..... 'FGBLEKPBHKNfiEANDSNNBEN 1N ACQUERED IMMUNE” T0 UBSHMWNUkDONUWMfl MfumDegreeofPhJo MiGHlGAN STATE UNIVERSITY CLARE 8.. SKW 1974 r ~.\£‘ 1. . LIFEARY mW!"I[umlllllfljljlllllllljflfllfll"ll 7 Whig“ 5”” Unimsity This is to certify that the ' Ithesis entitled T CELL DEPENDENCE AND SYNERGY IN ACQUIRED IMMUNITY TO LEISHMANIA DONOVANI presented by CLARE B. SKOV has been accepted towards fulfillment of the requirements for Ph. D. Microbiology and Public Health degree in “I Major professor Dateilz'éfnj /7, /7 7/7‘ ( 0-7639 ABSTRACT T CELL DEPENDENCE AND SYNERGY IN ACQUIRED IMMUNITY TO LEISHMANIA DONOVANI By Clare B. Skov Several recent studies have suggested that thymus-dependent (T) im- munity is primarily responsible for acquired resistance to Leishmania, but the mechanisms of this immunity have not been elucidated. In this investi- gation, a system was developed such that mice were immunosuppressed and reconstituted to determine if acquired immunity to L. donovani in mice was T cell dependent, and if so, whether the mechanism of acquired resistance involved interaction between different populations of T cells. Inbred CS7Bl/6J mice were given various combinations of immunosup- pressive treatments and infected intravenously with L. donovani amasti- gotes. Liver-parasites were enumerated during the course of infection from parasite counts on liver impression slides. In normal mice the parasite p0pu1ation reached its peak about 24 days post infection, after which parasite numbers gradually declined but were still detectable on day 60. The course of infection was not altered in mice which had been thymectomized, lethally irradiated, and reconstituted with syngeneic marrow cells. Moderate suppression of acquired resistance resulted when thymectomized—irradiated mice were reconstituted with marrow cells previously treated with anti-brain associated theta serum, and/or when recipient mice were treated with azathioprine on the same day they G 6579? Clare B. Skov were infected. Splenectomy, either alone or together with irradiation failed to suppress immunity. However, when mice were suppressed by combining splenectomy with all the above treatments, a long-lasting high level of immunosuppression resulted, and the mice so treated were desig— nated as deprived mice. To determine if T cells were required and/or if T cells interacted synergistically in acquired immunity, deprived mice were infected and reconstituted with thymocytes and/or lymph node cells (LNC). The capacity to resist infection was restored by LNC or thymocytes. LNC were 20-25 times more immunocompetent than thymocytes. Reconstitution by both cell types was inhibited by in viva treatment of recipient mice with azathio- prine or anti-brain associated theta serum. "Limiting dilution" experi- ments showed that constant minimal numbers of LNG combined with graded numbers of thymocytes, or vice versa, conferred greater protection than expected from the additive effect of both cell types acting independently. Cultured peritoneal macrophages from infected convalescing control and reconstituted mice suppressed parasite multiplication. MacrOphages from infected deprived mice were unable to prevent parasite multiplication in vitro. The results of this investigation provide convincing evidence that 1) acquired resistance of mice to L. donovani is predominantly dependent on thymus-derived lymphocytes, and that 2) the mechanism of cellular immunity to this parasite involves synergistic interaction of different T cell subpopulations and parasiticidal activity by macrophages. T CELL DEPENDENCE AND SYNERGY IN ACQUIRED IMMUNITY TO LEISHMANIA DONOVANI By Clare B. Skov A THESIS Submitted to Michigan State University in partial fulfillment of the requirements fer the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1974 ACKNOWLEDGEMENTS I wish to express my sincere appreciation to the members of my advisory committee, Dr. Virginia H. Mallmann, Dr. Harold C. Miller, Dr. Jeffery F. Williams, and especially my advisor, Dr. Donald W. Twohy, for their critical guidance and encouragement. In addition, I wish to thank Dr. D. E. Schoenhard as representative of the support received from state and federal sources in the form of assistantships and NDEA and NIH training grants. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . 3 Cell Interactions in Immunity . . . . . . . . . . . . . . . 3 Thymus-dependent Immune Responses . . . . . . . . . . . . . 3 Cellular Immunity . . . . . . . . . . . . . . . . . . . . . 6 Immunity to Leishmania . . . . . . . . . . . . . . . . . . . 13 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 ARTICLE 1 CELLULAR IMMUNITY T0 LEISHMANIA DONOVANI. I. THE EFFECT OF T CELL DEPLETION 0N RESISTANCE TO L. DONOVANI IN MICE . . . . . . . . . . 27 ARTICLE 2 CELLULAR IMMUNITY TO LEISHMANIA DONOVANI. II. EVIDENCE FOR SYNERGY BETWEEN THYMOCYTES AND LYMPH NODE CELLS IN RECONSTITUTION 0F ACQUIRED RESISTANCE T0 L. DONOVANI IN MICE . . . . . 54 iii LIST OF TABLES Table Page ARTICLE 1 CELLULAR IMMUNITY TO LELSHMANIA DONOVANI. I. THE EFFECT OF T CELL DEPLETION ON RESISTANCE TO L. DONOVANI IN MICE 1 Relative amounts of IgG and IgM in pooled sera from deprived and control mice during the convalescent stage of infection . . . . . . . . . . . . . . . . . . . . . 43 ARTICLE 2 CELLULAR IMMUNITY To LEISHMRNIA DONOVANI. II. EVIDENCE FOR SYNERGY BETWEEN THYMOCYTES AND LYMPH NODE CELLS IN RECONSTITUTION OF. ACQUIRED RESISTANCE T0 L. DONOVANI IN MICE 1 Inhibition of protection conferred by transplanted LNC or thymocytes by in viva treatment of recipients with A2 or aBAG serum . . . . . . . . . . . . . . . . . . . . . . 74 iv LIST OF FIGURES Figure Page ARTICLE 1 CELLULAR IMMUNITY TO LEISHMANIA DONOVANI. I. THE EFFECT OF T CELL DEPLETION ON RESISTANCE TO L. DONOVANI IN MICE 1 Growth curve of L. donovani in the livers and spleens of control, irradiated (Ir), and thymectomized- irradiated—bone marrow reconstituted (TxIrBM) mice infected i.v. with 20 x 106 LD bodies . . . . . . . . 44 2 Growth curve of L. donovani from 24 to 60 days post infection in livers of untreated controls compared to mice receiving various treatments including thymectomy (Tx), irradiation (Ir), reconstitution with theta— -negative bone marrow cells (0 BM), and azathiOprine (Az) . . . . . . . . . . . . 