential regulatory function of HVEM in different cellular contexts. Therefore, although further investigations will be required, HVEM is emerging as a major player in mucosal host defense, capable of regulating several cellular 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.

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 beta 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.

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

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 (IC(50)) 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-gamma1-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.

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-gamma1 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 14-3-3tau (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 14-3-3tau. Our results indicate that HPK1 negatively regulates TCR signaling and T cell-mediated immune responses.

Germline transmission and efficient DNA recombination in mouse embryonic stem cells mediated by adenoviral-Cre transduction.

Following gene targeting, a loxP-neo-loxP cassette was introduced into ES cells. The presence of a selectable marker such as neo in the targeted allele may result in gene interference in flox mice or unexpected phenotypes due to genetic ambiguity in direct knockout mice. Typically, the neo cassette is selectively removed by transient expression of the Cre recombinase in targeted ES cell. However, this method involves a tedious process of selecting, expanding, and screening ES cell clones which may compromise germline competency. Here, we describe a novel method of combining adenovirus-Cre mediated gene recombination with ES gene targeting to facilitate efficient loxP-neo-loxP removal in ES cells. We demonstrate that adenovirus-Cre infected ES cells can retain their germline competency. The procedures described here facilitate a rapid genetic manipulation of ES cells to obtain neo-free knockout animals, multiple gene targeting, homozygous mutant ES cells ideal for in vitro characterization, or Rag-deficient blastocyst complementation.

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.