Are lytic granules and perforin 1 involved in lysis induced by in vivo-primed peritoneal exudate cytolytic T lymphocytes?

We examined whether in vivo-primed, alloimmune, peritoneal exudate CTL obtained directly from the animal kill TC by perforating their membranes with C-like lesions produced by P1 or P2 originating in cytolytic granules of the CTL. In control experiments lytic granules were isolated from the formerly lytic CTLL-2 line as well as from Con A-activated lymphoblasts but not from the in vivo-primed CTL. The use of anti-P1 of CTLL-2 origin supports the above results in that immunoblots were positive with CTLL-2 extracts and isolated granules thereof but negative with extracts and isolated (nonlytic) granules from in vivo primed CTL; immunofluorescence of permeabilized cells gave similar results. We observed typical "ring structures" on EL4 membranes attacked by Ab + C as well as after exposure to lytic granules extracted from the formerly lytic CTLL-2 line; however, no such "structures" were observed after attack by the in vivo-primed CTL. These results are at variance with the recent suggestion of a common lytic mechanism--employed by CTL--of secreted lytic granules containing P1 (and P2) thereof that acts analogously to C9. An alternative mechanism for CTL-induced lysis not involving initial perforation of the TC membrane by 160A "holes" (Poly P1) has been presented elsewhere. It is important to note that our results with in vivo-primed CTL do not preclude involvement of a secreted, pore (channel)-forming agent(s) that is not observed by negative staining EM.