Gamma delta T cell immunotherapy against breast cancer and overcoming mechanisms of immune evasion

• Author / Creator

  Dutta, Indrani

• Gamma delta T cells (Tc) are immunosurveillance cells garnering great interest for their anti-tumoral activity. Target cell recognition is mediated by the T cell antigen receptor (TCR) and/or the natural killer receptor NKG2D, whose ligands are upregulated on cancer cells. Tumor cell lysis is mediated in part by Fas ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which induce apoptosis in target cells. Clinical trials have proven the safety of Tc immunotherapy and increased circulating Tc levels correlate with improved patient outcome. We investigated the impact of hypoxia on Tc and target breast cancer cells. We also investigated the efficacy of Tc against breast cancer stem-like cells (BCSC). Hypoxia and BCSC are two major factors associated with therapy resistance and cancer recurrence. Firstly, we discovered that using blocking antibodies to the TCR induces Tc apoptosis. Blocking assays require the use of antibodies against specific receptors and ligands in cytotoxicity assays. Gamma delta T cell apoptosis was further increased in the presence of IL-2, which is often included in cytotoxicity assays. However, we found that adding IL-2 does not significantly contribute to Tc cytotoxicity against breast cancer cells. These findings informed us to not use IL-2 in our cytotoxicity assays and to test Tc viability when using blocking antibodies in parallel with blocking cytotoxicity assays. Secondly, we investigated the impact of hypoxia on Tc cytotoxicity. Hypoxia or low oxygen levels are characteristic of the breast cancer micro-environment and can promote therapy resistance by inducing cellular plasticity. We found that hypoxia activated Tc and enhanced their ability to kill breast cancer cells. However, hypoxia also resulted in breast cancer cell resistance to Tc killing by increased MICA shedding. Soluble MICA can bind to the NKG2D receptor on Tc and competitively block its interaction with NKG2D ligands on the target cell surface. This can reduce NKG2D-mediated target recognition by Tc, ultimately reducing Tc cytotoxicity.
  Hypoxia is known to promote cellular plasticity and BCSC generation. Hence, finally, we investigated whether Tc can target BCSC. BCSC are a small population of cancer cells that are highly tumorigenic and can self-renew as well as differentiate into multiple lineages. Our study demonstrated that BCSC are less sensitive to Tc cytotoxicity than non-stem cells (NSC). We explored several mechanisms that could orchestrate this resistance. The most compelling results were the expression and shedding of NKG2D ligand MICA. The BCSC exhibited lower surface expression of MICA and higher MICA shedding than NSC. Mass spectrometry analysis, which indicated global differences in BCSC and NSC secretomes, also confirmed enhanced MICA shedding by BCSC. Accordingly, we demonstrated that blocking MICA shedding using inhibitors against proteases ADAM10 and ADAM17 lead to significantly enhanced cytotoxicity of Tc against BCSC. Notably, we observed that treatment with ADAM inhibitors brought breast cancer stem cell lysis by Tc to the same level as NSC killing by Tc alone without inhibitors. This suggests a complete reversal of BCSC resistance. Moreover, Tc killing of NSC was further enhanced using the ADAM inhibitor in combination compared to Tc alone. Hence, we concluded that a combination of Tc and the ADAM inhibitor GW280264X is an effective strategy to target resistant BCSC. In summary, we discovered that hypoxia and BCSC population can lead to breast cancer resistance against Tc and determining how these target cells evade Tc killing and devising strategies to overcome this resistance will improve Tc immunotherapy for cancer.

• Subjects / Keywords

  • Gamma Delta T cells
  • Cancer immunotherapy
  • Breast cancer stem cells
  • immune evasion
  • ADAM inhibitors

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-5dyb-1x34

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.