Article Text

Download PDFPDF

975 Characterization of signaling and metabolic differences between γδ and αβ CAR T cells
  1. Xiomar Bustos,
  2. Daniel Abate-Daga and
  3. Leticia Tordesillas
  1. H. Lee Moffitt Cancer Center & Research, Tampa, FL, United States


Background γδ T cell-based immunotherapies have emerged as an alternative to the traditional αβ T cell-based products. For example, our group has shown that γδ CAR T cells can reduce tumor burden and mitigate tumor-induced bone deterioration in a preclinical model of bone metastatic prostate cancer. In the present study, we investigated the signaling events triggered by a second-generation CAR (originally designed for αβ T cells) in γδ T cells, to test the hypothesis that CAR-induced signaling varies depending on the T cell subset. The ultimate goal of our study is to gain mechanistic insight into the biology of γδ T cells and to inform future CAR design for improved γδ-based adoptive cell therapies.

Methods We designed our analysis as a side-by-side comparison of phosphorylation events, resulting from CAR activation, between γδ and αβ T cells. These were expanded in parallel from a healthy donor and transduced with a retroviral vector (MSGV1) to express a second-generation anti-PSCA CAR (PSCA-8t28z). CAR-T cells were then cocultured independently with metabolically labelled C4-2B-PSCA tumor cells, for 1 hour, and protein phosphorylation was quantified using Liquid Chromatography–Tandem Mass Spectrometry (LC/MS-MS). Statistically significant differences in phosphorylation were defined using a Welch’s t-test (fold-change ≥ 1.5 and p-value < 0.05). Using Qiagen’s Ingenuity Pathway Analysis software, we identified canonical pathways that were significantly overrepresented in the population of proteins that displayed differential phosphorylation in γδ CAR T cells relative to αβ. We used the SeaHorse XF kits for Glycolytic Rate Assay and T Cell Metabolic Profiling for functional characterization of the metabolic properties of CAR-T cells. Finally, flow cytometry was used to analyze Glut-1 expression (anti-Glut1, clone 202915), glucose intake (2NBDG), mitochondrial mass (MitoTracker Green), and mitochondrial membrane polarization (TMRE).

Results We identified 323 phosphorylation events that were differentially abundant between T cell subsets. Within this group, glycolysis and gluconeogenesis were within the top overrepresented canonical pathways. Stimulated γδ T cells showed significantly lower glycolytic rate compared to αβ. CAR expression was accompanied by higher glycolytic rate and expression of Glut-1 receptor in both T-cell types. Finally, oxidative phosphorylation (OXPHOS) was lower in γδ CAR T cells, potentially related to their also lower mitochondrial mass.

Conclusions CAR-induced signaling varies among T cell subsets, and γδ T cells display lower glycolytic and OXPHOS rates upon activation. Ongoing efforts are focused on delineating molecular causes and functional consequences of the metabolic differences between αβ and γδ T cells.

Acknowledgements This work has been supported in part by the Bioinformatics, the Proteomics, and the Flow Cytometry Core Facilities at the Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We would also like to thank Dr. Paulo Rodriguez, and Dr. Gina DeNicola for their input in the analysis of the results.

Ethics Approval All animal experiments were performed under University of South Florida IACUC approval (R1762; R7429) and in accordance with the Guidelines for the Care and Use of Laboratory Animals manual published by the National Institutes of Health.

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.