Intravenous infection of six inbred mouse strains with small doses of dispersed cells of Mycobacterium bovis BCG (15.5 x 103 or 15.5 x 104 colony-forming units) separated them into resistant (C3H/HeCr, A/J, and DBA/2) and sensitive (B10.A, C57BL/6, and BALB/c) strains as assessed by the magnitude of bacterial multiplication in the spleens at 28 days. The two groups were more sharply separated after infection with the lower dose of BCG (15.5 x 103 colony-forming units), which allowed for true multiplication of the bacteria in the spleens of permissive hosts, expressed as the ratio of the number of BCG recovered from the spleens to the number of BCG injected. This coefficient of increase was less than 1 in resistant strains, whereas it was higher than 2.5 in sensitive strains. Significant splenomegaly developed only in mice of the sensitive strains infected with BCG when compared with uninfected controls. There was no correlation between the magnitude of the delayed-type hypersensitivity (DTH) to BCG and susceptibility to infection: DTH was absent in both the sensitive and the resistant strains when the smaller dose of BCG was used for infection. Moreover, significant DTH was detected in animals of the most sensitive (BALB/c) as well as of the most resistant (C3H/HeCr) strain when the higher dose of BCG (15.5 x 10W) was used for immunization. These results document significant genetic differences in the ability of inbred mice to inhibit bacterial multiplication after infection with small dispersed doses of BCG. Resistance to BCG multiplication, in this model, does not appear to be related to the establishment of DTH.

Spleen cells from female C57BL/6 mice infected intravenously with 1 mg (about 107 viable units) of bacillus Calmette-Guerin (BCG) were shown to suppress the blastogenic responses induced by the T-cell mitogens phytohemagglutinin and concanavalin A and by the B-cell mitogen lipopolysaccharide in spleen cells from normal syngeneic mice. By using various separation procedures or cellular treatments, evidence was found for two distinct populations of splenic suppressor cells. One population belonged to the monocyte-macrophage lineage on the basis of their adherence to plastic surfaces, their removal after treatment with carbonyl iron, and their resistance to gamma irradiation. The other population of suppressor cells belonged to the T lymphocytes due to their sensitivity to an anti-Thy 1 antiserum and complement and to gamma irradiation. After separation on nylon wool columns, inhibitory activity was found in both the nonadherent and the adherent spleen cell populations. Both populations of suppressor cells were present in the spleens 14 days after BCG inoculation and persisted for at least 40 days after infection.

SUMMARYSpleen cells hat^ested from tnice infected intraperitoneally with M. lepraemurium 11 17 weeks prior to harvest acquired the capacity lo inhibit concanavalin A (Con A) induced proliferation of normal spleen cells when precullured for up to 24 h in mitogen-frec medium. The in vitro induced suppressor activity correlated with the length of the prccullure period, the time post-infection and ihc infecting dose. These lindings were interpreted as an indication thai suppressor cell precursors aeeumulated in the spleen of infected mice during the early phase of the disease. The interaction of infectiondependent adherent suppressor eell preeursors and infection-independent, non-adherent regulatory cells is necessary lor the suppressor activity to develop. Both the cells which transmit the inductive signal and the precursor eells which mature into active suppressor eells are radiosensitive, whereas suppressor activity itself is a funetion of radioresistanl adherent cells. Preculture of cells for a short period.before they were coeuttured with Con A-stimulaied normal spleencells. allowed the deteetion of suppressor cells hefore they were deleterious to the infected hosl and also turned out to be a relevatit //; vitro model for characterization of suppressor cell development during M. lepractmirium infection.

Significant variations in the protein and carbohydrate contents of bacterial cells and of mycobacterial fractions occurred during the growth cycle of three strains of Mycobacteria (H37Rv, H37Ra, and BCG).In whole bacilli or their water-soluble extracts, a peak of proteins occurred early during the exponential phase of growth, whereas polysaccharides reached a maximum 2 to 3 weeks later. In culture filtrates, the amount of both proteins and carbohydrates increased with the aging of tubercle bacilli; the increase of carbohydrates, however, was larger. Few quantitative differences in the yield and chemical composition of mycobacterial fractions were noted between the three tubercle bacilli.

C57BL/6 mice were immunized intravenously (i.v.), intraperitoneally (i.p.), or subcutaneously with one dose of Bacillus Calmette-Guérin (BCG). At various time intervals after injection, the lymphocyte response, as measured by thymidine incorporation into DNA, and the number of immunoglobulin-secreting cells were determined in vitro before and after mitogenic stimulation with phytohemagglutinin, concanavalin A, or lipopolysaccharide. In unstimulated cultures, the spontaneous thymidine incorporation and immunoglobulin synthesis of spleen cells were increased to some extent in mice infected i.p. or i.v. with BCG, as compared with noninfected mice. In contrast, after mitogenic stimulation, a marked depression of the proliferative response of spleen cells to both T- and B-cell mitogens and a marked inhibition of LPS-induced immunoglobulin secretion were observed in mice infected i.v. and to a lesser extent in those infected i.p. The depression of lymphoblastogenesis in spleens was fully established 15 days after infection and persisted for a long period of time. When unfractionated or plastic-adherent spleen cells from BCG-infected mice were cultured with normal spleen cells, a strong depression of their reactivity to phytohemagglutinin, concanavalin A, and lipopolysaccharide was observed. After the removal of cells adherent to plastic, the response was partially restored in the nonadherent population from mice infected i.p., but not in that from mice infected i.v. After mitogenic stimulation, lymph node cells of mice inoculated subcutaneously showed a response to mitogen higher than that of normal cells. These results thus demonstrate that, depending on the route of administration, BCG exerts very different effects.