Figure 4.
T-PLL cells show a marked defect in the execution of AICD. (A) Apoptosis induction (Annexin V/7AAD flow cytometry) upon repeated T-cell activation. Healthy-donor pan–T cells (n = 3), healthy-donor CD4+ memory T cells (n = 7), and T-PLL cells (n = 7) were cultured in the presence of 10 U/mL IL-2 and stimulated once with 1 µg/mL PHA (either on day 1 or day 6) or repeatedly on day 1 and day 6. T-PLL cells and normal CD4+ memory T cells show a similarly diminished capacity to undergo AICD as compared with age-matched healthy-donor pan–T cells (unpaired Student t test, SEM). (B) CD95L (n = 70 T-PLL) and CD95 (n = 68 T-PLL) expression detected by flow cytometry in healthy-donor T cells (n = 10) and T-PLL cells. Although heterogeneously distributed, CD95L-positive cells are increased in T-PLL samples (P = .0011, unpaired Student t test, SEM). sCD95 (right) reveals a broader range in T-PLL than in healthy controls, with an obvious clustering of cases at the low and high percentages. Color-coded low (<50% cells, blue) vs high (≥50%, red) sCD95 expression is reiterated in panels C, D, and H. (C) Apoptotic response of T-PLL cells (12 cases) to CD95 engagement by an agonistic antibody (Annexin V/7AAD staining, flow cytometry). T-PLL cells are resistant to extrinsically induced apoptosis via Fas ligation, irrespective of FasR (sCD95) expression status. Positive controls: Hut78 mature T-cell lymphoma line and healthy-donor pan–T cells (n = 3; unpaired Student t test, SEM). (D) Lack of Caspase-3 cleavage in T-PLL cell lysates upon CD95-ligation (agonistic antibody), whereas Venetoclax (targeting the intrinsic apoptosis pathway) induced processing of this distal apoptotic executioner. (E) Viability (MTT-based metabolic activity) of Hut78 and Jurkat T cells (functionally Fas/L competent) upon exposure to the CD95 agonistic antibody was decreased to a lesser degree in the presence of TCL1A (unpaired Student t test, SEM). (F) Introduction of TCL1A in Jurkat cells reduced the CD95-ligation induced levels of processed Caspase 3 and PARP. (G) TCL1A-mediated resistance to apoptotic CD95-ligation was alleviated by steric antagonization of the prosurvival TCL1A-AKT interaction (similar susceptibility to anti-CD95 agonistic antibody between Hut78-TCL1A cells and Hut78-GFP controls; unpaired Student t test, SEM). TCL1A surface model (yellow, hydrophobic; green, hydrophilic) with the aligned stretch of interphase-mimicking decoy peptides (red) that were linked to a TAT protein transduction domain. (H) Levels of soluble Fas in plasma of patients with T-LGL (n = 5)and T-PLL (n = 30) were higher than in healthy-donor plasma samples (n = 4; P = .032 and P < .0001, respectively, unpaired Student t test with Welch’s correction), irrespective of CD95 expression (blue/red).

T-PLL cells show a marked defect in the execution of AICD. (A) Apoptosis induction (Annexin V/7AAD flow cytometry) upon repeated T-cell activation. Healthy-donor pan–T cells (n = 3), healthy-donor CD4+ memory T cells (n = 7), and T-PLL cells (n = 7) were cultured in the presence of 10 U/mL IL-2 and stimulated once with 1 µg/mL PHA (either on day 1 or day 6) or repeatedly on day 1 and day 6. T-PLL cells and normal CD4+ memory T cells show a similarly diminished capacity to undergo AICD as compared with age-matched healthy-donor pan–T cells (unpaired Student t test, SEM). (B) CD95L (n = 70 T-PLL) and CD95 (n = 68 T-PLL) expression detected by flow cytometry in healthy-donor T cells (n = 10) and T-PLL cells. Although heterogeneously distributed, CD95L-positive cells are increased in T-PLL samples (P = .0011, unpaired Student t test, SEM). sCD95 (right) reveals a broader range in T-PLL than in healthy controls, with an obvious clustering of cases at the low and high percentages. Color-coded low (<50% cells, blue) vs high (≥50%, red) sCD95 expression is reiterated in panels C, D, and H. (C) Apoptotic response of T-PLL cells (12 cases) to CD95 engagement by an agonistic antibody (Annexin V/7AAD staining, flow cytometry). T-PLL cells are resistant to extrinsically induced apoptosis via Fas ligation, irrespective of FasR (sCD95) expression status. Positive controls: Hut78 mature T-cell lymphoma line and healthy-donor pan–T cells (n = 3; unpaired Student t test, SEM). (D) Lack of Caspase-3 cleavage in T-PLL cell lysates upon CD95-ligation (agonistic antibody), whereas Venetoclax (targeting the intrinsic apoptosis pathway) induced processing of this distal apoptotic executioner. (E) Viability (MTT-based metabolic activity) of Hut78 and Jurkat T cells (functionally Fas/L competent) upon exposure to the CD95 agonistic antibody was decreased to a lesser degree in the presence of TCL1A (unpaired Student t test, SEM). (F) Introduction of TCL1A in Jurkat cells reduced the CD95-ligation induced levels of processed Caspase 3 and PARP. (G) TCL1A-mediated resistance to apoptotic CD95-ligation was alleviated by steric antagonization of the prosurvival TCL1A-AKT interaction (similar susceptibility to anti-CD95 agonistic antibody between Hut78-TCL1A cells and Hut78-GFP controls; unpaired Student t test, SEM). TCL1A surface model (yellow, hydrophobic; green, hydrophilic) with the aligned stretch of interphase-mimicking decoy peptides (red) that were linked to a TAT protein transduction domain. (H) Levels of soluble Fas in plasma of patients with T-LGL (n = 5)and T-PLL (n = 30) were higher than in healthy-donor plasma samples (n = 4; P = .032 and P < .0001, respectively, unpaired Student t test with Welch’s correction), irrespective of CD95 expression (blue/red).

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