Characterization of a non-granule associated pore-forming protein in agranular lymphocytes

Recently, two populations of small lymphocytes (SL) have exhibited non-major histocompatibility complex (MHC) restricted lysis. Recent studies by numerous laboratories have demonstrated that resting T cells triggered through CD3 and CD28 costimulations can result in immediate, non-MHC restricted killing. Our recent studies with CD3-, CD56+ SL demonstrated that although these cells contained no cytoplasmic granules detected with electron microscopy, they mediated potent NK and ADCC activity. In the present study, we have used a Western blotting technique that allows for the detection and quantitation of total cellular levels of pore-forming protein (PFP). We have found that freshly isolated peripheral non-granulated lymphocytes (both CD3+ and CD3-) contain PFP. In addition, CD3-, CD56+ SL contain levels of PFP similar to those of the highly granular CD3- LGL. In search of non-granule PFP, we exploited the rat NK (RNK) cell lines as a source of other potential cytotoxic factors. A membrane associated PFP, based on Western blotting, was isolated from rat RNK cells. Unlike PFP isolated from granules, this PFP was active after culture in Ca(2+)-containing medium. However, the lytic activity isolated from the non-granule PFP of RNK cells was inhibited by monoclonal antibodies to PFP. Collectively, these studies indicate that PFP is present in small agranular lymphocytes (both NK and T cells) and that it is not stored in large cytoplasmic granules. The implication of our results for the acquisition and activation of lytic ability in NK and T cells will be discussed.