Impaired Actin Polymerization Results in Defective Immunological Synapse Formation in T Cells in Chronic Lymphocytic Leukemia.

Cancer is associated with immune deficiency, but the molecular basis for this is poorly defined. We have previously demonstrated that multiple gene expression abnormalities are induced in patients with chronic lymphocytic leukemia (CLL) including defects within the actin cytoskeleton formation pathways. Based on this data, we hypothesized that failure of actin polymerization would result in defects in the formation of the immunological synapse (IS) which is critical for T cell activation and effector function. To assess this, actin polymerization at the IS in T cells in response to superantigen-pulsed B cells (APCs) was visualized using confocal microscopy. We observed significantly reduced ability to polymerize actin at the IS (> 50% reduction) in autologous CD4 and CD8 T cells from previously untreated CLL patients compared to age-matched healthy donors (p<0.05). Since reduced IS formation could result from defects in T cells, APCs or both, we examined IS formation in mixing experiments using T cells or APCs from leukemic patients with healthy allogeneic cells. These experiments demonstrated impaired IS formation using T cells from patients with CLL (p<0.01) or CLL cells as APCs (p<0.01), in keeping with defects in both T cells and APC function of CLL cells. We further postulated that interaction of CLL cells with healthy T cells would induce similar changes. Healthy allogeneic T cells were co-cultured for 48 hours with either allogeneic CLL cells or healthy B cells. Co-culture with CLL cells resulted in subsequent significant impairment in IS formation of the T cells with healthy superantigen pulsed APCs (p<0.01). Blocking experiments using anti-LFA-1 and anti-ICAM1 monoclonal antibodies with CLL B cells prevented subsequent actin remodelling impairment at the IS in the healthy allogeneic donor T cells. Further evidence that direct cell contact with CLL cells and not soluble factors is required to induce this T cell immune defect was provided by the finding that there was no impairment on IS formation when the T cells were co-cultured with CLL cells in transwell culture assays. The finding that direct contact of CLL cells with allogeneic T cells induces impairment in IS formation is relevant for the use of donor lymphocyte infusions in the setting of bulk disease. Co-localization experiments assessed by confocal microscopy suggest that the molecular basis for the defective T cells function stems from inability in T cells from CLL patients to recruit key proteins to the IS efficiently compared to healthy donor T cells. Greater than 50% reduction in co-localization at the IS was seen for dynamin 2, filamin A and LFA-1 integrin (p<0.05). These assays provide a rapid and simple method to assess T cell impairment in cancer and can be used to determine if steps to attempt to improve defective T cell function in cancer are successful. The finding of impaired IS formation as a key T cell defect in these cancer bearing patients has implications for both autologous and allogeneic immunotherapy approaches and identify both IS formation and the molecules regulating its organisation as potential functional markers and targets for the reversal of immune deficiency in cancer.