Figure 4.

Hsp70/Bag-4–positive exosomes derived from Colo+ and CX+ carcinoma sublines stimulate cytolytic activity of CD94⁺ NK cells against Hsp70/Bag-4–positive tumor targets. CD94⁺ NK cells were incubated either with IL-2 alone (control), with IL-2 plus TKD (TKD), or with IL-2 plus exosomes (10 μg protein/mL) derived from Colo−/Colo+ or CX−/CX+ carcinoma sublines for 4 days. A, cytolytic activity was measured using Colo+/Colo− and CX+/CX− carcinoma sublines as target cells in a standard ⁵¹Cr release assay. A comparable, strong lysis was detected against Colo+ and CX+ carcinoma sublines if NK cells were stimulated either with IL-2 plus TKD (TKD) or with exosomes derived from Colo+ and CX+ carcinoma sublines. After stimulation with IL-2 alone or with exosomes derived from Colo− and CX− carcinoma sublines, lytic activity of Hsp70/Bag-4–positive tumor target cells was weaker. Mean values of three to four independent experiments ± SE, at one distinct E/T ratio of 10:1, were summarized in Table 2A. B, lytic activity of CD94⁺ NK cells against CX+/CX− tumor target cells was also measured after stimulation with exosomes derived from Colo−/Colo+ carcinoma sublines. Again, lysis of CX+ tumor targets was significantly enhanced by stimulation with exosomes derived from Colo+ but not of Colo− exosomes. These data revealed that Hsp70 reactivity in NK cells is inducible by Hsp70/Bag-4 surface-positive exosomes, independent of the tumor cell origin. Mean values of four independent experiments ±SE, at one distinct E/T ratio of 10:1, were summarized in Table 2B. C, Hsp70/Bag-4–positive exosomes derived from Colo+ and CX+ carcinoma sublines stimulate secretion of granzyme B by CD94⁺ NK cells. CD94⁺ NK cells (2 × 10⁶ cells/mL) were incubated with different concentrations (0.1, 1, and 10 μg protein/mL) of Hsp70/Bag-4 surface-negative and surface-positive exosomes, generated from Colo− and Colo+ carcinoma sublines. As a control, NK cells were incubated with low-dose IL-2 (100 IU/mL) alone. After 4 days, the cell-free culture supernatants were harvested and the amount of released granzyme B was quantified by ELISA. Columns, mean values of three independent experiments; bars, SE. *Significantly different values from control levels (P < 0.05).