Article Figures & Data
Figures
- Figure 1.
Anti-BCMA CAR T cytolytic activity and cytokine production increased with lenalidomide in a concentration-dependent manner. Anti-BCMA CAR T materials from 3 healthy donors and 1 patient donor were assessed for cytokine production using 2 multiple myeloma cell lines. Cytolytic activity (A and C) and cytokine production were measured after 24 hours (B and D) against OPM-2 (A and B) and RPMI-8226 (C and D). Cultures were incubated at a ratio of 0.3:1 (effector to target) for cytolytic activity and 1:1 for cytokine production. Data were normalized to DMSO vehicle control; error bars, SEM. For all functional assessments, dose response modeling indicated a significant effect for lenalidomide for each donor and across all donors (P < 0.001), except healthy donors for RPMI-8226 cytolytic assay. *, P < 0.05 for lenalidomide across each donor.
- Figure 2.
Anti-BCMA CAR T cytokine production was increased by lenalidomide (Len) within 24 hours and across a range of stimulation intensities. Analysis of CD25 and intracellular cytokine levels (left, white bars indicate baseline effects of bead stimulation) for healthy CAR T donors after 24 hours of BCMA bead stimulation (gated on transduced live CD3+ CAR+) for CD4+ (A) and CD8+ (B) subsets. Gray bars demonstrate relative change for (Len) compared with vehicle alone. C and D, Analysis of effector cytokine production following CAR-specific stimulation on 50 μg BCMA and 50 μg PD-L1 beads for 24 hours in the presence of 1 μmol/L Len. *, P < 0.05 effect of Len for each stimulation condition.
- Figure 3.
Anti-BCMA CAR T cell count, cytokine production, and activation were increased by lenalidomide after repeated stimulation in vitro. A, Analysis of cell counts following serial stimulation in an MM1.S cell line in the presence or absence of 0.1 μmol/L Len. Data represent population doublings across 5 donors; error bars, SDs across 3 technical replicates. Linear mixed effects modeling indicated a significant effect for Len for each donor across time (P = 2.8 × 10−8). B–D, Analysis of bulk cytokine production 24 hours following serial stimulation replating at the indicated time points for 5 separate donors. Cytokine production was normalized to cell number at each reset to account for differences in cell replating density; error bars, SDs. Linear-mixed models indicated a significant effect for Len across donors and time points for IFNγ (P < 2.9 × 10−10), IL2 (P = 1.3 × 10−13), and TNFα (P = 1.2 × 10−9). *, P < 0.05 compared with vehicle for each donor.
- Figure 4.
Lenalidomide reduced functional exhaustion and altered surface phenotype of anti-BCMA CAR T cells. Cells were treated for 7 days on 50 μg BCMA-coated beads in the presence or absence of 1 μmol/L Len. A–D, Representative healthy donor–derived, freshly thawed anti-BCMA CAR T cells [vehicle (Veh), Len] or CAR T cells prestimulated with 7 days of BCMA bead stimulation and then cultured with RPMI-8226 cells to measure cytolytic activity (over 7 days; A) and cytokine production (24 hours; B–D). Percentage killing was normalized to anti-BCMA CAR T cells prestimulated on beads in the presence of vehicle. Prestimulated CAR T cells showed decreased cytolytic activity (P = 2.1 × 10−4) and cytokine production (P = 0.03 for IFNγ) compared with freshly thawed anti-BCMA CAR T cells. Len during the prestimulation period increased cytolytic function (P = 0.04). Significance was determined using t tests from linear regression coefficients. Three anti-BCMA CAR T donors (each column) were assessed for overall viability (E) and cell count and by flow cytometry (F) for median fluorescence intensity (MFI; CD25 and TIM3) or percentage positive PD-1 and LAG3 on the surface of T cell markers in CD4+ CAR+ and CD8+ CAR+ subsets (gated on live CD3+ cells). Values shown are percentage baseline (Veh) MFI, viability, or count. *, P < 0.05.
- Figure 5.
Anti-BCMA CAR T RNA-seq and ATAC-seq profiles were altered by lenalidomide after short- and long-term stimulation. A and C, Principal component analysis of expression (A; RNA-seq) and chromatin accessibility peaks (C; ATAC-seq). B and D, Volcano plots of differentially expressed genes (B) or peaks ± Len at 24 hours and 7 days (D). Directionality and significance of expression changes in selected, enriched biological pathways at 24 hours (E) and 7 days (F) in CAR T cells ± 1 μmol/L Len. G, RNA expression compared with chromatin accessibility changes for selected T cell loci. H, Top enriched motif predictions in ATAC-seq loci with increased accessibility along with their enrichment significance and prevalence at 7 days ± 1 μmol/L Len. FC, fold change; Ox, oxidative.
- Figure 6.
In vivo efficacy of subcurative dose of anti-BCMA CAR T and blood anti-BCMA CAR T count was altered by lenalidomide. A, Two Len dosing regimens, concurrent (C) or delayed (D) daily dosing, were tested in a disseminated NSG mouse OPM-2 tumor model with a single, subcurative dose of anti-BCMA CAR T cells from 2 separate donors (8 mice per group). B–E, Tumor bioluminescent measurement (B and C) and animal survival (D and E). Error bars, SEM. Concurrent Len led to a significant decrease in tumor burden for donor 1 (P = 0.02) and increased survival (log-rank test) for donor 1 (P = 0.057) and donor 2 (P = 0.04) compared with vehicle (Veh)-treated animals injected with anti-BCMA CAR T alone. Linear mixed-effects models (accounting for repeated mouse measurements over time) were used to estimate treatment effects for the tumor burden analysis, and log-rank testing was used for significance testing for the survival analyses. F and G, Flow cytometric analysis of blood CAR T cells gated on CD45+ CD3+ CAR+. Error bars, SEM. Concurrent Len showed significantly increased CAR T expansion after 7 days in vivo (P = 7.3 × 10−6, t test). *, P < 0.05 for concurrent lenalidomide compared with vehicle control.
Additional Files
Supplementary Data
- Supplementary Figure 1 - Supplementary Figure 1 shows information about the differential effects of lenalidomide with additional scfvs and the selected scfv at an additional E:T ratio with OPM-2 and RPMI-8226 cell lines
- Supplementary Figure 2 - Supplementary Figure 2 shows the effect of lenalidomide on BCMA expression of the cell lines used in the experiments in this manuscript.
- Supplementary Figure 3 - Supplementary Figure 3 shows the effect of lenalidomide on cytokine production by anti-BCMA CAR T cells stimulated with BCMA beads for 24 hours.
- Supplementary Figure 4 - Supplementary Figure 4 shows the effects of lenalidomide on CD27, CCR7, CD45RA, and CAR positivity.
- Supplementary Methods - Supplementary Methods and Figure Legends
- Supplementary Table 1 - anti-BCMA CAR T amino acid sequence
Volume 18, Issue 12
