Cyclophosphamide enhances the antitumor potency of GITR engagement by increasing oligoclonal cytotoxic T cell fitness
Daniel Hirschhorn, Allison Betof Warner, Rachana Maniyar, Andrew Chow, Levi M.B. Mangarin, Adam D. Cohen, Linda Hamadene, Gabrielle A. Rizzuto, Sadna Budhu, Nathan Suek, Cailian Liu, Alan N. Houghton, Taha Merghoub, Jedd D. Wolchok
Daniel Hirschhorn, Allison Betof Warner, Rachana Maniyar, Andrew Chow, Levi M.B. Mangarin, Adam D. Cohen, Linda Hamadene, Gabrielle A. Rizzuto, Sadna Budhu, Nathan Suek, Cailian Liu, Alan N. Houghton, Taha Merghoub, Jedd D. Wolchok
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Research Article Immunology Oncology

Cyclophosphamide enhances the antitumor potency of GITR engagement by increasing oligoclonal cytotoxic T cell fitness

Abstract

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8+ T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8+ T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

Authors

Daniel Hirschhorn, Allison Betof Warner, Rachana Maniyar, Andrew Chow, Levi M.B. Mangarin, Adam D. Cohen, Linda Hamadene, Gabrielle A. Rizzuto, Sadna Budhu, Nathan Suek, Cailian Liu, Alan N. Houghton, Taha Merghoub, Jedd D. Wolchok

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Figure 8

Combination therapy decreases the TCR repertoire by promoting clonal expansion of highly activated CD8+ T cells.

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Combination therapy decreases the TCR repertoire by promoting clonal exp...
Cohorts of mice were implanted with B16. On day 8, CTX was injected. The next day, mice were treated with anti-GITR antibody or rat IgG. Additional cohorts of mice were injected with B16 on day 8; on day 15 mice were injected with anti-GITR Ab or IgG. After 1 week, all cohorts of mice were sacrificed, and single-cell suspensions were prepared. Asynchronous B16 challenge was needed for the CTX groups a week apart given the lack of immune infiltrates caused by chemotherapy treatment. Single-cell suspensions were stained with anti-CD5 and hashtag multiplex antibodies. The cells were sorted by FACS based on CD5+, and next-generation TCR coupled with 10x Genomics sequencing was performed. (A) UMAP displaying different clusters. (For a list of genes included in each subclusters refer to Supplemental Table 2.) (B) UMAP showing gene expression of the top 10 clones present in each treatment. Number below each colored circle represents the total number of cells in each clone. (For clone sequences refer to Supplemental Table 1.) (C) UMAP on selected cell cycle and proliferation genes. (D) Heatmap of selected cell cycle and proliferation genes per condition.