Effects of <i>in vivo</i> Ag85B stimulation and 4-1BB ligation on CD4⁺ and CD8⁺ T cells of NP-mice.

<p>Spleen cells from NP-mice were adoptively transferred into C57BL/6Ly5.1 recipient mice accordingly to the scheme shown in panel A. 10⁷ splenocytes from NP-mice (CD45.2⁺ cells) were adoptively transferred i.v. into C57BL/6Ly5.1 recipient mice (expressing CD45.1 leukocyte antigen). On p.i. day 1, mice were inoculated s.c. with 10 µg of recombinant Ag85B protein and then i.p. with 150 µg agonistic anti-mouse 4-1BB mAb or with 150 µg rat IgG2a isotype control mAb. On p.i. day 3, mice were again injected with 150 µg agonistic anti-mouse 4-1BB mAb or with 150 µg rat IgG2a isotype control Ab. As controls, a group of mice adoptively transferred with splenocytes from NP-mice was treated with neither antigen Ag85B nor mAbs, and two groups of mice, not inoculated with splenocytes from NP-mice, received only the treatment with Ag85B and mAbs as descrived above. On p.i. day 10, fresh spleen cell recovered from mice adoptively transferred with NP-mice and treated <i>in vivo</i> with Ag85B and mAb were labelled with CD45.2-FITC, CD4-PE, CD8-PerCP, 4-1BB-PE, CD4-PerCPCy5.5 and analyzed by flow cytometry. In panel B, the percentage indicates the frequency of CD4⁺ and CD8⁺ T cells originating from NP-mice and in panel C the percentage of those CD4⁺ and CD8⁺ T cells expressing 4-1BB. Plots, gated on CD45.2⁺ cells in the lymphocyte-gate, are representative of 1 out of 3 mice per group. Spleen cells of C57BL/6Ly5.1 were then used for antigenic recall <i>ex vivo</i> experiments. Cells were re-stimulated <i>ex vivo</i> with Ag85B protein (5 µg/ml). In panel D spleen cells, labelled with CFSE at day 0 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a>, after 4 days of culture were stained with CD4-PE, CD8-PerCP and CD45.2-APC and analyzed by flow cytometry. The percentage indicates the number of replicating CD4⁺CD45.2⁺or CD8⁺CD45.2⁺ T cells. In panel E the frequency of IFN-γ-producing CD4⁺CD45.2⁺ T cells were measured by FACS analysis after 4 days of culture and an overnight incubation with brefeldin A on cells labelled with CD4-PerCP-Cy5.5, CD45.2-FITC and then intracellular stained with PE anti-IFN-γ Ab. Cells were then analyzed by flow cytometry. In panel F the amount of IFN-γ released in the culture supernatants after 4 days was detected by a commercial ELISA kit. The data has been reported as the mean of 3 individual determinations for each group. Error bars indicate SEM. The level of statistical significance for differences between unstimulated and <i>ex vivo</i> antigen-stimulated spleen cells of mice adoptively transferred with cells from NP-mice and treated <i>in vivo</i> with Ag85B protein in the presence of IgG2a control Ab or agonistic anti-4-1BB mAb were determined by a two-tailed Student <i>t</i> test (*, p<0.05; **, p<0.01).