Immune landscape of T-PLL, T-LGL, and... | American Society of Hematology

$Immune landscape of T-PLL, T-LGL, and healthy donor PBMCs upon IAP inhibition measured by spectral flow cytometry. (A) Schematic overview of the spectral flow cytometry experiments and gating strategy. In the stimulated experiment T-PLL (n = 10), T-LGL (n = 1), and healthy age-matched PBMC (n = 4) samples were treated with either birinapant (0.2 μM) or DMSO for 40 hours ex vivo, followed by 4 hours incubation with cytokine secretion block and stimulation with phorbol 12-myristate 13-acetate (PMA; 0.1 μg/mL) and ionomycin (1 μg/mL). In the unstimulated experiment, the same patient samples were treated with either selinexor (1 μM), birinapant (1 μM) with and without Q-VD-OPh (12.5 μM), or DMSO for 40 hours ex vivo, followed by 4 hours incubation with cytokine secretion block. In both experiments, cells were subsequently stained with 30 fluorescently labeled antibodies and dyes and processed for spectral flow cytometry. After acquisition, events were gated for single cells based on FSC-A and FSC-H. (B) UMAP representation of the spectral flow cytometry data derived from patient and healthy donor samples treated with birinapant (0.2 μM) or DMSO followed by subsequent stimulation. Major cell types are indicated by color. Corresponding UMAP representations colored by diagnosis (C), treatment (D), and cell death (E). (F) Box plot showing the fraction of proliferating cells per cell type for birinapant (0.2 μM) or DMSO treatment followed by subsequent stimulation. (G) Heat map showing the effect of treatment with birinapant (0.2 μM) on median cytokine expression per cell type compared with DMSO. Colors show increased (red) and decreased (blue) median expression, indicated by adjusted P values (linear mixed model; FDR < 10%). Significant effects are indicated with a star. (H) UMAP representation of the spectral flow cytometry data derived from patient and healthy donor samples treated with selinexor (1 μM), birinapant (1 μM) with and without Q-VD-OPh (12.5 μM), or DMSO. Diagnosis is indicated by color. Corresponding UMAP representation colored by treatment (I), cell death (J), and major cell types (K). (L) Box plot showing the fraction of apoptotic (cleaved caspase 3 positive) dying cells per cell type for patient and healthy donor samples treated with selinexor (1 μM) or birinapant (1 μM) with and without Q-VD-OPh (12.5 μM) relative to the DMSO negative controls. Each dot represents a patient, observations outside the plotting range are censored and shown as triangles. (M) Corresponding plot showing the fraction of nonapoptotic dying cells (cleaved caspase 3 negative). (N) Bar plot showing the mean frequency of cell subtypes among all living cells in selinexor-treated (1 μM) healthy donor samples relative to DMSO. (O) Corresponding bar plot for birinapant (1 μM). FSC-A, forward scatter area; FSC-H, forward scatter height; h, hour; p.adj., adjusted P value; Rel., relative.$

Immune landscape of T-PLL, T-LGL, and healthy donor PBMCs upon IAP inhibition measured by spectral flow cytometry. (A) Schematic overview of the spectral flow cytometry experiments and gating strategy. In the stimulated experiment T-PLL (n = 10), T-LGL (n = 1), and healthy age-matched PBMC (n = 4) samples were treated with either birinapant (0.2 μM) or DMSO for 40 hours ex vivo, followed by 4 hours incubation with cytokine secretion block and stimulation with phorbol 12-myristate 13-acetate (PMA; 0.1 μg/mL) and ionomycin (1 μg/mL). In the unstimulated experiment, the same patient samples were treated with either selinexor (1 μM), birinapant (1 μM) with and without Q-VD-OPh (12.5 μM), or DMSO for 40 hours ex vivo, followed by 4 hours incubation with cytokine secretion block. In both experiments, cells were subsequently stained with 30 fluorescently labeled antibodies and dyes and processed for spectral flow cytometry. After acquisition, events were gated for single cells based on FSC-A and FSC-H. (B) UMAP representation of the spectral flow cytometry data derived from patient and healthy donor samples treated with birinapant (0.2 μM) or DMSO followed by subsequent stimulation. Major cell types are indicated by color. Corresponding UMAP representations colored by diagnosis (C), treatment (D), and cell death (E). (F) Box plot showing the fraction of proliferating cells per cell type for birinapant (0.2 μM) or DMSO treatment followed by subsequent stimulation. (G) Heat map showing the effect of treatment with birinapant (0.2 μM) on median cytokine expression per cell type compared with DMSO. Colors show increased (red) and decreased (blue) median expression, indicated by adjusted P values (linear mixed model; FDR < 10%). Significant effects are indicated with a star. (H) UMAP representation of the spectral flow cytometry data derived from patient and healthy donor samples treated with selinexor (1 μM), birinapant (1 μM) with and without Q-VD-OPh (12.5 μM), or DMSO. Diagnosis is indicated by color. Corresponding UMAP representation colored by treatment (I), cell death (J), and major cell types (K). (L) Box plot showing the fraction of apoptotic (cleaved caspase 3 positive) dying cells per cell type for patient and healthy donor samples treated with selinexor (1 μM) or birinapant (1 μM) with and without Q-VD-OPh (12.5 μM) relative to the DMSO negative controls. Each dot represents a patient, observations outside the plotting range are censored and shown as triangles. (M) Corresponding plot showing the fraction of nonapoptotic dying cells (cleaved caspase 3 negative). (N) Bar plot showing the mean frequency of cell subtypes among all living cells in selinexor-treated (1 μM) healthy donor samples relative to DMSO. (O) Corresponding bar plot for birinapant (1 μM). FSC-A, forward scatter area; FSC-H, forward scatter height; h, hour; p.adj., adjusted P value; Rel., relative.

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