F. Ito1,2,3, T. Hoki2, C. A. Eppolito2, A. J. Francois2, K. Odunsi2,4,5, T. Yamauchi2 3State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Center For Immunotherapy,Buffalo, NY, USA 4Roswell Park Cancer Institute,Gynecologic Oncology,Buffalo, NY, USA 5Roswell Park Cancer Institute,Immunology,Buffalo, NY, USA
Introduction: Successful immunotherapeutic treatment of chronic infectious diseases and cancer requires the generation of a strong cellular immune response. Combined CD40 antibody and toll-like receptor (TLR) (CD40/TLR) stimulation has been found to mediate potent cellular immunity in the context of tumor immunology and cancer immunotherapy. However, the mechanisms of enhanced antitumor efficacy by CD40/TLR stimulation remain elusive.
Methods: To investigate phenotype and function of CD40/TLR vaccine-stimulated antigen-specific T cells, we used MC38 colon adenocarcinoma and B16 melanoma models. To evaluate endogenous T cell response, MC38 tumor-bearing C57BL/6 mice were treated with mutated-neoantigen peptide, agonistic CD40 antibody, and poly I:C (TLR3). To assess phenotype and function of adoptively-transferred T cells, we used pmel-1 T cell receptor (TCR) transgenic CD8+ T cells specific for the gp100 melanocyte differentiation antigen expressed on B16 melanoma. In vitro–activated Pmel-1 CD8+ T cells were adoptively transferred into C57BL/6 mice bearing subcutaneous B16 melanomas. Systemic administration of IL-2 and vaccination with the gp100, anti-CD40 antibody, and Imiquimod (TLR7) were used to enhance antitumor immunity of transferred T cells. Tissues including blood, spleen, and tumors were harvested for further analysis. In both tumor models, control mice were treated with no vaccination, CD40 antibody alone or TLR agonist alone.
Results: In both tumor models, mice treated with combined CD40/TLR vaccination had significantly decreased tumor growth and improved survival compared to no vaccination, CD40 antibody alone or TLR agonist alone. Interestingly, vaccination with the cognate antigen and CD40/TLR not only expanded antigen-specific CD8 T cells in both tumor models, but also facilitated them to express the chemokine receptor, CX3CR1. CX3CR1+ CD8+ T cells were found to express higher levels of killer-cell lectin like receptor G1 (KLRG1), TNF-related apoptosis-inducing ligand (TRAIL), perforin, and granzyme, suggesting terminally-differentiated subset. The generation of CX3CR1+ CD8+ T cells was greatly facilitated by CD40 antibody while TLR agonist increases the expansion of total number of antigen-specific CD8 T cells. Importantly, total number and frequency of CX3CR1+ CD8+ T cells were significantly decreased in tumor-bearing CD40 knockout (KO) mice, indicating that host expression of CD40 is required for generation and expansion of CX3CR1+ CD8+ T cells.
Conclusion: Effective vaccination with the cognate antigen and CD40/TLR accompanies generation of tumor-specific terminally-differentiated CX3CR1+ CD8+ T cells dependent on CD40 signaling.