Expression of B7-H1 in Mantle Cell Lymphoma Leads to Inhibition of T Cell Response to Tumor Cells

Abstract 2643

Mantle cell lymphoma (MCL) is a unique subtype of incurable B-cell, non-Hodgkin lymphoma, and its overall survival currently remains only 4–5 years. Management of relapsed or refractory MCL patients is still challenging. Immunotherapy may provide an alternative treatment for patients with MCL. Recent studies demonstrated that PD-1/B7-H1 signaling plays a crucial role in T-cell regulation in various immune responses and is involved in peripheral tolerance, autoimmunity, infection, and antitumor immunity. In the present study, we examined whether B7-H1 plays an important role in immune evasion in MCL. We demonstrated that B7-H1 gene and protein were expressed in most MCL cell lines and primary MCL cells from all patients examined. CD3⁺ T cells were cultured with irradiated MCL cell lines and primary cells, which were pre-incubated with or without anti-B7-H1 monoclonal antibody or control antibody. The presence of anti-B7-H1 blocking antibody, but not control antibody, increased CD3⁺ T cell proliferation. We confirmed the effect of B7-H1 in suppression of T cell proliferation by knockdown of B7-H1 gene expression using B7-H1 specific and non-specific control shRNA lentiviral particles. Upon transfection, the B7-H1-specific shRNA reduced both B7-H1 gene and surface protein expression, while the control shRNA did not. The B7-H1 specific shRNA, but not control shRNA, augmented CD3⁺ T cell proliferation. To address whether B7-H1 contributed to the suppression of host antitumor immunity in MCL, allogeneic CD3⁺ T cells isolated from normal donors were cocultured with irradiated MCL cell line SP53, control shRNA SP53 (SP53-ctl), or B7-H1 targeted shRNA SP53 (SP53-kd), respectively. After 7 days of coculture, CD3⁺ T cells were harvested and restimulated with newly irradiated SP53, SP53-ctl or SP53-kd cells. After at least 3 repeated cycles of in vitro restimulation, three cytotoxic T lymphocyte (CTL) lines were generated, and named CTL-SP53, CTL-SP53-ctl and CTL-SP53-kd. CTL-SP53-kd showed increased killing of target cells as compared with CTL-SP53 (P <.01) or CTL-SP53-ctl (P <.01). We further showed that B7-H1 targeted shRNA MCL cell line (SP53-kd cells) displayed more specific lysis than SP53 (P <.01) or SP53-trl (P <.01). When SP53 cells were pre-incubated with a blocking anti-B7-H1 monoclonal antibody, it also showed more specific lysis as compared to the control antibody pre-treated cells. In these experiments, purified autologous blood B cells and PBMCs were used as target cells to demonstrate whether T cell lines were cytolytic to normal cells. The results showed that the three CTL cell lines did not kill B cells or PBMCs. Intracellular cytokine staining and ELISA assay demonstrated that T-cell lines express IFN-γ, but not IL-4, IL-6, IL-10 or IL-17, and were thus type I T cells. Moreover, T cell lines stimulated by SP53-kd cells express more IFN-γ than SP53 and SP53-ctl. The T cells also expressed CD45RO, CD28, CD44, but not CD45RA, CD27 or CD62L, indicating that they were memory effector cells. In conclusion, B7-H1 expression may be involved in immune evasion mechanism of MCL. Therefore, B7-H1 may be a promising target for immunotherapy in MCL.