Supplementary MaterialsSupplementary File. into other memory T Neferine cell populations, we next assessed their capacity for homeostatic proliferation and long-term survival. Through three-way comparisons, we found that terminal-Tem expressed lower levels of signature T cell proliferation genes (52) as well as memory signature genes, but elevated levels of characteristic terminal effector genes relative to Tem and Tcm (Fig. 2expressing OVA. We found that terminal-Tem conferred comparative protection compared to Tem despite limited recall growth (Fig. 2challenge, likely due to constitutively elevated expression of granzymes, perforin, and important migratory molecules (Figs. 1and ?and2and = 3 to 4 4 per group; * 0.05, *** 0.005. Graphs show mean SEM, Neferine symbols represent an individual mouse (or OT-I cells at 1:1 ratio into congenically unique recipient mice subsequently infected with LCMV-OVA (Fig. 4heterozygosity resulted in a Neferine reduced frequency of standard Tem (3, 56) (Fig. 4or memory populations given the input ratio of transferred cells was 1:1. With this analysis, we found that heterozygosity resulted in enhanced accumulation of total splenic OT-I cells at a memory timepoint compared to OT-I cells (Fig. 4heterozygosity resulted in an approximately threefold increase in Tem (gray bars) but nearly a complete loss of terminal-Tem (reddish bars, Fig. 4and or (control) shRNA encoding retroviruses, mixed 1:1, and transferred into recipient mice subsequently infected with LCMV. Frequency of splenic memory CD8 T cell subsets on day 25 of contamination. (P14 and P14 cells were mixed 1:1 and transferred to recipient mice subsequently infected with LCMV. Quantification of the frequency of splenic memory CD8 T cell subsets on day 35 RPS6KA6 of contamination (P14 and P14 cells were mixed 1:1 and transferred to recipient mice subsequently infected with LCMV. Frequency of splenic memory CD8 T cell subsets on day 30 of contamination (= 4 to 6 6 per group ( 0.01, *** 0.005. Graphs show mean SEM, and symbols represent an individual mouse. A distinguishing characteristic of terminal-Tem included elevated expression levels of Blimp1 compared to Tem and Tcm (Fig. 3RNAi resulted in a reduced percentage of CD62Llo standard Tem (Fig. 4resulted in a greater accumulation of total P14 cells in the spleen compared to control P14 cells at a memory timepoint (Fig. 4and expression in the terminal-Tem populace, knockdown did not impair the overall formation of terminal-Tem (reddish bars) and actually enhanced the accumulation of Tem (gray bars) in the spleen (Fig. 4or resulted in a reduced frequency of Tcm (3, 25C28) (Fig. 4 and deficiency resulted in the enhanced frequency of Tem and terminal-Tem, whereas deficiency resulted in a Neferine lower frequency of Tem but increased frequency of terminal-Tem. Converse to the effects of diminished expression of T-bet or Blimp1, the relative large quantity of donor cells revealed conditional deletion of or resulted in a loss of total memory P14 cells, including fewer Tcm Neferine (blue bars) compared to control P14 cells (Fig. 4 and also resulted in a reduced large quantity of CD127hiCD62Llo Tem (Fig. 4 and and and and and 6 donors ( 0.05, *** 0.005. Graphs show mean SEM, and symbols represent an individual donor. NES, normalized enrichment score; FDR, false discovery rate. As there is often a disconnect between markers used to distinguish murine memory populations and human populations, we utilized single-cell RNA-seq analysis for unbiased identification of human terminal-Tem. We performed single-cell RNA-seq analysis on PBMCs from 10 healthy individuals (Fig. 5and and as well as relatively low levels of (Fig. 5 contamination (53). We found that terminal-Tem and redefined CD127hiCD62Llo Tem conferred comparative protection to contamination, but terminal-Tem conferred the most strong protection on a per cell basis. Although terminal-Tem displayed higher expression of cytolytic genes and up-regulation of granzymes, they also exhibited limited recall proliferation, decreased lymphoid tissue presence, and impaired cytokine production compared to Tem. Conversely, Tcm had been most protecting inside a tumor vaccine model in comparison to both terminal-Tem and Tem, likely because of a sophisticated lymph.