Abstract 911: Butyrophilin 3A2 expression plays a critical role in phosphoantigen-mediated γδ T cell cytotoxicity of neuroblastoma cells
Authors: Ho Andrew , Jonus Hunter C. , Goldsmith Kelly C. 
Published: April 2023

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&lt;NLF&lt;IMR5. Using a high-risk NB gene expression dataset, expression of important BTN members was queried, revealing IMR5 to have BT3.2low expression, NLF to have BT3.2mod expression, and SK-N-AS to have BT3.2high expression. BT3.2 expression levels was confirmed via western blotting. It has been postulated that BT3.2 may be a decoy receptor that interacts with γδ T cells without intracellular signaling, given it lacks the intracellular pAg sensor. Due to its high expression in SK-N-AS, a lentiviral system was used to knockdown BT3.2. SK-N-A-S shBT3.2 models had 48-55% lysis at a 5:1 E:T when untreated and 65-72% lysis when pre-treated with ZOL.

In summary, the anti-NB γδ T cell response can be augmented with ZOL pre-treatment. Additionally, the expression of BT3.2 may be responsible for differential sensitivity of NB cell lines to γδ T cells.

Citation Format: Andrew Ho, Hunter C. Jonus, Kelly C. Goldsmith. Butyrophilin 3A2 expression plays a critical role in phosphoantigen-mediated γδ T cell cytotoxicity of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 911.