Fig. 2. EOMES⁺ CD8⁺ T cells showing features of exhaustion accumulate in the Eµ-TCL1 mouse model of CLL.

A Splenocytes of primary heterozygous Eµ-TCL1 leukemic mice (n = 8) and wild-type (WT, n = 8) littermates at a median age of 65 weeks were analyzed by flow cytometry. Representative histogram and percentage of EOMES⁺ cells out of total CD8⁺ T cells are depicted. B Percentage of EOMES⁺ cells among CD8⁺ T_EF cells (left) and CD8⁺ T_M cells (right). C Pearson correlation of absolute counts of EOMES⁺ CD8⁺ T cells and CLL cells (left), as well as PD-1⁺ LAG3⁺ CD8⁺ T cells (right) in spleen of Eµ-TCL1 leukemic mice (n = 48). D EOMES expression in splenic CD8⁺ T cells of mice after adoptive transfer of TCL1 leukemia (TCL1 AT, n = 5) with end-stage disease and WT littermates (n = 7) was analyzed by flow cytometry. Representative histogram and percentage of EOMES⁺ cells out of total CD8⁺ T cells are shown. E Percentage of EOMES⁺ cells out of CD8⁺ T_EF cells (left) and of CD8⁺ T_M cells (right). F Splenic CD8⁺ T cells from TCL1 AT mice (n = 4) were concatenated and clustered using t-SNE algorithm based on the expression of CD127, CD44, EOMES, PD-1, LAG3, TIGIT, and CD244. Effector, memory, and naïve subsets were defined based on CD127 and CD44 expression (upper left panel, see “Materials and methods”). EOMES, PD-1, LAG3, TIGT, and CD244 protein levels in clustered CD8⁺ T cells are shown (middle and right upper panels, and lower panels). Graphs show mean with SEM, with each dot representing one mouse. Statistical analysis was performed using the Mann–Whitney’s test. *p value < 0.05, **p value < 0.01, ***p value < 0.001.