Chronic Lymphocytic Leukemia Cells Co-Opt CD200, CD270, CD274 and CD276 to Induce Impaired Actin Polarization At the T Cell Immune Synapse

Abstract 802

We have previously demonstrated that impaired formation of the T cell immunological synapse in response to autologous (auto) antigen-presenting cells (APCs) is a global immunosuppressive mechanism in chronic lymphocytic leukemia (CLL) (J Clin Invest. 2008;118(7):2427-2437). Polymerization of F-actin beneath the area of the T cell:APC contact site generates a structural support for signaling molecules to assemble and regulate appropriate CD4⁺ T cell activation and cytolytic CD8⁺ T cell (CTL) effector function. Importantly, direct contact interaction with tumor cells was shown to induce defective actin polarization at the synapse in previously healthy allogeneic (allo) peripheral blood (PB) T cells. Here we have extended our functional screening coculture assays and show that CD200, CD270 (TNF receptor, TNFR-superfamily 14, SF14), CD274 (programmed death ligand 1, PD-L1), and CD276 (B7-H3) are co-opted by primary CLL cells (n=25) to induce impaired actin polymerization at the CD3⁺ T cell synapse. Antibody neutralizaton of these CLL ligands significantly increased allo T cell synapse actin polymerization with APCs compared to isotype control treated cells (P<.01). Counteracting the combined activity of all four inhibitory proteins on CLL cells showed the largest increase in F-actin synapse polymerization. Importantly, we further demonstrate that direct contact coculture with CLL cells further augmented F-actin polymerization defects in auto PB patient T cells (isolated from low white blood cell count CLL patients), that was prevented by the prior blockade of these CLL inhibitory ligands (P<.01). Next we analyzed the in situ expression of inhibitory ligands and receptors by immunohistochemistry using a CLL lymphoid tissue microarray (TMA). Significantly higher expression of CD200⁺ CD270⁺ CD274⁺ CD276⁺ CD20⁺ CLL cells, and CD272⁺ (B and T lymphocyte attenuator, BTLA) CD279⁺ (PD-1) CD3⁺ T cells were detected compared to healthy counterpart cells from reactive control lymph node samples (P<.0001). Notably, higher expression of CD200⁺ CD274⁺ CLL cells correlated with poor disease outcome (P<.01). Flow cytometric analysis of peripheral blood patient cells showed that these inhibitory ligands were up-regulated on circulating CLL cells and also their receptors on auto T cells compared to age-matched healthy donor cells (P<.05). Next we investigated the impact of lenalidomide on CLL immunosuppressive signaling interactions with T cells. Both pretreatment of CLL cells with lenalidomide prior to primary coculture and direct addition of drug significantly increased (P <.01) subsequent allo T cell F-actin synapse polarization compared to control treated experiments. Flow cytometric analysis identified that lenalidomide downregulated the expression of these CLL inhibitory ligands and cognate receptors on allo T cells during intercellular contact interactions (P <.01), but not when age-matched healthy B cells were used. We next investigated the effect on cytolytic synapse function and demonstrated that allo CD8⁺ T cell killing function was significantly enhanced (P <.05) following combinational antibody blockade of CLL inhibitory ligands or lenalidomide treatment compared to control treated leukemic cells. Importantly, lenalidomide treatment blocked further augmented synapse impairment in auto T cells from CLL patients following coculture with CLL tumor cells. As members of the Rho family of GTPases, including RhoA, Rac1 and Cdc42 have been described as key regulators of actin polymerization, we measured their activated GTP-bound state in T cells following direct-contact interaction with CLL tumor cells. We demonstrate decreased active RhoA and Rac1 levels in TCR-stimulated allo T cells on coculture with CLL cells compared with primary coculture with healthy B cells (P <.05). In contrast, combinational antibody blockade of the CLL inhibitory ligands or lenalidomide treatment increased T cell Rho GTPase activity including Cdc42 (P <.05). In conclusion, our findings identify a new mechanism of cancer immunoescape in which CLL tumor cells co-opt multiple inhibitory B7-related molecules that can mediate global immunosuppressive actin defects in both auto and allo T cells.