Project description:In order to determine transcriptional differences between cells in the draining lymph node and those recently emigrating from the skin, we performed single cell RNA sequencing on KaedeGreen and KaedeRed CD8 T cells 12 days post infection with vaccinia-GP33. CD8+ cells were MACS enriched. Cells were stained with two tetramers (H2-Kb-B8R20-27 and H2-Db-GP33-41) conjugated to APC. Cells were then incubated with TotalSeq anti-APC (Biolegend, 408009)
Project description:Altered CD8 T cell differentiation and functional exhaustion prevent control of chronic virus infection and cancer. Yet, how fate commitment and exhaustion are determined and dynamically modulated throughout persistent infection are unclear. We compared the activation and differentiation of LCMV GP33-specific CD8 TCR transgenic cells (P14) primed at the onset versus in the midst of established persistent LCMV-Clone 13 viral infection. LCMV GP33-specific CD8 TCR transgenic (P14) cells were injected into naïve mice immediately infected with LCMV-Cl13 (Early priming) or into mice that had been infected 21 days earlier with LCMV-Cl13 (Late Priming). Sixty hours post-priming P14 cells were sorted from mice and subjected to RNA seq. We show early primed cells very rapidly exhibit a transcriptional profile of robust activation, effector differentiation and dysfunction, while late primed cells have increased expression of genes involved in memory differentiation and maintenance.
Project description:Exposure to inflammatory cytokines driven by bystander cytokines can have profound effects on the function of memory CD8 T cells. Here we transferred a 1-5X10^4 of P14 TCR-tg T cells (specific for gp33-41 of LCMV) on day -1 followed by infection with 2X10^5 PFU of LCMV Armstrong ip to generate memory P14 populations. Mice were rested for >50 days before further challenge. Mice containing memory P14 populations were either mock-infected with saline, infected with 2X10^6 Pichinde Virus ip (no cross-reactivity wtih LCMV gp33-41) or infected with 2X10^6 Pichinde Virus ip together with daily injections of 200 micrograms of anti-CD122 antibody (clone TM-b1). On day 4 following indicated treatments P14s were flow sorted and RNA was extracted from 1X10^6 cells using an RNeasy kit (Qiagen). Each group had 3 biological replicates. Transcriptomes were compared by DAVID analysis (p<0.01, Fold-Change > 1.5) 3 biological replicates per group. Groups included P14 from mock-infected mice, P14 from Pichinde virus infected mice and P14 from Pichinde virus and anti-cd122 treated mice.
Project description:Exposure to inflammatory cytokines driven by bystander cytokines can have profound effects on the function of memory CD8 T cells. Here we transferred a 1-5X10^4 of P14 TCR-tg T cells (specific for gp33-41 of LCMV) on day -1 followed by infection with 2X10^5 PFU of LCMV Armstrong ip to generate memory P14 populations. Mice were rested for >50 days before further challenge. Mice containing memory P14 populations were either mock-infected with saline, infected with 2X10^6 Pichinde Virus ip (no cross-reactivity wtih LCMV gp33-41) or infected with 2X10^6 Pichinde Virus ip together with daily injections of 200 micrograms of anti-CD122 antibody (clone TM-b1). On day 4 following indicated treatments P14s were flow sorted and RNA was extracted from 1X10^6 cells using an RNeasy kit (Qiagen). Each group had 3 biological replicates. Transcriptomes were compared by DAVID analysis (p<0.01, Fold-Change > 1.5)
Project description:Transcriptome profiling of lung tissues and lung CD8+ TRM during early phase of infection to investigate the mechanism of lung TRM cells during antigen re-exposure within 48hours. After P14 T cell transfer, PR8 infection would not induce P14 TRM cells, while PR8-gp33 would. Comparing the gene expression of lung tissues after LCMV Armstrong challenge at day 30 after PR8 or PR8-gp33 infection would help us understand the function of lung CD8+ TRM cells. Sorted TRM cells from mice challenged with LCMV Armstrong at day 30 after PR8-gp33 infection would reveal the activation mechanism of TRM cells.