T Cell Immune Deficiency in zap70 Mutant Zebrafish

Abstract

j ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP ؉ thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency. Z AP70 [zeta-chain (TCR) associated protein kinase, 70 kDa], is an important mediator of T cell differentiation and function. ZAP70 is a cytoplasmic protein kinase consisting of two aminoterminal Src homology 2 (SH2) domains and a carboxy-terminal kinase domain that plays a key role in regulating the T cell receptor (TCR) phosphorylation cascade. TCRs are activated by interacting with peptide antigens bound to major histocompatibility complexes (MHCs), causing the recruitment of CD4 or CD8 costimulatory receptors. The CD4 and CD8 receptors are bound by intracellular lymphocyte-specific protein kinase (LCK), which, in turn, phosphorylates the TCR immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3. Phosphorylated ITAMs recruit ZAP70 by binding its SH2 domains with high affinity. LCK phosphorylates ITAM-bound ZAP70, releasing it from an autoinhibited conformation and promoting its catalytic activity. Further phosphorylation of the TCR-associated scaffolding proteins by ZAP70 allows for the recruitment of additional signaling molecules and ultimately results in T cell activation, proliferation, and differentiation (1).Human ZAP70 deficiencies result in severe combined immune deficiency (SCID) with specific defects in T cell maturation (2-4). Patients with inactivating mutations in ZAP70 lack mature CD8 ϩ cytotoxic T cells and produce nonfunctional CD4 ϩ helper T cells. ZAP70 null CD4 ϩ T cells exit the thymus, yet they have dysfunctional T cell signaling and cannot mount effective T cell responses. Zap70 mutant mice also have T cell deficiencies, but they exhibit key differences compared with humans (5, 6). Zap70 mutant mice have a more severe block in thymocyte maturation, with T cells arresting at the CD4 ϩ /CD8 ϩ corti...

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