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. 2023 Mar 14;11(3):e006292. doi: 10.1136/jitc-2022-006292

Figure 3.

Figure 3

Rapamycin promotes selective expansion of CAR T cells. (A) PBMCs were transduced with UB-VV100 at an MOI of 5 for 3 days before vector removal. Cells were transferred to wells containing the indicated concentration of rapamycin and cultured for an additional 18 days. On study day 21, CAR+ and CAR-negative T-cell expansion was enumerated by flow cytometry. n=5, pooled results from two independent experiments using three separate PBMC donors. Data points indicate mean±1 SEM. (B) PBMCs treated as described (A) at the 10 nM rapamycin concentration were analyzed by flow cytometry to determine the absolute CD4 and CD8 CAR T-cell counts. n=6 per group per time point, pooled results of two independent experiments using three PBMC donors. Axis is split to allow separation of CD4 and CD8 T cells to be seen on the same scale. Symbols (*) indicate statistical significance for two-way ANOVA, main column analysis of effect of rapamycin treatment on cell expansion or enrichment over time series. Data points indicate mean±1 SEM; some error bars not visible due to eclipse by data symbol. PBMCs were transduced with MOI=5 with UB-VV100 and expanded for 18 days with rapamycin (study day 21 after transduction). T cells were assessed by flow cytometry for (C) representative flow staining of surface FMC63 detection and (D) memory phenotype. Representative plots show a single donor. CAR+ T cells are overlaid in red over total T-cell population. Memory phenotype was quantified for CD8+ T cells for day 7 no rapamycin, day 21 no rapamycin, day 21+10 nM rapamycin, in both the CAR+ and CAR-negative fractions. Naïve (CCR7+CD45RA+). Symbol (*) indicates p value of < 0.05 for two-way ANOVA, Tukey’s multiple comparison’s test for comparing the ‘naïve’ population of the CAR+ fraction to the naïve population of the CAR-negative fraction on study day 21+10 nM rapamycin. n=6 from replicate measurements of three unique PBMC donors per condition, results from one experiment. ANOVA, analysis of variance; CAR, chimeric antigen receptor; CM, central memory (CCR7+CD45RA−); EM, effector memory (CCR7−CD45RA+); EMRA, effector memory re-expressing CD45RA (CCR7−CD45RA+); MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell.