Activation priming and cytokine polyfunctionality modulate the enhanced functionality of low-affinity CD19 CAR T cells

Abstract

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and antitumor efficacy compared with the high-affinity (FMC63-based) CAR used in tisagenlecleucel, in preclinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a phase 1 clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T cells, we performed a systematic in vitro characterization of the transcriptomic (RNA sequencing) and protein (cytometry by time of flight) changes occurring in T cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared with FMC63 CAR T cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B cells present in the manufacture.

Graphical abstract

Figure 1.

Generation and phenotypic characterization of CAR T cells from HD-PBMCs. (A) Experimental workflow. PBMCs were isolated from HDs and LV transduced to express CD19 CAR construct (FMC63 or CAT) following overnight activation with CD3/CD28 beads. Six days after transduction, CAR T cells were cultured without (unstimulated) or with target cells (NALM6) at a 1:1 ratio (stimulated). Unstimulated and stimulated cells were analyzed by flow cytometry and sorted for RNA-seq 24 hours poststimulation. Mass cytometry analysis was performed on unstimulated and stimulated cells 24 hours poststimulation. Activated UNTR T cells were used as a control throughout the experiment. (B) (Left) spaghetti plots showing transduction levels of CAR T cells as percentage of mCherry⁺ (in CD3⁺) and (right) as MFI of mCherry in unstimulated transduced T cells measured by FACS 7 days posttransduction. Lines connect results from individual donors (n = 12 HDs, n = 3 independent experiments). (C) Spaghetti plot showing the percentage of surface CAR expression (in CD3⁺) in unstimulated transduced T cells measured by FACS 10 days posttransduction. Lines connect results from individual donors (n = 4 HDs, n = 1 independent experiment). (D) Variation (log2 fold change) of CD4 and CD8 proportion in unstimulated UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 7 days posttransduction. The dotted horizontal line (0) represents the conditions in which CD4 = CD8. Data represent mean ± SEM (n = 12 HDs, n = 3 independent experiments). (E) Bar plots showing the percentage of T_CM (CD45RA⁻CD62L⁺) (left), T_EM (CD45RA⁻CD62L⁻) (middle), and T_EMRA (CD45RA⁺CD62L⁻) (right) in unstimulated CD3⁺ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 10 days posttransduction. Data represent mean ± SEM (n = 7 HDs, n = 2 independent experiments). (B-E) Statistical significance was calculated by paired t test; ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .0001. Each experimental condition is indicated by a specific color code (UNTR, light gray; FMC63, light blue; CAT, orange). MFI, mean fluorescent intensity; SEM, standard error of the mean.

Figure 2.

RNA-seq and mass cytometry analyses of unstimulated UNTR and CAR-transduced T cells. (A) PCA of the top 500 variable genes from RNA-seq analysis across all experimental conditions (n = 6 HDs, n = 2 independent experiments). (B) PCA of mass cytometry EMD scores computed at 24 hours poststimulation in CD3⁺ cells across all experimental conditions (n = 4 HDs, n = 1 independent experiment). (C) Fuzzy clustering analysis of RNA-seq data across all experimental conditions (n = 6 HDs, n = 2 independent experiments). (D) Volcano plot showing differentially expressed genes between unstimulated FMC63 and CAT CAR T cells (top). The dashed horizontal line represents the statistical significance threshold (FDR <0.1). The bar plots show the expression of selected differentially expressed genes (FDR <0.1) in unstimulated UNTR and transduced T cells (bottom). Data represent mean ± SEM (n = 6 HDs, n = 2 independent experiments). (E) Bar plots showing the expression of mass cytometry EMD scores for granzyme B, perforin B, HLA-DR, CD25, NFAT1, pZAP70, and pS6 in unstimulated CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (0) represents the expression of a specific marker in unstimulated CD3⁺ UNTR T cells. Data represent mean ± SEM (n = 7 HDs, n = 2 independent experiments). Statistical significance was calculated by paired t test; ∗P < .05 and ∗∗P < .01. (A-E) Each experimental condition is indicated by a specific color code (unstimulated conditions: UNTR, light gray; FMC63, light blue; and CAT, orange; stimulated conditions: UNTR, gray; FMC63, blue; and CAT, red). FDR, false discovery rate; SEM, standard error of the mean.

Figure 3.

