To develop more effective combination immunotherapies lacking immune-related adverse events, we investigated the nature of the intratumoral immune response and systemic immune response induced by immunotherapies.
B16 melanoma cells were subcutaneously inoculated into IFNγ venus mice that express venus fluorescent protein as a reporter of IFNγ production. On days 5 and 9 after tumor inoculation, mice received intraperitoneal injections of 200 μg anti-PD-1 (RMP1-14), anti-CTLA-4 (9H10), anti-4-1BB (3H3), anti-CD4 (GK1.5) mAbs or a combination of anti-PD-1 and anti-4-1BB mAb. Fourteen days after tumor inoculation, cells in the tumor, draining lymph node, spleen were analyzed by flow cytometry and CDR3 region of TCR α and β chains sequencing. We decomposed TCR diversity into richness and evenness. Diversity Evenness 50 (DE50) was calculated as the ratio of how many clonotypes amongst the most frequent were necessary to account for 50% of the total read counts divided by the total number of read counts present.
The tumor growth suppression was achieved only by anti-PD-1, anti-PD-1/4-1BB combined, or by anti-CD4 treatment, but not by anti-CTLA-4 or anti-4-1BB monotherapy in our model. IFNγ venus signals of intratumoral CD8+ T cells was higher than those of spleens, suggesting that CD8+ T cells in the tumor were activated and expanded to a greater or lesser extent by these immunotherapies. Anti-tumor effects were associated with increased CD8+ T cell effector function and TCR diversity with enrichment of certain TCR clonotypes in the tumor. In the tumor of mice where the tumor growth was well-controlled by the combination of anti-PD-1/4-1BB or anti-CD4 treatment, DE50 values were significantly lower than those of control. Activation of tissue-reactive T cells were also examined. IFNγ venus+ T cells were increased in the spleens of all mice with the expansion of many clonotypes in mice receiving immunotherapies. Mononuclear cell infiltrations into liver, kidney, heart, lung and pancreas were observed particularly in mice that received anti-4-1BB and the anti-PD-1/4-1BB combination. DE50 values in the spleen were reduced in these mice.
In conclusion, effective immunotherapies increase TCR diversity with extended activation of selective CD8+ T-cells specifically in the tumor but not in the periphery, while polyclonal activation of T cells and emergence of dominant clones in the periphery was associated with tissue damage. We propose DE50 for the evaluation of diversity evenness of TCR repertoire that reflects anti-tumor activity and immune-related adverse events.
Citation Format: Akihiro Hosoi, Hirokazu Matsushita, Kazuyoshi Takeda, Ryuji Suzuki, Kazuhiro Kakimi. Diversity evenness of TCR repertoire in the tumor and periphery reflects antitumor activity and immune-related adverse events, respectively [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1721.