T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers

Abstract

Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3⁺ cells and the CD8⁺ subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4⁺ and CD8⁺ cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients (P=0.0003 and P=0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4⁺ and CD8⁺ subsets, with a significantly higher PD-1 expression. Higher numbers of CD4⁺ and CD8⁺ cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67⁺) and activated (CD69⁺) CD4⁺ and CD8⁺ cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls (P<0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways.

Figure 1.

PD-1 expression in T cells from chronic lymphocytic leukemia (CLL) patients and controls. Absolute numbers of (A) PD-1⁺CD4⁺ and (B) PD-1⁺CD8⁺ T cells from progressive (P) and non-progressive (NP) CLL patients compared to controls. Box plots display cumulative data with line at median. *P<0.05, **P<0.005, ***P<0.0005. Correlation of (C) PD-1⁺CD4⁺ and (D) PD-1⁺CD8⁺ T cells with total lymphocyte count at the time of testing (n=80).

Figure 2.

Comparative analysis of T-cell memory subsets in chronic lymphocytic leukemia (CLL) patients compared to controls. Absolute numbers of (A) CD4⁺, (B) CD8⁺, (C) CD4⁺PD-1⁺ and (D) CD8⁺PD-1⁺ naïve, central memory (CM), effector memory (EM) and effector (EMRA) cells in healthy controls (n=9) were compared to non-progressive (n=13), untreated progressive (n=8) and pre-treated progressive (n=12) CLL patients. Box plots display cumulative data with line at median. Only significant statistical values are reported. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

Figure 3.

Intracellular CTLA-4 expression in T cells from chronic lymphocytic leukemia (CLL) patients and controls. Absolute numbers of (A) CD4⁺ and (B) CD8⁺ T cells with intracellular (i.c.) CTLA-4 expression in CLL patients and controls. Box plots display cumulative data with line at median. Only significant statistical values are reported. NP: non-progressive; P: progressive. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

Figure 4.

CD69 and intracellular Ki67 expression in T cells from chronic lymphocytic leukemia (CLL) patients and controls. Absolute numbers of (A) CD69⁺CD4⁺, (B) CD69⁺CD8⁺, (C) Ki67⁺CD4⁺, (D) Ki67⁺CD8⁺ T cells from progressive (P) and non-progressive (NP) CLL patients and healthy controls. Box plots display cumulative data with line at median. Only significant statistical values are reported. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

Figure 5.

Comparative analysis of functional CD4⁺ T-helper subpopulations (Th1/Th2/Th17) and regulatory T cells (Tregs) in chronic lymphocytic leukemia (CLL) patients and controls. Absolute numbers of (A) Th1, Th2, Th17 and (B) Tregs cells in non-progressive (NP) (n=13), untreated progressive (P) (n=8), and pre-treated progressive (n=12) CLL patients compared with healthy controls (n=9). Box plots display cumulative data with line at median. Only significant statistical values are reported. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001.

Figure 6.

Relative change in absolute numbers of different T-cell subpopulations and T cells expressing immune checkpoints or activation / proliferation markers in chronic lymphocytic leukemia (CLL) patients compared to healthy controls. Relative change in the (A) CD4⁺ subset and (B) CD8⁺ subset calculated as median value patients/median value controls in non-progressive (green bars), untreated progressive (yellow bars) and pre-treated progressive (black bars) CLL patients.