Harold C. Miller

GM1 ganglioside isolated from mouse brain was found to be capable of inhibiting the thymocytotoxicity of AKR anti-Thy 1.2 antiserum and to a lesser extent anti-BAtheta antiserum. GD1b ganglioside of mouse brain and thymocytes inhibited the thymocytotoxicity of rabbit anti-BAtheta anti-serum for mouse thymocytes. When GM1 ganglioside, in the form of cholesterol-lecithin liposomes, was incubated with spleen cell cultures in the presence of SRBC, depressed anti-SRBC hemolytic plaque responses were observed. This effect could be neutralized by first absorbing the GM1 liposomes with anti-Thy-1.2 antibodies. The kinetics of GM1 numbers when the ganglioside was added even as late as the 4th day of culture. Results of experiments in which GM1 ganglioside was incubated with either bone marrow cells or with thymocytes suggest that the target cell for GM1 liposomes is the B lymphocytes.

Abstract Mice immunized with either 109 or 106 SRBC were used as a source of T suppressor (Ts) cells and T helper (Th) cells, respectively. T cells were prepared by purification on glass wool and nylon wool columns and were cultivated at 37°C for 3 days. The suppressive or helper effects of the conditioned medium obtained from the T cells were determined in spleen cell cultures by a Jerne-plaque assay. Addition of suppressor medium (Ts-M) to a spleen cell assay was modulatory in that it delayed the normal response time by several days. Thy-1 and GM1 ganglioside were associated with the modulatory substance because a normal response could be obtained by treatment of the Ts-M with anti-Thy-1.2 or anti-GM1 ganglioside sera. Addition of helper T cell medium (Th-M) significantly enhanced anti-SRBC PFC response but anti-Thy-1 or anti-GM1 ganglioside sera had no effect on the helper activity of Th-M. Glycolipids were isolated from Ts-M and Th-M, formulated into liposomes, and were added to PFC assay. A ganglioside isolated from Ts-M with the thin layer mobility (Rf) of brain GM1 ganglioside was found to inhibit anti-SRBC-plaques. Other glycolipids from Ts-M were not inhibitory and none of the glycolipids isolated from Th-M were inhibitory under our assay conditions. The modulatory activity of Ts-M and the isolated glycolipid was not antigen specific and could be neutralized with anti-Thy-1 or anti-GM1 sera. These findings demonstrate that the modulatory (or static) capacity of Ts-M isolated from cultures of partially purified T cells is associated with a cell product, probably a membrane complex, containing Thy-1 antigen and modulatory glycolipid.

Macrophages from superinfected mice show a resistance to intracellular forms of L. donovani in culture. The mice were given 2 to 4 intraperitoneal injections of live parasites at 20to 30-day intervals. Peritoneal macrophages from the superinfected and normal control mice were cultured in Leighton tubes. After inoculation with LD bodies of L. donovani, cover slips were removed at intervals to determine the number of intracellular parasites present. In macrophages from normal mice the parasites usually multiplied for the first 72 hr, but in macrophages from superinfected hosts the parasites either failed to multiply or decreased in numbers. When serum from superinfected mice was added to the culture, there was no significant change in either the rate of parasite multiplication in macrophages from normal mice nor in the rate of destruction in cells from infected mice. Resistance was associated with the cells from the superinfected host. There was no evidence that serum antibodies participated in this cultural demonstration of cellular immunity. Cellular immunity to Leishmania seemed to resemble the cellular immunity described as a factor of resistance to several other microorganisms which parasitize macrophages, such as Toxoplasma, Salmonella, and tubercle bacilli. Although humans and animals have a long lasting resistance to reinfection after recovery from most forms of leishmaniasis, the nature of the immunity is not known. Stauber (1963) stated, concerning humoral aspects of resistance, there is yet no evidence for an antibody basis of acquired resistance to either dermal or visceral Serological tests that have been employed to date are neither highly specific nor do they demonstrate protective antibodies. Adler (1963, 1964), Manson-Bahr (1963), and Stauber (1963) have summarized our limited knowledge of immunity to leishmaniasis. In recent years only limited attempts have been made to demonstrate cell associated systems of resistance. Adler and Nelken (1965) failed to transfer the delayed hypersensitivity reaction by injecting washed leukocytes from the blood of one hypersensitive donor into norisensitive human recipients. Bray and Lainson (1965) were unsuccessful in similar experiments on humans, monkeys, rabbits, and guinea pigs. More recently, Boysia (1967) was able Received for publication 14 May 1968. * This investigation was supported in part by Contract DADA17-67 C-7142 from the U. S. Army Medical Research and Development Command, by Public Health Service Fellowship 1-F1-GM34,783 from the National Institute of General Medical Sciences and by the Michigan State University Agriculture Experiment Station. Michigan Agriculture Experiment Station Journal Article No. 4398. t transfer passively the delayed hypersensitivity reaction using lymph node cells from sensiized guinea pigs. None of these authors tested the resistance of the recipients to infection. Cellular immunity has been demonstrated to be an important factor in the host's resistance to a number of intracellular parasites including: Mycobacterium, Salmonella, Brucella, Listeria, and Toxoplasma. All of these organisms are known to be intracellular parasites of macrophages. It is this host cell which expresses the resistance to the parasite that has been termed cellular immunity. The subject has been reviewed by Suter and Ramseier (1964) and Shands (1967). Since Leishmania has a predilection for macrophages, it seemed promising to determine what role cellular immunity plays in the host immunity to leishmaniasis. MATERIALS AND METHODS

SYNOPSIS. Peritoneal macrophages from hamsters were monolayered on coverslips in Leighton tubes. Twenty‐four hours later these were transferred to a perfusion chamber. Leptomonads were added with fresh medium and the infection process observed with the aid of phase contrast. In the perfusion chamber free‐swimming leptomonads attached to the macrophage by the tip of their flagella. Shortly after this initial attachment the macrophage extended a narrow pseudopodium around the flagellum which eventually reached and enveloped the body of the parasite. Upon complete envelopment the pseudopod containing the leptomonad was retracted into the central body of the macrophage. When first seen in the granular endoplasm of the macrophage, most of the leptomonads appeared to be surrounded by vacuoles. In most cases these vacuoles disappeared in a few minutes making it difficult to distinguish the parasite from the host cell cytoplasm. Leptomonads also were added directly to Leighton tube cultures, and the coverslips with the adherent macrophages and parasites were removed, fixed and stained periodically during the infection process. In these preparations most of the parasites were in clumps in the vicinity of macrophages. Details of the ingestion of the clumps could not be seen, but occasionally a single organism was seen with its flagellum and part of its body enclosed by an extended pseudopod. Most of the intracellular leptomonads were in large vacuoles. Forms intermediate between elongate leptomonads and LD bodies were surrounded by smaller vacuole‐like spaces. The halo‐like vacuoles most frequently seen around LD bodies may have been fixation artifacts. Under favorable conditions the leptomonads transformed to LD bodies in 1–4 hours, but it was 48 hours before a population increase could be found.