Donor-Derived Variable Delta 2 (Vδ2) Gamma Delta (γδ) T Cells: A Promising Off-the-Shelf Therapeutic Strategy for Acute Myeloid Leukemia

Background: Gamma delta(γδ) T cells have been associated with improved graft-versus-leukemia effect, leukemia-free-survival, and reduced risk of relapse in acute myeloid leukemia (AML). Variable delta 2 (Vδ2) cells are a subset of γδ T cells that are known to have a strong anti-cancer effect in a variety of malignancies, and increased Vδ2 cells have also been associated with better outcomes in chronic lymphocytic leukemia (CLL) and B cell acute lymphocytic leukemia. Earlier studies have indicated that AML blasts can disrupt T cell development. However, the effect of AML on γδ T cell memory phenotypes is not known. Naïve γδ cells are double-positive for CD27 and CD45RA and can differentiate into CD45RA-negative central memory (CM) γδ T cells in response to pAg stimulation. CM γδ T cells have the greatest potential for proliferation, and additional pAg stimulation causes them to further differentiate into effector memory (EM) γδ T cells. EM γδ T cells do not express CD27 or CD45RA, and though they are less proliferative than CM γδ T cells, they express more cytokines. EM γδ T cells can further differentiate into terminally differentiated effector memory (TEMRA) γδ T cells, which exhibit the highest cytotoxicity but the lowest proliferation. TEMRA γδ T cells also regain CD45RA expression. Imbalances of these γδ T cell memory phenotypes have been associated with poorer treatment responses and outcomes in CLL and myeloma. In this study, we have investigated γδ T cell memory phenotypes in AML patients. Additionally, we have tested ex-vivo expanded donor-derived Vδ2 γδ T cells for their effect on AML cells. We hypothesized that AML alters the memory phenotypes of γδ T cells and that ex-vivo expanded donor-derived Vδ2 γδ T cells induce a potent apoptotic effect on AML cell lines and primary AML cells.

Methods: To examine γδ T cell memory phenotypes in AML patients, we have developed a multi-parameter flow cytometry assay (8-color panel) using a Miltenyi Biotec MACSQuant16. Peripheral blood mononuclear cells from healthy donors (n=10) and AML patients (n=14) were used in this study. To investigate the potential of donor-derived γδ T cell therapy for AML, we co-cultured freshly thawed Vδ2 γδ T cells (GMP-grade frozen γδ T cells were obtained from CytoMed Therapeutics Limited, Singapore) overnight at effector-to-target cell ratios (E:T) of 2:1, 5:1, and 10:1 with 8 AML cell lines (OCI-AML2, OCI-AML3, U937, THP1, Kasumi-1, MV4-11, Molm-13, and Molm-14), as well as primary AML cells derived from patient samples. We analyzed the effect of the γδ T cells on apoptosis induction in the AML cells using flow cytometry.

Results: We did not observe any significant changes in the percentages of naïve or EM γδ T cell memory phenotype populations between samples from AML patients and healthy donors. However, we found significantly lower CM and significantly higher TEMRA γδ T cell populations in AML patients than in healthy individuals, with the mean CM percentage dropping from 52.47% ± 13.62% to 22.09% ± 10.07% (p<0.0001) and the mean TEMRA percentage increasing from 15.61% ± 10.08% to 35.55% ± 24.07% (p<0.05). As CM cells are the most proliferative memory phenotype and TEMRA cells are the least, this decrease indicates that γδ T cell populations have less capacity to proliferate in AML patients than in healthy individuals. This also suggests that adoptive transfer of donor-derived γδ T cells may provide a therapeutic opportunity for AML. Next, we determined the cytotoxic effect of donor-derived Vδ2 γδ T cells. When co-cultured with AML cells, the donor-derived γδ cells exerted a potent E:T-dependent apoptotic effect on all leukemic cell lines tested, including the TP53-mutant Kasumi-1 and FLT3-mutant Molm-13, Molm-14, and MV4-11 cells. The mean percentage of apoptotic AML cells was 41.30% ± 22.74% for the 2:1 E:T ratio, 67.52% ± 22.94% for the 5:1 ratio, and 77.54% ± 14.07% for the 10:1 ratio. One-way ANOVA confirmed an E:T ratio-dependent increase in apoptotic AML cells (p<0.0001). Experiments investigating the effect of Vδ2 γδ T cells on primary AML cells and in vivo studies are ongoing.

Conclusion: γδ T cell memory phenotype profiles from AML patients showed a marked decrease in CM γδ T cells and increase in TEMRA γδ T cells, suggesting a more limited proliferative capacity in AML. Furthermore, expanded donor-derived Vδ2 γδ T cells induced E:T-dependent apoptosis in AML cell lines and may be a promising off-the-shelf treatment option for AML patients.

Zeng:CytoMed Therapeutics: Current Employment. Tan:CytoMed Therapeutics: Current Employment. Maiti:Chimeric Therapeutics: Research Funding; Lin Biosciences: Research Funding; CytoMed Therapeutics: Research Funding; Inspirna: Research Funding; Hibercell Inc.: Research Funding; Indapta Therapeutics: Research Funding. Battula:Y-mAbs Therapeutics: Research Funding; Daiichi Sankyo Company, Ltd: Research Funding; InCyte Corporation: Research Funding; Inspirna: Research Funding; CytoMed Therapeutics: Research Funding; FATE Therapeutics: Research Funding.