Immune checkpoint blockade has revolutionized cancer immunotherapy, and combination treatment with antibodies against multiple inhibitory receptors promises to improve efficacy substantially. However, the spatiotemporal and combinatorial expression profile of various checkpoint receptors on effector T cells and the effect of combined blockade on immune function remain poorly defined. In this study we set out to determine the co-expression kinetics and functional impact of multiple checkpoint receptors (PD-1, LAG-3, TIM-3, and BTLA) on tumor antigen-specific effector T cells in controlled in vitro culture systems and in the tumor environment.
To elicit tumor antigen-specific responses we immunized mice with irradiated MC38 tumor cells engineered to express chicken ovalbumin (OVA; MC38.OVA) and used pentamer reagents to identify OVA257-264-specific CD8 T cells. Repeated stimulation with irradiated MC38.OVA cells or irradiated OVA-pulsed splenocytes ex vivo resulted in robust proliferation and activation of antigen-specific T cells as determined by BrdU incorporation and production of IFN-γ. Most OVA-specific cells expressed PD-1 ex vivo and maintained PD-1 expression during repeated cycles of in vitro stimulation. In contrast, antigen-specific T cells did not express TIM-3 or LAG-3 ex vivo, yet both receptors were induced on the majority of PD1 positive, but not PD-1 negative, cells upon repeated stimulation ex vivo, resulting in double and triple positive cells. The frequency of BTLA expressing cells remained low under all conditions. The addition of PD-1 and TIM-3 blocking antibodies during ex vivo restimulation enhanced T cell activation, demonstrating that these receptors mediate inhibitory function. Consistent with our in vitro data demonstrating that repeated antigen stimulation promotes the coordinated expression of checkpoint receptors, we found that the vast majority of MC38.OVA or Colon26 tumor infiltrating T cells (TILs) express PD-1, and most PD-1 positive cells also co-express TIM-3 and LAG-3, whereas these receptors are largely absent from T cells in secondary lymphoid organs. Importantly, combination treatment of tumor-bearing mice with anti-PD-1 and anti-TIM-3, or anti-PD-1 and anti-LAG-3 antibodies enhanced the immune control of tumor growth in both models as compared to the respective monotherapies. Finally, in vitro activation of human PBMC-derived T cells resulted in a similar sequential upregulation of checkpoint inhibitors ultimately resulting in their co-expression.
Here, we have established a culture system to delineate the hierarchical expression of inhibitor checkpoint receptors on tumor antigen-specific murine T cells during repeated antigen stimulation. Defining the inhibitory checkpoint receptor landscape will guide the process of identifying the most promising combinations of checkpoint blockers in cancer immunotherapy.
Citation Format: Robert J. Durso, Jerry Pei, Michelle Russell, Pratha Budhani, Elena Burova, Chandrika Taduriyasas, Ella Ioffe, Markus Mohrs, Gavin Thurston, Jie Dai. Multiple immune checkpoint receptors are co-expressed on tumor antigen-specific T cells and contribute to tumor immune evasion [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B129.
- ©2016 American Association for Cancer Research.