Fig. 4: Pre-treatment of T cells with IL-2 enhances ImmTAC-redirected tumor killing and overcomes pro-tumoral macrophage suppression.

a–d Untreated or IL-2 pre-treated T cells were used in ImmTAC-redirected co-cultures with or without M2 macrophages for 20 h. CD69 MFI quantification of CD3⁺CD8⁺ T cells (mean ± SEM, n = 7 biological replicates, Wilcoxon matched-pairs single rank test, exact two-sided p values are given) (a), IFN-γ levels in co-culture supernatants (mean ± SEM, n = 4 biological replicates, Mann Whitney test, two-sided p values < 0.05 are shown) (b), representative tumor killing flow cytometry plots (c), quantification of early and late apoptotic cells in ImmTAC-redirected cultures (mean ± SEM, n = 7 biological replicates, Wilcoxon matched-pairs single rank test, two-sided p values < 0.05 are shown) (d). e Bulk RNA-seq analysis of effector T cells sorted from co-cultures. Venn diagram shows common and uncommon differentially expressed genes in untreated or IL-2 pre-treated T cell co-cultures in the presence of M2 (blue, upregulated; red, downregulated) (n = 4 biological replicates, adj p < 0.05, absolute log₂ fold change >0.75, Wald test, DESeq2). f Bulk RNA-seq heatmap plot showing the expression levels of activated CD8⁺ T cell-associated genes in different conditions (light green, ImmTAC-redirected; dark green, ImmTAC-redirected in the presence of M2; light purple, IL-2 pre-treated T cells in ImmTAC-redirected cultures; purple, IL-2 pre-treated ImmTAC-redirected T cells in the presence of M2 (log₂ (TPM + 1), n = 4 biological replicates, genes with a log₂ fold change >2 are shown). g Comparison of gene expression of T cell activation markers, cytokines, and chemokines across different donors (n = 4 biological replicates, box plots represent the interquartile range with the line set at median and whiskers show minimum and maximum values, Tukey’s multiple comparisons test, two-sided p values < 0.05 are shown). Abbreviations: E = Effectors, T = Targets, M2 = M2 macrophages, (UT) = Untreated T cells, (IL-2) = IL-2 pre-treated T cells, MFI = Mean Fluorescence Intensity, TPM = Transcripts Per Million, ns = not significant.