Bridging T Cell Receptor and Integrin Signaling Pathways with SKAP2 - Functional Insights and Biochemical Characterization

Inflammatory bowel disease (IBD) refers to chronic inflammatory disorders of the gastrointestinal tract resulting from complex interactions between the individual genetic make-up, the immune system, and environmental risk factors (1, 2). The importance of the genetic component is highlighted by the high monozygotic twin concordance rate, familial aggregation, and by the identification of ~200 risk loci in genome-wide association studies (GWAS) (2, 3). We focused our attention on an unusual locus linked to increased risk of Crohn’s disease (CD) (4-6), type I diabetes (6, 7) and multiple sclerosis (8). This locus contains 194 non-coding single-nucleotide polymorphisms (SNPs) in high linkage disequilibrium, with SKAP2 being the sole protein-coding gene in it (9). The minor allele is associated with heightened risk of CD and increased SKAP2 expression specifically in human CD4+ T lymphocytes (4), hinting at a direct link between SKAP2 levels in T cells and CD pathogenesis. Despite such evidence indicating a dose-dependent effect of SKAP2 expression on both T cell responses and disease susceptibility, its specific role in governing T cell functions remains unexplored. SKAP2 is an adapter protein involved in modulating receptor signaling. Our unpublished data established its importance in regulating human T cell cytokine production and adhesion, suggesting a role in T cell receptor signaling and integrin activation. We also found that SKAP2 undergoes caspase-mediated cleavage during T cell activation, and that abolishing this cleavage enhances T cell responses. With this project, we now aim at: 1) elucidating the impact of SKAP2 dosage on T cell responses; 2) dissecting the biochemical mechanisms governing SKAP2 regulation; and 3) establishing animal models with modified SKAP2 expression to investigate its role in disease and its potential as a clinically relevant target. The following objectives will be addressed:

Aim 1. To explore the functional consequences of SKAP2 dosage in human T lymphocytes. The expression quantitative trait loci (eQTL) in SKAP2 link the minor allele with increased levels of SKAP2 expression and CD susceptibility. In this aim, we will determine how SKAP2 dosage impacts T cell responses. We will use CRISPR-activation (CRISPRa) to modulate endogenous SKAP2 expression in primary human CD4+ T cells, followed by RNA-sequencing and functional analyses, overall linking gene dosage to T cell function.

Aim 2. Biochemical characterization of SKAP2-dependent signalling and regulation. We will determine the impact of caspase-mediated SKAP2 cleavage during T cell responses, and we will characterize the interactome of full-length and cleaved SKAP2 fragments in response to triggers of cell activation and adhesion. We will use CRISPR-Cas9-mediated engineering to generate T lymphocytes lacking SKAP2 and then transduce them with lenti-vectors encoding the different versions of this protein. Mass-spectrometry experiments, T cell functional analyses and RNA-sequencing will be performed to determine how the uncleavable SKAP2 affects T cell signalling and functions.

Aim 3. Functional analyses within disease-relevant in vivo models. We found that both SKAP2 expression dosage and its proteolytic cleavage influence T cell responses and adhesion properties. To assess the importance of these processes in disease, we will generate an animal model that expresses an uncleavable version of Skap2. Models of gut inflammation will then be used to interrogate the effect of Skap2 expression and regulation on disease.

Overall, the impact of this project will be twofold: it will advance our knowledge into the regulation of T cell activation and signaling, while establishing the mechanistic basis for the causal link between a genetic locus associated with an increased risk of CD and T lymphocyte inflammatory responses.