INFECTION

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Vol. 20, No. 3

IMMUNITY, June 1978, p. 714-720

0019-9567/78/0020-0714$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Trypanosomt hodesiense Infection in Congenitally Athymic (Nude) Mice GARY H. CAMPBELL,t* KLAUS M. ESSER, AND S. MICHAEL PHILLIPStt Department of Immunology, Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20012 Received for publication 19 December 1977

Athymic nude mice (nu/nu), heterozygous litter mates (nu/+), and thymic reconstituted homozygous animals (nu/nu TxR) were infected with Trypanosoma rhodesiense. A reduced parasitemia and prolonged survival were observed in the nu/nu animals as compared with controls. Active immunity could be observed in nu/nu animals after infection and cure or after immunization with irradiated organisms. These studies indicate that resistance of mice to T. rhodesiense infection is relatively independent of thymic lymphocyte function.

Infection with Trypanosoma rhodesiense, the causative agent of African sleeping sickness, is characterized by a succession of peaks of parasitemia (36). Each distinct peak consists of organisms demonstrating unique immunological determinants. Thus, the immune system must sequentially face a series of rapidly changing forms, each of which is resistant to an immune response that has been stimulated by the previous parasitemia peak. These discrete peaks or "variants" are thought to contribute to the prolonged clinical course and relative ineffectiveness of the immune system in combating the disease. However, many of the mechanisms, relating both to the immunologically mediated resistance to trypanosomiasis and the pathogenesis of the disease, remain to be elucidated. Recent studies have indicated that the host response to trypanosomiasis is mediated primarily through B lymphocyte-dependent mechanisms. This has been demonstrated through adoptive transfer of variant-specific resistance to T. rhodesiense (4) and T. gambiense (33) with B lymphocytes and serum. B cell-deficient mice cannot resist infection with T. rhodesiense (3). In addition, extracts of T. rhodesiense have been shown to be primary B cell mitogens in vitro (unpublished observations; reference 11), fulfilling another criterion for relative thymic independence (15). However, the importance of T lymphocytes in mediating resistance against T. rhodesiense has not been excluded. For example, they may perform valuable helper functions (34) in collaboration with B cells in the initial imt Present address: Malaria Research Program, University of New Mexico, Albuquerque, NM 87131. tt Present address: Allergy and Immunology Section, Department of Medicine, University of Pennyslvania School of Medicine, Philadelphia, PA 19174.

mune response and provide cells for cooperative effector mechanisms and memory (manuscript in preparation); or, alternatively, they may provide a regulatory function through suppressor activities (9, 12). The involvement of T cells in the immune response to trypanosomes has been demonstrated by in vitro antigen-specific blastogenic responses of immune T lymphocytes to trypanosome antigens (manuscript in preparation) and by observation of delayed hypersensitivity responses to trypanosomal immunogens during infection (35). In addition, the mechanisms of host morbidity and mortality due to trypanosomiasis have not been elucidated. Although some attention has been paid to the histopathological events associated with trypanosomiasis (16) and certain biochemical aberrations (31) occurring during infection, there is no obvious direct relationship between the level of parasitemia, tissue infiltration by parasites, and morbidity and mortality. The congenitally athymic, or nude, mouse represents a useful model in which to study immunity to T. rhodesiense, since it is a model of welldefined T cell immunodeficiency (21). This report describes the use of these mice to study contributions of the thymic component of the immune system to resistance to infections with T. rhodesiense. MATERIALS AND METHODS Animals. Congenitally athymic (nu/nu) and littermate heterozygous (nu/+) mice, backcrossed to the BALB/c line for seven generations, were obtained from the National Institutes of Health, Bethesda, Md. Animals were maintained in sterile isolators with autoclaved water, food, and bedding. Mice were removed from the environment only for experimental manipulations, which were performed under aseptic conditions. All control groups were age and sex matched. 714

