High-dimensional mass cytometry analysis of murine splenocytes. (A) viSNE plots (Cytobank) allowing the visualization of CD45⁺ splenocytes from healthy controls (HC) C57BL/6 (left) and AT-TCL1 (right, CLL) mice on 2 dimensions based on the expression of all markers. The main plot depicts CD19 expression. The lower left box displays PD-L1 expression on B and CLL cells, respectively. Blue indicates the absence of expression; red indicates the highest expression among all cells and all samples. (B) Heatmap plotting the median signal intensity of discriminative markers between normal B and CLL cells (top panel, n = 6). Heatmap plotting the median expression of markers between PD-L1^low (MSI<20) and PD-L1^high (MSI>90) CLL cells (bottom panel, n = 6). Expression in CD4⁺ T cells, CD8⁺ T cells, and CD11b⁺ myeloid cells from C57BL/6 mice was plotted as references. (C) viSNE plots illustrating splenic live CD45⁺CD19⁻B220⁻ immune cell populations from AT-TCL1 mice (left) and characteristic marker expression (right). (D) The 15 enriched clusters (P < .01) in AT-TCL1 mice (CLL) compared with C57BL/6 were overlaid on the viSNE plots. The color scale depicts the cluster number and the related percentage of cells within live CD45⁺CD19⁻B220⁻ cells. (E-H) Heatmaps plotting the median signal intensity of discriminative markers between different clusters of immune cells in AT-TCL1 mice. Selected clusters show a significant enrichment of CD8⁺ T cells (E), Treg cells (F), cDCs (G), and monocytes (H) in AT-TCL1 compared with C57BL/6 mice (fold change [FC], P value). Bar charts represent percentages of cells or ratios observed between C57BL/6 (HC, n = 6) and AT-TCL1 (CLL, n = 6) mice. CM central memory; EM, effector memory; EX exhausted; INF inflammatory; INT intermediate. (I) Mean signal intensity (MSI) of PD1 and LAG3 expression on relevant immune cell subpopulations. NK, natural killer. For all panels, *P < .05, **P < .01, ***P < .001, and ****P < .0001.