Mechanisms for the rapid recall response mediated by memory T cells remain unknown. In this study, we present a novel, multiparameter analysis of TCR-coupled signaling and function in resting and activated naive and memory CD4 T cells, revealing a biochemical basis for immunological recall. We identify a striking elevation in expression of the proximal tyrosine kinase Zap70 in resting Ag-specific and polyclonal mouse memory vs naive CD4 T cells that is stably maintained independent of protein synthesis. Elevated Zap70 protein levels control effector function as IFN-γ production occurs exclusively from the Zap70high fraction of activated T cells in vitro and in vivo, and specific down-modulation of Zap70 expression in memory CD4 T cells by small interfering RNA or protein inhibition significantly reduces rapid IFN-γ production. Downstream of Zap70, we show quantitative differences in distal phosphorylation associated with effector function in naive and memory subsets, with low accumulation of phosphorylation in memory T cells producing IFN-γ at early time points, contrasting extensive phosphorylation associated with IFN-γ production following sustained activation of naive T cells. Our results reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficacious responses through increased Zap70 expression and reduced accumulation of downstream signaling events.

Memory T cells are distinguished from naive T cells by their rapid production of effector cytokines, although mechanisms for this recall response remain undefined. Here, we investigated transcriptional mechanisms for rapid IFN-γ production by antigen-specific memory CD4 T cells. In naive CD4 T cells, IFN-γ production only occurred following sustained antigen activation and was associated with high expression of the T-bet transcription factor required for Th1 differentiation, and T-bet binding to the IFN-γ promoter as assessed by chromatin immunoprecipitation analysis (ChIP). By contrast, immediate IFN-γ production by antigen-stimulated memory CD4 T cells occurred in the absence of significant nuclear T-bet expression or T-bet engagement on the IFN-γ promoter. We identified rapid induction of NFκB transcriptional activity and increased engagement of NFκB on the IFN-γ promoter at rapid times following TCR stimulation of memory compared to naive CD4 T cells. Moreover, pharmacologic inhibition of NFκB activity or peptide-mediated inhibition of NFκB p50 translocation abrogated early memory T cell signaling and TCR-mediated effector function. Our results reveal a molecular mechanism for memory T cell recall through enhanced NFκB p50 activation and promoter engagement, with important implications for memory T cell modulation in vaccines, autoimmunity and transplantation.

The functional coupling of T cell receptor (TCR)-mediated signaling events in primary human T cells remains undefined. We demonstrate here that alterations in the expression of proximal TCR-coupled signaling subunits are associated with distinct effector capacities in differentiated human CD4 T cells. Analysis of proximal signaling profiles using biochemical and single cell approaches reveals decreased CD3zeta and ZAP-70 expression correlating with functional anergy, with increased CD3zeta/ ZAP-70 expression and phosphorylation connoting acquisition of effector capacity. By contrast, the FcRgamma signaling subunit known to be expressed in human effector cells and in T cells from the autoimmune disease SLE is up-regulated upon activation, yet does not correlate with functional capacity in effector cells, and does not alter signaling or function in primary FcRgamma transfectants. Our results have implications for targeting signaling molecules in immunotherapy and evaluating the functional consequence of signaling alterations associated with autoimmunity and chronic diseases.

Mechanisms for the rapid effector responses mediated by memory T cells remain unknown. In this study, we used multiparameter analysis of intracellular signaling and function in naive and memory T cells in vitro and in vivo to determine a biochemical basis for immunological recall. We identify here a striking and specific elevation in expression of the ZAP-70 protein tyrosine kinase in resting memory compared to naive CD4 T cells that is stably maintained independent of protein synthesis, whereas low ZAP-70 expression in naive cells increases with sustained antigenic stimulation requiring new protein synthesis. We demonstrate that ZAP-70 levels control effector function, as specific downregulation of ZAP-70 expression in memory CD4 T cells abrogates rapid effector function, and acquisition of effector function occurs exclusively from activated T cells that have upregulated ZAP-70. Moreover, rapid IFN-γ production from ZAP-70hi memory CD4 T cells occurs via a quantitative bypass in distal signaling in vitro and in vivo, contrasting the increased distal phosphorylation events that precede late IFN-γ production from activated naive CD4 T cells. Our findings reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficient functional coupling through increased ZAP-70 expression and reduced downstream signaling events. This work was supported by NIH AI42092