Extended Data Fig. 7: Treatment with TNC-1 induces T cell activation and gene expression changes associated with T cell-mediated killing.
(a) T cell activation in response to in vitro treatment with TNC-1 in co-cultures with primary T cells and HNT-34 cells (left; p = 0.041), KG-1 cells (middle; p = 0.314) and THP-1 cells (right; p = 0.364), as determined by surface marker expression of CD25 and CD69. Mean ± SEM of four (KG-1) or six (HNT-34, THP-1) T cell donors, normalized to isotype control (two-sided Mann-Whitney U test). (b) AML cell killing in response to treatment with TNC-1 in suspension cultures without T cells, containing only HNT-34 cells (left; p = 0.999), KG-1 cells (middle; p = 0.700) and THP-1 cells (right; p = 0.999). Mean ± SEM of four experiments, normalized to isotype control (two-sided Mann-Whitney U test). (c) T cell-mediated killing of AML cells over time in co-cultures with T cells expressing the NY-ESO T cell receptor and HNT-34 AML cells expressing the corresponding peptide-MHC complex. Mean ± SEM of four technical replicates for each time point and treatment condition for one T cell donor (two-sided Mann-Whitney U test; p = 0.029 for all time points). (d) T cell activation after 72 h in the NY-ESO co-culture model, as determined by surface expression of CD25. Mean ± SEM of three T cell donors (two-sided Mann-Whitney U test; p = 0.999). (e) UMAP projection and cell type classifications for co-cultures with T cells and HNT-34 AML cells after treatment with TNC-1 (left; n = 16254 cells) or an isotype control antibody (right; n = 15723 cells), based on single-cell RNA sequencing. Joint projection for co-cultures with two independent T cell donors. MAIT: mucosal-associated invariant T cell, Treg: regulatory T cell, gdT: gamma delta T cell, NK: natural killer cell. (f) Volcano plot of differentially expressed genes between HNT-34 cells treated with TNC-1 or an isotype control antibody in T cell co-cultures (n = 23886 genes). Y axis represents significance denoted as the negative logarithm of the p value (two-sided t test, without multiple testing correction). Grey: P value > 10−5. Blue: P value < 10−5. (g) The 15 most significantly downregulated (left) and upregulated (right) gene sets in HNT-34 cells treated with TNC-1 compared to treatment with an isotype control antibody, based on gene set enrichment analysis (GSEA) using the Reactome database (fgsea test with multiple testing correction and threshold p < 0.05). (h) Distribution of T cell populations in two independent donors after treatment with TNC-1 or an isotype control antibody, based on single-cell RNA sequencing analysis.
