Antigen availability determines CD8+ T cell-dendritic cell interaction kinetics and T cell fate decisions.. Mus musculus

This experiment compares the transciptional changes in antigen specific murine CD8 T cells (P14 T cells) after exposure in vivo to dendritic cells (DC) pulsed with low dose cognate peptide (1uM KAVYNFATC), high dose cognate peptide (100uM KAVYNFATC) or no antigen. Splenic dendritic cells were freshly isolated, peptide pulsed, washed and then adoptively transferred s.c. to the right footpad of C57BL/6 hosts. After 18h, freshly isolated P14 CD8 T cells were labelled with CMFDA and adoptively transferred iv. Two hours after T cell transfer, anti-L selectin antibody was given iv. At 12 and 24 hours, recipients were sacrificed and The right popliteal LN was harvested at 12 or 24h post T cell transfer and a single cell suspension was created and stained with PE CD4, B220 and CD19 (dump channel). Cells were then sorted on a FacsARIA for being non-doublets, CMFDA positive and dump channel negative. Overall design: This experiment compares the transciptional changes in antigen specific murine CD8 T cells (P14 T cells) after exposure in vivo to dendritic cells (DC) pulsed with low dose cognate peptide (1uM KAVYNFATC), high dose cognate peptide (100uM KAVYNFATC) or no antigen. Splenic dendritic cells were freshly isolated, peptide pulsed, washed and then adoptively transferred s.c. to the right footpad of C57BL/6 hosts. After 18h, freshly isolated P14 CD8 T cells were labelled with CMFDA and adoptively transferred iv. Two hours after T cell transfer, anti-L selectin antibody was given iv. At 12 and 24 hours, recipients were sacrificed and The right popliteal LN was harvested at 12 or 24h post T cell transfer and a single cell suspension was created and stained with PE CD4, B220 and CD19 (dump channel). Cells were then sorted on a FacsARIA for being non-doublets, CMFDA positive and dump channel negative. The experiment was conducted for 39 samples out of which 35 passsed transcriptional quality control tests. The phenotypic distribution for the 35 samples includes: (1) high dose (100uM KAVYNFATC ) cognate peptide pulsed samples harvested at 12h post T cell transfer: 6 biological replicates (2) high dose (100uM KAVYNFATC ) cognate peptide pulsed samples harvested at 24h post T cell transfer: 7 biological replicates (3) low dose (1uM KAVYNFATC) cognate peptide pulsed samples harvested at 12h post T cell transfer: 6 biological replicates (4) low dose (1uM KAVYNFATC) cognate peptide pulsed samples harvested at 24h post T cell transfer: 9 biological replicates (5) no antigen pulsed samples harvested at 12h post T cell transfer: 3 biological replicates (6) no antigen pulsed samples harvested at 24h post T cell transfer: 4 biological replicates.