Relationship between antibody formation in cultures of lymphoid cells and incubation temperature]

An increase of the plaque-forming cells (PFC) and of the 3H-thymidine incorporation in the spleen cell cultures of immunized and nonimmunized C57BL/6 mice were studied in increase of the incubation temperature from 2 to 37 degrees C. An exponential rise of the PFC cells with the elevation of temperature and the presence of the "breaking" temperature, above which the rate of increase of the PFC cells displayed a sharp elevation, was demonstrated. The curves of intensification of the 3H-thymidine incorporation with the temperature elevation failed to follow the pattern of the PFC growth curve in a number of cases. Cultivation of immune cells at low temperatures led to the accumulation in the medium of some factors simulating the AFC formation.     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

Dissociation between proliferation and antibody formation by old mouse spleen cells in response to LPS stimulation.

Both lipopolysaccharide (LPS)-induced proliferation and antibody formation by C57B1/6 spleen cells from old mice were studied by measuring thymidine incorporation and plaque-forming cells (PFCs) to the 2,4-dinitrophenyl group (DNP). There was no significant difference in the proliferative response of spleen cells from young or old mice. Anti-DNP antibody formation by spleen cells from the old mice was greatly reduced. The reduced PFC response could not explained by a shift in kinetics of the responding cells. A similar dissociation could be obtained with LPS-stimulated spleen cells from young mice by using an anti-μ serum or a low concentration of hydroxyurea in the culture medium.     

Study of the inductive phase of antibody formation in tissue cultures

Автор учился синтеза ан тител в submerse культуры ткан ей изолированн ого клет ок селезенк и, в синтетич еских Паркер в средн еср очной содержащ его 0.5% гом ологичных альбумин. Эт о обеспечило хорошу ю п роизводства анти тел, е сли ячеек были и золиров аны от приви вки доноров уже форм ирование антит ел. Когда Клетки селе зе нки explanted 24 часов после иммун изации доноров, антител а никогда не были образ ована в тканях куль тур а; очень изредка, после к онцентрации среде, форм ирование антит ел на тк ани удаляются из орг ан изма 48 часа после им муни зации, была опре делена Когда антиген был доба влен в vitro к селе зенки кле тки от нормальных, не сд еланы прививки доноров или доноров, ранее стим улировали с lipopolysaccharide, антите ла. формирование ник ог да не найдено. В цел ях оп ределения меха низма ан тител синте з с тревого й в тканевых культур, вы ращивания селезен ки кл еток в сочетани и с посл едующим перед ача куль тивированных клеток (с мешанная с антиген) для молодых кроликов. Было обнаружили, что клетки, способные поб удить ан титела форми рование в ыжила в тече ние 6, 12, 24 и Ино гда за 48 часо в без куль ту ры потере ими спосо бно сти формировать анти т ела кроликов. Это по каз ывает, что ткань культу ры не поврежд ение клет ок, но что он а делает не о беспечи т необходимые услов ия для биохимиче ских и морфологичес ки х изменений диффе ренц иации, которые п роисхо дят в vivo при индуктивной фазы антитела синтез.     

Stromal fibroblasts of the hematopoietic organs and antibody formation in cultures

The thymus, bone marrow- and spleen-derived stromal mechanocytes from the monolayer cultures (3rd--6th passages) when added to the suspension cultures of rabbit spleen cells according to Mishell and Dutton produced a considerable effect on the plaque-forming cells (PFC) accumulation by the 4th day of cultivation. Their action distinctly depended on the dose. Bone marrow-derived stromal mechanocytes in doses of 2.1 X 10(3)--6.25 X 10(5) caused inhibition of PFC formation in cultures. Thymus-derived stromal mechanocytes in doses of 2.75 X 10(3)--8 X 10(5) cause an increase in number of PFC; spleen-derived stromal mechanocytes in doses of 2.1 X 10(3)--1.3 X 10(4) failed to bring about any significant changes, but when the dose was increased to 8 X 10(4)--6.25 X 10(5) the inhibition of PFC formation took place. Most of the live cells and PFC were found in the free cells fraction.     

Induction of antibody to foot-and-mouth disease virus in presensitized mouse spleen cell cultures.

Cultures of spleen cells from immunized mice were stimulated in vitro by soluble preparations of purified foot-and-mouth disease virus. Virus-specific antibody, as detected by an enzyme-linked immunosorbent assay, was produced by immune spleen cells but not by normal, nonimmune cells. The optimal specific response was obtained with 1 microgram of virus per ml of culture; as the virus concentration was increased, the production of specific antibody was reduced. For very low concentrations of virus (less than 0.01 microgram per culture), there was tentative evidence of suppression of the specific antibody response. The levels of specific antibody induced were dependent on the source and number of plastic-adherent cells present in the cultures. We intend to use this model system to study further the basis of immunity to foot-and-mouth disease virus.