Background/Purpose: ZAP70, a cytoplasmic protein tyrosine kinase, is critical for TCR signaling and T cell development. Complete loss of function of ZAP70 in humans causes severe immunodeficiency. Autoimmune disease due to mutant ZAP70 alleles in humans has not been reported until recently. Our previous study has shown that compound heterozygous mutations R192W/R360P in ZAP70 are responsible for a new familial autoimmune syndrome manifested by bullous pemphigoid, colitis, proteinuria and autoantibody to factor VIII in early life. Cell line data showed that the R192W allele leads to reduced binding of mutant ZAP70 to the phosphorylated TCR zeta-chain, thus functions as a null-allele. In contrast, the R360P allele in the catalytic domain causes weak constitutive activation of the TCR pathway that is suppressed by the wild type (WT) but not the R192W allele. Therefore, the R360P allele is likely responsible for the disease, but the mechanism remains unclear. We hypothesize that the R360P allele interferes with ZAP70 autoinhibition, results in an increase in T cell antigen sensitivity, enhanced follicular helper T (Tfh) cell differentiation, and excessive auto-reactive T helper cell activity for B cells, thereby leading to a primarily autoantibody mediated disease.

Methods: Using Zap70 R360P knock-in mice we generated and biochemical and functional assays, we assessed how the R360P mutation affects lymphocyte development, TCR signaling, and Tfh cell differentiation following LCMV infection.

Results: The structural location and biochemical signaling effects of the R360P mutation were consistent with weakening of the autoinhibitory conformation of ZAP70. Mice with a ZAP70 R360P mutation and polyclonal TCR repertoires exhibited relatively normal T cell development but showed evidence of increased signaling, as evidenced in the increase in ZAP70 Y319 phosphorylation and faster calcium mobilization following TCR stimulation. Additionally, the R360P mutation resulted in enhanced follicular helper T cell expansion following LCMV infection. When R360P or WT CD8⁺-OTI T cells were stimulated by antigen presenting cells preloaded with peptides of different agonist potency, we observed marked augmentation of CD69 induction in R360P cells relative to that of WT cells in the responses to weak (Q4H7, G4) agonists, contrasting with only slight to no enhancement in the responses to strong (OVA, Q4R7) agonists. There was also enhanced proliferation of R360P CD8⁺-OTI T cell than WT controls in response to weak and self peptides.

Conclusion: Our findings suggest that the R360P mutation is a weak hypermorphic allele of ZAP70, resulting in enhanced TCR signaling through disruption of ZAP70 autoinhibition, and enhanced Tfh cell expansion. The R360P mutation may allow increased mature T cell sensitivity to weak and self-antigens that would normally be ignored by WT T cells, a mechanism that may contribute to the break of tolerance in human patients with R360P mutation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/disease-mutation-that-weakens-zap70-autoinhibition-enhances-responses-to-weak-and-self-ligands/