Impacto fenotípico y funcional de quimeras de cd3gamma y cd3 delta en el complejo tcr/cd3 humano deficiente de cd3gamma

Resumen

The CD3 subunits of the antigen-specific T-cell receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3g have a potentially lethal immunodeficiency, reduced surface TCR expression levels and abolished ph orbol ester (PMA)-induced, but not anti-CD3-induced, TCR downregulation. We are investigating delivery of CD3 chains to treat this type of immunodeficiency and to understand TCR dynamics in vivo. The response to PMA has been clearly mapped to the int racellular (IC) domain of CD3?. However, the molecular cause of the reduced TCR surface expression in g- lymphocytes is still not known. We developed retroviral vectors carrying normal CD3? or CD3d or the following chimeras with different domains of both chains (EC-extracellular, TM-transmembrane and IC):d ECgTMgIC (dgg for short), ggd,gdd and gg-. ggg is thus full CD3?, and --- is the empty vector. The constructs were initially delivered into a gamma- T cell line called JGN (for Jurkat Gamma Ne gative), which lacks surface TCR. The results confirmed that expression of the TCR in JGN was dependent on the EC domain of CD3g since ggg, ggd, gdd and gg-, but not ddd, dgg or ---, scored positive for this assay and could be analysed further. Among the former four constructs, gg- induced higher TCR expression than any other, indicating that IC CD3g is required for normal TCR dynamics in this cell line. Signalling assays in stable clones demonstrated that all of them were able to transduced sig nals to the intracellular side. Paradoxically ggd, gdd and gg- did it slightly better than ggg, it could be due to an inhibitory effect of the CD3g tail, for example via the di-LL motif. All four were otherwise functionally normal for anti-CD3-induce d downregulation and conformational change. In contrast, only ggg was down-regulated by PMA, as expected. In g- peripheral blood T lymphocytes and their immortalized counterparts, which have reduced rather than absent surface TCR, the tested chimeras (including dgg, which could not be analyzed in JGN because it did not restore surface TCR) confirmed that the response to PMA maps to the IC domain of CD3g. However, ???, ???, and ???, and unexpectedly also dgg, but not gg- (or ---), normalized surf ace TCR levels in both primary and immortalized g- T lymphocytes. Since protein homology explains these results better than domain structure, we conclude that CD3g contributes conformational cues which improve surface TCR expression, likely at the as...