Redox index and capacity analysis (RICA) reveals NAD biology changes in T cell responses in vitro and ex vivo

The importance of NAD metabolism in T cell differentiation and function has gained attention in recent years. However, technical limitations impede the specific interrogation of NAD dynamics in living immune cells. In this report, we present the redox index and capacity analysis (RICA) assay, a novel technique 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 phenotypic subsets of immune cells. We validated this technique using metabolic modulators and used it to examine murine CD8 T cell subsets in vitro and ex vivo. Consistent with previous findings, we observed that metabolically active, 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 a vaccinia viral immunization model. Mitochondrial NAD content fluctuated considerably in response to fuel availability and metabolic modulators, even within short treatment timeframes. Finally, tumor localization and differentiation status dramatically affected the mitochondrial NAD pool but not the NADH:NAD+ ratio of adoptively transferred CD8 T cells in a B16 melanoma model. This study 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 an important and novel element of CD8 T cell biology.