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We studied the specificity of protection conferred by Toxoplasma gondii immune lymphocytes and their supernatants on infected hamster kidney cells, using Besnoitia jellisoni immune lymphocytes as a nonspecific control. The intracellular growth of the organisms was measured by [3H]uracil incorporation, and inhibition of multiplication was used as a measurement of immunity. Although the immune lymphocytes restricted principally the multiplication of homologous organisms, partial protection, expressed against the heterologous organism, was found. This was true for either parasite with intact lymphocytes or their supernatants. Exposure of immune lymphocytes to antigen for 18 to 24 h and treatment of kidney cells with supernatant fluids for 18 to 24 h were required for maximal protection. The specific protective mediator in supernatants of immune lymphocytes was characterized by dialysis as having a molecular weight between 3,000 and 12,000 and was found in the 3,000 to 5,000 peak after Sephadex G-50 chromatography. Nonspecific protective activity was greater than 12,000 by dialysis; it chromatographed in the excluded peak, measuring over 43,000, and was destroyed by exposure to pH 2. In vitro production of lymphokines from toxoplasma immune lymphocytes was first detected 7 to 10 days after vaccination of hamsters. At about the same time, hamsters began to resist challenge infection with the pathogenic RH strain of T. gondii and were able to prevent its multiplication in lungs, liver, spleen, and the subcutaneous infection site. The expression of tissue immunity and the production of toxoplasma-immune lymphokines appear to be time-related events.

Lymphocytes from mice immunized against Toxoplasma gondii protected T. gondii-infected macrophage and kidney cell cultures. After contact with antigens, supernatants of such immune lymphocytes, also contained a factor protective for T. gondii-infected macrophages and kidney cells. Supernatants were protective only when the lymphocytes and kidneys cells were isogeneic. Protection was specific in that supernatants from only T. gondii-immune, but not Besnoitia jellisoni-immune, lymphocytes provided protection against toxoplasmosis. Sixteen to 24 h were required for an appreciable amount of protective factor to be secreted; a similar absorption time was necessary for kidney cells to be protected. Peritoneal lymphocyte lysates, prepared as transfer factor, contained protective substances with a potency similar to that of lymphocyte supernatants, which were also strain restricted in their effect.

Mice challenged with a pathogenic strain of Toxoplasma gondii develop fatal infections. However, if such mice are initially treated with sulfadiazine (SD), they develop immunity and survive with chronic infections. The role of antibody (Ab) in establishing protective immunity against acute parasitemias and in maintaining chronic infections was investigated using B-cell-deficient (immunoglobulin M-suppressed), T-cell-deficient (athymic), and normal BALB/c mice. All mice not receiving SD treatment rapidly died (mean 7.5 days) after infection, but the majority (80%) of intact mice developed immunity during SD treatment and survived for over 5 months with chronic toxoplasmosis. Athymic mice rapidly died (mean 6.0 days) after the removal of SD treatment. Although all SD-treated immunoglobulin M-suppressed mice eventually died, they lived considerably longer (18 to 83 days) in the complete absence of antitoxoplasma Ab than unprotected mice (7 to 9 days). Histopathological sections of liver, lung, brain, and other tissues showed that toxoplasma organisms gave rise to fatal lesions in all nonsurviving animals. The injection of Ab into acutely infected and athymic mice imparted no protection, but transfer of antitoxoplasma Ab (titer greater than 1:8,000) to immunoglobulin M-suppressed mice after SD treatment resulted in elimination of the parasites in 50% of the mice. Results of this study suggest that Ab may not be decisive in acute infections, but may be important in controlling long-term toxoplasmosis.

Antigen-treated lymphocytes from immune hamsters specifically protected not only macrophages, but also cultured fibroblasts and kidney cells infected with Toxoplasma gondii or Besnoitia jellisoni. Macrophages were not necessary for the protection of fibroblasts and kidney cells. A mediator that inhibited the intracellular proliferation of these microbes was obtained from immune lymphocytes in contact with specific antigen. Again, macrophages were not necessary for the elaboration of this mediator or its activity in kidney cells or fibroblasts. The mediator was microbe and host specific, had a molecular weight between 4,000 and 5,000, was resistant to heating at 56 degrees C for 30 min, and was sensitive to chymotrypsin, but resistant to ribonuclease and deoxyribonuclease. A single injection of Besnoitia mediator afforded better protection to hamsters infected with Besnoitia than did antibody. Whereas antibody lysed extracellular organisms, the microbe-specific mediators conferred immunity not only on macrophages, but also on other cells of the body, apparently the first such demonstration.

The inhibitory effects of cortisol on cellular immunity were studied in vitro by using hamster peritoneal exudate cells. Two obligate, intracellular protozoa--Toxoplasma gondii and Besnoitia jellisoni-- were used to control for specificity of effects. Results indicate that immune lymphocytes specifically confer immunity to (or "arm") macrophages that specifically express immunity. This arming can be inhibited by 5 microng of cortisol per ml. Macrophages that have been armed already will continue to express immunity (by limiting parasite growth specifically) in the presence of 5 microng of cortisol per ml. Cortisol levels of 20 microng/ml are required to inhibit the expression of immunity by armed macrophages. It was also found that lymphocytes, from hamsters given 20 mg of cortisol subcutaneously 2 days before the harvest of cells, did not arm macrophages, whereas macrophages from these same animals could be armed by immune lymphocytes from untreated hamsters. Therefore, it was concluded that in relation to cellular immunity, lymphocytes are more sensitive to cortisol than are macrophages. Since antibody to these parasites is almost always present in vivo, we also tested the effects of cortisol on the disposition of antibody-modified organisms by activated (not armed) macrophages, and found that 50 microng of cortisol per ml was needed to inhibit macrophage effects on antibody-treated organisms.

When tested for their activity in suppressing the acquisition of immunity during acute Besnoitia infection of hamsters, antilymphocyte serum (ALS), aminopterin, cyclophosphamide, cortisol, and whole-body irradiation were the most active agents and effectively blocked the development of immunity during 4 to 12 days of immunization. Actinomycin D and chlorambucil were moderately active, and nitrogen mustard, 6-mercaptopurine, and vinblastine exhibited slight immunosuppressive activity. Established immunity was especially labile to cortisol and cortisone administration with generalized Besnoitia relapse occurring consistently in all hamsters; this occurred infrequently during cyclophosphamide treatment. Focal relapse was seen in chronically irradiated and ALS-treated hamsters, but the location of the lesions differed. Irradiated hamsters had lesions in the lungs and brain, whereas ALS-treated hamsters showed splenic relapse. Acquisition of immunity was more sensitive to suppression than established immunity and did not necessarily parallel antibody development and vice versa, emphasizing the importance of cellular over humoral factors in immunity to this intracellular parasite.