Polarity of CD4+ T cells towards the antigen presenting cell is regulated by the Lck adapter TSAd

Abstract

Polarization of T cells towards the antigen presenting cell (APC) is critically important for appropriate activation and differentiation of the naïve T cell. Here we used imaging flow cytometry (IFC) and show that the activation induced Lck and Itk adapter T cell specific adapter protein (TSAd), encoded by SH2D2A, modulates polarization of T cells towards the APC. Upon exposure to APC presenting the cognate antigen Id, Sh2d2a−/− CD4+ T cells expressing Id-specific transgenic T cell receptor (TCR), displayed impaired polarization of F-actin and TCR to the immunological synapse (IS). Sh2d2a−/− T-cells that did polarize F-actin and TCR still displayed impaired polarization of PKCξ, PAR3 and the microtubule-organizing center (MTOC). In vitro differentiation of activated Sh2d2a−/− T cells was skewed towards an effector memory (Tem) rather than a central memory (Tcm) phenotype. A similar trend was observed for Id-specific TCR Sh2d2a−/− T cells stimulated with APC and cognate antigen. Taken together our data suggest that TSAd modulates differentiation of experienced T cells possibly through polarization of CD4+ T cells towards the APC.

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

“…In some experiments requiring IS formation, Raji cells attached to ibidi microwell culture dishes using fibronectin (ibiTreat, for paraformaldehyde fixing) or poly-L-lysine (glass bottom, for acetone-fixation) were labelled with CMAC and pulsed with 1 µg/ ml SEE, mixed with transfected Jurkat clones and ISs analysed as described [15,31,32]. Acetone fixation required for γ-tubulin staining of MTOC was compatible with phalloidin labelling [33]. In other experiments, transfected Jurkat clones were stimulated with plastic-bound anti-TCR UCHT1 (10 µg/ml) or in suspension with phorbol myristate acetate (PMA, 100 ng/ ml).…”

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

“…Acetone fixation required for γ-tubulin staining of MTOC was compatible with phalloidin labeling [33]. In other experiments, transfected Jurkat clones were stimulated with plastic-bound anti-TCR UCHT1 (10 µg/ml) or in suspension with phorbol myristate acetate (PMA, 100 ng/ml).…”

Adaptor proteins: Flexible and dynamic modulators of immune cell signalling

“…Upon viral challenge, TSAd‐deficient mice displayed reduced clearance of murine cytomegalovirus in the spleen 181 . Murine SH2D2A −/− CD4 + T cells exhibited impaired polarization in the immunological synapse of multiple molecules involved in TCR signalling 190 . Moreover, TSAd KO mice displayed accelerated rejection of heart transplants in an MHC class II‐mismatched model, and resistance to graft‐prolonging therapy of costimulatory blockade in the fully mismatched model 191 .…”