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It has been previously established that an intravenous injection of a protein antigen solution into mice primed with the same antigen in the form of a protein-cellulose complex induces an intensive antibody production (up to 10,000 antibody-forming cells/10(6) splenocytes and up to 3 mg of antibodies/ml of serum). The present study has shown that secondary immune response can be considerably enhanced if large amounts of the antigen are administered intraperitoneally in a protein-cellulose complex during secondary immunization. In these experiments the mean number of antibody-forming cells was 50.000/10(6) splenocytes and the antibody serum level averaged 10 to 12 mg/ml. The effect persisted for a long time: as late as on day 80 the antibody concentration was 2 mg/ml of serum.

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The protein-cellulose complex prepared by covalent immobilization of single protein molecules on insoluble cellulose particles was used for priming C57BL/6 and BALB/c mice. The serum antibody content and the number of spleen AFC were assayed after animals' boosting with the soluble protein. Such a complex was shown to have marked advantages over the same protein injected both in complete Freund's adjuvant and in a soluble form, in particular. The immune response of BALB/c mice was more than 10-fold higher than that of C57BL/6 animals.

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The role of surface antigens in the density-dependent inhibition of primary immune response in mouse spleen cell cultures was investigated. For this purpose Fab-fragments of rabbit IgG obtained after immunization with mouse splenocytes were used. Such Fab-fragments alone had no effect on immune response in both optimal and dense cultures. However, successive treatment of cells with Fab-fragments and with ass antibodies against rabbit IgG dramatically augmented the density-dependent inhibition of antibody formation.

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The appearance of antibody- and nonspecific immunoglobulin-forming cells (AFC and nIFC) in spleen cell suspensions from normal mice and from those immunized with sheep red blood cells (SRBC) was studied. The cell suspensions were cultivated in the modified Mishell-Dutton system. The induction of the immune response in vitro resulted in a sharp increase of nIFC formation. Their amount reached 51 218 per 10(6) spleen cells, i. e exceeded the number fo AFC 25-172-fold. Much more was the increase in the absolute number of nIFC during cultivation in the presence of antigen of spleen cells from animals immunized with SRBC 3--4 days before experimentation. In this case, the maximum number of nIFC reached 165 000 per 10(6) spleen cells, i. e. 33% of the total number of B-cells. The kinetics of AFC and nIFC formation and regulatory mechanisms of the populations varied under different conditions. The nature and potential mechanisms of nIFC formation under the influence of the antigen are discussed.

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The cultivation of normal mouse spleen cells in a modified Mishell-Dutton system for 1-4 days in the presence of a water-soluble antigen of sheep red blood cells results in a sharp increase in the number of cells secreting non-specific immunoglobulins (nIFC). This increase is much more visible if spleen cells from mice primed with the same antigen 3-4 days before cultivation, are used. The rise in NIFC becomes apparent on day 1 and runs up to maximum on day 3. At this time a peak of 165 X 10(3) nIFC per 10(6) cells is attained, i.e. the nIFC quantity reaches approximately 33% of total B-cells. Kinetics of the antibody-forming cells and nIFC appearance under varying conditions is different. Clearcut differences are also revealed between the mechanisms of regulation of both these populations. The initial population of cells destined to form non-specific immunoglobulin is estimated to be 363/10(6) cells during a primary immune response in vitro; if splenocyte donors are primed with a homologous antigen, this population become approximately 800-1,900/10(6) cells.

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A study was made of the effect of mitogens on general proliferation and primary immune response to sheep red blood cells in density-inhibited cultures of mouse spleen cells. The mitogens applied included fetal calf serum and both B cell- and T cell-specific mitogens (dextran sulfate, LPS and ConA). Experiments with 3H-thymidine incorporation demonstrated that the proliferation was equally enhanced by any mitogen in both optimal and density-inhibited cultures. The mitogens did not remove the density inhibition of antibody formation.

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Incorporation of uridine into the cells was studied under conditions of rapid increase in density of the cell suspension either freshly-prepared from spleen of mice strain C57BI/6 or subjected to precultivation. Absorption of uridine by the cells after an increase in their density was rapidly decreased within several minutes. The phenomenon was not due to presence of dead cells in the culture. An inhibitor of protein synthesis puromycin partially removed the suppression of uridine absorption in dense cultures.

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The authors previously reported local intercellular interactions suppressing the increase of the antibody-forming cell (AFC) count in the suspension of the spleen cells of nonimmunized mice and sharply elevating with increase in density of the cultivated suspensions. This work showed that preliminary immunization of mice with an antigen eliminated or sharply reduced the accretion inhibition of the AFC cells against the given antigen, but failed to influence the accretion inhibition of cells producing antibodies against another antigen, and the proliferation inhibition of the dividing bulk cells in the culture.

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There was a sharp decrease in the number of antibody producing cells formed in induction of primary immune reaction in vitro with increase of surface density (number of cells per 1 cm2 of the bottom of the incubation vessel); the cause of this phenomenon was studied. It was shown that depression of antibody formation in dense cultures was associated not with disturbance of the induction of the clone of antibody-forming cells, but with the inhibition of the subsequent proliferation of the cells of this clone. Disturbance of proliferation was reversible: a reduction of the density of the cultures 1 or 3 days after the beginning of the incubation led to the restoration of the growth of the number of antibody-producing cells. The depressive effect could not be transferred by humoral way from the cultures with a high density to the cultures with a low density; it could not be eliminated by daily replacement of the incubation medium in dense cultures for a fresh nutrient medium.

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Stromal fibroblasts from the monolayer cultures of human bone marrow, guinea pig bone marrow, spleen, thymus and peripheral blood suppressed the response of the plagueforming cells against sheep erythrocytes in the suspension cultures of mouse spleen cells. Combined cultivation of 20 X 10(6) fibroblasts from all the mentioned sources led to complete suppression of the immune response. This suppression was less in mice immunized three days before the spleen cell explantation into the suspension cultures and was absent entirely in case the pre-immunization of spleen cell donors was accomplished nine days before the explantation.

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A relationship was revealed between the number of cells in the suspension of the splenic tissue taken for induction of primary immunization of the reaction outside the organism and the number of the plaque-forming cells (PFC) formed by the end of incubation. It was shown that with increase of the cell density in the culture in the bottom of the incubation flask there was a 10-100 fold decrease of the PFC response; as to the cell viability--it was not affected or decreased but slightly. This effect was observed in using sheep red blood cells and water-soluble antigen extracted from them as an antigen. The effect was independent of shortage of the antigen or of the nutrient substances; it was accompanied by a general reduction of the 3H-thymidine incorporation into cultured cells.

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