T. Yamauchi1, T. Hoki1, C. A. Eppolito1, A. Francois1, K. Odunsi1,2,3, F. Ito1,4,5 1Roswell Park Cancer Institute,Center For Immunotherapy,Buffalo, NY, USA 2Roswell Park Cancer Institute,Gynecologic Oncology,Buffalo, NY, USA 3Roswell Park Cancer Institute,Immunology,Buffalo, NY, USA 4State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 5Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA
Introduction: Cancer immunotherapies that target the T-cell immune checkpoints, such as programmed cell death-1 (PD-1) and its ligand (PD-L1) have shown unprecedented success for the treatment of a variety of malignancies including melanoma. Although a significant number of cancer patients benefit from immune checkpoint inhibitors (CPIs), many fail to have clinical responses. A better understanding of the mechanisms that regulate CD8+ T-cell responses in the tumor microenvironment is required to improve immunotherapies that restore function in exhausted CD8+ T cells. Although heterogeneity of effector CD8+ T cells in the tumor microenvironment (TME) has been recognized, their functions and roles are ill-defined.
Methods: We have evaluated phenotypical and functional heterogeneity of tumor-infiltrating lymphocytes (TILs) after adoptive transfer of ex vivo primed 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 that had been established for 11-14 days. Systemic administration of IL-2 and vaccination with anti-CD40 antibody and toll-like receptor (TLR) agonist were used to enhance antitumor immunity of transferred T cells. Tumor and spleen were harvested for functional analysis of adoptively transferred T cells.
Results: We found that the chemokine receptor, CX3CR1 identified three distinct effector CD8+ T-cell subsets, CX3CR1 negative (-), intermediate (int), and high (hi) in blood, spleen and the TME. A CX3CR1hi subset contained terminally-differentiated CD8+ T cells that expressed higher levels of killer-cell lectin like receptor G1 (KLRG1), TNF-related apoptosis-inducing ligand (TRAIL), perforin, and granzyme. Significantly more CX3CR1int CD8+ T cells expressed CD25 compared to other subsets, suggesting this is the subset that is rapidly proliferate and preferentially generate terminally-differentiated T cells. Unexpectedly, despite their terminally differentiated status, a CX3CR1hi CD8+ T-cell subset expressed significantly lower levels of co-inhibitory receptors, PD-1, LAG3, and TIGIT compared to CX3CR1– and CX3CR1int CD8+ T-cell subsets in the TME. In line with this, proliferation and cytokine production of CX3CR1– and CX3CR1int CD8+ T-cell subsets were significantly decreased in the TME compared to CX3CR1hi CD8+ T-cell subset. Importantly, PD-1/PD-L1 blockade significantly improved effector functions of CX3CR1– and CX3CR1int CD8+ T-cell subsets in the TME.
Conclusion: The chemokine receptor, CX3CR1 defines distinct effector CD8+ T-cell subsets in periphery and in the TME. Tumor-infiltrating CX3CR1– and CX3CR1int CD8+ T-cell subsets express high levels of co-inhibitory receptors, PD-1, LAG3, and TIGIT, and their effector functions are improved by PD-1/PD-L1 blockade.