Characterization of an immunostimulatory factor associated with spontaneously regressing subline of murine L1210 lymphoma

Abstract

The cell free ascitic fluid from X-irradiated DBA/2J mice, bearing the spontaneously regressing subline (L1210/MR or MR) of L1210 murine lymphoma, contains a potent immunostimulatory (IST) factor that is capable of stimulating a specific secondary IgG antibody response, when injected into normal mice 3-4 days before priming with an antigen. When the ascitic fluid was fractionated by Ultrogel AcA-22 column chromatography, the IST activity was localized in the high-molecular-weight fraction (>1000 Kd), designated as UG-IST. UG-IST, on the other hand, suppressed the primary IgM antibody response. Further purification of UG-IST by ultracentrifugation in KBr at densities of 1.25, 1.063, and 1.006 g/ml revealed that IST behaved like a high-density lipoprotein (HDL-IST), and was more potent than complete Freund's adjuvant in stimulating the secondary IgG response. Extraction of UG-IST by chloroform-methanol revealed that the IST activity was mostly associated with the aqueous protein phase. When the extracted lipids were separated by thin-layered chromatography, compounds with Rf values similar to that of cholesterol esters were the only major lipids present. The effect of IST on the production of IgM-bearing cells in the mouse lymphoid organs (the bone marrow, spleen, and the lymph nodes) was also tested. When mice were treated with MR ascitic fluid, UG-IST or HDL-IST 3-4 days prior to organ removal and with or without antigen priming, only the bone marrow showed an increase in the percentage of IgM-bearing cells; all other lymphoid organs tested had no such increase.

We conclude from this study that this unique factor, that is capable of stimulating secondary IgG response, is a high-density lipoprotein containing almost all neutral lipids, and the active moiety may reside in the protein part of this complex. It also increases the percentage of IgM-bearing cells in the bone marrow, even without antigen priming. This IST may also be the same factor which suppresses the primary IgM response, but needs further investigation. We postulate that IST is possibly a specific B-lymphocyte mitogen, which causes the proliferation of certain precursor B-lymphocytes, and it promotes the differentiation and maturation of B cells into plasma cells by enhancing class-switching and the generation of more memory B cells in an animal when primed with a T-dependent antigen.