Figure 2.
HSPCs differentiated in the presence of the supernatant of activated CAR-T cells presented less mature phenotypes. (A) Schematic representation of the ex vivo myeloerythroid differentiation model employed. CD34+ HSPCs were harvested and subjected to differentiation under 3 conditions, namely the addition of supernatant produced by the coculture of untransduced lymphocytes (spUTD), BCMA CAR-T cells (spCAR) to the MM tumoral cell line U266 for 48 hours, and control condition without the addition of supernatant. Cytokine production was measured in the supernatant. The phenotype obtained after 12 and 24 days of ex vivo differentiation of the HSPCs was studied using next generation flow cytometry. scRNA-seq was performed after 24 days of ex vivo differentiation. (B) Concentration of IFN-γ, TNF-α, IL-2, and IL-6 cytokines in the spUTD (blue) and activated BCMA CAR-T cells (red) after 48 hours of coculture with the MM cell line U266 at a 1:1 effector-to-target ratio (n = 3). (C) Proportion of HSPCs differentiated (n = 3) in the control (green), spUTD (blue), and spCAR (red) conditions. Analysis of less differentiated (upper panel) and more differentiated (lower panel) cells is shown for the 3 lineages, namely neutrophilic (CD10−; CD10+CD16+), monocytic (CD14−CD64+; CD14+CD35+), and erythroid (CD71+CD36−; CD71+CD36+) lineages. The proportion of cells that achieved mature myeloerythroid phenotypes was significantly lower in the spCAR group. (D) FACS gating results of HSPCs that were differentiated in the presence of spUTD (blue) or spCAR (red) at day 24 of differentiation. Gates of more differentiated cells are shown for neutrophilic (CD10+CD16+), monocytic (CD14+CD35+), and erythroid (CD71+CD36+) lineages, respectively. (E) The proportion of HSPCs after differentiated for 24 days (n = 2) in the control (green), spUTD (blue), spCAR (red), and spCAR with inhibitors mix (yellow) conditions. The mix included IL-6 inhibitor at working concentration of 0.1 mM, TGF-β inhibitor at 1 mM, IFN-γ inhibitor at 1 mg/mL, IL-17a inhibitor at 1 mM; and TNF-α-TNF-β inhibitor at 0.1 mg/mL. Welch tests for panel B and unpaired t tests for panel C were used. ∗P < .05; ∗∗P > .01.

HSPCs differentiated in the presence of the supernatant of activated CAR-T cells presented less mature phenotypes. (A) Schematic representation of the ex vivo myeloerythroid differentiation model employed. CD34+ HSPCs were harvested and subjected to differentiation under 3 conditions, namely the addition of supernatant produced by the coculture of untransduced lymphocytes (spUTD), BCMA CAR-T cells (spCAR) to the MM tumoral cell line U266 for 48 hours, and control condition without the addition of supernatant. Cytokine production was measured in the supernatant. The phenotype obtained after 12 and 24 days of ex vivo differentiation of the HSPCs was studied using next generation flow cytometry. scRNA-seq was performed after 24 days of ex vivo differentiation. (B) Concentration of IFN-γ, TNF-α, IL-2, and IL-6 cytokines in the spUTD (blue) and activated BCMA CAR-T cells (red) after 48 hours of coculture with the MM cell line U266 at a 1:1 effector-to-target ratio (n = 3). (C) Proportion of HSPCs differentiated (n = 3) in the control (green), spUTD (blue), and spCAR (red) conditions. Analysis of less differentiated (upper panel) and more differentiated (lower panel) cells is shown for the 3 lineages, namely neutrophilic (CD10−; CD10+CD16+), monocytic (CD14−CD64+; CD14+CD35+), and erythroid (CD71+CD36−; CD71+CD36+) lineages. The proportion of cells that achieved mature myeloerythroid phenotypes was significantly lower in the spCAR group. (D) FACS gating results of HSPCs that were differentiated in the presence of spUTD (blue) or spCAR (red) at day 24 of differentiation. Gates of more differentiated cells are shown for neutrophilic (CD10+CD16+), monocytic (CD14+CD35+), and erythroid (CD71+CD36+) lineages, respectively. (E) The proportion of HSPCs after differentiated for 24 days (n = 2) in the control (green), spUTD (blue), spCAR (red), and spCAR with inhibitors mix (yellow) conditions. The mix included IL-6 inhibitor at working concentration of 0.1 mM, TGF-β inhibitor at 1 mM, IFN-γ inhibitor at 1 mg/mL, IL-17a inhibitor at 1 mM; and TNF-α-TNF-β inhibitor at 0.1 mg/mL. Welch tests for panel B and unpaired t tests for panel C were used. ∗P < .05; ∗∗P > .01.

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