Skip to main content
. 2013 Jul 1;210(7):1463–1479. doi: 10.1084/jem.20112495

Figure 7.

Figure 7.

N-ras–deficient CD8+ T lymphocytes have an intrinsic deficiency in intracellular expression of Eomes transcription factor during the primary and secondary antiviral responses in vivo and in vitro. (A) Around 200 N-ras+/+ or N-ras−/− OT-I cells, either treated or not with rapamycin, were cotransferred as in Figs. 5 (A and B) and 6 (A and B) into the same N-ras+/+ recipients, which were then infected with rVACV-OVA and treated or not with rapamycin as in Fig. 6 (A and B) for 8 d. CD45.1+ OT-I cells were stained for intracellular T-bet and Eomes expression at 7 d p.i. and discriminated as WT or N-ras−/− according to CD45.2 expression (n = 3, two experiments). The graphs below represent mean ± SEM. (B) To study the primary response, WT and N-ras−/− mice were infected as in Fig. 2 A and treated or not with rapamycin as before. On day 7 p.i., Eomes expression was determined by FACS in CD8+ T lymphocytes and analyzed with respect to their staining with MHC H-2KbB8R pentamer (n = 3, two experiments). (C) For measuring the secondary response, WT and N-ras−/− mice were vaccinated with peptide-pulsed mDC and then infected as in Figs. 4 A and 6 (E and F). On day 5 p.i., Eomes expression and staining with H-2KbB8R pentamer was determined by FACS in CD8+ T lymphocytes (n = 3). The graphs below in B and C represent mean ± SEM Eomes expression in H-2KbB8R pentamer-positive CD8+ T lymphocytes. (D and E) Purified CD45.1+ N-ras+/+ and N-ras−/− OT-I CD8+ T lymphocytes were stimulated in vitro for 72 h at an E/T ratio of 1/6 with CD45.1− N-ras+/+ splenocytes loaded or not with nonlimiting OVA peptide concentration (10−9 M; D), or with OVA and the weak agonists Q4R7 and Q4H7 peptides at concentrations that induced either the maximal (EC100) or half-maximal (EC50) CD69 expression (as determined in Fig. 1 C; E). Cultures were then stained for CD45.1 and CD25 for gating and for transcription factors T-bet and Eomes and analyzed by FACS (three experiments). Results in E are expressed as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.