Dissecting the cellular components of ex vivo γδ T cell expansions to optimize selection of potent cell therapy donors for neuroblastoma immunotherapy trials

Abstract

γδ T lymphocytes represent an emerging class of cellular immunotherapy with preclinical promise to treat cancer, notably neuroblastoma. The innate-like immune cell subset demonstrates inherent cytoxicity toward tumor cells independent of MHC recognition, enabling allogeneic administration of healthy donor-derived γδ T cell therapies. A current limitation is the substantial interindividual γδ T cell expansion variation among leukocyte collections. Overcoming this limitation will enable realization of the full potential of allogeneic γδ T-based cellular therapy. Here, we characterize γδ T cell expansions from healthy adult donors and observe that highly potent natural killer (NK) lymphocytes expand with γδ T cells under zoledronate and IL-2 stimulation. The presence of NK cells correlates with both the expansion potential of γδ T cells and the overall potency of the γδ T cell therapy. However, the potency of the cell therapy in combination with an antibody-based immunotherapeutic, dinutuximab, appears to be independent of γδ T/NK cell content both in vitro and in vivo , which minimizes the implication of interindividual expansion differences toward efficacy. Collectively, these studies highlight the utility of maintaining the NK cell population within expanded γδ T cell therapies and suggest a synergistic action of combined innate cell immunotherapy toward neuroblastoma.

Directing the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells

“…To further investigate the chemoattraction properties of γδ T-cell migration, a neuroblastoma NSG mouse model was developed using IMR5-luciferase cells ( Figure 6B ), as we previously published ( 41 , 46 ). IMR5 tumors were established in NSG mice, and MSCs or γMSCs were administered intratumorally.…”

“…This human neuroblastoma NSG mouse model has been previously published by our group as a bona fide model in which to study γδ T-cell characteristics in targeting cancer ( 41 , 46 ). NSG mice were administered IMR5-luciferase cells subcutaneously.…”

The yes-associated protein (YAP) is associated with resistance to anti-GD2 immunotherapy in neuroblastoma through downregulation of ST8SIA1

“…γδ T cells were expanded under our 12-day protocol as previously described with αβ T cell depletion on day 6 of culture from healthy donor peripheral blood mononuclear cells. 32 The expanded γδ T cell population was profiled by flow cytometry with antibodies: CD3-BV421, CD56-APC-R700, CD16-BV480, and αβ-TCR-PE or γδ-TCR-PE and used between days 12 and 14 in the cytotoxicity assays described below. 32 See Supplemental Table S2 for antibody information.…”

“… 32 The expanded γδ T cell population was profiled by flow cytometry with antibodies: CD3-BV421, CD56-APC-R700, CD16-BV480, and αβ-TCR-PE or γδ-TCR-PE and used between days 12 and 14 in the cytotoxicity assays described below. 32 See Supplemental Table S2 for antibody information.…”

Human Vδ2 T Cells and Their Versatility for Immunotherapeutic Approaches

“…Expansion protocols provided an initial exposure to N-BP or p-Ag in combination with IL-2 (100 and 1000 U/mL), replenished every 2–3 days, led to increased expression of cytotoxicity and activation markers like CD16, CD56, Nkp44, NKp30, and the production of IFN-γ, granzymeB, perforin, or TNF-α [ 22 , 28 , 141 , 142 , 146 , 150 , 151 ]. The anti-tumor functions of IL-2-expanded Vδ2 T cells against several tumor cancer cell lines have been investigated including human neuroblastoma, myeloma, glioblastoma, melanoma, and renal adenocarcinoma and Burkitt´s lymphoma [ 29 , 140 , 141 , 142 , 146 , 147 , 148 ].…”