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Mice were studied initially between 8 to 12 weeks of age. Thymic reconstitution (TxR) was accomplished via four newborn (0 to 36 h) thymic grafts obtained from BALB/c mice inserted subcutaneously beneath the axillary skin fold (13). Reconstitution was performed 6 weeks before exposure to T. rhodesiense and was confirmed in selected animals by the demonstration of a mitogenic response to concanavalin A in tissue culture (22) by lymphocytes obtained from the reconstituted animals or by a significant rise in y G2A immunoglobulin, measured by radial immunodiffusion techniques. Trypanosomes. The East African Trypanosomiasis Research Organization (EATRO) strain 1886 of T. rhodesiense was used for the majority of these experiments. EATRO 1886 was isolated from a human demonstrating significant clinical disease in 1971 and subsequently has been maintained by rodent passage. The organism was passed at 3-day intervals in BALB/c mice before a standardized infectious inoculum or "stabilate" of organisms was produced by freezing the parasites to -70'C in M199 (Microbiological Associates) and 10% glycerol. Organisms from this frozen stabilate were used to infect additional BALB/c mice 3 days before their sacrifice. The parasites were separated from the cellular elements of blood via DE 52 (Whatman) column chromatography (14), washed in M199, and diluted to a concentration of 104 parasites per ml. Organisms (103) were injected intraperitoneally into the experimental and respective control animals to produce infection. The 3-day passage procedure was necessary to ensure the presence of a vigorous parasitic source of consistent antigenic composition. Parasite counts. Blood (4 to 10 gil) was collected daily from the tails of infected mice. Blood was diluted with 0.05% Nile blue A (Matheson, Coleman, and Bell) in sodium citrate buffer (pH 7.4). The trypanosomes were counted in a hemocytometer chamber. Parasitemia termination. Parasitemia was terminated through the use of a combination of normal human serum (1 ml intraperitoneally [i.p.]) on each of two occasions 6 days apart followed by administration of 0.5 mg of Berenil on two occasions 1 day apart. After multiple passages in mice, the 1886 strain of T. rhodesiense is destroyed by exposure to normal human serum. Parasitemia was considered to be terminated when no parasites were observed for a period of 7 days after treatment termination. Normal uninfected BALB/c animals were similarly treated to serve as controls for any effects of serum or drug per se. Immunizaton procedures. The more virulent Wellcome CT strain (26) of T. rhodesiense was used in these experiments. Organisms were harvested as previously described for the EATRO 1886 strain for stabilate production. A portion of these was separated from cellular elements of the blood by DE 52 chromatography and was irradiated with 60,000 rads, utilizing a 'Co y-source (Gamma Cell-C) (8) before freezing. Immunization was accomplished through the i.p. injection of 107 irradiated organisms. The live challenge infection consisted of 103 organisms obtained from a 3-day mouse passage of nonirradiated stabilate

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Determination of antibody response. Antibody response to T. rhodesiense was determined through a modification of an indirect fluorescent-antibody assay (39). DE 52 column-separated trypanosomes of the Wellcome CT strain were allowed to air dry on microscope slides. Slides were stored at -20'C until used. Slides were washed once in phosphate-buffered saline (pH 7.2) before overlaying with serum dilutions for antibody determination. After incubation at 370C for 15 min and washing in phosphate-buffered saline, fluorescein-labeled goat anti-mouse immunoglobulin M (IgM) or immunoglobulin G (IgG) (Meloy) was added. After incubation at 370C for 15 min and additional washing, the parasites were observed with ultraviolet microscopy for fluorescence. The reciprocal of the highest dilution of sera that resulted in fluorescence of the trypanosomes was determined as the titer of antisera for the respective immunoglobulin class.

RESULTS Course of infection of nu/nu and nu/+ mice with T. rhodesiense. The parasitemia in nu/nu and nu/+ mice rose quickly and in parallel until day 6 (Fig. 1). Thereafter the mean number of trypanosomes in nu/+ mice decreased slowly until day 15 and then rose again terminally. All nu/+ mice were dead by day 28. In contrast, the parasitemia observed in nu/nu animals decreased more dramatically and subsequently rose more slowly in a cyclical fashion. In addition to demonstrating significantly lower (P < 0.05) parasitemia levels between days 10 and 23, the survival time of nu/nu mice was prolonged, with the last athymic mouse dying on day 45. Effect of thymic reconstitution on the course of infection with T. rhodesiense. Because the preceding experiments suggested a significant difference between the course of disease in nu/nu and nu/+ animals, the effect of thymic reconstitution was next studied. Organisms obtained from the same stabilate which was previously used to define the normal course of infection were injected i.p. into groups of nu/nu, nu/+, and nu/nu TxR mice (Fig. 2). Although the differences in parasitemia between nu/nu and nu/+ animals were not as dramatic as that shown in the experiment depicted in Fig. 1, the athymic mice were again observed to control parasitemias and survive as well as or better than nu/+ mice. The nu/nu TxR animals demonstrated a course intermediate between the nu/nu and nu/+ animals; however, their pattern more closely resembled the nu/+ animals, both in terms of parasitemia and death. Development of immunity to infection by T. rhodesiense in nu/nu and nu/+ mice. To study the ability of nu/nu mice to develop resistance to infection, mice were infected, cured

