Antithymocyte Serum Suppression of Immunity in Mice Immunized to Leishmania donovani*

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1 ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 20, No. 4 Copyright 1990, Institute for Clinical Science, Inc. Antithymocyte Serum Suppression of Immunity in Mice Immunized to Leishmania donovani* G. L. RUNEY, PH.D.,t J. C. TARECKI-BLACK, Ph.D.,$ M. W. RUNEY, Ph.D.,H and A. B. GLASSMAN, tdepartment of Biology, The Citadel, and tdepartment of Laboratory Medicine, Medical University of South Carolina, and 11Department o f Biology, College o f Charleston, ABSTRACT M ice im m unized w ith a glucan-killed parasite vaccine ex h ib ited enhanced resistance to Leishmania donovani infection as evidenced by decreased hepatic parasite burdens when com pared to unvaccinated controls. This resistance was not seen in mice immunized with killed parasites alone. Glucan vaccination resulted in increased resistance at day 6, but this effect was no longer present by day 20 of the experiment. Treatment of vaccinated and control mice w ith antithym ocyte sera abrogated protection against infection, w hether such resistance was vaccine induced or the result of acquired immunity. Introduction Visceral leishmaniasis, caused by the protozoan parasite Leishmania donovani, is a significant cause of m ortality and m orbidity w orldw ide. C u rren t therapy * Send reprint requests to: G. L. Runey, Ph.D., Department of Biology, The Citadel, Charleston, SC Current address: Department of Zoology, University of Georgia, 724 Biological Sciences Building, Athens, GA Current address: National Reference Laboratory, 1400 Donelson Pike, Nashville, TN relies upon chem otherapeutic intervention with pentavalent antimonials,20 but such drugs are relatively toxic and treatm ent failure of 10 to 25 percent has also b een re p o rte d.2 R ecent research has resulted in the developm ent of potential new drug regimens21 and approaches to tre a tm e n t,15 but vaccine-induced resistance w ould provide a powerful alternative in the control of visceral leishm aniasis. O u r lab o rato ry has prev io u sly re p o rte d pro tectio n in m ice induced w ith a subcutaneous im m unization protocol com bining formalin killed parasites, /90/ $00.90 Institute for Clinical Science, Inc.

2 2 6 4 RUNEY, JARECKI-BLACK, RUNEY, AND GLASSMAN adm inistered with glucan.6 It has also been shown by us that the resistance elicited with this vaccine can be transferred to naive, syngeneic mice with 5 X 108 spleen cells from immunized mice.8 In an attem pt to determ ine if T-lymphocytes were responsible for the increased resistance observed in vaccinated mice and in recipients of adoptively transferred spleen cells, antithymocyte serum (ATS) were used specifically to suppress cell m ediated immunity. M aterials and Methods E x p e r im e n t a l M o d e l a n d P a r a sit e Fem ale C57BL/6 m ice, eight to 10 weeks of age, w ere obtained com m ercially* and m aintained in standard caging under the supervision of a veterinarian. A com m ercial m ouse d ie tf was provided ad libitum. Leishmania donovani, strain WR371, was m aintained in our laboratory in axenic culture. Growth media consists of M edia 199 su p p lem en ted 10 p ercen t with lysed, defibrinated rabbit blood and 14.6 mg L-glutamine per litre. Cultures w e re in itia te d from m in c e d m ouse spleen and passaged at four-to-five day intervals. Prom astigotes for im m unization or challenge were harvested within 10 serial passages of culture initiation. I m m u n iz a t io n P r o t o c o l Prom astigotes for im m unization or challenge w ere harvested during stationary phase growth by centrifugation (900 g, 15 min) as a previous study has shown that stationary-phase parasites are more immunogenic,9 as well as more infective than those harvested from log phase cultures. The parasites were washed three tim es in E arles Balanced Salt Solution (EBSS), and killed in 0.1 percent formalin for 30 m inutes at room tem perature. Formalin killed parasites (P) were kept at 4 C o v e rn ig h t, w ashed th re e tim es, counted in a hem ocytom eter and resuspended in EBSS at a concentration of 108 p e r m l. G lu c a n w as p r e p a r e d as d e s c rib e d p re v io u s ly.4 M ice w ere divided into groups (14 mice each) and immunized according to a standard protocol, consisting of three subcutaneous injections given at four-day intervals. G roups received e ith e r 107 form alin killed parasites com bined with six mg glucan (GP), glucan alone (G), dead parasites alone (P), or were untreated (NT). H alf of the m ice in each group w ere tre a te d w ith antithym ocyte se ru m.$ Three subcutaneous injections, two ml each, w ere given on an alternate day basis for the first week and then continued twice weekly for the duration of the experim ent. All mice w ere challenged intravenously 21 days post-immunization with 107 prom astigotes per mouse. M o n it o r in g o f I n f e c t io n C o u r s e G roups of m ice (7 mice each) w ere sacrificed in chloroform vapor at day 6 and day 20 post-challenge. Body, spleen, and liver weights w ere recorded, and impression slides were made from liver and spleen. Slides were fixed with m ethanol, stained with giemsa, and hepatic parasite burdens determ ined according to Staube s m ethod.19 The hepatic amastigote burden was used as the indicator of resistance since splenic parasites are rapidly cleared in this model. Results were analyzed using the Mann-W hitney U-test10 with a probability of p < 0.05 considered significant. All data w ere expressed as mean ± standard error of the mean. * Jackson Laboratory,?? t Purina. $ Calbiochem-Behring Corp., La Jolla, CA.

