Fig 2: Single cell RNA-seq analysis reveals vaccine-associated reduction of LPL B-cell but not plasma cell-like subpopulations in the bone marrow.A Schematic of personalized chemokine-idiotype DNA vaccines and treatment schedule. B Swimmer plot illustrating clinical responses (patients are designated as LPL-001 through −009) (C) UMAP of B-lineage cell populations extracted from the total dataset (left). Total cell numbers per cluster are shown for normal B-lineage cells and, specifically, tumor B cells, based on idiotype sequences (right). D Cell frequencies by total B-lineage cluster, comparing pre- and post-vaccine bone marrow for individual patients (n = 8; mean values +/- SD). Two-sided Wilcoxon matched-pairs signed rank test was used (E) Cell frequencies of BCR clonotypes in all B-cell clusters pre- and post-vaccine. Blue = dominant tumor idiotype clone isolated for vaccine production; all other colors indicate normal B-cell clonotypes, except for patient LPL-002 (see Results). F Pooled data from all patients. Volcano plots of differentially expressed genes (adjusted p-value < 0.05) pre- vs. post-vaccine for the major B-lineage clusters containing tumor cells (top). Ingenuity Pathway Analysis of differentially expressed genes (z-score > 2, adjusted log p-value < − 1.3) of canonical pathways (bottom left) and biological processes (bottom right) contrasting tumor mature B-cell (0-2) and plasma cell-like clusters (5,10), respectively. Wilcoxon rank-sum test was used. P-values were adjusted with Bonferroni correction. Colors represent z-scaled pathway activity scores (G) Heatmap of HLA class II gene expression by clonal tumor cells pre- and post-vaccine relative to total cells, grouped by B-lineage cluster (left). Dot plots of HLA family gene expression by clonal tumor cells pre- and post-vaccine in cluster 2 only. H Dual staining immunohistochemistry of post-vaccine bone marrow slides for HLA class II and either CD79a or CD138 to identify mature B- cell and plasma cell-like LPL subpopulations. Normal human tonsils served as a positive control. All images are 200X magnification Scale bar = 50 μm (left). Flow cytometry analysis of HLA-DR expression on plasma cell-like (CD38highCD19low) and mature B cell (CD38lowCD19high) subpopulations of MWCL-1 and BCWM.1 LPL cell lines (right). Representative result of three independent experiments. Source data are provided as a Source Data file.

Fig 3: BCR pathway perturbations drive changes in cell mass and stiffness in Jeko-1 cells.(A) Schematic illustrating BCR inhibition via acalabrutinib treatment (top panel), BCR stimulation through anti-IgM treatment (middle panel) and BCR pathway enhancement through BLK and CD79A overexpression (bottom panel) in Jeko-1 wild type (wt) cells for biophysical profiling. BCR inhibition was confirmed by western blot, and stable overexpression in cell lines was validated using RT-PCR, western blot, and flow cytometry. Functional assays (cell cycle and proliferation) further assessed GFP, BLK or CD79A-expressing cells. Created with BioRender. (B) Median single-cell mass and stiffness measurements obtained using the SMR from >500 Jeko-1 wild type cells after 1h or 24h of ex vivo treatment with DMSO, 0.25 μM, or 0.5 μM acalabrutinib. Data are presented as the median ± SD of three biological replicates. (C) Median single-cell mass and stiffness measurements from >500 Jeko-1 wt cells, with or without IgM stimulation for 10min or 24h, assessed using the SMR. Data represent the median ± SD of three biological replicates. (D) Relative mRNA expression of BLK and CD79A measured by RT-qPCR in Jeko-1 overexpressing GFP, BLK, or CD79A. Results are presented as fold change normalized to GAPDH mRNA. Each bar represents the mean ± SEM of two independent stable cell lines. (E) Representative images of western blot showing BLK, CD79A, GFP and α-actinin protein in Jeko-1 overexpressing GFP, BLK or CD79A. (F) Median single-cell mass and stiffness measurements obtained using the SMR from >500 cells of Jeko-1 wild type (WT) and Jeko1 cells stably overexpressing GFP, BLK or CD79A. Data are presented as median ± SD of three biological replicates. NS: non-significant, *P < 0.05, **P < 0.01 as compared between indicated groups (Student’s t-test).