VAV1 as a putative therapeutic target in autoimmune and chronic inflammatory diseases.

The guanine nucleotide exchange factor (GEF) VAV1, a previously 'undruggable' protein integral to T/B lymphocyte antigen-receptor signaling, promotes actin polymerization, immunological synapse formation, T cell activation and differentiation, and cytokine production. With the development of novel modalities for targeting proteins, we hypothesize that interventions targeting VAV1 will have therapeutic potential in T and T/B cell-mediated autoimmune and chronic inflammatory diseases. This opinion is supported by recent CRISPR-Cas9 studies showing VAV1 as a key positive regulator of T cell receptor (TCR) activation and cytokine production in primary human CD4 and CD8 T cells; data demonstrating that loss/suppression of VAV1 regulates autoimmunity and inflammation; and promising preclinical data from T and T/B cell-mediated disease models of arthritis and colitis showing the effectiveness of selective VAV1 targeting via protein degradation.

Immune Landscape of Epstein-Barr Virus-Associated Gastric Cancer: Analysis From a Western Academic Institution.

Introduction: Epstein-Barr virus-associated gastric cancer (EBVaGC) may be a meaningful biomarker for potential benefit from immunotherapy. Further investigation is needed to characterize the immune landscape of EBVaGC. We assessed our institutional frequency of surgically treated EBVaGC and analyzed the immunologic biomarker profile and tumor-infiltrating lymphocyte (TIL) phenotypes of a series of EBVaGC compared to non-EBVaGC cases.

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry.

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry.

Interplay between IL-10, IFN-γ, IL-17A and PD-1 Expressing EBNA1-Specific CD4 and CD8 T Cell Responses in the Etiologic Pathway to Endemic Burkitt Lymphoma.

Children diagnosed with endemic Burkitt lymphoma (eBL) are deficient in interferon-γ (IFN-γ) responses to Epstein-Barr Nuclear Antigen1 (EBNA1), the viral protein that defines the latency I pattern in this B cell tumor. However, the contributions of immune-regulatory cytokines and phenotypes of the EBNA1-specific T cells have not been characterized for eBL. Using a bespoke flow cytometry assay we measured intracellular IFN-γ, IL-10, IL-17A expression and phenotyped CD4 and CD8 T cell effector memory subsets specific to EBNA1 for eBL patients compared to two groups of healthy children with divergent malaria exposures.

Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK.

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2-inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable.

Optimized Detection of Acute MHV68 Infection With a Reporter System Identifies Large Peritoneal Macrophages as a Dominant Target of Primary Infection.

Investigating the dynamics of virus-host interactions remains an important challenge, often limited by the ability to directly identify virally infected cells. Here, we utilize a beta-lactamase activated fluorescent substrate to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response.

Activation of the Tec Kinase ITK Controls Graded IRF4 Expression in Response to Variations in TCR Signal Strength.

TCR signal strength is critical for CD8 T cell clonal expansion after Ag stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through the induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood.

CD8 T Cells Require ITK-Mediated TCR Signaling for Migration to the Intestine.

The Tec kinase IL-2-inducible T cell kinase (ITK) regulates the expression of TCR-induced genes. T cell responses are impaired but not absent. ITK inhibition prevented colitis disease progression and impaired T cell migration to the colon in mice.

TCR signaling: it's all about the numbers.

Quantitative mass spectrometry applied to T cell activation reveal key insights into signal transduction pathways. These data identify selective alterations in activated T cell protein concentrations, and detail previously undescribed protein-protein interactions.

The Tec kinase ITK is essential for ILC2 survival and epithelial integrity in the intestine.

Innate lymphoid cells (ILC) are lymphocytes that lack an antigen-specific receptor and are preferentially localized in non-lymphoid tissues, such as mucosal barriers. In these locations ILC respond to tissue perturbations by producing factors that promote tissue repair and improve barrier integrity. We show that mice lacking the Tec kinase ITK have impaired intestinal tissue integrity, and a reduced ability to restore homeostasis after tissue damage.

The Transcription Factor Runx2 Is Required for Long-Term Persistence of Antiviral CD8 Memory T Cells.

During acute lymphocytic choriomeningitis virus infection, pathogen-specific CD8 cytotoxic T lymphocytes undergo clonal expansion leading to viral clearance. Following this, the majority of pathogen-specific CD8 T cells undergo apoptosis, leaving a small number of memory CD8 T cells that persist long-term and provide rapid protection upon secondary infection. Whereas much is known about the cytokines and transcription factors that regulate the early effector phase of the antiviral CD8 T cell response, the factors regulating memory T cell homeostasis and survival are not well understood.

Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8 T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei.

Circulating naive T cells exist in a quiescent state. After TCR contact with the cognate peptide presented by APCs in secondary lymphoid structures, T cells undergo a period of rapid transcriptional changes that set the stage for fate-determining effector or memory programming. We describe a novel method to analyze TCR signaling pathway activation in nuclei isolated from primary mouse naive T cells after stimulation with natural peptide Ags.

