Radiotherapy in Combination with αOX40 and Actla-4 Immunotherapy Reverses Anergy and Prevents Development of Functional Exhaustion within Tumor-Specific CD8 T Cells

Abstract

The immunosuppressive tumor microenvironment and chronic T cell stimulation that occurs in the presence of cancer results in CD8 T cell dysfunction that has been difficult to reverse with immunotherapy (IT) alone. This dysfunction can be separated into two categories of anergy and exhaustion. Anergy is a lack of proliferative response to stimulation with cytokine or antigen resulting from inadequate co-stimulation or suboptimal T cell receptor (TCR) stimulation during T cell priming. Functional exhaustion is the inability of T cells to kill or produce effector cytokines in response to stimulation. We hypothesized the addition of ionizing radiotherapy (RT) to IT with agonist αOX40 & blocking αCTLA-4 antibodies would reverse tumor associated CD8 T cell anergy and exhaustion. We utilized an established model of CD8 T cell anergy in which transgenic, OVA-specific, OT-I CD8 T cells are adoptively transferred into centrally tolerant POET-1 mice bearing OVA expressing tumors. The development of anergy/exhaustion was monitored within this T cell population and animals were treated with a single 20 Gy fraction of RT using a SARRP, αOX40/αCTLA-4, or combined IT/RT 21 days after transfer. To assess anergy/exhaustion in endogenous CD8 T cell responses CT26 colon carcinoma bearing BALB/c mice were treated with IT, RT, or combined IT/RT either 10 or 17 days after tumor implantation. The frequency/function of tumor-specific (AH1 or OT-I) CD8 T cells was monitored in blood, lymph node (LN), and tumor infiltrating lymphocytes (TIL) in these models. Reporter Nur77-GFP-OT-I CD8 T cells expressing green fluorescent protein in response to TCR ligation were used in to measure the presence/strength of TCR signaling following IT or RT. We demonstrate OT-I cells become anergic/exhausted and produce few effector cytokines 21 days after adoptive transfer. Combination therapy with IT/RT resulted in increased TCR stimulation (measured by GFP expression) and reversed anergy (measured by proliferation) within OT-I T cells in LN and TIL compared to IT or RT alone. Combination therapy was able to reverse functional exhaustion (measured by cytokine production) within LN but not TIL in this model. In CT26 tumor bearing mice combination IT/RT significantly increased (up to 4-fold) the frequency of tumor-specific endogenous AH1 CD8 TIL and prevented development of functional exhaustion within TIL when compared to IT or RT alone. Combined ablative RT and IT with αOX40/αCTLA-4 results in increased CD8 TCR signaling, reversal of T cell anergy, and can prevent or reverse functional exhaustion. These novel results suggest the addition of RT to IT can effectively reverse tumor-associated T cell dysfunction where IT alone is insufficient and provide rationale for early phase clinical trial design.