Abstract 2762: Inhibition of murine myeloid suppressor cells increases CD8+ T cell activation in Vitro
Authors: Zaitouna Anita J. , Lapinski Philip , Rowse Amber , Draper David , Wise Scott 
Published: April 2023

Tumor microenvironments (TME) are rich in cells that potentially inhibit T cell function, such as regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs). Many TMEs are dominated by myeloid cells, making targeting these cells an area of interest. The purpose of this study was to develop an in vitro assay system to assess the ability of novel drugs to overcome MDSC-mediated immune suppression by monitoring changes to T cell function. Arginase1 (ARG1), an enzyme that catalyzes L-arginine into L-ornithine and urea, is highly expressed in MDSCs. An ARG1 target is currently in clinical trials (INCB001158, CB-1158 from Calithera Biosciences) and was used as a target of interest in this study.

MDSC generation from bone marrow cells (BM) was compared using GM-CSF and IL-6 or GM-CSF, IFNγ, and LPS. α-Difluormethylornithine (DFMO), a 2nd generation ARG1 inhibitor that irreversibly binds ARG1, which leads to increased availability of L-arginine, known to modulate T cell activity, was tested during MDSC generation. BM were harvested and characterized with a myeloid flow cytometry panel. GM-CSF, IFNγ, and LPS generated more reproducible MDSCs that express ARG1. M-MDSC-derived BM expressed 2 times more ARG1 as compared to DFMO cultured MDSC-derived BM.

The cytokines and other growth factors produced by MDSC-derived BM have the potential to alter the activation state of CD8+ T cells; therefore, the effect of a suppressive TME on CD8+ T cells was monitored by co-culturing CD8+ T cells with MDSCs in vitro. MDSC-derived BM were co-cultured for 4 days with CD8+ T cells (purified from C57BL/6 splenocytes) and activated with anti-CD3/anti-CD28 beads. CD8+ T cells were characterized for proliferation using CellTraceTM violet, surface activation markers (CD69 and PD-1), and intracellular cytokines (IFNγ, TNFα, and IL-2). CD8+T cells co-cultured with DFMO-inhibited MDSC-derived BM were more proliferative, had greater surface activation, and greater intracellular cytokine of CD8+ T cells as compared to when co-cultured with MDSC-derived BM. CD8+ T cell proliferation was measured at multiple ratios of CD8+ T cells and MDSC-derived BM with a proliferation range of 88-7% when CD8+ T cells were co-cultured with MDSC-derived BM and 91-30% when co-cultured with DFMO-inhibited MDSC-derived BM. Similar trends were observed with CD69, PD-1, IFN-y, TNF-a and IL-2 expression in CD8+ T cells. Overall, the ability to monitor changes in T cell activation following MDSC has broad applications in in vitro screening of novel drug compounds that alter the TME as well as sets the experimental framework for ex vivo analysis of cells isolated from TME following treatment.

Citation Format: Anita J. Zaitouna, Philip Lapinski, Amber Rowse, David Draper, Scott Wise. Inhibition of murine myeloid suppressor cells increases CD8+ T cell activation in Vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2762.