46 3 Growth curve of L. donovani from day 24 to 40 in control, Splenectomized (Sx), and splenectomized- irradiated-bone marrow reconstituted (SxIrBM) mice injected i.v. with 20 x 106 LD bodies . . . . . . . . 48 4 Growth curve of L. donovani from day 24 to 60 in control6and deprived mice infected i.v. with 20 x 10 LD bodies . . . . . . . . . . . . . . . . . . . . SO 5 Liver section from a deprived mouge about 4 wk after infection i.v. with 10 x 10 LD bodies . . . . . . . 52 ARTICLE 2 CELLULAR IMMUNITY TO LEISHMANIA DONOVANI. II. EVIDENCE FOR SYNERGY BETWEEN THYMOCYTES AND LYMPH NODE CELLS IN RECONSTITUTION OF ACQUIRED RESISTANCE TO L. DONOVANI IN MICE 1 Experimental design . . . . . . . . . . . . . . . . . . . . 75 2 Relative immunocompetence of thymocytes and LNC . . . . . . 77 3 Synergistic effect of reconstitution with graded numbers of thymocytes mixed with 104 LNC . . . . . . . . . 79 4 Synergistic effect of reconstitution with graded numbers of LNC mixed with 10S thymocytes . . . . . . . . . 81 V Figure Page Survival of parasites in peritoneal macro- phages from control, reconstituted, and deprived mice 44 days after infection and harvested 2 days after i.p. injection of 5 x 106 LB bodies . . . . . . . . . . . . . . . . . . . . . 83 Protection conferred by cells from non- immune and immune donors . . . . . . . . . . . . . . . . . . 85 vi INTRODUCTION Immunologic responses toward infectious or foreign agents and modi- fied self-antigens are classically categorized into two systems; the humor— al and the cellular immune systems. Natural activation of one system alone rarely occurs. Depending on the nature of antigen and other factors, either system may be relatively ineffective or predominantly responsible for de- fense against a particular antigenic agent. The immune response to many antigens may require c00peration between the two systems. Experimental evidence suggests that the interaction of lymphoid cells with antigen and with each other, and their consequent immune activation, usually depends on a variety of cell-derived chemical mediators or lymphokines (62). Although specific antibodies may be induced, they may be ineffective against intracellular infectious agents including many viruses, bacteria, protozoa, and fungi. Host resistance against these organisms depends pri- marily on activation of thymus-derived (T) lymphocytes. T cells may re- cruit and activate other lymphocytes and/or macrophages. Both activated lymphocytes and macrophages may lyse target cells in the absence of anti- body. The expression of cellular immunity to infection resides principally in activated macrophages while lymphocytes which lyse target cells directly have been demonstrated in transplantation immunity. This study was designed to elucidate the cellular aspects of immunity to Leishmania donovani. In man, L. donovani causes visceral leishmaniasis (kala azar), a zoonotic tr0pical disease which is usually fatal if untreat- ed. Leishmania exist in two morphological forms. The promastigote is a 2 flagellated form found both in culture and in the gut of its vector, the sandfly (Phlebotomines). This stage of the parasite transforms into an intracellular amastigote (also called the Leishman-Donovan or LD body) after ingestion by a macrOphage, the host cell. Two major clinical forms of leishmaniasis exist, cutaneous and visceral leishmaniasis. Other forms of the disease, post-kala-azar dermal leishmaniasis, and metastasizing forms such as mucocutaneous and diffuse cutaneous leishmaniasis occur less fre— quently. A large range of host responses occur in man. Spontaneous recov- ery and solid immunity usually occur after a case of the chronic cutaneous disease. Delayed hypersensitivity usually develops but circulating anti- bodies are absent during the course of cutaneous leishmaniasis. In con- trast, the visceral disease usually does not induce delayed hypersensitiv- ity until after healing occurs, but may induce antibodies and cause hepato- splenomegaly, nonspecific hyperglobulinemia, and death without treatment. A small portion of human victims apparently recover spontaneously and suc- cessfully treated cases usually result in life-long immunity. However, the mechanisms of this resistance to visceral leishmaniasis are poorly under— stood. The purpose of this investigation was to determine both the nature and the role of the lymphoid cells which confer protection against intra- cellular L. donovani in mice. A system was deve10ped in which the capacity of the mice to immunologically eliminate the parasite was significantly suppressed. Immunosuppressed mice were used in reconstitution experiments to determine 1) if normal T cells from the thymus (T1) or peripheral sources (T2) could protect mice against L. donovani; 2) if T1 and T2 lymphocytes act synergistically in their protective roles; and 3) if T2 cells from immune convalescing donors are more immunocompetent thantxflls from non-immune donors. LITERATURE REVIEW Cell interactions in immunity. Recognition of the heterogeneous nature of the cellular components of the immune system has been the most significant advance in modern immunology. The mode of expression of the effector branch of the immune response has become the basis for the arbitrary division of immune phenomena into humoral and cellular cate- gories. It is now realized that many immune responses depend on lymphocyte- lymphocyte and/or lymphocyte-macrophage interactions. Many such inter— actions involve the production of antigen induced lymphokines which regu— late the expression of the immune response. Macrophages (93,53), bone marrow derived (B) lymphocytes (115,129), and thymus—derived (T) lympho— cytes (6,37,40,49,65) have all been reported to produce soluble mediators. In addition, B—B cell (35), T—macrophage-B cell (23,43,44,87,105), and T-T cell (20,128) interactions have been reported. However, the major role in the immune system's cell interactions has been ascribed to T cells. Immune responses to most antigens are probably thymus dependent. The subject of T cell regulation of B cell responses was recently reviewed by Katz and Benacerraf (62). Thymus—dependent immune responses. Thymic function and the mechanism of cell interactions are neither simple nor well understood. Thymus-dependency shows great variation in relation to the nature of antigenic stimulus and the organism in which the response takes place. The clearest division of immune function, related to