Fig 2: Flow cytometry analysis of T cell-/lymphocyte-specific markers on normal and malignant B cells from CLL patients. a Summary scheme representing functional implications of CLL-specific candidate genes selected for flow cytometric analysis. b Flow cytometric analysis of expression of CTLA-4, TIGIT, CD276, LILRB4, and CD2 on peripheral blood B cells of CLL patients. The expression was determined for non-malignant B cells (“Normal”; CD19+ CD5- B cells, represented in green) and neoplastic B cells (“CLL”, CD19+ CD5+ B cells, represented in orange) detected in the same samples. “Co,” no antibody staining control; “Ab,” staining with the antibody of interest as indicated. c Normalized median fluorescence intensities (target MFI - MFI of negative control [Co]; nMFI). d ? normalized median fluorescence intensities between CLL cells and normal B cells (?nMFI (CLL-normal)) for each patient tested

Fig 3: T cells from CLL patients display elevated TIGIT expression. (a-b) Peripheral blood samples from CLL patients (CLL) and age-matched healthy controls (HC) were analyzed by flow cytometry (FACS) with regard to TIGIT, 2B4 and PD-1 expression on CD4 or CD8 T cells. (c) Distribution of inhibitory receptors on TIGIT- or TIGIT+ T cells. (d) Correlation of TIGIT and PD-1 expression on CD4+ or CD8+ T cells. (e) Distribution of CD4+TIGIT+ cells in patients divided according to clinical markers of disease burden (Rai/ Binet stage or treatment status).

Fig 4: TIGIT+ cells display a distinct cytokine profile. (a) Representative dot plots showing intracellular cytokine production after cultivating CLL PBMCs for 24 h with CD3/CD28 activating beads. (b) Cytokine production of TIGIT- or TIGIT+CD4+ T cells in 14 samples. (c) Mean fluorescence intensity ratio (MFIR) of CD155 and CD112 on CD5+CD19+ CLL (top) or CD5+ T cells (bottom). The histograms show representative FACS plots of CLL cells (gated for CD5+CD19+cells) and T cells (CD5+ cells) stained with isotype controls (in gray) and CD112/CD155 specific antibodies (in black). The dot plots show results from n = 14 samples.

Fig 5: CD4+ TIGIT+ cells provide a supportive microenvironment for CLL cells. (a) Representative dot plots showing gating strategy for flow cytometric cell sorting. (b) PBMCs have been depleted of TIGIT+, PD-1+ or TIGIT+PD-1+ CD4+ or CD8+ cells followed by incubation with CD3/CD28 activating beads. After 5 days in culture, CLL viability was measured and corresponding T/ CLL ratios were analysed (n = 6) (c).