LAUSR

The clinical use of antibodies targeting the diaslganglioside GD2 improved survival and clearly identified a role for immunotherapy in the aggressive pediatric solid tumor neuroblastoma (NB). Even so, the 5-year survival rate for high risk patients remains 50%, warranting novel immunotherapeutic approaches. Given their MHC-independent cytotoxic activity towards transformed cells, γδ T cells are attractive tools for adoptive cellular immunotherapy. γδ T cell cytotoxicity is mediated through the expression of various activating receptors that contribute to multiple mechanisms of anti-tumor activity. Specifically, their expression of the γδ-TCR works in concert with tumor expression of members of the butyrophilin-3 (BTN3A) subfamily for cytotoxicity in response to phosphoantigens (pAg). In other cancers, stimulation of pAg production through zoledronate (ZOL) supplementation promotes γδ T cell recognition/killing. We have identified that γδ T cells are differentially cytotoxic towards NB cell lines, suggesting variations in expression of key players responsible for γδ T cell susceptibility. This work serves to understand the significance of BTN3A members in NB, and their impact on the susceptibility of NB to γδ-TCR mediated killing. It is hypothesized that the pAg-mediated response can be augmented by increasing intracellular pAg via use of ZOL or by manipulating BTN3A members. To test this, NB cells were pre-treated with either vehicle or ZOL for 24 hours and then co-incubated with expanded γδ T cells for 4 hours at various effector:target (E:T) ratios. Apoptotic death was then determined for NB cells using Annexin-V/7-AAD staining. Across 8 tested NB cell lines, Kelly, NB-1643, NGP, SK-N-AS, SH-SY5Y, NLF, and SMS-SAN cells were resistant to γδ T cells at a 10:1 E:T ratio when untreated with ZOL. IMR-5 demonstrated 12-18% lysis at a 5:1 E:T. When NB cells were pre-treated with ZOL, γδ T cell cytotoxicity was enhanced towards all models except SK-N-AS. We identified three NB cell lines with differential susceptibility to γδ T cell killing, SK-N-AS