Fig. 2: Pro-tumoral macrophages suppress ImmTAC-redirected T cells at the transcript level.

a Validation of ImmTAC-redirected T cell activation using bulk RNA-seq analysis. Ridge plot showing the fold change of T cell activation genes in response to ImmTAC redirection grouped in proliferation (log₂ fold-change = 4.44 ± 0.26), IFN-γ response (log₂ fold-change = 2.98 ± 0.24) and CD8-related cytotoxic and cytokine genes (log₂ fold-change = 1.5 ± 0.36) (n = 6 biological replicates of unstimulated T cells and n = 4 biological replicates for ImmTAC-stimulated T cells). b T cell activation gene expression heatmap plot of untreated T cells (n = 6 biological replicates) and ImmTAC-redirected T cells sorted from E:T + ImmTAC co-cultures (n = 4 biological replicates, the 30 most up- or down-regulated genes with an adjusted (adj) p-value < 0.05 are shown using Wald test, DESeq2). c Bulk-RNA-seq analysis of effector T cells sorted after 20 h of co-cultures. Volcano plot shows differentially upregulated or downregulated genes of T cells in ImmTAC redirected co-cultures in the presence of M2 macrophages (n = 4 biological replicates, adj p < 0.05, absolute log₂ fold-change > 0.75, using Wald test, DESeq2). d Fold-change of the top 15 downregulated T cell genes in the presence of M2 macrophages. CD8⁺ T cell activation (cytotoxic response/cytokine release, IFN-γ mediated- response) (blue), proliferation (orange) genes are shown (n = 4 biological replicates, box plots represent the interquartile range with the line set at median and whiskers show minimum and maximum values, adj p < 0.05, absolute log₂ fold-change >0.75, Wald test, DESeq2). Abbreviations: E = Effectors, T = Targets, M2 = M2 macrophages, (UT) = Untreated T cells, TPM = Transcripts Per Million.