Fig 2: EVs isolated from NHL and AIDS-NHL cell lines sequester rituximab and inhibit apoptosis in Ramos cells. (A) Characterization of extracellular vesicles isolated from NHL and AIDS-NHL cell lines. Western blots of EVs demonstrate the presence of classic exosome markers: HSP70, TSG101, CD81, CD9, and (B) CD63. 20 µg of EV protein lysate was loaded into each well. Imaging/exposure times of each blot: CD81, 60 s; HSP70, 75 s; CD9, 60 s; TSG101, 40 s; and CD63, 60 s. (C) CD20 concentrations measured in EVs by ELISA. Data is for one Luminex assay run. (D) Ramos cells were treated with rituximab (3 µg/ml) in the presence or absence of EVs isolated from NHL (Raji, Ramos) or AIDS-NHL cell lines (2F7, RRBL, R) cell lines, including OY6, a lymphoblastoid cell line of AIDS-NHL. 30 µg of total EV protein was used for each treatment. Shown are representative flow cytometry plots of Ramos cells double stained with Propidium iodide (PI) and Annexin V-FITC after treatment with rituximab in the presence of 2F7 EVs or Raji EVs to evaluate subpopulations of cells undergoing apoptosis. Percentage values are shown in each quadrant of each plot. (E) Quantitative data presented as bar graphs showing the fold increase of apoptosis: Ratio of % double positive cells (PI + and Annexin V+) after treatment with rituximab/untreated Ramos cells. Results are from two to four independent experiments.

Fig 3: Baseline plasma levels of CD20+ EVs are significantly elevated in AMC-034 trial participants with DLBCL tumor subtype and IPI scores of 2 to 3. The box plot shows the distribution of CD20 concentration (ng/ml) on EVs according to IPI scores 0 to 1 (N = 18) and 2 to 3 (N = 29) for DLBCL tumor subtype. A two-sided Wilcoxon two-sample test was conducted, where p = 0.050.