Single cell gene expression profile of WT and Opa1 knockout OT-I T cells

The goal of our study was to compare the gene expression of both naïve and activated T cells deficient in the mitochondrial protein Optic atrophy 1 (Opa1) using single cell RNA sequencing. A growing amount of evidence has begun to illustrate that the requirements driving effector- and memory-destined CD8 T cells during an infection are not shared with adjuvanted vaccine responses. Short-lived effector cells are characterized metabolically by a highly glycolytic state driving energy and biomass production whereas vaccine-elicited T cells can meet these requirements through mitochondrial metabolism. Here we show that the mitochondrial protein Optic atrophy 1 (Opa1) is critical for rapidly dividing CD8 T cells in vivo. This requirement, contrary to prior published data, is most pronounced in effector CD8 T cells. More specifically, Opa1 supports proper cell cycle initiation, progression, and supports the viability and survival of CD8 T cells during immune responses to both infections and vaccine challenges Overall design: Opa1fl/fl mice were crossed to OT1 TCR transgenic mice (ovalbumin-specific) expressing Cre under the distal Lck (dLck) promoter to produce OT1 x Opa1fl/fl Cre+ (Opa1-/- OT1) mice. Congenically distinct WT (CD45.1/2) and Opa1-/- (CD45.1/1) OT-I T cells were co-transferred into WT B6 recipients (CD45.2/2) and challenged with ActA-deficient LM (LM.Ova.ActA). On day 5, T cells were isolated and pooled from 4 mice prior to cell sorting of CD45.1+ CD8+ CD19- cells. Naïve OT-I T cells cells from unimmunized WT or Opa1-/- were isolated using Biolegend CD8 isolation kit. Naïve and day 5 activated cells for each genotype were pooled at a 1:1 ratio prior to sequencing, resulting in two samples, one WT and one Opa1 -/-, which were then captured and processed for single cell squencing.