derivation of lymphocytes from distinct pri— mary lymphoid organs, has been observed in birds. Bursectomy of chickens 4 removes most of the antibody producing potential without altering the abil- ity to reject grafts, which is lost following thymectomy (29,130). In con- trast, in utero thymectomy of lambs at the end of the first trimester, has little effect on either antibody production (26) or rejection of grafts (27). In man, immunological deficiency diseases have been linked to separate B and T stem-cell defects. Patients with congenital agammaglobulinemia pos- sess thymus-dependent cellular responses whereas children with congenital thymic aplasia produce antibodies to some antigens while having markedly impaired cellular responses (56). The dichotomy of the immune system among mammals has been most thor- oughly investigated in mice. The production of antibodies in mice was first shown to be dependent on T-B cooperation by Claman et a1. (23) and Miller et a1. (88). Humoral responses to most antigens tested have re- quired T-B collaboration (71). T cells have been shown to be responsible for carrier-molecule recognition as "helper cells" (100,118) while differ- entiated B cells synthesize hapten specific antibody (34). Different im- munoglobulin classes appear to vary in their relative dependency on T cells. Taylor and Wortis (117) and Torrigiani (124) found that IgM and IgGZ pro- duction were relatively less thymus—dependent than was IgGl. Although re- cent reports by Tingle and Shuster (122) and Kagnoff et a1. (61) have pre- sented evidence which questions the thymus—independence previously ascribed to brucellin and lipopolysaccharide, some antigens may be thymus-independent. Only the IgM response increased when high doses of a polyvalent hapten- carrier conjugate was injected into thymus-deprived mice (4). And, even in the presence of an intact thymus, polyvalent conjugates and lipOpolysaccha- rides were shown to give rise to higher IgM/IgG ratios than do monovalent conjugates (33,78). Macrophages may also play a vital role in T-B c00peration. Feldmann 5 and his collaborators (42,43,44) have prOposed that specific and nonspecif- ic factors secreted by T cells mediate antibody synthesis by B cells. In their model, the specific factor, an IgM-antigen polymer, is presented to the cells via macrophage intermediates while T cell derived nonspecific mediator(s) augment B cell proliferation. It has been postulated that two signals are required to trigger B cells. Dukor and Hartman (39) proposed that triggering of antibody-secreting precursor cells involves specific binding of an antigen to the immunoglobulin receptor and the interaction of activated C3 with the B cell's C3 receptor. Schrader (105) presented an in vitro model which indicated that the second signal required to trigger B cells is a macr0phage product while the T cell product only has an amplify- ing effect. The "allogeneic effect" provides complementary evidence for the existence of a thymus-dependent amplifier factor (96). The concept of a three cell system is indirectly supported by two other lines of evidence. Although it is still a controversial issue, there is increasing evidence that T cells, like B cells, possess antigen-specific receptors and that these receptors on T cells are immunoglobulin-like or IgM (6,7,39,79,101). In addition, macr0phage-derived lymphocyte-activating factors have been re- ported (53,93). The regulatory role of T cells is complex and involves a wide range of immune phenomena. T cells have been shown to suppress and/or enhance the activity of both B and T cells (10,20,38,50,52,95,136). T and B cells may both be responsible for immunological memory (89) or rendered tolerant (22). Terman et al. (118) has introduced evidence which suggests that neo- natal tolerance may be a result of active suppression. Perhaps a clearer definition of T-B interaction will arise from experiments using hapten- specific tolerant B cells (116) and carrier-specific tolerant T cells (51). Identification of the specific roles of the thymus and T cells is 6 central to an understanding of the inductive influences which determine the differentiation and immunocompetence of T cells. In mice, precursors of T and B lymphoid cells apparently originate from marrow stem cells. T cells, after leaving the thymus, may migrate to secondary lymphoid organs or be- come recirculating peripheral T cells (121, 131). T cells have been identi- fied by their capacity to restore antibody production to sheep erythrocytes and graft rejection in thymectomized mice; they are also identified by their sensitivity to anti-theta or anti-lymphocyte serum (ALS). However, it is not clear whether the T "helper" fUnction in antibody production and T cell functions in cellular immunity are different functions of the same or dis- tinct cells. In addition, the mechanism by which the thymus affects immuno- competence is not well defined. There is good evidence that thymosin, the putative thymic hormone, can replace some of the biological functions as- signed to the thymus (54,123,135). Miller et a1. (86), Goldstein (55), and Bach and Bach (8) have demonstrated induction of theta antigen and "helper" function in marrow cells via thymic factor(s) in vitro. There seems to be much promise in the experimental success in restoration of immunological function with thymic factor(s). Interpretation of these results is still guarded with respect to the mode of action since polyions and other agents also appear to be capable of restoring some responses in T cell deprived mice (36). Kook and Trainin (64) have recently found that thymic factor(s) may exert its effect via cyclic adenosine monophosphate which suggests that thymic factor(s) functions as a hormone (55). Cellular immunity. T cells are primarily re5ponsible for cell- mediated immune functions. The term cell-mediated immunity has evolved to encompass a broad spectrum of immune phenomena in which propitiation of effector mechanisms, following exposure of the immune system to antigen, usually involves interaction of two or more lymphoid cell types and may be 7 expressed by either of the two major effector systems. Mechanisms control- ling production of antibody include mediation via collaborating lymphoid cells and a well characterized antibody-feedback inhibition system (125). Mechanisms governing cellular