136 A metabolic assay to assess human T-cell activation in squamous cell carcinoma

Background: T cells exhibit distinct metabolic profiles based on their activation state. Naïve T cells are metabolically quiescent and use oxidative phosphorylation (OXPHOS) as their primary pathway of ATP production. Upon T cell antigen receptor (TCR)-mediated recognition of antigens and costimulatory signals, T cells become activated and preferentially utilize glycolytic and glutaminolytic metabolism. Methods: An Agilent Seahorse XFp Analyzer was used to assess T cell activation in vitro. T cells are exposed to anti-CD3/CD28 immunogenic beads. Then, extracellular acidification is measured in real time as a readout of glycolytic activity. Basic assay parameters, including the immunogenic bead to cell ratio and timing, were established using Jurkat T lymphocytes. CD4 and CD8 T cells were isolated from homogenized squamous cell carcinoma (SCC) tumor samples obtained from patients (n=3) via flow cytometry using a PE-conjugated anti-human α/β T cell receptor antibody. Results: Rapid induction of extracellular acidification was observed 5 minutes after stimulation of 300,000 naïve Jurkat T cells upon exposure to anti-CD3/CD28 immunogenic beads in a 2:1 bead to cell ratio. These effects persisted for the duration of the 2-hour study. CD4 and CD8 T cells obtained from patient samples exhibited similar activation kinetics upon stimulation with anti-CD3/CD28 immunogenic beads. Assessment of the activation potential of T cells obtained from patient samples can provide insight into a patients clinical status given that exhausted T cells are correlated with a poorer prognosis. In addition, the non-destructive nature of the assay permits further correlative studies using patient-derived activated T-cells. Conclusion: This is the first report of a rapid kinetic assay to assess activation of SCC patient-derived T cells. This information could potentially be used to obtain a comprehensive clinical profile of the patients tumor microenvironment to tailor patient-specific treatment regimens.