Rescue of humoral immune response by adoptive transfer of ex vivo cultured and in vivo primed donor T cells.Figure 6A Schematics of adoptive transfer of in vitro activated WT CD45.1+ CD4+ T cells into CD45.2 Cd40lg-/- mice pre-conditioned or not with different lymphodepleting regimens and immune response studies. B Total counts of recipient CD3+ CD45.2+ cells in PB of mice NC (n=13) or pre-conditioned with 300 mg/kg CPA (n=19), 200 mg/kg CPA (n=6), anti-lymphocyte serum (ALS; n=5) or anti-CD4 antibody (n=6). Three independent experiments. Comparisons at day 3\4 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0009 and ****p<0.0001 in all comparisons). Mean ± SEM. C Total counts of engrafted donor CD45.1+ cells in PB of mice from (B). Three independent experiment. Comparisons at day 18\24 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. Mean ± SEM. D Analysis of the relationship between total counts of recipient CD3+ cells gated within CD45.2+ cells at day 3, 4 and engrafted donor CD45.1+ T cells at 18\24 days. This relationship was modeled with an asymptotic NLME model (Appendix Supplementary Statistical Methods), accounting for the different groups and multiple experiments. From the estimated model, a lower value of recipient CD3+ T cells corresponds to a higher value of engrafted donor CD4+ T cells (p=0.0006). E TNP-KLH specific IgG concentration in sera of mice from (B) collected before (day 35; pre) and after (day 49; post) the first boost (n=13 NC, 19 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4; three independent experiments) and at day 81 (pre) and 89 (post) for the second boost (n=4 NC, 4 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4). Sera from vaccinated WT (n=5) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. For early challenge data, comparisons were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. WT group, not indicated in the figure, is significantly different from all groups (p<0.0001 in all comparisons). Of note, regarding the comparisons between time-points, the groups CPA 300 and WT show a significant increase between pre and post values (p<0.0001 for both), while all other groups show a significant decrease between pre and post values (p<0.0001 for all except p=0.0022 for ANTI-CD4). Median ± IQR. F Total counts of recipient CD3+ cells gated within CD45.2+ cells after transfer of primed donor T cells in PB of mice treated with 300 mg/kg CPA (n=11), 200 mg/kg CPA (n=11) or NC (n=6). Longitudinal comparisons were performed by LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 4 (****p<0.0001 in all comparisons). Mean ± SEM. G Total counts of engrafted donor CD45.1+ cells in PB of mice from (F). Longitudinal comparisons were performed with an LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 25. At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 (****p<0.0001 for both). Mean ± SEM. H TNP-KLH specific IgG concentration in sera of mice from (F) collected before (day 36, pre) and after (day 50, post) the first boost (n=6 NC, 11 CPA 300, 11 CPA 200) and at day 212 (pre) and 219 (post) for the second boost (n=6 NC, 11 CPA 300, 9 CPA 200). Sera from vaccinated WT (n=11) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. Comparisons were performed with an LME model followed by an appropriate post-hoc analysis, separately for each challenge data (Appendix Supplementary Statistical Methods). Reported statistical comparisons refer to the overall difference among groups, for early challenge data (*p=0.0282 and **p=0.0037), while to differences between time-points within each group, for late challenge data (**p=0.0018 and ****p<0.0001 in both comparisons). Of note, in the late challenge data, no significant overall differences were observed among the groups. Median ± IQR.