immunity have not been elucidated. Cellular immune reactions include those responses in which the main expression of the effector branch is carried out by either specifically sensitized T cells and/or macr0phages which have been armed by T cells. Such expressions of immunity may be made independent of humoral antibody and passively transferred only by sensitized lymphoid cells. The delayed skin response has long been used as an in vivo indicator of cellular immu- nity. Both events are putatively related to or dependent on common mecha- nisms. Both are apparently initiated by antigenic stimulation of T cell receptors, either directly or via presentation by macr0phages, which results in transformation and clonal proliferation of specific antigen reactive cells. ConSequent events, although less clear, include the production of solUble mediators, the suppression or recruitment and activation of other cells, and possibly the differentiation of T cells into memory and/or cytotoxic lymphocytes, depending on the microenvironment and the nature of the antigen. In contrast to the above concept, several in vitro models have indicated that nonsensitized B cells and macrophages may be cytotoxic to antibody coated erythrocytes (105,126) or tumor cells (63,134). Whether these phenomena occur in vivo or play a significant role in cellular immu- nity has not been established. Immune rejection of grafts is commonly viewed to be dependent mainly on cellular immunity because adoptive transfer of immunosensitive lympho- cytes results in accelerated rejection of allografts (12). The role of humoral antibody in graft rejection has been questioned on the basis of the failure of immune sera to passively accelerate graft rejection (16). 8 Silverstein and Kraner (108) have demonstrated that allograft rejection can occur in sheep in the absence of serum antibody. However, antibodies are frequently induced by grafts and may play a role in transplantation immuni- ty depending on the circumstances, i.e., the nature of the antigen or pre- sensitization (84). Lance and Batchelor (66) found that treatment of mice with ALS selectively depletes the T cell population and prolongs the sur— vival of skin allografts. Nevertheless, these experiments failed to dif— ferentiate between lysis by antibody and T cells because both may be T cell dependent. Several reviews of transplantation immunology have been written (90,102,132). The analogy between graft and tumor immunology was made evident by Mitchison (91) who showed that rejection of either kind of tissue graft depended on adoptive transfer of specifically sensitized lymphoid cells. It has become evident that similar mechanisms may be involved both in immunological rejection or enhancement of grafts, tumors, and normal tis- sues (12,24,25,58,97). Existing evidence suggegts a role for both humoral and cellular responses in graft, tumor, and autoimmunity (57,58,92); which mechanism predominates may be decided by microenvironmental condi- tions. Particularly interesting is the fact that in vivo, lymphoid cells are apparently ”tolerant” to distinct transplantation antigens possessed by both tumor and normal cells, but can express immunological sensitivity to the same cells in vitro (ll,24,25,58). The existence of a serum ”block- ing" factor is apparently related to the inability to reject tumors. An analogous serum factor appears to operate in suppression of both in vivo and in vitro autosensitization (24,25,58,97). T cells may be responsible for homeostatic control as exemplified by the NZB mouse model in which autoimmune disease and associated lymphoid malignancies follow progressive loss of T cell function (47). 9 Cellular immunity also plays a major role in defending against intra- cellular parasites. In 1942, Lurie (72) first demonstrated that tubercle bacilli survived less well in macrophages from immunized donors as compared to macrophages from nonimmunized donors. Activated macrophages have been shown to possess enhanced microbicidal or growth inhibiting activity against a variety of intracellular parasites including Brucella (41), Pasterella (119), Listeria (73), Salmonella (104), Tbxoplasma (127), and Leishmania (8S). Delayed hypersensitivity to tuberculin was first passively trans- ferred with cellular exudates by Chase (21). The first successful transfer of cellular immunity to an intracellular parasite (mycobacteria) was report- ed by Sever in 1960 (107). During the past decade, adOptive transfer of cellular immunity has been accomplished with cells from animals immunized to a wide variety of intracellular parasites (5,46,77,103,104). Passive transfer of immunity with lymphoid cells, but not with serum, has become the major criterion of cellular immunity. Two major hypotheses have been proposed to define the nature of acquired cellular resistance to infectious intracellular parasites. From their studies of the cellular basis of immunity to mycobacteria and Listeria, Mackaness and his collaborators have advanced the concept of a two stage mechanism of defense (75,76). Mackaness (73,74) found that macrophages were primarily responsible for enhanced microbicidal activity in immune mice, both homologous and heterologous organisms being destroyed. But only homologousorganisms were able to evoke recall of the immune mechanism for the enhanced microbicidal activity. The expression of nonspecific immunity, but not delayed hypersensitivity, by adoptively transferred immune cells was also feund to require the presence of specific antigen (77). Thus, according to their model, activated macr0phages have been shown to be non- specifically armed and to exhibit increased physical and metabolic activity 10 (22,32). The first stage of cellular resistance requires the specific sensitization of lymphocytes which respond by transforming, proliferating, and producing nonspecific soluble mediators. Control over intracellular parasites follows consequent activation of macrophages in the effector stage. This interpretation of evidence is consistent with the placement of cellular immunity and delayed hypersensitivity as manifestations of a common first stage immune mechanism. Alternatively, Youmans and Youmans (133) have fractionated mycobac- terial preparations into cell-wall and ribosomal preparations for the pur- pose of comparing their potential for eliciting specific and nonspecific cellular responses. When coupled with tuberculoprotein, the stable cell- wall fractions were found to elicit transient nonspecific protection togeth- er with strong tUberculin hypersensitivity (31). The nonspecific protec- tion to Listeria was similarly transient when heat-killed cells were used for vaccine. In contrast, immunization of mice with Listeria provided long-lasting protection. Similarly, Collins (28) reported that anamnestic immunity to Salmonella was also specifically recalled and that the expres- sion of specific immunity was greater and longer lasting than the nonspecif- ic expression of immunity. The labile ribsomal fraction did not produce tuberculin hypersensitivity but only evoked specific immunity against virulent mycobacteria (30). Circulating antibodies had no apparent role. Thus cellular resistance to tUberculosis was completely dissociated from delayed hypersensitivity. 