3 ATS SUPPRESSION OF IMMUNITY IN VISCERAL LEISHMANIASIS 265 Results Mice immunized with the glucan and killed p arasite vaccine (GP) dem onstrated significant reductions (p < 0.02) in hepatic parasite burdens as compared to untreated mice at both day 6 and day 20 post-challenge (table I). Mice receiving killed parasites alone (P) at either e x p e r im e n ta l d a te o r v a c c in a te d w ith glucan (G) alone at day 20 w ere not p ro te c te d against infection w ith L. donovani. Parasite burdens in mice vaccinated with glucan alone were lower than those in untreated mice (p < 0.05) at day 6. This is consistent with previous results in o u r lab o rato ry w hich show th a t th e administration of glucan, a non-specific imm unom odulator, may result in a transitory increase in im m une responsiveness in the early stages of infection. The protection induced by the GP vaccine, h o w ev er, is c o n siste n t and is seen th ro u g h o u t th e course of the experim ent. By day 20 post-challenge, results T A B L E Course of Leishmania donovani Parasite Infection (x!07) in Immunized Mice and Mice Receiving Antithymocyte Serum I Hepatic Parasite Burden Group Day 6 Day 20 GP Gpats G Gats P ± pats NT NTats Mice received three subcutaneous immunizations at four day intervals combining glucan and killed parasites (GP); glucan alone (G) ; killed parasites alone (P) or were untreated (NT). Mice received 0.2 ml anti-thymocyte sera (ATS) on alternate days throughout the course of the experiment. N = 7 animals per group; data is given as the mean ± standard error of the mean. indicate there is no longer any difference in p a ra site b u rd en s b e tw e en glucan treated mice and controls. Administration of anti-thymocyte sera appears to abrogate the vaccine-induced resistance seen in GP mice. Parasite burdens are significantly increased in GP vaccinated animals receiving ATS (p < 0.001) at b oth day 6 and day 20 po stchallenge. Comparison of GP vaccinated m ic e tr e a te d w ith ATS (G P ats) to u n tre a te d controls also receiving ATS (NTats) shows that at day 6, NTats animals exhibited significantly fewer parasites (p < 0.001) than GPats mice. By day 20, however, parasite burdens did not differ in the two groups. U ntreated controls exhibited no differences in parasite proliferation at day 6, w hether or not the mice were treated with ATS. However, by day 20, NTats m ice showed higher levels of infection than untreated mice not receiving ATS. Discussion Infection with Leishmania donovani is characterized by a wide range of host immunological responses. In m an, leishmaniasis often leads to unrestricted parasite proliferation, with death being the eventual outcom e.17 Chem otherapeutic regimens can interrupt this process leading to the acquisition of a strong immune response w hich p revents reinfection w ith the hom ologous species of parasite.11 C urrent evidence indicates that cell m ediated immunity (CMI) is responsible for the acquired resistance to visceral leishmaniasis seen in spontaneous or drug cured individuals.14 Although the C57/BL m ouse is considered to be a valid model for the study of experim ental leishm aniasis, infection with L. donovani is eventually cured, even in untreated m ice.18 Such acquired resistance is genetically determ ined.3 In our hands, peak parasite levels are seen about 14 days post-challenge and th en

