Abstract 3663: Immune suppressive myeloid cells expansion in vitro requires a simulated tumor microenvironment

Abstract

Abstract Elevated levels of immune suppressor cells induced by tumor derived factors are associated with inhibition of immune responses. One such group of immune suppressor cells is Myeloid Derived Suppressor Cells (MDSCs). They are phenotypically defined in humans as HLADR low or negative and CD33, CD11b, and CD15 or CD14 positive and functionally by their ability to suppress the activation of other immune cells such as T cells. We hypothesized that MDSCs generated in vitro could be propagated but would lose their suppressive ability on the action of T cells in the absence of a simulated tumor microenvironment. MDSCs were first differentiated in vitro from normal donor peripheral blood mononuclear cells (PBMCs) using a simulated tumor microenvironment consisting of either cytokines (GMCSF and IL-6) or tumor cell lines (pancreatic adenocarcinoma SW-1990 or head and neck cancer CAL-27). The MDSCs generated in vitro suppressed T cell proliferation of autologous T cells using the carboxyfluorescein succinimidyl ester (CFSE) assay (31%, 46% and 75% suppression for cytokines, CAL-27 and SW-1990, respectively). Given the MDSCs obtained from co-culture of PBMCs with SW-1990 were the most suppressive on the proliferation of T cells, we attempted to proliferate these cells in vitro to expand the number of MDSC available for experimentation rather than having to purify MDSC from clinical samples. In vitro generated MDSC were propagated using a Rho-associated kinase (ROCK) inhibitor. Generated MDSC collected via magnetic bead isolation (CD33) were cultured with irradiated fibroblast feeder cells (± 3000 rad) in F medium [3:1 (v/v) F-12 Nutrient Mixture (Ham)-Dulbecco's modified Eagle's medium, 5% fetal bovine serum, 0.4 µg/mL hydrocortisone, 5 µg/mL insulin, 8.4 ng/mL cholera toxin, 10 ng/mL epidermal growth factor (or 10 ng/mL GMCSF), and 24 µg/mL adenine]. The ROCK inhibitor was added at 10 µM and the media was exchanged every 3 days. The generated MDSC did propagate as measured by CFSE staining of the generated MDSC; however, the generated MDSCs completely lost their ability to inhibit T cells by day 11 in culture. In conclusion, generated MDSCs can be propagated in culture but lose their ability to suppress T cells in the absence of a simulated tumor microenvironment and the presence of a Rho kinase inhibitor. Citation Format: Joseph Markowitz, Taylor R. Brooks, William E. Carson. Immune suppressive myeloid cells expansion in vitro requires a simulated tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3663. doi:10.1158/1538-7445.AM2014-3663