0n the basis of their work with mycobacteria, the Youmans proposed the hypothesis that distinct nonspecific (granuloma- tous) and specific immune responses occur together following immunization with viable attenuated cells while only the nonspecific response is acti- vated by killed cells. If this model is correct, then data involving microbicidal activity of cultured macrophages harvested by methods including 11 injections of homologous antigen shortly before their harvest should be questioned because under such conditions, one would not be able to dis- tinguish between specific immunity and delayed hypersensitivity related nonspecific immunity. Acquired immunity to Listeria and mycobacteria involves the interac- tion of lymphocytes and macrophages. The lymphocytes re5ponsible for pas— sive transfer of immunity to Listeria were shown by Lane and Unanue (69) to be T cells by their sensitivity to anti-theta serum. T cells were also shown to be responsible for resistance to mycobacteria by North (94) who demonstrated that T cell deficient mice were unable to prevent bacterial growth. However, apparent differences exist in the lymphocytes responsible for adoptive transfer of resistance to these organisms. Cells which convey protection against Listeria were found to be radiation-resistant residual T lymphocytes in thymectomized, irradiated, and bone marrow reconstituted (TxIrBM) rats (82) and mice (94). In response to Listeria, most of the proliferating cells in the liver of TxIrBM mice were macrophages and their proliferative response was not reconstituted by thymocytes (94). McGregor and Logie (83) found that adoptive transfer of resitance to Listeria was dependent upon rapidly proliferating, large lymphocytes, which had a short circulating lifespan, and were no longer detectable by passive transfer of thoracic duct cells eleven days post infection. In contrast, Lefford et a1. (70) found that lymphocytes which confer adoptive immunity to tubercle bacilli in rats were initially a population of large cells similar to those described by McGregor and LOgie (83). However, the large cells either matured or were replaced by small recirculating, long-lived cells which conferred long—lasting immunity as detected by passive transfer. The capac- ity of lymphocytes from TxIrBM rats to passively transfer immunity to tuber- cle bacilli was reconstituted by injection of normal thoracic duct 12 lymphocytes (82). Similarly, resistance to tuberculosis and the prolifer- ative response of lymphocytes in the livers of TxIrBM mice were reconsti- tuted by thymocytes (94). Cantor, Asofsky, and their collaborators have investigated the nature and roles of lymphocytes involved in the graft-vs-host (GVH) response, a classical model for cellular immunity. They tranSplanted various popula- tions of parental lymphocytes (i.e., thymocytes and peripheral blood lymphocytes) into neonatal F1 hybrids, either together or separately, in limiting dilution experiments. Their results demonstrated that both donor cell types caused GVH reactions but that peripheral blood lymphocytes were approximately 20 times more reactive than thymocytes (20). When injected together, the two cell componentsacted synergistically only when both cell types were allogeneic to the host. The cells in excess in the thymus were apparently precursors of GVH effector celk;(Tl) while the predominant cell type among peripheral lymphocytes (T2) appeared to amplify the reac- tivity of the first cell. Tigelaar and Asofsky (120,121) also found T2 cells to be relatively more active in producing lethal runting than spleno- megaly, the opposite of T1 activity in GVH reactions. Both cell types were shown to be T cells (19) and to possess different homing potentials (121). Raff and Cantor (99) proposed the hypothesis that distinct cell types may be responsible for initiating different expressions of cellular immunity. Evidence supporting the functional heterogeneity of T cells is be- coming increasingly abundant. Marked differences in the relative responses to phytohemagglutinin and concanavalin A, as well as differences in cell surface markers was demonstrated between central and peripheral T cells by Stobo et al. (113) and Stobo and Paul (112). Stobo et al. (114) also found differences in T cell radiation sensitivity in the spleen. Altman et a1. (6) reported a dissociation between lymphocyte proliferation and 13 production of monocyte chemotactic factor. Similarly, Rocklin (100) dis- sociated between antigen induced lymphocyte profiferation and the produc- tion of MIF (macrophage inhibition factor). Tigelaar and Asofsky (120) de— scribed a population of cortisone-resistant thymocytes which had reduced GVH reactivity; these cells could not act synergistically with either normal thymocytes or peripheral lymphocytes. Segal et al. (106) also distinguished between two populations of T cells by differential cortisone sensitivity. Recently, Wagner (128) reported T-T synergy in an in vitro allograft system in which T2 cells provided the major source for precursor cells of cytotoxic lymphocytes, while Tl cells acted mainly as amplifier cells. Results ob- tained by Baker et al. (9,10) indicate that suppressor T cells, possibly Tl, limit B cell proliferation while peripheral T cells, probably T2, amplify antibody production by B cells. Finally, the results contained in this dissertation are consistent with the model preposed by Raff and Cantor (99). Immunity to Leishmania. Speciation