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. 2015 Aug 5;4:e09017. doi: 10.7554/eLife.09017

Figure 1. CD8+ T cell proliferative response is IL-12 independent while effector cell differentiation is IL-12 dependent.

(A) Absolute numbers of tgd057-specific CD8+ T cells in spleen after CPS vaccination in wild-type (WT) and IL-12p35−/− mice. (B) tgd057-specific CD8+ T cells were analyzed for CD44, CD62L and KLRG1 cell surface expression. Pie charts represent frequency averages of F1 (CD62L+ KLRG1−), F2 (CD62L−, KLRG1−), F3 (CD62L−, KLRG1+), and F4 (CD62L+, KLRG1+) subsets of tgd057-specific CD8+ T cells from WT and IL-12p35−/− spleens D0–D7 post CPS vaccination. Data shown include only CD44hi cells on D4–D7 and include all tgd057-specific CD8+ T cells on D0–D3. (C) Absolute numbers of tgd057-specific CD8+ T cells in peritoneal exudate cells (PECs) of WT and IL-12p35−/− mice after CPS vaccination. Cells were characterized based on tgd057-specific CD8+ T cells. (D) Average of frequencies of F1 (CD62L+ KLRG1−), F2 (CD62L−, KLRG1−), F3 (CD62L−, KLRG1+), and F4 (CD62L+, KLRG1+) subsets of tgd057-specific CD8+ T cells in PECs D0–D7. Data represent 5 independent experiments with 3–5 mice per group per experiment. Data were analyzed using two-way ANOVA and (A) Holms-Sidak or (C) Bonferroni post-hoc tests; *p < 0.05. See Figure 1—figure supplements 1, 2 for the methodology employed to enumerate absolute numbers of tgd057-specific CD8+ T cells.

DOI: http://dx.doi.org/10.7554/eLife.09017.003

Figure 1.

Figure 1—figure supplement 1. Tetramer-based enrichment increases detection of epitope-specific naïve CD8+ T cells in spleen.

Figure 1—figure supplement 1.

Samples from pre- and post-tetramer-based enrichment of D4 and D5 post CPS vaccinated spleens, showing a 2-log increase in detection of tgd057:H-2Kb+ specific CD8+ T cells. Data represent 5 independent experiments with 3–5 mice per group per experiment.
DOI: http://dx.doi.org/10.7554/eLife.09017.004
Figure 1—figure supplement 2. Accurate determination of endogenous naïve tgd057:H-2Kb-specific CD8+ T cells.

Figure 1—figure supplement 2.

To quantify absolute numbers of endogenous tgd057-specific CD8+ T cells, naïve B6 spleens were pulsed with 50 naïve monoclonal Thy1.1+ SCNT CD8+ T cells. Representative flow cytometry plots of tetramer binding cells (doubly stained with APC and PE-labeled tetramers) partitioned according to Thy1.1 staining are shown. Absolute numbers of naïve endogenous tgd057-specific CD8+ T cells in a naïve spleen was calculated based on the ratio of endogenous CD8+ T cells to the pulsed Thy1.1+ cells. Total numbers of tgd057-specific CD8+ T cells in a naïve spleen were found to be 2093 ± 273 in WT hosts and 1200 ± 138 cells in IL-12p35−/− hosts. Data represent 5 independent experiments with 3–5 mice per group per experiment. Scatter plots are mean ± SEM, data were analyzed using paired t test and Holms-Sidak post-hoc test; **p ≤ 0.01.
DOI: http://dx.doi.org/10.7554/eLife.09017.005