A novel combination strategy for effectively targeting cancer stem‐like cells

Immunotherapy is now established as the fourth pillar of cancer therapy alongside surgery, radiation and chemotherapy. The major success of cancer immunotherapy to date has been the use of therapies targeting immune checkpoints such as PD-1 and CTLA-4, which act to enhance the ability of tumor-antigen specific CD8+ T cells to target cancer cells.1 Similarly, adoptive cellular therapy of expanded tumor-infiltrating lymphocytes or vaccines based on recognition of tumor (neo)antigens are also promising approaches, which make use of the cytotoxic activity of CD8+ T cells. Although these strategies have shown considerable promise in some patients, the effectiveness of anti-tumor CD8+ T cells is limited by several factors, including local tumor-induced immunosuppression and the requirement of tumors to express MHC-I, which is often downregulated as a tumor escape mechanism.2 Furthermore, the cancer stem cell (CSC) population, reported to be important for tumor progression and metastasis, is known to be resistant to anti-tumor T cells (Figure 1). Therefore, there is a pressing need to develop strategies to further enhance the activity of CD8+ T cells in patients who would otherwise be non-responsive to immunotherapy. The recent publication by Chen et al.3 highlights the potential for utilizing γδ T cells to enhance the sensitivity of CSCs to CD8+ T-cell mediated killing. First, Chen et al.3 developed a unique model to isolate CSCs from ras/SV40 large T-antigen transformed cells and compared their phenotype to the non-CSC population. The CSCs were identified as mesenchymal-like cells through their lack of expression of epithelial markers cytokeratin-14/18 and their positive staining for vimentin. Moreover, the stem cell properties of the identified CSCs were also shown by their renewal properties in spheroid cultures and their ability to form tumors in NOD scid gamma mice, which was significantly enhanced above and beyond the non-CSC population. Using an intricate co-culture system in which FluM1/ CMV-specific CD8+ T cells were co-cultured with cancer cells that were either retrovirally engineered to express the endogenous FluM1 or pulsed with peptides to the immunodominant epitopes of FluM1/ CMV, the authors show that the CD8+ T cells were significantly less able to lyse the CSC population. Since the CSCs express less MHC-I and ICAM-1 (CD54) than their non-CSC counterparts, Chen et al.3 postulated that the restoration of MHC-I and ICAM-1 expression on CSCs could enhance their susceptibility to T-cell mediated killing. One potential way to achieve this therapeutically is induction of an innate immune response which can consequently enhance tumor immunogenicity to CD8+ T cells via a number of mechanisms, including the production of IFN-γ which is known to regulate MHC-I.4 The use of γδ T cells has previously been postulated in cancer immunotherapy due to their high cytotoxic potential and their ability to target tumors in a non-MHC-restricted fashion.5 One method to enhance human Vγ9Vδ2 T cell activity is through the use of aminobisphosphonates such as zoledronate,5 which interfere with phosphoantigen-processing enzymes and consequently enhance the presentation of phosphoantigens recognized by Vγ9Vδ2 T cells. In the current study, the use of zoledronate was shown to enable γδ-mediated killing of both non-CSC and CSC populations and to synergistically enhance the killing activity of CD8+ T cells through the IFN-γ-driven upregulation of MHC-I and ICAM-1 (Figure 1). The findings in this study demonstrating effective targeting of CSCs could have major implications for the effective treatment of metastatic disease, given the current problem of the resistance of this population to standard therapies such as chemotherapy, often leading to cancer relapse in patients. Another important escape mechanism used by cancer cells following conventional treatments is the downregulation of MHC-I rendering them ignorant to CD8+ T-cell recognition.2 The two-pronged attack involving stimulation of γδ T cells resulting in increased production of IFN-γ and upregulation of MHC-I may potentiate CD8+ T cell activity in patients that otherwise remain unresponsive. To extend the clinical relevance of this approach described in the paper, there are a number of important aspects that need to be addressed in future studies. The HMLER cell line utilized in the experiments was derived from transformed mammary epithelial cells. It will be important to investigate whether the synergy between γδ T cells can be demonstrated for primary CSCs. Another important experiment to undertake in future work will be to investigate whether this synergistic combination is effective in vivo. It would be interesting to determine whether the stimulation of endogenous γδ T cell activity or the adoptive transfer of activated γδ T cells may trigger cytokine-induced upregulation of MHC-I and ICAM on both CSC and non-CSC populations, sensitizing these cells to CD8+ T-cell mediated cytotoxicity. The possible role of other potential γδ T cell cytokines such as IL-17 in the synergistic effect would also be of interest. It is currently unclear whether stimulation of other innate cell populations may also sensitize CSCs to CD8+ T cells in vivo. Previous studies have shown effective anti-tumor responses can be achieved via antigen stimulation of other innate cell populations such natural killer T (NKT) cells. NKT cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells, and recognize the non-polymorphic antigen-presenting CD1d molecule that binds both self and foreign lipids and glycolipids. Activation of NKT cells with the ligand α-GalCer results in augmenting their function, including production of large quantities of cytokines such as IFN-γ. Therefore, it would be interesting to examine whether the observations described in this study for γδ T cells may extend to other innate cell types. One potential extension of this approach is the use of checkpoint blockade inhibitors such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1). The administration of these inhibitors in patients has shown some remarkable responses against some cancers, including melanoma and lung cancer.1 Thus, it would be interesting to investigate in future preclinical studies whether the synergistic effects observed by γδ T cells and CD8+ T cells may be potentiated further in the presence of checkpoint blockade. This may represent a novel strategy to broaden the utility of this combination approach. However, one reported escape mechanism utilized by cancer cells is through intrinsic resistance to the effects of IFN-γ,6, 7 which has been shown to limit the effectiveness of checkpoint immunotherapy.6, 7 This may also similarly reduce the ability of innate immune cells to enhance the effects of cytotoxic CD8+ T cells in patients with tumors harboring mutations in this pathway. In conclusion, the authors have described for the first time a powerful synergism between non-MHC and MHC-restricted T cells in targeting CSC-like cells. The studies fully support combination approaches that harness both innate and adaptive responses. Activation of innate cell types such as γδ T cells using stimulants such as zoledronate may also find utility in enhancing adoptive T-cell regimens including chimeric antigen receptor (CAR T cells) in the future. PAB is supported by a National Breast Cancer Foundation Fellowship (ID# ECF-17-005). PKD is supported by an NHMRC Senior Research Fellowship (APP1041828). The authors declare no conflict of interest. Effective targeting of cancer like stem cells through the synergistic action of γδ and CD8+ T cells. Cancer stem cells (CSCs) are generally resistant to the action of anti-tumor T cells, often leading to tumor escape and metastasis following therapy (a). CSCs can be sensitized to killing in a two-pronged attack involving both innate and adaptive T cells (b). Activation of γδ T cells using a stimulant, such as zoledronate enables their killing of CSC populations and to synergistically enhance cytotoxicity by CD8+ killer T cells through IFN-γ-driven upregulation of MHC-I and ICAM-1 on CSCs.