Analysis of apoptotic response to BNZ-1 of CD4⁺ T cells and T-LGLL cells. (A) Flow cytometric analysis of CD4⁺ T cells and T-LGLL cells to BNZ-1. A representative flow pattern (annexin-V-FITC, BioLegend) of apoptotic cells in the CD4 T-cell subset (defined by CD45⁺CD3⁺CD4⁺) and T-LGLL cells (defined by CD45⁺CD3⁺CD8⁺CD5^dimCD57⁺CD45RO^loCCR7^lo) before and on day 2 of the BNZ-1 treatment (patient ID: UVA-001; dose cohort 1, 0.5 mg/kg) is shown. PBMCs were purified from blood using Ficoll-Paque, and 2 million cells were stained as described in the text. After staining with fluorochrome-conjugated antibodies (BioLegend; see “Methods” for details), each target cell was gated using the markers (top), and their annexin V staining is shown as histograms. Staining of fluorescein isothiocyanate (FITC)–annexin V in phosphate-buffered saline was used as the negative control to set the photomultiplier tubes (PMT) of the flow machine (BD FACSAria II) and gating windows with the FlowJo analysis. (B.) JC-1 (mitochondrial membrane potential) assessment of apoptotic response of the T-LGLL cells (a representative staining pattern). PBMCs from a patient (UVA-001; before and on day 2 of BNZ-1 treatment at 0.5 mg/kg) were purified using Ficoll-Paque, and 2 million PBMCs were stained with JC-1 dye at 37°C for 10 minutes per the manufacturer’s instructions, followed by staining with fluorochrome-conjugated antibodies including allophycocyanin (APC) annexin V. T-LGLL cells were defined by CD3⁺CD8⁺CD5^dimCD57⁺CD45RO^loCCR7^lo, and the JC-1 (excited by 488 nm laser, signal collected in the 575/25 channel) and annexin V staining of gated T-LGLL cells are shown. The majority of annexin V–positive cells (those which have lost the polarity of the plasma membrane) showed lowered mitochondrial membrane potential. (C) Cleavage of caspase 9 in CD4 and T-LGLL cells after BNZ-1 treatment (a representative result). PBMCs were collected from a patient with LGLL (UVA-001) before and on day 2 of BNZ-1 treatment. CD4 T cells and T-LGLL cells were sorted using the aforementioned marker definition using BD FACSAria II and lysed radioimmunoprecipitation assay (RIPA) buffer. Cellular lysates were then resolved in a sodium dodecyl sulfate gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with anticaspase-9 antibody (Cell Signaling Technologies); cleavage of caspase 9 was only seen in T-LGLL cells after BNZ-1 treatment. The images were visualized using a standard electrochemiluminescence (ECL) (Femto-substrate, Thermo Fisher) and collected using a Gel Doc system (BioRad). (D) Longitudinal analysis (kinetics) of the apoptosis of CD4 T cells from 15 patients with T-LGLL. Of the 20 enrolled patients, 5 were excluded because their T-LGLL cells showed extremely high apoptosis before BNZ-1 treatment. The annexin V staining of the CD4 T cells (target cells were defined by surface markers as shown earlier) in PBMCs from the remaining 15 patients with T-LGLL are shown. Samples were collected at indicated time points (day 1, 2, 8, 15, and 29; weeks 9, 13, and 17) and 3 washout points (2, 4, and 6 weeks after the termination of the treatment: Z2, Z4, and Z6, respectively). The average and 95% confidence interval are shown. There was no statistical difference in annexin V positivity between day 1 (pretreatment) and any other time point; D01/day 1 is “predose.” (E) Longitudinal analysis (kinetics) of the apoptosis of T-LGLL cells (target cells were defined by the surface markers shown above) from 15 patients with T-LGLL. Of the 20 enrolled patients, 5 were excluded because their T-LGLL cells showed extremely high apoptosis before BNZ-1 treatment. Annexin V staining of the CD4 T cells in PBMCs from the remaining 15 patients with T-LGLL are shown. Samples were collected at indicated time points (days 1 (D01), D02, D08, D015, and D029; weeks 9 [W09], 13 [W13], and 17 [W17]), and 3 washout points (Z2, Z4, and Z6). D01/day 1 is predose. The average and 95% confidence interval are shown. P values from the comparison of the average (%) from ∼D2-D29 in comparison with that of D1 were all < .05. memb, membrane.