Fig. 5: Declining role of TCR-mediated signaling in active-stage T-PLL cells.

A Heatmap of scaled TCR-pathway transcription factor signatures in indolent and active-stage T-PLL cells as derived from the DoRothEA gene regulatory network⁴³. Samples were arranged by their mean enrichment across all TCR transcription factor signatures. B Mean gene expression of negative TCR-signal regulating checkpoint molecules CTLA4 and LAG3 shows significant downregulation in indolent and active-stage T-PLL cells as compared to healthy-donor derived CD3 + T cells⁷³ (permutation test, B = 10,000, two-sided). Box plots: center: 50th percentile, box bounds: 25th and 75th percentiles (IQR), whiskers: smallest and largest observations within 1.5×IQR of the box. C–D Downregulation of TCR-signaling molecules in active T-PLL cells of patient pair 3. C Differential gene expression of TCR-signaling cascade components. Genes and gene relationships were derived from KEGG³⁷. Gene color represents the log2 expression fold-change between active and indolent-stage T-PLL cells (red: upregulation in active-stage T-PLL, white: no difference, blue: downregulation in active-stage T-PLL). P-values from two-sided MWW with correction for multiple testing. D Single-cell gene expression values of TCR-signaling associated genes in patient pair 3. Cell stage (indolent: blue, active: red) and scaled gene expression values (red: maximum expression, light gray: minimum expression) are color-coded on the t-SNE representation of pair 3. E Single-cell gene expression values of highly deregulated oncogenes and tumor-suppressor genes in patient pair 1: T-PLL stage (indolent: blue, active: red) and scaled gene expression values (red: maximum expression, light gray: minimum expression) are color-coded on the t-SNE representation of pair 1 T-PLL cells. F Surface expression of activation markers upon TCR stimulation via anti-CD3/CD28 crosslinking in CD45+ and CD45- T-PLL cells of patient pair 1 assessed by flow cytometry. Cells were stratified by T-PLL disease stage and by their expression of CD45. CD45- T-PLL cells showed diminished expression of surface activation markers upon TCR activation. CD45 + T-PLL cells of the active T-PLL stage displayed a reduced response to TCR-signaling compared to indolent CD45 + T-PLL cells. See Supplementary Fig. 12 and Supplementary Fig. 13 for gating strategies. G Representative Western blot (n = 1) showing reduced phosphorylation of TCR-downstream kinases in active-stage T-PLL samples of patient pair 1 upon CD3/CD28 crosslinking. Densitometric quantification via normalization to β-actin signals of unstimulated indolent T-PLL sample. Source data and complete summaries of statistical analyses are provided in the Source Data file.