Specific detection of galectin-9–containing exosomes in the plasma of NPC patients. (A) Captures with anti–HLA class II beads were performed on low-density vesicles (110-kg pellets) derived from human plasma samples. Initially, plasmas were collected from 2 NPC patients (NPC A and B; samples 2, 3, 4, and 5), 1 patient with a non-NPC head and neck carcinoma (non-NPC TA; samples 6 and 7), and 1 healthy donor (samples 8 and 9). In each case, beads coated with nonspecific Ig (NS Ig) were used as negative controls. In addition to plasma material, a protein extract from the C17-xenografted tumor was used as a positive control for Western blot detection (no. 1). Clinical and pathologic data on donor patients are provided in Tables S1 and S3. (B) Numerous bilamellar vesicles approximately 70 nm in diameter are visualized by HLA class II capture and electron microscopy when these procedures are applied to plasma vesicles from NPC patients but not control subjects. Scale bar represents 500 nm. In the inset, 2 vesicles at original magnification (× 4). (C) In parallel experiments, Western blot analysis revealed expression of galectin-9, CD9, CD63, and the α chain of the DR molecule in NPC plasma vesicles. In contrast, none of these proteins is detected when the same procedure is applied to control plasmas. Gp96 is only detected in the tumor extract. (D) Galectin-9–carrying exosomes are captured by anti–HLA class II beads from 4 additional samples of NPC plasmas (NPC C, D, E, and F) but not from 2 control subjects with non-NPC tumors (non-NPC TB and TC). Clinical and pathologic data on donor patients are in Tables S1 and S3. Consistent results were obtained for 11 NPC and 5 non-NPC additional plasma samples (see Tables S1, S2, and S4 and Figures S1 and S2).