Multidomain Control Over TEC Kinase Activation State Tunes the T Cell Response.

Signaling through the T cell antigen receptor (TCR) activates a series of tyrosine kinases. Directly associated with the TCR, the SRC family kinase LCK and the SYK family kinase ZAP-70 are essential for all downstream responses to TCR stimulation. In contrast, the TEC family kinase ITK is not an obligate component of the TCR cascade.

Transient expression of ZBTB32 in anti-viral CD8+ T cells limits the magnitude of the effector response and the generation of memory.

Virus infections induce CD8+ T cell responses comprised of a large population of terminal effector cells and a smaller subset of long-lived memory cells. The transcription factors regulating the relative expansion versus the long-term survival potential of anti-viral CD8+ T cells are not completely understood. We identified ZBTB32 as a transcription factor that is transiently expressed in effector CD8+ T cells.

High pathogen burden in childhood promotes the development of unconventional innate-like CD8+ T cells.

Cellular and humoral constituents of the immune system differ significantly between children and adults, yet very little is known about the impact of early-life pathogen exposure on this immunologic transition. We examined CD4+ and CD8+ T cell subsets defined by CCR7 and CD45RA expression in two longitudinal pediatric cohorts experiencing divergent levels of pathogen burden. Using multiparameter flow cytometry, along with serological, cytokine, and transcriptomic data, we show that cumulative pathogen burden promotes the development of atypical CD8dim T cells with an innate-like profile (Granzyme Bhi, IFNγlow, TNFαlow, PLFZhi, ID2hi, IKZF2hi) in contrast to age-matched children residing in a low pathogen-exposure area who display a more conventional CD8bright profile (IFNγ+, TNFα+, CCL4+).

NKG2C/E Marks the Unique Cytotoxic CD4 T Cell Subset, ThCTL, Generated by Influenza Infection.

CD4 T cells can differentiate into multiple effector subsets, including ThCTL that mediate MHC class II-restricted cytotoxicity. Although CD4 T cell-mediated cytotoxicity has been reported in multiple viral infections, their characteristics and the factors regulating their generation are unclear, in part due to a lack of a signature marker. We show in this article that, in mice, NKG2C/E identifies the ThCTL that develop in the lung during influenza A virus infection.

Type 1 interferon licenses naïve CD8 T cells to mediate anti-viral cytotoxicity.

Naïve T cells, unlike memory T cells, exhibit very limited effector function in response to cognate antigen, but exposure to type 1 interferon (IFN) prior to cognate antigen allows for rapid manifestation of effector functions. A full assessment of the functions of these IFN-sensitized otherwise naïve T cells has not been made, nor has their capacity to be effector cells in vivo. We describe here that IFN-sensitized naïve T cells in the absence of cognate antigen adopt a partial activated phenotype distinguished by the upregulation of the surface activation marker CD69, effector-associated transcription factors Eomes and IRF4, and cytotoxicity effector molecule granzyme B.

T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells.

Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength.

IRF4 Regulates the Ratio of T-Bet to Eomesodermin in CD8+ T Cells Responding to Persistent LCMV Infection.

CD8+ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation.

A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression.

In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4(+) T cells from Itk(-/-) and Itk(-/-)Rlk(-/-) mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice.

TCF1 Is Required for the T Follicular Helper Cell Response to Viral Infection.

T follicular helper (TFH) and T helper 1 (Th1) cells generated after viral infections are critical for the control of infection and the development of immunological memory. However, the mechanisms that govern the differentiation and maintenance of these two distinct lineages during viral infection remain unclear. We found that viral-specific TFH and Th1 cells showed reciprocal expression of the transcriptions factors TCF1 and Blimp1 early after infection, even before the differential expression of the canonical TFH marker CXCR5.

Suppression of systemic autoimmunity by the innate immune adaptor STING.

Cytosolic DNA-sensing pathways that signal via Stimulator of interferon genes (STING) mediate immunity to pathogens and also promote autoimmune pathology in DNaseII- and DNaseIII-deficient mice. In contrast, we report here that STING potently suppresses inflammation in a model of systemic lupus erythematosus (SLE). Lymphoid hypertrophy, autoantibody production, serum cytokine levels, and other indicators of immune activation were markedly increased in STING-deficient autoimmune-prone mice compared with STING-sufficient littermates.

Jarid2 is induced by TCR signalling and controls iNKT cell maturation.

Jarid2 is a reported component of three lysine methyltransferase complexes, polycomb repressive complex 2 (PRC2) that methylates histone 3 lysine 27 (H3K27), and GLP-G9a and SETDB1 complexes that methylate H3K9. Here we show that Jarid2 is upregulated upon TCR stimulation and during positive selection in the thymus. Mice lacking Jarid2 in T cells display an increase in the frequency of IL-4-producing promyelocytic leukemia zinc finger (PLZF)(hi) immature invariant natural killer T (iNKT) cells and innate-like CD8(+) cells; Itk-deficient mice, which have a similar increase of innate-like CD8(+) cells, show blunted upregulation of Jarid2 during positive selection.