Aging promotes acquisition of naive-like CD8+ memory T cell traits and enhanced functionalities

Chemokine Signatures Of Pathogen-Specific T Cells I: Effector T Cells

“…In humans, a reported preferential association with the content of cytolytic granules (GzmA, perforin, granulysin) [17,64] contrasts with the identification of a unique subcelluar CCL5 compartment [65], and the frequent use of T cell clones or blasts, the differential regulation of cytolytic effector gene and protein expression in primary murine CTL [44,[66][67][68], and the previously reported absence of constitutive CCL5 expression by mouse CD8 + TMP in particular [69,70] further complicate resolution of this issue. Our direct ex vivo FC analyses of LCMVspecific CD8 + TE now demonstrate a clear association of CCL5 and GzmB expression while GzmA, as reported previously by us (and also similar to influenza-specific CD8 + TE [34,44]), was expressed by only ~60% of the CD8 + TE population ( Fig.4D). These observations indicate that CCL5 co-localization studies in murine CD8 + TE should be extended beyond the visualization of GzmA.…”

“…In order to refine these analyses within the context of infectious diseases and to define the complete range of chemokines produced by pathogen-specific CD8 + TE, we first employed the established "p14 chimera" system to quantify chemokine mRNA and protein expression by virus-specific CD8 + TE with a combination of gene arrays and FC-based assays [33,34]. In brief, p14 chimeras were generated by transducing congenic B6 mice with a trace population of naïve TCR transgenic CD8 + T cells (p14 TN) specific for the dominant LCMV-GP33-41 determinant; after challenge with LCMV, p14 TE populations rapidly differentiate, expand and contribute to efficient virus control before contracting and developing into p14 memory T cells (TM) ~6 weeks later [34][35][36]. At the peak of the effector phase (d8), p14 TE were purified, RNA was extracted either immediately or after a 3h in vitro TCR stimulation, and processed for gene array hybridization.…”

“…To generate p14 chimeras, naïve p14 T cells were enriched by negative selection and ~5x10 4 cells were transferred i.v. into sex-matched B6 recipients that were challenged 24h later with LCMV [34]; to assure reliable detection of cytolytic effector molecules [109], additional p14 chimeras were generated with lower numbers (~10 3 ) of p14 cells.…”

“…For microarray analyses (Figs.1A, S2 & S3A), p14 TE were purified (>99%) from individual p14 chimeras on d8 after LCMV challenge by sequential magnetic and fluorescence activated cell sorting as Cancer Center according to standard protocols; further experimental and analytical details are provided in ref. [34], and the data can be retrieved from the GEO repository accession number GSE143632. Note that the files deposited therein also contain data for ex vivo p14 TE previously uploaded in the context of a related study (GSE38462) as well as data on ex vivo and aCD3/aCD28-stimulated p14 TM to be discussed in a separate manuscript in preparation (though all ex vivo and stimulated p14 TE and TM data were generated in the same set of experiments).…”

“…and FasL expression as well as degranulation were quantified as described [34], and perforin stains were performed with antibody clone S16009B (Biolegend, not shown).…”