Abstract 2856: Unique changes in the TCR repertoire of tumor-infiltrating lymphocytes underlie the synergy of radiotherapy with CTLA-4 blockade
Authors: Pilones Karsten , Koelwyn Graeme , Emerson Ryan , Formenti Silvia , Robins Harlan , Demaria Sandra 
Published: August 2015

Abstract
BACKGROUND: Monoclonal antibodies (mAb) that target CTLA-4, a negative regulator of T-cell activation, have significantly extended survival of patients with metastatic melanoma. However the number of responders remain low. We have previously shown in the 4T1 mouse breast tumor model that resistance to α-CTLA-4 therapy can be overcome by concurrent local radiotherapy (RT) (Demaria et al Clin Can Res 2005). Multiple effects of RT on the tumor microenvironment contributed to its ability to sensitize tumors to anti-CTLA-4 treatment, including enhanced homing of effector T cells to the tumor and formation of stable immune synapses between CTL and tumor cells.Importantly, CD8 T cell responses to the endogenous tumor antigen gp70 were seen only in mice treated with the combination, consistent with the hypothesis that RT generates an in situ vaccine and CTLA-4 expands primed T cells. Here we used next generation sequencing of complementarity determining region 3 (CDR3) from rearranged T cell receptor (TCR) variable beta (Vβ) chains to obtain a comprehensive picture of the changes in the repertoire of infiltrating lymphocytes that occur during CD8 T cell-mediated tumor rejection. METHODS: BALB/c mice were inoculated with 4T1 cells and thirteen days later, when tumors became palpable, randomly assigned to one of 4 treatment groups (N = 5 mice/group): control, RT alone, α-CTLA-4 alone or RT+anti-CTLA-4. RT was given in 2 fractions of 12 Gy on days 13 and 14 post-tumor inoculation. α-CTLA-4 mAb (Clone 9H10) was given i.p. on days 15, 18 and 21. Tumors were harvested on day 22 for high throughput sequencing of TCRβ CDR3 regions performed using the ImmunoSEQ platform. RESULTS: We observed distinct and non-overlapping effects of RT and immunotherapy on TIL repertoire. CTLA-4 blockade increased clonality and significantly expanded the top 5 most frequent clonotypes. On the other hand, RT increased TIL numbers and broadened their repertoire by selectively expanding the top 6-20 clonotypes. Importantly, analysis of Vβ/Jβ usage landscape showed that the combined treatment generated the most dramatic changes from baseline with the expansion of several unique Vβ/Jβ combinations not otherwise seen in tumors treated with RT or CTLA-4 blockade as single agents. Significantly reduced diversity of the TCRβ profile seen only with combination therapy is consistent with antigen-driven TIL clonal expansion.CONCLUSIONS: Overall, tumor rejection induced by RT+anti-CTLA-4 is associated with both quantitative and qualitative changes in TIL repertoire. Data suggests that a broader repertoire of tumor-specific T cells may be critical for tumor rejection, and is achieved by coupling the effects of RT, which promotes priming of T cells specific for endogenous tumor antigens, and CTLA-4 blockade, which drives expansion of selected clones. These results have important implications for the therapeutic synergy of radiation and CTLA-4 blockade.
Citation Format: Karsten Pilones, Graeme Koelwyn, Ryan Emerson, Silvia Formenti, Harlan Robins, Sandra Demaria. Unique changes in the TCR repertoire of tumor-infiltrating lymphocytes underlie the synergy of radiotherapy with CTLA-4 blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2856. doi:10.1158/1538-7445.AM2015-2856