Tyrosine Phosphatase SHP-1 Acts As a Key Regulator Of Alloresponses In Primary Murine T Cells

T cell-mediated alloresponses may result in severe transplantation-related morbidity and mortality such as graft rejection or graft versus host disease. After the adhesive interaction of a T cell and an allogeneic antigen-presenting cell an integrin-mediated, detrimental response against tissues expressing the respective alloantigen is being initiated. So far, the molecular processes underlying signaling events between T cell receptor activation and cellular adhesion are not fully understood. The vast majority of published studies was done with various tumor-derived cell lines, thus producing controversial results depending on the cell line used.

To overcome these drawbacks, we used in vitro generated polyclonal primary T cells based on the priming of naïve T cells from B10.A mice (responder) with denditic cells from C57BL/6 mice (allogeneic stimulator) or from B10.A mice (syngeneic stimulator) to study the lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion.

We identified the protein tyrosine phosphatase SHP-1 as a key regulator of LFA-1-mediated adhesion. Upon alloactivation of primary T cells LFA-1-mediated adhesion was significantly higher when compared to syngeneically stimulated cells. Furthermore, elevated adhesion of alloactivated cells was accompanied by a strong decline of SHP–1 activity. To reinforce these findings, we examined the influence of a siRNA-based knockdown of SHP-1 from syngeneically and allogeneically stimulated T cells. Most notably, transfection of syngeneically stimulated cells with SHP-1-specific siRNA resulted in a two-fold increase of adhesion, when compared to cells transfected with nonsense siRNA under identical conditions. In contrast, transient overexpression of wild–type SHP-1 in allostimulated cells strongly reduced LFA–1-mediated adhesion.

Since our experiments have ascertained SHP–1 as a negative regulator of the adhesion-associated signaling cascade SLP-76 → ADAP → LFA–1, we investigated the tyrosine phosphorylation of ADAP. Western blots revealed strong tyrosine phosphorylation of ADAP in allogeneically stimulated cells, but not in syngeneically stimulated cells. Furthermore, SHP-1 binding with ADAP was significantly stronger in syngeneically stimulated cells, when compared to allogeneically stimulated cells. Besides these experiments, substrate specificity of SHP–1 to ADAP confirmed by in vitro dephosphorylation asssays with GST-SHP–1 and GST-SHP–2 fusion proteins indicate that SHP–1 modulates the binding of SLP–76 with ADAP by tyrosine dephosphorylation of ADAP.

These findings might be of clinical relevance, since the decisive role of SHP–1 in the regulation of LFA–1-mediated adhesion in alloresponses allows the development of novel target-specific immunosuppressive agents.

No relevant conflicts of interest to declare.