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. 2011 Aug 25;108(37):15336–15341. doi: 10.1073/pnas.1102855108

Fig. 2.

Fig. 2.

Absorption of anti-CD20 antibody rituximab and consumption of complement on lymphoma-derived exosomes in vitro and in vivo. (A) Exosomes from Su-DHL-4 cells were added to medium supplemented with rituximab at an initial concentration of 35 ng/100 μL for 1 h at 21 °C. Soluble rituximab was measured by ELISA, and mean values of triplicates from a representative experiment were expressed as a percentage of controls treated with nonexosome-labeled beads only. Asterisks mark significant reduction, based on repeated measure ANOVA with Bonferroni's post test, for CD20-negative exosomes from K562 leukemia cells as control (Fig. S3B). (B) Exosomes were prepared from the plasma of patients at 3 h after the end of infusion, and rituximab binding was detected with a specific antibody (MB2A4) by flow cytometry (for further examples, see Fig. S3A). (C) For quantification of free versus exosome-bound antibody, rituximab was measured by ELISA, documenting approximately one-third to one-half of plasma rituximab bound to exosomes. Rituximab in the exosomal pellet is represented by dark shading, soluble rituximab from the supernatant by light shading. (E) Latex beads were coated with lymphoma exosomes and exposed to rituximab in the presence of 20% human serum for 30 min. Formation of the terminal complement complex (TCC) was detected with the anti–SC5b-9 primary monoclonal antibody WI3/I5, a phycoerythrin (PE)-labeled secondary antibody and measured by flow cytometry (E, Upper panels). As control for exosome labeling, the beads were stained for the exosome marker flotillin-2 (E, Lower panels). TCC formation was also detected on exosomes from patient samples taken 3 h after exposure to rituximab (F). (D) For an estimate of complement consumption, C3d levels were measured by ELISA using a polyclonal rabbit antibody (I3/15) following addition of beads labeled with exosomes from Su-DHL-4 lymphoma cells in the presence of rituximab (D, bars on right), with PBS and unlabeled beads as negative controls and zymosan as positive control for maximal complement fixation. Error bars indicate SDs of triplicates from a representative experiment of three replicates (repeated measure ANOVA with Bonferroni's post test).