Figure 2.
CLL progression drives T-cell transcriptional program toward an exhausted-like phenotype. Analysis of patient samples (left section, A-D). (A) Quantification of CD8+ T-cell memory subsets at baseline in HD and CLL PBMCs, based on the surface expression of CD45RA and CD27: naïve, CD45RA+CD27+; central memory (CM), CD45RA–CD27+; effector memory (EM), CD45RA–CD27–; and terminally differentiated EM (EMRA), CD45RA+CD27–. (B) Quantification of CD8+ T-cell subsets based on Tbet and Eomes expression in HD and CLL. (C) TOX expression levels within the EomeshiTbethi and EomesloTbetlo populations. (D) Ratio of mitochondrial membrane potential to mitochondrial mass (MTO:MTG) within naïve, CM, EM, and EMRA CD8+ T-cell subsets in samples from HDs and patients with CLL. Analysis of murine samples (right section, E-I). (E) Flow cytometry t-distributed stochastic neighborhood embedding (tSNE) and Xshift clustering analyses of splenic CD8+ T cells from WT or AT Eμ-TCL1 mice at different disease stages (left) with the quantification of the frequency of each cluster (right). (F) Expression levels of surface markers (PD-1, KLRG-1, CD44, and CD62L), TFs (Eomes, TOX, and TCF-1), and transcription coactivator (PGC1α) used in the tSNE analysis (left). Distinct expression of Eomes and TCF-1 within major clusters is shown (right). (G) Quantification of CD8+ T-cell populations in WT and at different disease stages based on the expression levels of Eomes and TCF-1. Quantitative results of exhaustion-related markers expression (H) and memory phenotypes:naïve, CD44–CD62L+; CM, CD44+CD62L+; EM, CD44+CD62L–; and effector, CD44–CD62L–. (I) within the Eμ-TCL1 T-cell Eomes/TCF-1 gates mentioned in panel G. Data are cumulative results from ≥3 experiments in panels H-I or representative of 3 independent experiments in panels E-G. Experiments were replicated using intermediate (interm)- and late-stage AT Eμ-TCL1 mice. Each symbol represents an individual animal in panels G-I. Data are presented as mean ± SEM in panels A-C,H-I; or mean ± SD in panels D,G. Differences were analyzed using a 2-way ANOVA with Sidak multiple correction test in panels A-D,G; or a 2-tailed, unpaired t test in panels H-I. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. AT, adoptive transfer; gMFI, geometric mean fluorescence intensity; ns, non-significant; PBMCs, peripheral blood mononuclear cells; TFs, transcription factors.

CLL progression drives T-cell transcriptional program toward an exhausted-like phenotype. Analysis of patient samples (left section, A-D). (A) Quantification of CD8+ T-cell memory subsets at baseline in HD and CLL PBMCs, based on the surface expression of CD45RA and CD27: naïve, CD45RA+CD27+; central memory (CM), CD45RA–CD27+; effector memory (EM), CD45RA–CD27–; and terminally differentiated EM (EMRA), CD45RA+CD27–. (B) Quantification of CD8+ T-cell subsets based on Tbet and Eomes expression in HD and CLL. (C) TOX expression levels within the EomeshiTbethi and EomesloTbetlo populations. (D) Ratio of mitochondrial membrane potential to mitochondrial mass (MTO:MTG) within naïve, CM, EM, and EMRA CD8+ T-cell subsets in samples from HDs and patients with CLL. Analysis of murine samples (right section, E-I). (E) Flow cytometry t-distributed stochastic neighborhood embedding (tSNE) and Xshift clustering analyses of splenic CD8+ T cells from WT or AT Eμ-TCL1 mice at different disease stages (left) with the quantification of the frequency of each cluster (right). (F) Expression levels of surface markers (PD-1, KLRG-1, CD44, and CD62L), TFs (Eomes, TOX, and TCF-1), and transcription coactivator (PGC1α) used in the tSNE analysis (left). Distinct expression of Eomes and TCF-1 within major clusters is shown (right). (G) Quantification of CD8+ T-cell populations in WT and at different disease stages based on the expression levels of Eomes and TCF-1. Quantitative results of exhaustion-related markers expression (H) and memory phenotypes:naïve, CD44–CD62L+; CM, CD44+CD62L+; EM, CD44+CD62L–; and effector, CD44–CD62L–. (I) within the Eμ-TCL1 T-cell Eomes/TCF-1 gates mentioned in panel G. Data are cumulative results from ≥3 experiments in panels H-I or representative of 3 independent experiments in panels E-G. Experiments were replicated using intermediate (interm)- and late-stage AT Eμ-TCL1 mice. Each symbol represents an individual animal in panels G-I. Data are presented as mean ± SEM in panels A-C,H-I; or mean ± SD in panels D,G. Differences were analyzed using a 2-way ANOVA with Sidak multiple correction test in panels A-D,G; or a 2-tailed, unpaired t test in panels H-I. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. AT, adoptive transfer; gMFI, geometric mean fluorescence intensity; ns, non-significant; PBMCs, peripheral blood mononuclear cells; TFs, transcription factors.

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