Venetoclax treatment does not alter T cell proliferation or effector function. (A) Schematic of the time points analyzed, early clinical benefit, and dose of venetoclax for each patient with breast cancer. Measurements were made at pre- (cycle 1) and post- (cycles 2 and/or 4-23); each cycle is 28 days. (B) Flow cytometry gating strategy to identify T-cell populations: lymphocytes, single cells, propidium iodide–negative live cells, CD3⁺ T cells, and either CD4⁺ or CD8⁺ cells. (C) Representative histograms showing expression of CD25 by CD4⁺ or CD8⁺ T cells from 1 patient during venetoclax treatment after incubation with anti-CD3/CD28 beads for 48, 72, or 96 hours. Graphs summarize CD25 expression by CD4⁺ or CD8⁺ T cells in patients during venetoclax treatment cycles. (D) Representative histograms showing cell trace violet (CTV) staining of CD4⁺ or CD8⁺ T cells from 1 patient during venetoclax treatment cycles after incubation with anti-CD3/CD28 beads for 48, 72, or 96 hours. Graphs show the percentage of proliferating cells (having undergone ≥1 division) in CD4⁺ or CD8⁺ T cells in patients at different venetoclax treatment cycles (n = 5). (E) Graphs showing the concentration of cytokines and cytotoxic molecules measured using the LEGENDplex CD8/NK panel produced by total T cells after 48 hours of culture with anti-CD3/CD28 beads for patients during venetoclax treatment cycles. In graphs in panels C-E, each symbol represents an individual patient, bar is at the mean, and error bars represent standard error of the mean. IFNγ, interferon gamma; IL-4, interleukin 4; NS, no stimulation; SSC, side scatter; TNF, tumor necrosis factor.