Figure 4.
Figure 4. Functionality of NY-ESO-1 SPEAR T cells postinfusion. (A) Cytokine production: cytokine levels per cell were measured by median fluorescent intensity. Cytokines interferon γ, interleukin 2, and tumor necrosis factor α were measured by intracellular cytokine staining after antigen-specific stimulation. Data were measured in 20 of 25 patients. Line graph shows the mean and standard deviation. Median fluorescent intensity was measured from the cytokine-positive population only (ie, median fluorescent intensity values ≥2000). (B) Functional characterization by antigen-specific cytokine responses: NY-ESO-1 SPEAR T cells produced cytokine in response to antigen stimulation, the level of which correlated with the transduction percentage. Before manufacturing, and therefore, in the absence of NY-ESO-1 SPEAR T cells (ie, day –50), there was no measurable response to antigen stimulation. Transduction efficiency of manufactured product, median (range): CD8+ T cells 18.85% (5.94%-46.5%), CD4+ T cells 14.83% (2.46%-44.83%). Data were measured in 22 of 25 patients. Day –50 = baseline peripheral blood sample. Box plot shows the mean and upper and lower quartiles. (C) Polyfunctionality: the percentage of CD4+ T cells and CD8+ T cells expressing interferon γ, interleukin 2, or tumor necrosis factor α; a combination of any 2 of the 3 cytokines; or all 3 cytokines in response to ex vivo stimulation with antigen-pulsed T2 cells were evaluated in paired PBMC and BM samples from a subset of patients (n = 11). The percentage of cytokine-positive cells producing 1 or more cytokines is displayed as a percentage of all cytokine-producing cells. Box plot shows the mean and upper and lower quartiles. (D) Expression of exhaustion markers: the percentage of CD4+ pentamer+ T cells or CD8+ pentamer+ T cells were evaluated for expression of 3 exhaustion markers (LAG3, PD1, and TIM3) in paired PBMC and BM samples from a subset of patients (n = 11). Line graph shows the mean and standard deviation. MP, manufactured product; PMA-IONO, phorbol myristate acetate + ionomycin (positive control); T2, T2 cells unpulsed (negative control); T2-NY-ESO, T2 cells pulsed with NY-ESO-1 peptide.

Functionality of NY-ESO-1 SPEAR T cells postinfusion. (A) Cytokine production: cytokine levels per cell were measured by median fluorescent intensity. Cytokines interferon γ, interleukin 2, and tumor necrosis factor α were measured by intracellular cytokine staining after antigen-specific stimulation. Data were measured in 20 of 25 patients. Line graph shows the mean and standard deviation. Median fluorescent intensity was measured from the cytokine-positive population only (ie, median fluorescent intensity values ≥2000). (B) Functional characterization by antigen-specific cytokine responses: NY-ESO-1 SPEAR T cells produced cytokine in response to antigen stimulation, the level of which correlated with the transduction percentage. Before manufacturing, and therefore, in the absence of NY-ESO-1 SPEAR T cells (ie, day –50), there was no measurable response to antigen stimulation. Transduction efficiency of manufactured product, median (range): CD8+ T cells 18.85% (5.94%-46.5%), CD4+ T cells 14.83% (2.46%-44.83%). Data were measured in 22 of 25 patients. Day –50 = baseline peripheral blood sample. Box plot shows the mean and upper and lower quartiles. (C) Polyfunctionality: the percentage of CD4+ T cells and CD8+ T cells expressing interferon γ, interleukin 2, or tumor necrosis factor α; a combination of any 2 of the 3 cytokines; or all 3 cytokines in response to ex vivo stimulation with antigen-pulsed T2 cells were evaluated in paired PBMC and BM samples from a subset of patients (n = 11). The percentage of cytokine-positive cells producing 1 or more cytokines is displayed as a percentage of all cytokine-producing cells. Box plot shows the mean and upper and lower quartiles. (D) Expression of exhaustion markers: the percentage of CD4+ pentamer+ T cells or CD8+ pentamer+ T cells were evaluated for expression of 3 exhaustion markers (LAG3, PD1, and TIM3) in paired PBMC and BM samples from a subset of patients (n = 11). Line graph shows the mean and standard deviation. MP, manufactured product; PMA-IONO, phorbol myristate acetate + ionomycin (positive control); T2, T2 cells unpulsed (negative control); T2-NY-ESO, T2 cells pulsed with NY-ESO-1 peptide.

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