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Experiments in vitro comparing normal mouse peritoneal macrophages with cells from Salmonella typhimurium-infected mice have shown that the "immune" macrophages have conspicuously enhanced microbicidal properties. Whereas normal macrophages could inactivate only 50 to 60% of intracellular S. typhimurium pretreated with immune serum, cells from infected animals killed virtually all ingested organisms and did so at an accelerated rate. Macrophages from Listeria monocytogenes-infected mice were shown to possess similarly enhanced microbicidal activity against S. typhimurium. Furthermore, the growth of S. typhimurium in the liver and spleen was more effectively restricted in Listeria-infected mice than in animals vaccinated with heat-killed S. typhimurium, even though the Listeria-infected animals possessed no demonstrable cross-reacting antibody to S. typhimurium. The lack of resistance in the mice vaccinated with heat-killed organisms could not be attributed to any deficiency of humoral factors, since the serum from these animals was as effective at promoting phagocytosis and killing by macrophages as serum from actively infected (and demonstrably resistant) mice. Conversely, Salmonella-infected mice were totally resistant to intravenous challenge with L. monocytogenes. The level of resistance in individual animals was related to the numbers of residual Salmonellae remaining in the tissues; mice with heavier residual infections being the more resistant. Specific antiserum from mice vaccinated with heat-killed S. typhimurium was found to be significantly protective only when the intraperitoneal route of challenge was employed. The foregoing studies have been interpreted to mean that enhancement of the microbicidal ability of macrophages is the mechanism of major importance in acquired resistance to S. typhimurium infection in mice.

Salmonella enteritidis is highly virulent for the mouse causing an infection resembling mouse typhoid. Survivors of the infection are completely resistant to reinfection and eliminate a large challenge dose of virulent organisms within 72 hr. The antigenically related Salmonella gallinarum was almost avirulent for the mouse but animals vaccinated with this organism were equally capable of eliminating a lethal dose of virulent S. enteritidis. Living Salmonella pullorum, on the other hand, was quickly eliminated from the tissues of normal mice. Vaccination with this organism failed to evoke an effective bactericidal mechanism. Alcohol-killed vaccines of these three Salmonellae all produced an increase in blood clearance rate, but gave only marginal protection against S. enteritidis. Liver and spleen counts on these mice revealed a 1 to 2 day delay before any net increase in the total bacterial population could be observed. Immunization of mice with increasing doses of living Salmonella montevideo resulted in progressively greater killing of a challenge dose of S. enteritidis despite the absence of common somatic antigens between the two strains. The degree of protection varied with the size of the residual population of S. montevideo in the vaccinated mice. The significance of these findings in assessing the importance of various factors involved in the development of acquired resistance to Salmonella infections is discussed.

Spleen cells from immunized mice were shown to have a strong antiviral action after transfusion into infected recipients. Spleen, liver, and lung titers in recipients were reduced within 24 hr, but there was no detectable effect on brain titer. Spleen cells were active if taken 6 days after infection of donors, when no antibody was detectable. Spleen cell activity was diminished, but by no means abolished, by treatment with potent anti-theta antibody. Immune spleen cells transferred to mice infected 3 days earlier induced early signs of sickness but no change in average survival time. Normal mice injected intracerebrally with a mixture of immune spleen cells plus virus showed unusually early illness and death.

Histological and immunofluorescence techniques showed that mononuclear cells invaded virus-infected foci in the livers of passively immunized mice within 10 hr of the receipt of immune spleen cells or hyperimmune serum; by 24 hr, marked destruction of virus antigens had occurred in these lesions. Immune cell transfer promoted denser packing of mononuclear cells in the foci and more efficient destruction of infectious material than immune serum. Similar liver lesions developed by the 6th day after sublethal, primary, subcutaneous infection in normal mice. In contrast, in mice with GVHR which were immunosuppressed but possessed hyperactive macrophages and unimpaired splenic interferon response, mononuclear cells did not invade liver lesions and the animals died. These results, together with data reported previously, indicated that mononuclear cell invasion of infected liver foci, triggered by CMI, was of key importance in recovery from primary mousepox. The roles of specifically sensitized lymphocytes and macrophages within lesions were not directly evaluated, but indirect evidence suggested that lymphocytes could cause no more than a halt in virus multiplication, and that macrophages were required for the inactivation of preformed virions. Possible augmentation of the efficiency of macrophages by locally-produced lymphocyte interferon, neutralizing antibody, or stimulation of their phagocytic and intracellular digestive capacity cannot be excluded.

