Heterogenity of memory CD4 T cells

Analysis of single cell gene transcription by mouse influenza virus specific CD4 T cells examined 40 days after infection. Overall design: 4 TRACE reporter mice, that enable identification of memory T cells by EYFP expression, were infected with influenza A virus (WSN) intranasally. After 40 days, the mice were injected with fluorescently labelled anti-CD45-PE to enable exclusion of memory CD4 T cells within the blood. Lung and spleen CD4 cells were isolated using magnetic beads and then co-cultured with bone marrow dendritic cells presenting influenza virus antigens. After 4 hours, the cells were stained with surface antibodies and AbSeq tagged anti-ICOS and anti-PD1 antibodies. EYFP+ memory CD4 T cells from the spleen and lung and naive (EYDP negative CD44low) CD4 from the spleen were FACS sorted and analysed by BD Rhapsody scRNAseq for gene expression (targeted) and CDR3 sequence of T cell receptors.