of Leishmania which are infective to man is still based on clinical criteria and therefore is not well defined. This grouping system is inadequate because of the large range in parasite virulence and/or host reSponses which is reflected by a broad spectrum of clinical forms. Adler (2) has provided the clearest separation of Leish- mania "Species" on the basis of the growth characteristics of promastigotes cultured in immune sera. He was able to distinguish between L. trapica major and L. tropica minor which cause "wet" and "dry" cutaneous leishman- iasis, respectively, in the old world. He also differentiated between L. donovani and L. trapica and between agents causing cutaneous disease in America including L. mexicana, L. braziliensis, and L. pifano. Although L. tropica and L. braziliensis may be used to induce anti- bodies in experimental animals, antibodies cannot be detected in most 14 cases of cutaneous leishamaniasis. Detection of species-specific antibod- ies in experimental animals is complicated by cross-reactivity caused by numerous group-specific antibodies. Most of the immunoglobulin in L. dono— vani induced hyperglobulinemia is not specific for the parasite and positive serological findings are not correlated with protection. As expected, pro- mastigotes have additional antigen not shared by amastigotes. Protective cross—reactivity does occur between certain groups within species. Generally, the more virulent strain confers complete cross- protection while the less virulent strain, even though it induces a posi- tive delayed (montenegro) reaction, may only afford partial or homologous protection. Examples of cross-immunity are the demonstrations by Lainson and Shaw (68) and Adler and Gunders (1) that L. t. major protected against L. t. aunor. Similarly, rodent strains of L. donovani protected man against human strains in experiments done by Manson-Bahr (80,81) and Southgate and Manson- Bahr (109). SUCCess at prophylaxis against visceralization of L. donovani may depend on the route of admistration and/or on the preparation of the "vaccine” parasites. Various manipulations of the parasites may decrease or increase virulence (3,110,111). While protective immunity may be established either following spon- taneous or chemotherapeutic cure, it is still not clear whether it is pre— munitive or sterile immunity. Heyneman (60) proposed an alternative hypoth- esis that immunity to visceral leishmaniasis resides largely in the skin where cell-bound antibodies, plasma cells, and sensitized lymphocytes are concentrated. As yet, there is no evidence that circulating or cyt0philic antibodies provide functional protection. Injection of L. donovani into immune people may produce an Arthus reaction (79) but attempts to passively transfer dermal hypersensitivity to cutaneous forms in man have failed (3, 15 15). By emulsifying promastigotes in adjuvant, Boysia (l4) induced delayed hypersensitivity in guinea pfigsand passively transferred the delayed re- sponse only with lymphoid cells. Both immediate and.dahyed.type reactions probably occur but neither has been shown to signify anything more than an indication of host contact with a wide rangelof group-specific antigens. Adoptive transfer of protection to L. donovani in mice has been reported by Miller and TWohy (85). Bryceson, Preston, and their colleagues have recently developed two experimental models which suggest that development and maintenance of immu- nity to Leishmania are predominantly thymus-dependent immune re5ponses (17, 18,98). Single cutaneous lesions in guinea pigs infected with L. enriettii ulcerate and heal in about three months. During infection, delayed hyper- sensitivity reactions become positive; they persist following recovery and are passively transferrable via lymphoid cells (17). Following recovery, macrophage migration inhibition, lymphocyte transformation, and lymphokine production by L. enriettii sensitive lymphocytes were demonstrated in vitro. Guinea pigs immunosuppressed with ALS demonstrated greater susceptibility to infection (18). Unlike the relatively quick healing lesions of controls, the metastasizing lesions in ALS treated animals contained few lymphocytes around heavily infected macrophages and numerous plasma cells. In the second experimental model, "thymus-deprived” (TxIrBM) mice were infected with L. t. major (98). Slow healing occurred in some deprived mice. Furthermore, lesions deve10ped relatively slowly but endured much longer than in controls. 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This is Journal Article No. 6790 from the Michigan Agricultural Experiment Station. 28 1This work was supported by Grant No. DADA l7—69C-9135 from the U.S. Army Medical Research and Development Command under sponsorship of the Commission on Parasitic Diseases of the Armed Forces Epidemiology Board, and with support from the Michigan Agricultural Experiment Station. Journal Article No. 6790 from the Michigan Agricultural Experiment Station. 2Abbreviations used in this paper: MEM, Minimal Essential Medium; Az, azathioprine; aBAO, anti-brain associated theta serum; BM, bone mar- row cells; G'BM, aBAG serum treated bone marrow cells; Tx, thymectomized; Sx, splenectomized; Ir, irradiated; TxIrBM, thymectomized, irradiated, and bone marrow reconstituted mice; TxIre'BM, thymectomized, irradiated, and reconstituted with aBAe serum treated marrow cells; TxIrG'BMAz, azathio- prine treatment of mice previously thymectomized, irradiated, and recan- e"BMAz, mice treated stituted with aBAO serum treated marrow cells; TxSxIr with azathioprine after being thymectomized, splenectomized, irradiated, and reconstituted with aBAO serum treated marrow cells; T, thymus-dependent; B, bone marrow derived; NMS, normal mouse serum. 