Deep profiling of circulating lymphocytes cells in patients with breast cancer after long-term venetoclax treatment reveals limited changes in T-cell subsets. (A) Schematic representation of immunophenotyping of the patients with breast cancer who received long-term venetoclax (VEN) treatment. Blood was collected from patients with breast cancer before (pre) and after (post) venetoclax treatment (median treatment, 88.6 weeks; range, 8.8-204.2 weeks). Pre- and posttreatment cells from each patient were analyzed by CITE-seq (n = 5 pairs). (B) Concentrations of B cells from the 5 samples selected for CITE-seq. (C) UMAP and annotation of circulating immune cells from 5 patients with breast cancer on long-term venetoclax treatment analyzed by CITE-seq. (D) Dot plots showing a curated list of key cell-surface makers expressed in naïve (cluster 12), transitional (cluster 13), and memory (clusters 14 and 15) B-cell subsets. Color intensity reflects relative level of protein expression and the size of the dot indicates the fraction of cells expressing that protein in that cluster. (E) Table of the numbers of DEGs (false discovery rate < 0.05) found by pseudobulk DEG analysis (postvenetoclax:prevenetoclax). (F) Violin plots of BCL-2 survival gene transcription from pseudobulk analysis of all clusters. (G) Intracellular protein expression of MCL-1 measured by flow cytometry in B cells from patients BC_01024 and BC_01028. (H) Changes in MCL-1 protein expression measured in immune cell populations expressed as log2 changes in post- vs pretreatment measured by flow cytometry. (I) The expression ratio of checkpoint genes on CD4⁺ and CD8⁺ T cells, calculated by mean (postvenetoclax)/mean (prevenetoclax) measured by CITE-seq. In graphs in panel H, each symbol represents an individual patient, bar is at the mean, and error bars represent standard error of the mean. mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell; Tcon, concentration of T cell; Treg, regulatory T cell.