ACIDIFICATION IS ESSENTIAL FOR MAINTAINING THE STRUCTURE AND FUNCTIONOF LYTIC GRANULES OF CTL - EFFECT OF CONCANAMYCIN-A, AN INHIBITOR OF VACUOLAR-TYPE H-ATPASE, ON CTL-MEDIATED CYTOTOXICITY()

An inhibitor of vacuolar type H+-ATPase, concanamycin A (CMA), inhibit ed the specific cytolytic activity of a CD8(+) CTL clone, OE4. The inh ibitory effect was observed when the effector cells, and not the targe t cells, were pretreated with CMA. CMA did not seem to inhibit early e vents, inasmuch as effector/target conjugate formation remained unaffe cted. Although CMA treatment of OE4 resulted in a slight decrease in t he efficiency of granule exocytosis in response to anti-CD3 stimulatio n, the most prominent effect was a marked reduction of perforin activi ty and DNA degradation activity in lytic granules. Western blotting an alysis indicated a drastic decrease in the amount of perforin in CMA-t reated cells. Fluorescent microscopic observation of OE4 stained with acridine orange indicated that CMA raised the pH of the lyric granules . Under transmission electron microscopy, striking morphologic changes in cytoplasmic granular structures were observed after CMA treatment of OE4. The lytic granules of OE4 had homogeneously stained large core s and numerous small vesicles that filled peripheral areas. In contras t, the lyric granules of CMA-treated OE4 showed irregular shapes with no small vesicles, but with cores that became rough and loose. Vacuole s with no structure in them were seen occasionally. These results sugg est that acidification through vacuolar type H+-ATPase is essential to maintain the structure and function of lytic granules.