29 ABSTRACT C57B1/6J mice were treated with varied combinations of immunosup— pressive treatments and infected intravenously with Leishmania dbnovani amastigotes. In normal mice the parasite population reached its peak about 24 days past infection, after which parasite numbers gradually de- clined but were still detectable on day 60. The course of infection was not altered in mice which had been thymectomized, lethally irradiated, and reconstituted with syngeneic marrow cells. Maderate suppression of acquired resistance resulted when thymectomized irradiated mice were re- constituted with marrow celkswhich were previously treated with anti- brain associated theta serum and/or when recipient mice were treated with azathiOprine on the same day they were infected. Splenectomy alone or to- gether with irradiation failed to suppress immunity, but a long-lasting high level of immunosuppression resulted when Splenectomy was combined with all the above treatments. These results suggest that 1) small num- bers of thymus-dependent cells which survive irradiation and thymectomy or which may be present in the bone marrow, may be sufficient to mount an immune response to L. danavani in mice, and 2) that T cells play an impor- tant role in acquired resistance to L. danavani in mice. INTRODUCTION In man, visceral leishmaniasis is characterized by nonspecific hyperglobulinemeia, hepatasplenomegaly, and an absence of delayed hyper— sensitivity (30). Although specific antibodies are produced, evidence for immune protection mediated by humoral mechanisms is lacking. Without treatment the disease is usually fatal, but recovery is generally followed by development of delayed hypersensitivity and long-lasting immunity (35). 30 The nature of the immunity has not been well defined. Literature dealing with the cellular nature of immunity to Leish- mania is scarce. Cutaneous leishmaniasis is characterized by spontaneous recovery accompanied with delayed hypersensitivity but an absence of a humoral response (9,10,11). Boysia (8) successtlly transferred delayed hypersensitivity to L. trapica using lymph node cells from guinea pigs immunized with promastigotes in adjuvant. Miller and Twahy (34) reported that the efferent branch of immunity to L. danavani, like cellular immuni— ty to mycobacteria (42), Brucella (18), Listeria (27,28), and Toxoplasma (4,5) is expressed by macrophages. Two experimental models have recently demonstrated that slaw-healing farms of cutaneous leishmaniasis in guinea pigs (12) and mice (37) can be induced by selective immunosuppression of thymus-dependent (T) immune responses. Twahy (unpublished) found C57Bl/6J mice to be a suitable host for investigating the mechanisms of immunity to L. donovani. This investigation was performed to determine 1) whether experimen- tal depletion of the thymus-dependent lymphoid system would inhibit the develapment of immunity to L. danavani in C57Bl/6J mice and 2) what treat- ments could be routinely employed to eliminate the development of this immunity. It was found that extensive and multiple treatments were re- quired to significantly suppress the expression of immunity to L. danavani. MATERIALS AND METHODS Experimental design. Inbred female C57Bl/6J mice were purchased from Jackson Laboratories (Bar Harbor) at approximately 6 to 8 wk of age and maintained on a standard pellet diet and acidified-chlorinated water (22). The mice were used to assess the effects of separate and combined immunosuppressive treatments on the course of L. danavani infection. 31 Control and immunosuppressed mice were infected intravenously (i.v.) through the tail vein with 10 or 20 x 106 amastigotes (LD bodies), usually within 24 hr but never later than 48 hr after the harvest. The level of infection was calculated from the number of parasites in liver impression smears according to the method of Stauber (40). Prior to the harvest of livers for each assay, the blood from each group was collected after decapitation. Sera was decanted from the pooled blood, sterilized by filtration, and stared at -20 C. Parasites. L. danavani (3S) amastigotes were maintained by serial intracardial passage in young male hamsters. Amastigotes were asceptically harvested by homogenizing the spleens of infected hamsters in cold NCTC 135 medium, centrifuging the hamagenate at 63g for 5 min, and collecting the LD bodies from the supernatant fluid by centrifugatian at 360g for 10 min. The pellet containing parasites was resuspended in cold NCTC 135 medium and counted in a Petroff-Hausser counting chamber by phase micros- copy. The parasites remained viable in suspension for 3 days at 5 C. Immunosuppressive procedures. Surgical procedures were performed on mice anesthetized with sodium pentabarbital. Seven to eleven week old mice were thymectomized (TX) by the method described by Miller (33). Splenectomy (Sx) was performed.ane natwo weeks after thymectomy. Both surgical wounds were closed with sterile autoclips (Clay Adams). About 35 days after thymectomy, the mice were placed into individual restraining chambers and irradiated (Ir) with 1000 R from a 60Ca gamma emitter. One day after irradiation, bone marrow (BM) cells were flushed from the femurs and tibias of normal or thymectomized syngeneic donors of the same age. Suspensions of single cells were obtained by progressive gentle passage of the cells through 20 to 27 gauge needles. Marrow cells were suspended in 32 cold MEM (Minimal Essential Medium) and held an ice until used. In later experiments, marrow cells were pretreated with anti—brain associated theta (aBAe) serum plus guinea pig complement (designated theta—negative bone marrow, G'BM). Irradiated mice were reconstituted i.v. with 5 x 106 viable untreated or aBAO serum treated marrow cells; marrow cells and LD bodies were usually injected together. Within 24 hr of the injection of LD bodies, designated groups also received azathiaprine (A2). Azathigprine. Az (Burroughs Wellcame) was dissolved in saline with 1 N NaOH immediately prior to use and 150 to 200 mg/kg of body weight was injected by the intraperitoneal (i.p.) route 18 to 20 hr after infection. Complement. Immediately before use, lyophilized guinea pig complement (GIBCO) was reconstituted and absorbed with agarose as described by Cohen and Schlesinger (15). Anti-brain associated theta serum. The protocol used by Golub (21) was used for the production and absorption of aBAO serum. Briefly, homo- genized mouse or rat brain was emulsified in Freunds' complete adjuvant (BBL) and 0.3 ml was injected subcutaneously at 10 sites in the legs, back, and neck of adult New Zealand White Rabbits. Booster injections were given by the same route two weeks later. The rabbits were bled by cardiac puncture 10 days later and the sera were collected from the blood by standard.pracedures. The sera were pooled, heat inactivated, and absorbed four times with a mixture of erythrocytes and homogenized livers and kidneys, such that 5 ml of aBAO