Figure 8. Generation of terminally differentiated CD8⁺ T cells occurs after cessation of proliferation in the splenic RP.

D5.5 post CPS vaccinated WT mice were first injected i.p. with EdU 2 hr prior to sacrifice, then injected i.v. with 4 μg of anti-CD8-FITC antibody and sacrificed within 2–3 min. The differential localization and fractional distribution of F1–F4 stages in EdU⁻ and EdU⁺ (A) for total CD8⁺ T cells and (C) tgd057-specific CD8⁺ T cells are shown. The rate of EdU-positivity as a function of differentiation stage (B, D left panels) or differentiation stage and i.v. anti-CD8 accessibility (B, D right panels) are shown for total CD8⁺ T cells (B) and tgd057-specific CD8⁺ T cells (D). Immunofluorescence images showing D5.5 splenic localization of EdU⁺ and double-positive for IV α-CD8 and ex vivo α-CD8 antibodies (E), and KLRG1 (F) staining. Splenic compartments are represented as follows: dashed line, MZ border; RP–red pulp; BC–bridging channel; MZ–marginal zone. Sections were stained for IV α-CD8 (green, CD8), ex vivo α-CD8 (red, EX CD8), EdU (blue), α-KLRG1 (white). Key to symbols: Yellow arrows: EdU⁺ IV CD8⁺ EV CD8⁺, Pink Arrows: EdU⁺ KLRG1⁺ IV CD8⁺ EV CD8⁺. Expression of CXCR3 in proliferating and non-proliferating CD8⁺ T cells (G). Data are from 2 experiments of 3–5 CPS vaccinated mice each. Immunofluorescence represents more than six sections per mouse taken from three mice. Mean ± SEM. Student's t-test or ANOVA analysis was done Holms-Sidak post hoc test, *p < 0.01, **p < 0.001 and ***p < 0.0001. Refer to Figure 8—figure supplement 1 for IHC image of EdU⁺ CD8⁺ T cells in splenic bridging channels.

DOI: http://dx.doi.org/10.7554/eLife.09017.013

Figure 8—figure supplement 1. CD8⁺ T cells in bridging channels are proliferating.

D4.5 post CPS vaccinated WT mice were injected i.p. with EdU 2 hr prior to sacrifice and then injected i.v. with 4 μg anti-CD8-FITC antibody and sacrificed within 2–3 min. 6 μm splenic sections were stained for α-CD8α antibody and developed for EdU. Immunofluorescence showing localization of double stained CD8α⁺ T cells that are proliferating (EdU⁺, yellow arrows) or non-proliferating (Edu⁻, blue arrows) within the bridging channel and MZ. Enlarge inset depicts proliferating and non-proliferating double stained CD8⁺ T cells in the bridging channel. Splenic compartments are represented as follows: dashed lines–MZ border; orange lines–bridging channel; RP–red pulp; BC–bridging channel; MZ–marginal zone. Sections were stained for IV CD8 (green, CD8), CD8 ex vivo (red, EX CD8), EdU (blue). Key to symbols: Yellow arrows: EdU⁺ CD8⁺ T cells, Blue arrows: EdU⁻ CD8⁺ T cells. Data are representative of more than 4 sections per mouse taken from three mice.

DOI: http://dx.doi.org/10.7554/eLife.09017.014