Longitudinal development of expression of PD-L1 and PD-L2 on CLL/normal B cells and PD-1 on CD3⁺CD8⁺ T cells. (A) Spleen cells from fully leukemic aged Eµ-TCL1 and AT mice meeting predefined end point criteria for CLL and age-matched WT mice were stained for CD19, CD5, and PD-L1; PD-L1 expression was compared between viable CD19⁺CD5⁺ CLL and CD19⁺ B cells (a representative overlaid flow plot is depicted on the left, cells were gated based on FMO and isotype controls). (B) Representative histogram and differences of MFI corrected for the MFI of the FMO/isotype control of PD-L1 between fully leukemic Eµ-TCL1 and age-matched WT mice. (C) ANs were determined by normalizing percentages of cells to overall cell counts for spleen. (D) Longitudinal development of PD-L1 and PD-L2 expression across organs on healthy B cells (hBC) from 3-, 6-, 12-month-old WT and 3- and 6-month-old Eµ-TCL1 mice and on CLL cells from 6- and 12-month-old Eµ-TCL1 and AT mice (in 6-month-old Eµ-TCL1 mice, both CD19⁺ normal and CD19⁺CD5⁺ malignant B cells exist next to each other and PD-L1/PD-L2 was gated on either population). (E) Heat map summary of P values describing statistical differences in MFI of PD-L1 and PD-L2 across organs. Groups were compared with corrected MFIs in 3-month-old mice. Blue, increase; red, decrease of MFI; ns, nonsignificant. *P < .05, **P < .001, ***P < .0001. (F) At least 2 cohorts of 3-, 6-, and 12-month-old Eµ-TCL1, WT, and AT mice containing ≥5 mice each were euthanized; splenocytes were stained for CD3, 4, 8, 62L, 44, and PD-1 and assessed in relation to CLL load (supplemental Figure 1). Quantification of PD-1 expression in the context of T-cell CD3⁺CD8⁺CD44⁺ subset changes. Stacked bar charts are used to visualize the loss of naive (light gray) and shift to Ag-experienced cells (dark gray). Longitudinal changes of (G) percentages and (H) ANs of PD-1⁺CD3⁺CD8⁺CD44⁺ cells. All graphs depict median ± interquartile range, and combine data from at all cohorts of mice at each age group.