GADS is required for TCR-mediated calcium influx and cytokine release, but not cellular adhesion, in human T cells

Abstract

GRB2 related adaptor protein downstream of Shc (GADS) is a member of the GRB2 family of adaptors and is critical for TCR-induced signaling. The current model is that GADS recruits SLP-76 to the LAT complex, which facilitates the phosphorylation of SLP-76, the activation of PLC-γ1, T cell adhesion and cytokine production. However, this model is largely based on studies of disruption of the GADS/SLP-76 interaction and murine T cell differentiation in GADS deficient mice. The role of GADS in mediating TCR-induced signals in human CD4+ T cells has not been thoroughly investigated. In this study, we have suppressed the expression of GADS in human CD4+ HuT78 T cells. GADS deficient HuT78 T cells displayed similar levels of TCR-induced SLP-76 and PLC-γ1 phosphorylation but exhibited substantial decrease in TCR-induced IL-2 and IFN-γ release. The defect in cytokine production occurred because of impaired calcium mobilization due to reduced recruitment of SLP-76 and PLC-γ1 to the LAT complex. Surprisingly, both GADS deficient HuT78 and GADS deficient primary murine CD8+ T cells had similar TCR-induced adhesion when compared to control T cells. Overall, our results show that GADS is required for calcium influx and cytokine production, but not cellular adhesion, in human CD4+ T cells, suggesting that the current model for T cell regulation by GADS is incomplete.

Glycerol monolaurate induces filopodia formation by disrupting the association between LAT and SLP-76 microclusters

“…Inhibition of actin polymerization by Latrunculin A substantially inhibits LAT clustering, but not SLP-76 clustering (53). Additionally, T cells deficient in Gads, an adaptor protein that canonically links SLP-76 to LAT, exhibit suppressed PLC-γ1-mediated responses because of the recruitment of PLC-γ1 to LAT microclusters (56), but no defect in SLP-76 phosphorylation or downstream actin polymerization (56). Finally, whereas both TCR stimulation and integrin activation lead to the formation of SLP-76 microclusters, only TCR stimulation results in the formation of LAT microclusters (57).…”

Optimization of methods for the genetic modification of human T cells

“…Therefore, caution must be taken if calcium assays and/or activation markers are to be analyzed shortly after CD4 + T‐cell electroporation. Owing to the above reasons, we have focused our studies upon retro/lentiviral mediated DNA delivery 15 , 16 …”

“…Owing to the above reasons, we have focused our studies upon retro/lentiviral mediated DNA delivery. 15,16 Transduction of antigen-inexperienced primary human CD4 + T cells Lentiviral gene delivery to primary T cells in the absence of TCR activation is more challenging compared with primary T cells activated through TCR and CD28 pathways. The main reason for this appears to be the reduced proliferative and metabolic status in cells that were not activated in vitro.…”

“…Owing to their efficiency, low toxicity, simplicity of production and relative safety, we have utilized retroviral transduction for the genomic modification of human CD4 + T cells. These methods have been successfully applied in recent studies utilizing human T‐cell lines as well as primary human CD4 + T cells 15 , 16 . In this study, we describe in detail the optimized methods for the transduction of human primary CD4 + T cells and human T‐cell lines.…”

“…These methods have been successfully applied in recent studies utilizing human T-cell lines as well as primary human CD4 + T cells. 15,16 In this study, we describe in detail the optimized methods for the transduction of human primary CD4 + T cells and human T-cell lines. Specifically, this manuscript includes specific details for producing retroviral particles carrying interference RNAs and reconstitution of mutated proteins.…”