Redox Index and Capacity Analysis (RICA) sheds light on NAD biology in tumor-infiltrating lymphocytes 2251652

Abstract

Abstract Introduction NAD metabolism is critical for T cell function; however, technical limitations impede interrogation of NAD biology in living immune cells. This is especially true for small, heterogenous cell populations, such as tumor-infiltrating lymphocytes (TILs). We developed the redox index and capacity analysis (RICA) assay, a novel method for measuring mitochondrial NAD content and redox balance. The RICA assay is a flow cytometry-based technique that uses NADH autofluorescence and mitochondrial inhibitors to assess NAD within specific subsets of immune cells. We used this to reveal nuances in NAD biology in T cells from in vitro and ex vivo contexts. Methods We generated CD8 and CD4 T cell subsets in vitro using standard culture conditions. We also assessed antigen-specific CD8 T cell responses ex vivo using an OT-I adoptive transfer system in the context of a vaccinia viral immunization and in a B16 melanoma model. T cell subsets were analyzed using the RICA assay, which measures NADH autofluorescence in specific cell populations at baseline and after rotenone or FCCP treatment to induce maximum and minimum mitochondrial NADH signal. Results We found that effector-like cells had a higher mitochondrial NADH:NAD+ ratio than quiescent cells. We discovered that cells with greater differentiation potential often possessed a larger pool of mitochondrial NAD than terminally differentiated cells in vitro and in our viral model. Tumor localization and differentiation status dramatically affected the mitochondrial NAD pool, but not the NADH:NAD+ ratio, of adoptively transferred CD8 T cells in our melanoma model. Conclusion Our work establishes a tool for evaluating mitochondrial NAD biology in living immune cells at a greater level of detail than previously possible. It also highlights dynamic changes in mitochondrial NAD pool size as a novel element of CD8 T cell biology, which holds significance for improving the efficacy of CD8 T cell-based immunotherapies. Funding Source NIH grants CA257954 and AI115015 to E.J.U., P30CA023108 to Dartmouth Cancer Center Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)