Figure 3. AML blasts alter viability and the expression of co-signaling molecules on CD8⁺ T cells.

Sorted PB T cells from AML patients (n = 20) and HCs (n = 18) were stimulated with anti–CD3/CD28 beads alone or with IL-2 (50 IU/ml) for 3 days. Stimulated T cells were either cultured alone or with autologous blasts (T cells + blasts; allogeneic blasts for HCs) in a 1:10 T cell/blast ratio. (A–C) Forest plots represent the ratio of log-scale mean percentages of CD8⁺ T cells expressing each marker as assessed by flow cytometry on day +3, comparing (A) AML patients to HCs, (B) CD8⁺ T cells cultured with or without IL-2, and (C) CD8⁺ T cells cultured with or without blasts. P values for the ratios being different from 1.0 are shown to the right and were calculated using (A) standard and (B and C) mixed-effects linear regression models. *P < 0.05 for the effect of (B) IL-2 and (C) blasts between the AML patients and HC. (D) CITRUS analysis of flow cytometry data on day +3 for AML patients and HCs. Depicted are cell abundances in clusters identified by CITRUS, stratified by the presence or absence of blasts. P values were calculated using Mann–Whitney U or Wilcoxon’s signed-rank test as appropriate. (E) bh-SNE map of PB CD8⁺ T cells expressing ICOS and OX40 upon 3-day stimulation in the presence or absence of blasts, both for HCs and AML patients. Each point in the bh-SNE map represents an individual cell, and the cells are colored according to the intensity of expression of the individual marker as indicated on the color scale. CIMminer software was used to summarize the MFI of ICOS and OX40 expression in heatmaps. P values were calculated using paired t test. (F) Fold expansion of AML (n = 10) and HC (n = 8) CD3⁺ T cells on day +6 upon culture with blasts and stimulation with anti–PD-1 mAb and OX40 ligand. Dashed horizontal line represents cell count on day 0. P values were calculated using mixed-effects linear regression model for the log-scale fold induction as a function of cell source (AML patients and HCs) and stimulator. (G) bh-SNE maps of Ki67 distribution on stimulated HC and AML PB CD8⁺ T cells upon culture alone, or with blasts ± anti–PD-1 mAb or ± OX40 ligand. The heatmap summarizes MFI of Ki67 expression. (H) Anti-CD33/CD3 BiTE–mediated cytotoxicity of CD8⁺CD57⁺ or CD8⁺CD57^– T cells towards primary AML blasts (E/T ratio 1:1). Primary patient samples (n = 5) were cultured with BiTE or control BiTE (c-BiTE) for 48 hours. Experiments were run in duplicate and P values were calculated using paired t test.