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Immunotherapies have transformed the treatment of cancer through the development of drugs that allow T cells to recognize and kill malignant cells. With this has come a need to evaluate how these drugs affect T cell function. We developed an imaging cytometry-based method that can simultaneously assess T cell activation and the functional ability of these activated T cells to kill cancer cells. We used the Nur77-GFP reporter, which expresses GFP upon T cell receptor signaling, as a readout of T cell activation. We combined this reporter system with an MC38 colon adenocarcinoma cell line expressing a TCR-specific antigen that is used as a model tumor antigen (TA). We show that in T cell-cancer co-culture systems, we are able to quantify the number of activated cytotoxic CD8+ T cells and their ability to recognize and kill cancer cells by imaging cytometry. We tested the sensitivity of this platform by quantifying the proportion of antigen-specific activated T cells in co-cultures with varying ratios of unresponsive T cells, mimicking heterogenous lymphocyte populations. To test our system in a pharmacological context, we evaluated the effects of a SRC family kinase inhibitor on T cell activation and cancer cell death. In each case, we were able to quantify the proportion of activated T cells and the extent of TA-specific cancer cell death. Our findings show that the Nur77-GFP reporter system combined with imaging cytometry is a fast and reproducible system to evaluate how different treatment conditions influence TA recognition by T cells using T cell-cancer co-cultures. The simultaneous analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics. Supported by The Elsa U. Pardee Foundation (AHC), the V Foundation Scholar Award (AHC), the Cancer Biology Training Program T32CA009676 (MKC), the Pharmacological Sciences Training Program T32GM140223 (CES), and a Charles W. Edmunds Pharmacology Fellowship (YZ). This study was also supported by resources provided by the U-M Flow Cytometry Core and the U-M Rogel Cancer Center (P30CA046592).

Blockading antibodies against PD-1/PD-L1 immune checkpoints are approved for melanoma treatment; however, only a minority of patients experience durable remission. Our previous work suggested that pegylated liposomal alendronate (PLA) targets tumor-associated macrophages, another mediator of immunosuppression, and polarized them towards an antitumor phenotype. We hypothesized that dual immunotherapy with PLA and PD-1 inhibitor would result in enhanced efficacy by targeting nonoverlapping immune escape mechanisms. In vitro cytotoxicity studies were performed in B16-OVA melanoma and murine bone marrow-derived dendritic cells. In vivo efficacy/toxicity studies were conducted in seven-week old C57BL/6 mice with 100,000 B16-OVA cells implanted on the rear flank. Once tumors were palpable, mice (n=4-5/group) were randomized to receive PLA (alendronate 4 mg/kg) + α-PD-1 mAb (10 mg/kg), vehicle + IgG-isotype, PLA + IgG-isotype, or vehicle + α-PD-1. Tumor growth was monitored, and at endpoint, tissues (tumors, lymph nodes, livers, kidneys, spleen, intestines, and lungs) were harvested for analysis by flow cytometry and histopathology. Neither PLA nor α-PD-1 were cytotoxic to melanoma or dendritic cells in vitro when compared to doxorubicin (cytotoxic chemotherapy). Mice treated with PLA + α-PD-1 had significantly smaller tumors compared to placebo controls (p=0.0015) and α-PD-1 monotherapy (p=0.0168).There were no significant pathologic changes in tissues. Assessment of immune responses in tumors and sentinel lymph nodes is ongoing. Dual immunotherapy targeting tumor-associated macrophages and the PD-1 checkpoint is safe and more efficacious than monotherapies solely targeting the PD-1 checkpoint in the treatment of murine melanoma. This is rapidly translatable to clinical trials, as the separate components are already approved for use in humans. Citation Format: Claire E. Shudde, Jalpa Patel, Robin Rajan, Hilary Shmeeda, Dung Nguyen, Alberto A. Gabizon, Ninh M. La-Beck. Dual immunotherapy targeting macrophages and PD-1 immune checkpoint for treatment of melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B21.