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FIG. 1. Comparison of parasitemia in nu/nu (--- ) and nu/+ ( ) mice infected with 10; T. rhodesiense organisms. The day of death of each individual mouse is marked by +. Each point is the geometric mean, and the vertical bars show the limits of the standard error. The parasitemias were significantly (P < 0.05) different between days 10 and 23 by Student's t-test (n = 6).

by a combination of serum and Berenil, and subsequently rechallenged (Fig. 3). A significant difference in the levels of parasitemia was again observed between the nu/nu and nu/+ animals. After treatment, no organisms were observed in either group of animals for a period of 14 days (days 35 to 49). The nu/nu and nu/+ mice were then rechallenged with 10' organisms on day 49 (after original infection). An additional control group of nu/+ mice (data not shown), which had not been previously infected but had received the normal human serum and Berenil, were also challenged at this time. These latter animals showed a significant parasitemia 3 days after challenge; however, no organisms were observed in either the nu/nu or the nu/+ animals that had been previously exposed to the viable T. rhodesiense. As a further confirmation of the ability of nu/nu mice to develop active immunity against T. rhodesiense, irradiated trypanosomes of the more virulent Wellcome CT strain were used as an immunogen in an attempt to immunize both nu/nu and nu/+ animals (Table 1). Both nu/nu and nu/+ animals developed significant resistance against challenge with live organisms. Both previously immunized groups survived for over 30 days after exposure, whereas the non-immunized group died within 5 days of challenge. Determination of antibody production

against T. rhodesiense by nu/nu and nu/+ mice. Serum was obtained from nu/nu and nu/+ mice on day 15 after initial infection with T. rhodesiense. Antibody titers obtained utilizing an indirect fluorescent-antibody procedure with organisms from the original trypsanosome inoculum as the antigen were determined (Fig. 4). Heterozygote (nu/+) mice showed significant IgM and IgG antibody responses against the trypanosomes. Athymic (nu/nu) animals demonstrated only an IgM antibody response. However, this antibody response was significantly higher than that shown by the nu/+ animals (P < 0.05). DISCUSSION These studies strongly suggest that resistance to T. rhodesiense infection in mice is relatively independent of T lymphocyte-dependent mechanisms. Athymic mice developed immunity to reinfection after an initial exposure to a viable infection and subsequent pharmacological cure, or after exposure to irradiated organisms. Moreover, nu/nu mice develop lower parasitemias and survive longer than normal animals. These studies add to our previously presented data (G. H. Campbell, S. M. Phillips, and K. M. Esser, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, E137, p. 86) and are compatible with the lower

VOL. 20, 1978

TRYPANOSOMIASIS IN ATHYMIC MICE

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parasitemias observed in nude mice by Jayawardena and Waksman (12). The apparent thymic independence of resistance to trypanosomiasis is presently among parasitological diseases. Several other studies involving a variety of parasites including the protozoans Babesia microta (6), Plasmodium berghei yoelii (38), or T. musculi (25, 27, 37), as well as helminths such as Schistosoma mansoni (23; S. M. Phillips and D. G. Colley, Prog. Allergy, in press), indicate a dependence on T-cell mechanisms. Relative independence of T-dependent mechanisms for resistance has been shown in certain bacterial infections such as Streptococcus pneumoniae (40); however, this phenomenon is not universal, as resistance to other bacteria such as Listeria monocytogenes (5, 10) apparently is under T-cell control. These studies agree with observations in B

cell-deficient mice (3) and strongly suggest that antibody, especially of the M class, is functionally of great importance in controlling parasitemia. They also tend to exclude T cell-dependent mechanisms as an obligate requirement for a protective immune response to trypanosomiasis. However, by no means do they exclude the participation of the thymic-dependent system in various other aspects of the immune response. For example, the maintenance of immunological memory may be under T-cell control. The ability of irradiated organisms used as antigen to elicit long-term protection as previously described (4) suggests that a memory cell response is operative. The involvement of the T cell in the maintenance of long-term protective immunity is currently under study. The observation that prolonged survival may be related to absence of T cell-dependent mech-

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