Next-generation sequencing of three memory OT-I cell subsets from mice infected with vesicular stomatitis virus

Purpose: RNA-seq analysis of three memory OT-I cell subsets (from a Klrg1-Cre fate reporter mouse model) isolated from the spleen of C57BL/6 mice infected with vesicular stomatitis virus. The hypothesis tested in the present study was that KLRG1+ effector CD8 T lymphocytes differentiate into KLRG1- memory CD8 T lymphocytes and provide long-lasting immunity against infectious diseases and malignancies. Methods: Total RNA was obtained from FACS-purified OT-I cell subsets isolated from spleen 70 days post infection with ovalbumin-expressing vesicular stomatitis virus (VSV-OVA) (experiment 3). Results: Using RNA-seq technology, we performed genome-wide transcriptional profiling of three memory OT-I cells (KLRG1+ Reporter+, KLRG1- Reporter+ (exKLRG1) and KLRG1- Reporter-) and identified 36 genes differentially expressed (> 1.5-fold) between exKLRG1 and KLRG1- Reporter- memory OT-I cells, and 132 differentially expressed genes between exKLRG1 and KLRG1+ Reporter+ memory OT-I cells. We then confirmed the expression of 15 genes/molecules by qRT-PCR and/or flow cytometry. Conclusions: Our study represents the first fate mapping analysis of KLRG1+ effector OT-I cells, demonstrates that KLRG1+ effector OT-I cells differentiate into all memory T cell lineages thereby promoting protective immunity. RNA-seq also identified CX3CR1 as a marker of circulating exKLRG1 early memory OT-I cells. Total RNA was obtained from FACS-purified OT-I cell subsets isolated from spleen 70 days post infection with ovalbumin-expressing vesicular stomatitis virus (VSV-OVA).