Phenotypic and molecular characterization of stimulated CAR-transduced T cells. (A) Variation (log2 fold change) of CD4 and CD8 proportion in stimulated UNTR T cells and FMC63 and CAT CAR T cells measured by FACS (n = 12 HDs, n = 3 independent experiments). The dotted horizontal line (0) represents the conditions in which CD4 = CD8. (B) Bar plot showing the percentage of T_CM (CD45RA⁻CD62L⁺) in stimulated CD3⁺ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 96 hours postantigen stimulation (n = 7 HDs, n = 2 independent experiments). (C) Bar plots showing the expression of T-cell exhaustion markers (PD1, TIM3, and LAG3) as MFI in stimulated CD3⁺ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS 96 hours postantigen stimulation (n = 4 HDs, n = 1 independent experiment). (D) Volcano plot showing differentially expressed genes between FMC63 and CAT CAR T cells upon NALM6 coculture (left). The dashed horizontal line represents the statistical significance threshold (FDR <0.1). Bar plots showing the expression of selected differentially expressed genes (FDR <0.1) in stimulated UNTR T cells and in CAR T cells (n = 6 HDs, n = 2 independent experiments) (right). (E) Bar plots showing the expression of mass cytometry EMD scores for CD25, HLA-DR, NFAT1, FOXP3, pZAP70, pS6, pp38, pCREB, pRB, and CD4 in stimulated CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (0) represents the expression of a specific marker in stimulated CD3⁺ UNTR T cells (n = 7 HDs, n = 2 independent experiments). (A-E) Bar plots show mean ± SEM. Each experimental condition is indicated by a specific color code (UNTR, gray; FMC63, blue; and CAT, red). Panels A-C,E show statistical significance calculated by paired t test; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. FDR, false discovery rate; MFI, mean fluorescent intensity; SEM, standard error of the mean.

Figure 4.

Cytokine polyfunctionality in stimulated CAR-transduced T cells. (A) Bar plots showing the expression of mass cytometry EMD scores for effector (granzyme B, IFN-γ, TNF-α) and stimulatory (GM-CSF, IL-2) cytokines in stimulated CAR T cells. The data shown are normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (0) represents the expression of a specific marker in stimulated CD3⁺ UNTR T cells. (n = 7 HDs, n = 2 independent experiments). (B) Bar plots showing the mean cytokine polyfunctionality in stimulated CAR T cells, normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (1) represents the mean polyfunctionality in stimulated CD3⁺ UNTR T cells. (n = 7 HDs, n = 2 independent experiments). (C) Stacked bar plots showing the percentage of stimulated CAR T cells (CD3⁺mCherry⁺) expressing 1 to 4 or ≥5 cytokines per cell as measured by mass cytometry. (D) Circos plots showing all the combinations of the 8 cytokines in stimulated FMC63 (left) and CAT (right) CAR T cells analyzed by mass cytometry. The numbers indicate patterns of cytokine coexpression (from 1-4 or ≥5 cytokines/cell). A specific color code has been assigned to each cytokine. (A-B) Data represent mean ± SEM. Statistical significance was calculated by paired t test; ∗P < .05 and ∗∗∗P < .001. Each experimental condition is indicated by a specific color code (FMC63, blue and CAT, red). GM-CSF, granulocyte-macrophage colony-stimulating factor; SEM, standard error of the mean; TNF-α, tumor necrosis factor α.

Figure 5.

Molecular characterization of CAR T cells generated from CD19-depleted PBMCs. (A) UMAP representation of the 4 cell populations (CD8, CD4, CD3 low/neg, and CD19) identified by FlowSOM analysis in 4 representative unstimulated samples analyzed by mass cytometry at 24 hours poststimulation (left) (n = 4 HDs, n = 1 independent experiment). Cell types are indicated by different colors. Percentage of residual B cells detected by mass cytometry in unstimulated samples at 24 hours poststimulation (right) (n = 7 HDs, n = 2 independent experiments). (B) Bar plots showing the percentage of T_CM (CD45RA⁻CD62L⁺) (left) and T_EM (CD45RA⁻CD62L⁻) (right) in unstimulated CD19-depleted CD3⁺ UNTR T cells and FMC63 and CAT CAR T cells measured by FACS (n = 4 HDs, n = 1 independent experiment). (C) Bar plots showing the expression of mass cytometry EMD scores for granzyme B, perforin B, HLA-DR, CD25, NFAT1, pZAP70, and pS6 in unstimulated CD19-depleted CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (0) represents the expression of a specific marker in unstimulated CD3⁺ CD19-depleted UNTR T cells. (n = 3 HDs, n = 1 independent experiment). (D) Bar plots showing the expression of mass cytometry EMD scores for CD25, HLA-DR, NFAT1, pZAP70, pS6, pp38, pRB, and CD4 in stimulated CD19-depleted CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (0) represents the expression of a specific marker in stimulated CD19-depleted CD3⁺ UNTR T cells (n = 3 HDs, n = 1 independent experiment). (E) Bar plots showing the mean polyfunctionality in CD19-depleted CAR T cells at 24 hours upon stimulation. The data shown are normalized to stimulated CD3⁺ UNTR T cells. The dotted horizontal line (1) represents the mean polyfunctionality in stimulated CD3⁺ CD19-depleted UNTR T cells (n = 3 HDs, n = 1 independent experiment). (A-E) Each experimental condition is indicated by a specific color code (unstimulated conditions: UNTR, light gray; FMC63, light blue; CAT, orange; stimulated conditions: FMC63, blue; CAT, red). Bar plots show mean ± SEM. Statistical significance was calculated by paired t test; ∗P < .05 and ∗∗P < .01. SEM, standard error of the mean.