Heterologous organisms (L. monocytogenes and S. typhimurium) were used to study the rate of development, magnitude, and persistence of the antimicrobial resistance engendered in mice by vaccination with BCG. These same methods were used to investigate the influence of prior vaccination on the host response to reinfection. The rate of onset and magnitude of the resistance produced by BCG varied with the vaccinating dose. Increased resistance was detected within 48 hr of injecting large numbers of BCG (approximately 10(8) viable units), but concurrent treatment with isoniazid interrupted its further development. An equal number of heat-killed organisms failed to influence host resistance significantly. The development of tuberculin sensitivity was also dependent upon the continued survival of the immunizing population of BCG. When vaccinated mice were reinfected with BCG, host resistance in spleen and liver was rapidly augmented to the accompaniment of striking changes in the morphology and microbicidal activity of the peritoneal macrophages. These changes occurred most rapidly in mice with a high level of delayed hypersensitivity at the time of reinfection.

The development of acquired resistance to Salmonella typhimurium has been studied in mice infected intravenously with small numbers of streptomycin-sensitive or streptomycin-resistant organisms. By the 14th day of a primary infection the mouse develops a mechanism capable of destroying completely a super infecting dose of organisms, but is unable to eliminate organisms of the primary infection. The latter are constantly returned to the circulation from necrotic foci at the sites of implantation. Passive transfer of serum from actively infected or vaccinated animals, and immunization with heat-killed organisms, increase the capacity of the host to clear organisms from the blood, but do not interfere to any significant extent with their subsequent multiplication in the tissues. It is concluded that the resistance of actively infected animals depends on a nonhumoral mechanism capable of destroying organisms from endogenous or exogenous sources.

Agglutination and immunofluorescence tests in vitro showed that the ATS used in these experiments cross-reacted with macrophages and RBC. However, ATS was not toxic in vivo, and small doses given subcutaneously depleted thymus-dependent areas of lymphoid tissues and selectively depressed blood lymphocyte counts without affecting other cell types in the blood. Furthermore, the function of littoral macrophages as indicated by the clearance of blood-borne virus and its subsequent behavior over a 48 hr period in the liver and spleen was not changed by ATS. Thus, the innate resistance of these vital target organs was not depressed. A similar regimen of subcutaneous ATS caused a highly significant increase in mortality from mousepox with an associated failure to control virus growth in the liver and spleen which was manifest by 6 days after infection. The interferon and neutralizing antibody responses were not impaired in ATS-treated mice, but the cell-mediated immune response was significantly suppressed. This evidence, and consideration of the timing of these host responses during the course of infection in relation to the control of virus growth in the liver and spleen, led to the conclusion that cell-mediated immunity probably contributed an essential acquired recovery mechanism. However, no evidence was obtained concerning the nature of this antiviral mechanism.

The following passive transfer experiments evaluated the contributions of the various host responses in recovery from mousepox. (a) Immune spleen cells transferred highly efficient antiviral activity, but preinfected recipients of these cells made no detectable splenic interferon or antibody in the 24 hr interval after cell transfer. (b) Passively administered interferon was ineffective. (c) Recipients of hyperimmune serum had much more antibody than recipients of immune spleen cells but significantly less antiviral activity. (d) Immune spleen cell populations with antiviral activity contained mediators of CMI to virus antigens. (e) The antiviral activity of immune spleen cells was specific; it was inhibited by in vitro treatment with ATS, anti-light chain serum, and anti-theta ascitic fluid, but not by removal of mononuclear phagocytes from the immune population. These results are interpreted to mean that recovery mechanisms conferred by immune spleen cells were triggered by specifically sensitized, thymus-derived lymphocytes, and that antibody and interferon responses were of less importance. A radiosensitive recipient component was necessary for the full expression of the antiviral activity of both immune cells and immune serum. It seemed likely that this component was the blood monocyte.