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 (O2)] on cells within the TME may lead to tumor evasion. Gamma delta T cells (γδTcs) naturally kill transformed cells and are therefore under development as immunotherapy for various cancers. Clinical trials have proven the safety of γδTc immunotherapy and increased circulating γδTc levels correlate with improved patient outcome. Yet, the function of γδTc tumor infiltrating lymphocytes in human breast cancer remains controversial. Breast tumors can be highly hypoxic, thus therapy must be effective under low O2 conditions. We have found increased infiltration of γδTc in areas of hypoxia in a small cohort of breast tumors; considering their inherent plasticity, it is important to understand how hypoxia influences γδTc function. In vitro, the cell density of expanded primary healthy donor blood-derived human γδTc decreased in response to hypoxia (2% O2) compared to normoxia (20% O2). However, the secretion of macrophage inflammatory protein 1α (MIP1α)/MIP1β, regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L by γδTc 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 γδTc 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 γδTc cytotoxicity, but that MIP1α, RANTES, and CD40L do not play a direct role in cytotoxicity. Hypoxia appeared to enhance the cytotoxicity of γδTc such that exposure for 48 h increased cytotoxicity of γδTc 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 γδTc 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 γδTc cytotoxicity. Despite enhanced γδTc 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 γδTc immunotherapies.

Cancer immunotherapy with γδ T cells: many paths ahead of us

“…Tumor-intrinsic mechanisms that have been identified to impair γδ T cell attack include the release of large amounts of prostaglandin E2 by tumor cells with strong expression of cyclooxygenase-2 (COX-2), 151 the activity of indoleamine-2,3-dioxygenase (IDO) and its metabolite kynurenine, 225 the release of galectin-3, 226 and the hypoxic tumor microenvironment. 227 Inhibitors for the respective pathways can enhance tumor killing by Vδ2T cells in vitro, and it seems reasonable to propose that these strategies can also work in vivo, given the availability of approved drugs such as COX2 inhibitors. In addition to galectin-3, other tumor-expressed galectins, such as galectin-9, also suppress T cell activation.…”

Hypoxia regulates the differentiation and anti-tumor effector functions of γδT cells in oral cancer

“…Sieger et al showed that the hypoxia‐exposed γδT cells failed to lyse the MCF7 breast tumor cells exposed to hypoxia, which concurs with our observation. They attributed it to the reduced expression of NKG2D and MICA, which is an indirect effect of hypoxia [31]. A recent study demonstrated that oxygen pressure in the tumor microenvironment orchestrates an anti‐ and pro‐tumoral γδT cell equilibrium through tumor‐derived exosomes which inhibited γδT cells proliferation and anti‐tumor cytotoxicity [32].…”

γδ T cells in cancer: a small population of lymphocytes with big implications

“…However, tumors in hypoxic environments begin to secrete soluble NKG2D ligands, rendering cd T cells incapable of killing these cells. 112 Having previously homed to target organs, adoptively transferred Vd1 + T cells should be capable of homing again to a target organ containing a tumor. Furthermore, protocols have been developed that allow the rapid expansion of highly cytotoxic donor Vd1 + T cells (DOT cells), which are able to control leukaemic cell growth.…”