Figure 94.
TDS assay for analysis of mouse blood CD8 T cells. BALB/c mice were vaccinated against HIV-1 gag used as a model Ag by prime with ChAd3-gag and boost with MVA-gag, as described [877]. TDS assay was performed on blood collected at day 3 and day 44 after boost. Blood from 3 mice was pooled to obtain enough cells for analysis. (A) Gating strategy. Example of gating of viable single CD8 T cells in 6 steps: 1) Single cells. Single cells having 2n≤ DNA content ≤4n were selected on the DNA-Area (A) versus (vs) DNA-Width (W) plot; 2) Time. Stable acquisition over time (seconds) was monitored on the time vs DNA-A plot and any events collected in case of pressure fluctuations were excluded; 3) Live cells. Live cells were selected on the FSC-A vs L/D eF780 plot; 4) “relaxed” FSC/SSC gate. A “relaxed” gate was used on the FSC-A vs SSC-A plot, to include highly activated and cycling lymphocytes [877]; 5) CD8 T cells. CD8 T cells were gated on the CD3 vs CD8 plot. 6) No “shadow” doublets. A few remaining doublets composed by one cell sitting on top of another (so called “shadow” doublets) were excluded as Ki-67int/− events having > 2n DNA content [878]. Numbers indicate cell percentages in the corresponding gate. This gating strategy was used as a base for CD8 T cell analysis. (B) gag-specific CD8 T cell frequency at day 3 and day 44 post-boost. Example of gag-tetr/ gag-pent plots, showing “gag-specific” and “not gag-specific” cell gates in untreated (left) and vaccinated (right) mice at day 3 (top) and day 44 (bottom) post-boost, as indicated. (C) Cell cycle of gag-specific and not gag-specific CD8 T cells from vaccinated mice at day 3 and day 44 post-boost. Cell cycle phases of “gag-specific” (left) and “not gag-specific” (right) cells from vaccinated mice at day 3 (top) and day 44 (bottom) post-boost were defined on DNA-A vs Ki-67 plot as follows: cells in G0 were identified as DNA 2n/ Ki-67− (bottom left quadrant); cells in G1 as DNA 2n/ Ki-67+ (upper left quadrant); cells in S-G2/M as DNA>2n/ Ki-67+ (TDS cells, top right quadrant). Ki-67 FMO controls are shown. Note that the “No shadow doublet” gate (Step 6 in A) cannot be applied to Ki-67 FMO samples. Unpublished data in relation to [877].