Figure 1.
T cells show functional, phenotypic, and transcriptional evidence of dysfunction during continuous blinatumomab. T cells collected at baseline (preblinatumomab), middle (day 14), and end time points (day 28) during a 28-day continuous blinatumomab infusion were analyzed. (A) Left plot: cytotoxicity of T cells. Line graph (right): time point-matched data; each line represents a patient. Green diamonds, cytotoxicity sustained; purple triangles, cytotoxicity not sustained. Vertical bracket: Mann-Whitney test comparison of baseline data. (B-E) Frequency of TSCM, TEMRA, TIM3+, IFN-γ+, TNF-α+, p-mTOR+, p-MEK+, and p-S6+ cells among all T cells. p-S6: marker of mTOR activity. (D-E) Data from intracellular flow cytometry analysis after coculture of T cells and B-ALL cells with CD19/CD3 BiTE antibody. Values relative to control cocultures (positive cells [%]with BiTE − positive [%]without BiTE) are shown. (F) Volcano plot of fold change vs statistical significance from DESeq differential gene expression analysis (day 14 vs day 0). Differentially expressed genes (FDR adjusted P < .15) in dark red. AFF3, a cofactor highly expressed in progenitors of exhausted T cells9; LAYN, MAGEH1, genes related to T-regulatory suppressive function10; LYST, lysosomal trafficking gene associated with T-cell dysfunction.11 Dotted vertical line: −log10 P value cutoff for defining differential expression. (G) Gene set enrichment analysis for T-cell exhaustion (acute lymphocytic choriomeningitis virus [LCMV] vs chronic [exhausted T cells] LCMV), TEMRA (naive T cells vs PD1 low [enriched for TEMRA] T cells), terminally differentiated effector (KLRG1 high [terminal effector] vs KLRG low12), and effector gene sets among genes ranked by day 14 vs day 0 expression. (A-E) Box plots: 25th to 75th percentiles. Whiskers: 1.5 × the interquartile range beyond 25th and 75th percentiles. Thick black horizontal line, median; red triangle, mean; blue circles, individual data points. P value (blue): Friedman test (TSCM, TIM3+, IFN-γ+, and TNF-α+) or 1-way repeated-measures analysis of variance (all others). Horizontal line (red): paired t test or Wilcox test (Holm-Sidak correction for multiple comparisons). ∗P < .05; ∗∗P < .01. N = 10 patients for panels A, D, and E and 11 patients for panels B, C, F, and G. DN, down; FDR, false discovery rate; GS, gene set; IFN-γ, interferon gamma; KLRG, killer cell lectin-like receptor subfamily G member; mTOR, mammalian target of rapamycin; NES, normalized enrichment score; PD1, programmed cell death protein 1; Tfh, T follicular helper; Th1, type 1 helper T cell; TNF-α, tumor necrosis factor alpha.

T cells show functional, phenotypic, and transcriptional evidence of dysfunction during continuous blinatumomab. T cells collected at baseline (preblinatumomab), middle (day 14), and end time points (day 28) during a 28-day continuous blinatumomab infusion were analyzed. (A) Left plot: cytotoxicity of T cells. Line graph (right): time point-matched data; each line represents a patient. Green diamonds, cytotoxicity sustained; purple triangles, cytotoxicity not sustained. Vertical bracket: Mann-Whitney test comparison of baseline data. (B-E) Frequency of TSCM, TEMRA, TIM3+, IFN-γ+, TNF-α+, p-mTOR+, p-MEK+, and p-S6+ cells among all T cells. p-S6: marker of mTOR activity. (D-E) Data from intracellular flow cytometry analysis after coculture of T cells and B-ALL cells with CD19/CD3 BiTE antibody. Values relative to control cocultures (positive cells [%]with BiTE − positive [%]without BiTE) are shown. (F) Volcano plot of fold change vs statistical significance from DESeq differential gene expression analysis (day 14 vs day 0). Differentially expressed genes (FDR adjusted P < .15) in dark red. AFF3, a cofactor highly expressed in progenitors of exhausted T cells9; LAYN, MAGEH1, genes related to T-regulatory suppressive function10; LYST, lysosomal trafficking gene associated with T-cell dysfunction.11 Dotted vertical line: −log10 P value cutoff for defining differential expression. (G) Gene set enrichment analysis for T-cell exhaustion (acute lymphocytic choriomeningitis virus [LCMV] vs chronic [exhausted T cells] LCMV), TEMRA (naive T cells vs PD1 low [enriched for TEMRA] T cells), terminally differentiated effector (KLRG1 high [terminal effector] vs KLRG low12), and effector gene sets among genes ranked by day 14 vs day 0 expression. (A-E) Box plots: 25th to 75th percentiles. Whiskers: 1.5 × the interquartile range beyond 25th and 75th percentiles. Thick black horizontal line, median; red triangle, mean; blue circles, individual data points. P value (blue): Friedman test (TSCM, TIM3+, IFN-γ+, and TNF-α+) or 1-way repeated-measures analysis of variance (all others). Horizontal line (red): paired t test or Wilcox test (Holm-Sidak correction for multiple comparisons). ∗P < .05; ∗∗P < .01. N = 10 patients for panels A, D, and E and 11 patients for panels B, C, F, and G. DN, down; FDR, false discovery rate; GS, gene set; IFN-γ, interferon gamma; KLRG, killer cell lectin-like receptor subfamily G member; mTOR, mammalian target of rapamycin; NES, normalized enrichment score; PD1, programmed cell death protein 1; Tfh, T follicular helper; Th1, type 1 helper T cell; TNF-α, tumor necrosis factor alpha.

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