CD69+ memory T lymphocytes of the bone marrow and spleen express the signature transcripts of tissue-resident memory T lymphocytes

To understand tissue resident features of memory CD4+ and CD8+ T lymphocytes of the bone marrow and/or spleen according to expressing or not the tissue retention marker CD69, we performed whole transcriptome profiling of ex vivo antigen-specific CD69+ and CD69- memory CD4+ T cells isolated from bone marrow and spleen, and ex vivo CD69+ and CD69- memory CD8+ T cells isolated from bone marrow. For CD4 analysis, 8-week-old C57BL/6 mice were primed at day 0 with LCMV.GP61-80-NP-MSA + poly(I:C) and immunized again at day 14 with LCMV.GP61-80 + poly(I:C). Sixty days after the last immunization mice were sacrificed and LCMV.GP66–77-specific CD69+ and CD69- memory CD4+ T cells were isolated from BM and spleen by FACS. The whole transcriptome profiling of purified cells was performed by RNA-seq. For CD8 analysis, single cell suspensions were prepared from bone marrow of 8-week old untreated C57BL/6 male mice. CD8+ T cells were pre-enriched with anti-CD8a microbeads and the CD69+ and CD69- subpopulations of memory CD8+ T cells were then FACS purified. The whole transcriptome profiling of sorted cells was analyzed by microarray analysis.