DNA vaccination against v-src oncogene-induced tumours in congenic chickens.

DNA vaccination is particularly efficient for induction of cytotoxic T-lymphocyte (CTL) response. In our experiments, we used MHC(B) congenic chicken lines CB and CC (regressors and progressors of v-src-induced tumours, respectively) and a mutated, non-oncogenic v-src gene construct as the DNA vaccine. A high degree of vaccine protection against oncogenic v-src challenge was achieved in the CB line chickens. CTL response was demonstrated in vitro and by adoptive transfer of immune cells to the syngeneic host and to the CC line chickens rendered tolerant to CB cells. In the CC line chickens we observed tumour growth retardation after a low-dose DNA vaccination administered to immature recipients while higher amounts of DNA vaccine in immunocompetent chickens exerted an enhancing effect.

Production of monoclonal antibodies specific to the carboxyl terminal region of the 85 kDa subunit of phosphatidylinositol 3-kinase: use of the antibodies in recognition of mutant p85.

We have established two hybridomas producing mAb to the carboxyl terminal region of phosphatidylinositol-3 kinase 85 kDa subunit type alpha (p85 alpha). Analysis using deletion mutants of p85 revealed that epitopes for the two mAb were located on the border of the src homology 2 (SH2) sequence located at the carboxyl end of p85. They immunoprecipitated free p85 efficiently, but reactivity to p85 bound to p110 was very weak. Together with the mAb which we have reported previously, a panel of mAb that covered the various parts of p85 alpha was obtained. Using this panel, we characterized two mutants of p85 (70 and 50 kDa) expressed in the human colon carcinoma cell line, HCC2998. No wild-type p85 was detected in these cells. A mAb specific to the carboxyl terminal region detected p70 but not p50, suggesting that this region is missing in p50. The panel of mAb is a useful tool to use to analyse mutant forms of p85.

src-specific immune regression of Rous sarcoma virus-induced tumors.

We have developed an oncogene-specific tumor regression system in chickens. Injection s.c. of chicken wing webs with either rASV1702 or rASV157, mutants of Rous sarcoma virus (RSV) that express nonmyristolyated src product containing novel N-terminal domains, results in noninvasive fibrosarcomas that regress fully. The ability of challenge infections with wild-type RSV to form tumors is suppressed. This protective effect was shown to be specific for determinants encoded or induced by v-src (I. Gelman and H. Hanafusa, J. Virol., 61: 2461-2468, 1989). In the current study, we used SC chickens, inbred for major histocompatibility complex Class I haplotype B2/B2, to investigate whether this protection results from active immunity. Preinoculation of chickens with either replication-defective rASV1702 virus or non-virus-producing syngeneic chicken embryo fibroblasts expressing 1702src conferred protection against challenge infections with RSV. Thus, viremia was not required for this protection. Splenic lymphocytes from rASV1702-infected donors could transfer protective immunity against RSV tumor challenge to naive chickens. These lymphocytes were cytotoxic in vitro against RSV- or rASV1702-infected SC-chicken embryo fibroblasts, but not against SC-chicken embryo fibroblasts infected with helper virus, suggesting a specificity for src-encoded or -induced determinants. In contrast, splenic lymphocytes from RSV-infected chickens transferred protective immunity poorly and exhibited low in vitro cytotoxic potential for src determinants, suggesting possible suppression mechanisms. Finally, murine cell lines expressing 157src or 1702src produced tumors in nude mice that failed to regress. Thus, although cells expressing 157src or 1702src are inherently tumorigenic, the tumors they induce most likely regress due to immune mechanisms. These results suggest that 1702src and 157src induce src-specific tumor Ag that potently prime an oncogene-specific protective cellular immunity.