Clonal derivatives 8 and 11 of the T47D human breast carcinoma cell line release particles that have the biochemical characteristics of a retrovirus. Particles recovered from cultures of [3H]uridine-labeled clone 11 had a density of 1.18 g/ml and contained 60-70S and 35S RNAs associated with reverse transcriptase activity. The production of these particles was steroid-dependent. Clone 8 particles had a higher density, 1.195 g/ml, and their production was independent of steroid hormone. By RIA, antigens crossreactive with the 52,000-dalton envelope glycoprotein gp52, the major external protein of mouse mammary tumor virus, were found associated with these particles and in the media. Most of the gp52-related antigen was in soluble form, but it was enriched in the particle preparation. A lesser amount of antigen was distributed within the cultured cells. Absorption of rabbit antibody to gp52 with clone 11 particle preparations eliminated the ability of this antibody to detect immunocytochemically a crossreactive antigen previously localized in tissue sections of human breast carcinoma. These results indicate that the particle isolates from T47D contain the same gp52-related antigen found in human breast carcinomas and constitute an excellent source for the purification and characterization of this antigen.Previous studies (1)(2)(3)(4) To this end, it was of interest to resolve certain issues regarding the nature of the crossreactivity observed between gp52 and the unique antigens found in human breast cancers. Our experience indicated that it was unlikely that the supply of surgically removed human tumors would provide sufficient antigen for the necessary biochemical studies. Therefore, we focused on the T47D cell line, which had been established from the pleural effusion of a patient with intraductal and invasive carcinoma of the breast (13).It is the purpose of the present paper to present data on the T47D cell line and its clonal derivatives that reproducibly secrete virus-like particles containing the gp52-related antigen. This situation solves the difficult logistic problem of making available a reliable source of material from which to purify the relevant breast cancer antigens with comparative ease. MATERIALS AND METHODSCells. The cells were grown in RPMI 1640 medium containing fetal calf serum (10%), insulin (0.2 unit/ml), glutamine (2 mM), streptomycin (100 pug/ml), penicillin (100 units/ ml), and mycostatin (25 ,g/ml). When the cells were grown in the presence of steroid hormones, the fetal calf serum was dialyzed.For virus collection, the cells were seeded in RPMI 1640 medium containing 10% fetal calf serum for 24 hr followed by medium containing 5% dialyzed fetal calf serum and 1 nM 17-estradiol. After one medium change, this medium was replaced by one containing 10 nM progesterone free of fetal calf serum (day 5 after passage). From day 6, medium was collected every day, and the cells were refed with progesterone-containing medium. The collected medium was clarified by centrifugation for 15 mi...

SUMMARYA mechanism is proposed which explains the perpetuation of B-cell immunological memory inde®nitely without requiring the presence of long-living memory cells or persisting antigen. The salient feature of this model is that immunological memory can be perpetuated inde®nitely through the mutual interaction of idiotypic and anti-idiotypic B cells. These cells mutually stimulate and clonally expand with either speci®c or bystander T-cell help. Because B cells can present antigen, they present`apparently foreign' idiopeptides to T cells. The idiopeptides of de novo synthesized antibody is presented to CD8 + T cells that recognize the idiopeptide-presenting cell as targets and regulate their population. The recycling of immunoglobulins from surface to endosomal compartment of B cells leads to the presentation of idiopeptides by major histocompatibility complex (MHC) class II to CD4 + T cells. Even if the majority of the clonally expanded cells die because of lack of stimulation, cytotoxic T lymphocyte (CTL) lysis or for other reasons, the surviving cells will be able to carry forward the memory. This mechanism also provides a means for af®nity maturation through idiotypic selection of somatically mutated high af®nity cells or those from the nai È ve pool. We have termed these two types of complementary B cells as Burnet B cells: those which recognize the antigen or antigen mimic, and Jerne B cells, which can recognize the idiotypes of antibody and carry antigen mimics. The proposed hypothesis can explain differential duration of memory for different antigens, the shelf space paradox, af®nity maturation, repertoire shift, etc.

No abstract

The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells. We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E. coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells. Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c). A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response. The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted. Based on the prediction for H-2(d)-restricted T-cell motifs we tested the lysis of transfected P815 (H-2(d)) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins. We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins. Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope. This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides.

No abstract