Determination of the antigen/epitope that is recognized by human monoclonal antibody CLN-IgG.

Ever since the development of human monoclonal antibody CLN-IgG in 1982, we anticipated the identification of the antigen that is recognized by this antibody. Despite its scarce expression on the cell surface, susceptibility to proteolytic enzymes and adherence to experimental equipment, we finally succeeded in determining the antigen moiety that is recognized by this antibody by means of CLN-IgG conjugated column affinity chromatography, two-dimensional electrophoresis, MALDI-TOF/MS and use of glioblastoma cell line U-251MG. The antigen was found to be vimentin, a cytoskeletal protein, and we succeeded in determining a 79 amino acids sequence of the epitope which turned out to comprise a part of the c2 (coil 2 of the central rod) domain of vimentin.

Enhancing effects of retinoic acid on monoclonal antibody production of human-human hybridomas.

The effects of retinoids on the production of monoclonal antibody of human-human hybridomas were examined. IgG antibody secretion of a hybridoma CLNH11 was enhanced up to about two- to fourfold by retinoic acid (RA) at concentrations ranging from 10(-9) to 10(-5) M, where RA had little effect on the growth rate and saturation density of the cell. Among other retinoids, retinol magnified the antibody production as well as RA. Retinal and retinyl acetate had weak effects. Retinyl palmitate showed no effect. RA also enhanced the production of monoclonal antibodies from other human-human hybridomas: SLNF10, IgG-producing; CoLNE10, IgA-producing; TOS/H8, IgM-producing. RA and human hybridomas provide a defined system to study the effects of retinoids on immune responses at a molecular level.

Identification of a malignant cell associated antigen recognized by a human monoclonal antibody.

A human X human hybridoma, CLNH11, derived from a lymphocyte of a patient with cervical carcinoma, produces a human monoclonal antibody of gamma 1 and kappa isotypes (CLN-IgG). Immunoperoxidase staining showed that CLN-IgG reacted with frozen tissue sections of human malignant tumors (cervical carcinoma, gall bladder carcinoma, glioblastoma), but not with their normal counterparts. Enzyme-linked immunosorbant assay also demonstrated that CLN-IgG reacted with various human tumor cell lines, but not with non-tumorigenic cells such as some fibroblasts, peripheral blood lymphocytes and red blood cells. Indirect and direct immunofluorescence staining indicated that the tumor antigens recognized by CLN-IgG were located in restricted areas close to the cell surface and exposed on the outer surface of the cell membrane. A protein antigen of Mr 226,000 was purified to homogeneity by affinity chromatography with CLN-IgG from the plasma membrane fraction of A549 lung tumor cell line. The antigen consisted of alpha (Mr 60,000) and beta subunit (Mr 53,000) which were linked by disulfide bond(s) (TA60K/53K). The TA60K/53k antigens were expressed commonly in other tumor cell lines originated from histologically different tissues.

Ontogeny of 'macrophage' function. IV. Newborn mouse macrophages strongly suppress tumour cell growth and readily acquire cytolytic activity in comparison with adult macrophages.

Peritoneal macrophages (PM) were prepared as adherent peritoneal exudate cells from newborn and adult mice injected i.p. with thioglycollate medium 4 days previously. In comparison with adult PM, newborn PM exerted a high suppressive effect on the in vitro growth of syngeneic and allogeneic tumour cells, though they did not manifest cytolytic activity. The high suppressive activity of newborn PM was maintained until about 2 weeks of age, and then declined rapidly until about 3 weeks of age toward the level of adult PM. The treatment with a high dose of LPS enhanced the suppressive effect of both newborn and adult PM, while a low dose of LPS was effective only for newborn PM. Necessary minimum dose of LPS to make PM significantly cytolytic was lower for newborn PM than for adult PM. Addition of a low concentration of LK to the culture for activating adult PM with LPS resulted in the augmentation of cytolytic activity and the reduction of necessary minimum dose of LPS. Activation of newborn PM by LPS was not affected by the addition of such a low concentration of LK. On the other hand, newborn PM were rapidly activated by LPS as compared with adult PM. LK accelerated the activation of adult PM by LPS. The activity of newborn PM to bind to tumour cells was higher than that of adult PM. These results seem to indicate that newborn PM are activated to some extent inherently or by some intrinsic agents.

Ontogeny of 'macrophage' function. III. Manifestation of high accessory cell activity for primary antibody response by Ia+ functional cells in newborn mouse spleen in collaboration with Ia- macrophages.

The ontogenesis of the responsiveness of murine whole spleen cells in the in vitro primary antibody response paralleled not only the development of competent lymphoid cells but also that of the accessory cell (A-cell) activity of spleen adherent cells (SAC). The Ia+ cell content of SAC (and also peritoneal exudate cells) was very low until 2 weeks of age. The phagocytic activity of macrophages in SAC was higher in newborns than in adults, though no significant difference was observed between Ia- and Ia+ macrophages in phagocytic activity. We attempted to reveal a high A-cell activity using cells in newborn spleen by means of our experimental strategy documented previously in adult mice (Inaba, Nakano & Muramatsu, 1981): though neither Ia- macrophage population (Ia- SAC) nor a temporarily adherent spleen cell population containing few phagocytic macrophages (crude non-macrophage cell fraction, CF) serves as an autonomous A-cell source, the collaboration of Ia+ non-macrophage cells in CF with Ia- SAC causes the manifestation of A-cell activity. Adult CF collaborated as well with newborn SAC as with adult Ia- SAC, indicating that newborn Ia- macrophages are functionally comparable with adult Ia- macrophages in the ability to collaborate with Ia+ non-macrophage cells. On the other hand, a high A-cell activity was generated by the combination of adult Ia- SAC with a large number of newborn CF cells, indicating that there exist competent Ia+ cells in newborn spleen, though much fewer than in adult spleen.