4 2 6 6 RUNEY, JARECKI-BLACK, RUNEY, AND GLASSMAN slowly are reduced until parasites can no lo n g er be d e te c te d in liv e r im p re s sion smears. O u r la b o r a to r y h a s p r e v io u s ly reported that resistance against L. donovani infection in mice can be induced w ith a subcutaneous im m unization protocol com bining glucan and form alin killed promastigotes as evidenced by a decrease in parasite levels during peak infection.9 It has also been reported by us th a t th e tra n sfe r of such vaccine induced resistance is possible by injecting naive, syngeneic recipients with 5 X 108 spleen cells h arv ested from im m u nized donors,6 thus indicating that CM1 may be im portant in m urine leishm aniasis as has been previously suggested.16 In order to determ ine if such vaccine induced im m unity is solely dependent upon T-lymphocytes, it was attem pted in our laboratory specifically to suppress cell m ediated im m unity by injecting antithym ocyte serum (ATS) after vaccination and throughout the course of the experim ent. Im m unosuppression with ATS h as b e e n show n to r e s u lt in th e grow th or in crease in th e n u m ber of metastases in a variety of human a n d a n im a l m a lig n a n t tu m o rs 5,7,13 and in th e prom otion of successful organ transplantation.12 F urther results reveal that the administration of ATS significantly increases parasite burdens in mice previously vaccinated with the G P imm unization protocol at both days 6 and 20 of the experim ent. By day 20 post-challenge, this increase in am astigote proliferation is apparent in all groups treated with ATS. It is possible that ATS adm inistration only affected the GP group because this group alone dem onstrates increased resistance to infection, p resu m ab ly through the stimulation of cell-mediated immunity. Groups receiving killed parasites alone or u n tre a te d m ice do not show increased resistance to infection at this early date. By day 20, however, all mice are beginning to m ount an immune response to L. donovani, w hether as a result of vaccine induced resistance or acquired immunity. Such immunity may thus be affected by ATS and result in the in c re a se d p a ra s ite b u rd e n s seen in our results. The use of ATS provides a specific p ro b e for th e s u p p re s s io n of cellm ediated im m unity. M ost of the evidence concerning infection by leishm ania organism s in d icates th at cell m ediated immunity, in general, and T- lymphocytes, in particular, are responsible for resistance to leishmaniasis. However, such a hypothesis does not rule out a role for B-lymphocytes. Alexander and Phillips showed that the transfer of both T and B-lymphocytes provided optimum protection against L. mexicana while B cells alone conferred no protection to infection.1 W ith L. major, however, the transfer of T-cells alone was as effective at conferring resistance as the transfer of B and T-lymphocytes. This study provides evidence substantiating the role of T-lymphocytes in promoting im m unity against visceral leishmaniasis, w hether or not such immunity is acquired as a result of infection or is induced by a vaccination protocol. Acknowledgments The authors gratefully acknowledge the help and encouragement of the late Dr. Thomas W. Holbrook, without whom this project would never have reached completion, and the editorial assistance of Pam Eidson. The studies were supported by UNDP/World Band/WHO Special Programme for Research and Training in Tropical Diseases. References 1. A l e x a n d e r, J. a n d P h il l ip s, R. S.: Leishmania mexicana a n d Leishmania tropica major: A d o p tiv e tra n s fe r o f im m u n ity in m ic e. E x p. P arasito l. 49:34-40, B e r m a n, J. D., R a in e y, P., and Sa n t i, D. V.: Metabolism of formycin by Leishmania amastigotes in vitro. J. Exp. Med. 158: , 1983.

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