Regulatory T cells Suppress Zap70 Phosphorylation in Responding T cells

Objective: Regulatory T cells (Treg) have a critical role in controlling responding T cells (Tresp), thereby preventing autoimmune manifestations including those observed in adenosine deaminase (ADA) enzyme-deficient patients and mice (ADA-KO). Treg suppress various functions of Tresp such as proliferation, cytokine production and expression of activation markers, yet it is not known if Treg suppress the early steps of Tresp activation, such as the phosphorylation of the Zeta-associated protein (Zap)70. Methods: Zap70 phosphorylation and CD69 expression in anti-CD3 and anti-CD28 stimulated mice CD4+CD25Tresp were measured by flow cytometry in the presence or absence of CD4+CD25+ Treg activated with anti-CD3 and anti-CD28 antibodies. Suppression of Zap70 phosphorylation in Tresp from normal mice cultured with Treg from ADA-KO mice, either treated with PEG-ADA enzyme replacement or untreated, was similarly measured. Results: Zap70 phosphorylation in activated Tresp was markedly (50 ± 13%) decreased 2 hours after culture with Treg, while 51 ± 8% suppression of CD69 expression was only detected after 7 hours. Suppression of Zap70 phosphorylation in activated Tresp correlated with the ratio of Treg to Tresp. Treg from ADA-KO mice had significantly reduced (p=0.012) ability to suppress Zap70 phosphorylation (16.2 ± 16.7%) compared to Treg from healthy littermates (51.6 ± 23.4%), while PEG-ADA treatment restored Treg suppressive ability (45 ± 10%). Conclusions: Treg suppress Zap70 phosphorylation in Tresp, a finding that might help better understand and assess Treg function.