Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia

Abstract

Interactions between immune and tumor cells in the tumor microenvironment (TME) often impact patient outcome, yet remain poorly understood. In addition, the effects of biophysical features such as hypoxia [ low oxygen (O-2)] on cells within the TME may lead to tumor evasion. Gamma delta T cells (gamma delta Tcs) naturally kill transformed cells and are therefore under development as immunotherapy for various cancers. Clinical trials have proven the safety gamma delta Tc immunotherapy and increased circulating gamma delta Tc levels correlate with improved patient outcome. Yet, the function of gamma delta Tc tumor infiltrating lymphocytes in human breast cancer remains controversial. Breast tumors can be highly hypoxic, thus therapy must be effective under low O-2 conditions. We have found increased infiltration of gamma delta Tc in areas of hypoxia in a small cohort of breast tumors; considering their inherent plasticity, it is important to understand how hypoxia influences gamma delta Tc function. In vitro, the cell density of expanded primary healthy donor blood-derived human gamma delta Tc decreased in response to hypoxia (2% O-2) compared to normoxia (20% O-2). However, the secretion of macrophage inflammatory protein 1 alpha (MIP1 alpha)/MIP1 beta, regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L by gamma delta Tc were increased after 40 h in hypoxia compared to normoxia concomitant with the stabilization of hypoxia inducible factor 1-alpha protein. Mechanistically, we determined that natural killer group 2, member D (NKG2D) on gamma delta Tc and the NKG2D ligand MHC class I polypeptide-related sequence A (MICA)/B on MCF-7 and T47D breast cancer cell lines are important for gamma delta Tc cytotoxicity, but that MIP1 alpha, RANTES, and CD40L do not play a direct role in cytotoxicity. Hypoxia appeared to enhance the cytotoxicity of gamma delta Tc such that exposure for 48 h increased cytotoxicity of gamma delta Tc against breast cancer cells that were maintained in normoxia; conversely, breast cancer lines incubated in hypoxia for 48 h prior to the assay were largely resistant to gamma delta Tc cytotoxicity. MICA/B surface expression on both MCF-7 and T47D remained unchanged upon exposure to hypoxia; however, ELISAs revealed increased MICA shedding by MCF-7 under hypoxia, potentially explaining resistance to gamma delta Tc cytotoxicity. Despite enhanced gamma delta Tc cytotoxicity upon pre-incubation in hypoxia, these cells were unable to overcome hypoxia-induced resistance of MCF-7. Thus, such resistance mechanisms employed by breast cancer targets must be overcome to develop more effective gamma delta Tc immunotherapies