serum was absorbed with tissues from four C57Bl mice. Aliquots of absorbed sera were stored at -20 C. Cytotoxicity and in vitro treatment of bone marrow. The cytotoxicity of aBAO serum was tested against thymus and marrow cell suspensions 33 according to the method described by Golub (21). A l/l6 dilution of ab- sarbed aBAO serum completely killed thymocytes (3 x 107 cells/ml) and about 40% of the bone marrow cells in vitro. The procedure for testing cytotox- icity was modified for the purpose of eliminating T cells from marrow prior to injection into irradiated recipients. In brief, a 1 ml suspension 7 cells in MEM, was incubated on ice of marrow cells, containing 1 to 3 x 10 with 0.4 m1 of a 1/4 dilution of absorbed aBAO serum for 30 min. Two-tenths ml of absorbed complement, diluted 1/5, was added and the mixture was in- cubated for 60 min at 35 C. Treated marrow cells were pooled, centrifuged for 6 min at 250g, resuspended in cold MEM, and the viability determined by trypan blue exclusion. Radial immunodiffusion assay. The relative IgG and IgM content of sera from control, suppressed, and normal (uninfected) mice were quantita- ted by radial immunodiffusion as described by Mancini et a1. (29) and Fahey and McKelvey (19). Goat anti-mouse IgG and anti-mouse_IgM (heavy chain) were purchased from Miles Laboratory and Cappel Laboratory, re- spectively. Histology. Histological sections were fixed in Bauin's Fluid and stained with hematoxylin and easin. RESULTS Effect of thymectomy and irradiation. The course of infection in normal mice was compared to irradiation and thymectamized-irradiated-bone marrow reconstituted (TxIrBM) mice (Fig. l). The peak of infection in controls was reached between day 21 and 28 past infection. The number of parasites in the spleen and liver had declined by day 60, to levels which were as law or lower than parasite levels on day 3. In some experiments, 34 peak parasite levels persisted langer in spleens, but generally the course of infection followed a parallel course in bath organs. In nanrecansti- tuted mice, irradiation did not affect parasite proliferation until 9 days after irradiation when these mice began dying from irradiation; all mice in this group died by day 14 past irradiation. The course and level of infection in TxIrBM mice was not different from that in non-treated controls. Attempts to eliminate residual T cells. The above results suggest- ed that if T cells were necessary for the expression of resistance to L. dbnavani, successfu1 elimination of parasites by TxIrBM mice depended on the presence of T cells in the marrow cells used to reconstitute TxIr recipients and/or of residual T cells in the recipients themselves. Attempts were made to eliminate T cells in marrow first by thymectomizing donors, and secondly by treating marrow cells from thymectomized donors with aBAO serum (G'BM). In some experiments Az was used to eliminate residual T cells in thymectomized and TxIre'BM mice. Reconstitution of TxIr recipients with marrow cells from thymectomized donors failed to re- duce resistance. However, moderate suppression of resistance resulted when TxIr mice were reconstituted with e"BM, and when Az was used to elim- inate residual T cells in thymectomized or TxIre'BM mice (Fig. 2). A2 e"BM mice afforded an apparent advantage over other treatment of TxIr regimens at 40 days past infection. Regardless of the combination of immunosuppressive measures used, the peak level of infection (day 24) in controls was not significantly different from the level of infection in suppressed groups on the same day. Although not significantly different from each other on day 60, all three immunoSuppressed groups were signif- icantly different from the control group (P<0.05). Because the spleen represented a major potential source of residual 35 T cells in the host, several experiments were performed to assess the ef- fect of splenectomy on the course of infection. SpleneCtomy, with or with- out IrBM treatment, did not appreciably suppress the immune elimination of parasites (Fig. 3). However, when combined with TxIre‘BMAz treatments (Fig. 4), significant immunosuppression resulted (P<0.001). The combined treatment, TxSxIre-BMAz (hereafter designated deprived mice), permitted parasite multiplication up to day 60. Without Splenectomy, the other combined treatments delayed the peak of infection from day 24 to about day 40, and the level of infection was significantly higher than in controls (P<0.01). Comparison of the slopes of parasite multiplication prior to day 24 (Fig. l) and in deprived mice from day 40 to day 60, indicates that the rate of proliferation was reduced in deprived mice after day 24. Effect of suppreSSion on immunoglobulin leVels. Results of radial immunodiffusion test used to determine the effects of Leishmania infection on the relative amounts of IgG and IgM in control and deprived mice are given in Table 1. Compared to the amounts of IgG and IgM in pooled sera from uninfected normal mice (NMS), infection of control mice resulted in a progressive increase in both IgM and IgG up to day 50 when parasite levels had been greatly reduced. Initially, the amount of IgM in deprived mice was not different form IgM levels in NMS while the amount of IgG was from 71 to 83 percent less than the amount of IgG in NMS. Two major precipitin bands were observed when NMS and control sera were reacted with goat anti-mouse IgG serum. These bands possibly repre- sented IgGl and IgGZ. The radii of both bands from control sera increased from day 24 to day 50. Sera from deprived mice formed a single band. 36 Histological observations. In histological sections from the liver, lung, and intestines of a deprived mouse there was a general depletion of lymphoid cells. Histological sections of liver (Fig. 5) had many macro- phages containing parasites in the absence of pathological manifestations. LD bodies normally associated with infection were not seen in alveaiar macr0phages in lung sections. DISCUSSION This experiment provides evidence that T cells play an important role in the development of immunity to the intracellular parasite, L. dono- vani, in mice. The results are consistent with those of Bryceson et a1. (12) and Preston et al. (37) who found that anti—lymphocyte serum and TxIrBM treatments increased susceptibility to cutaneous leishmaniasis in guinea pigs and mice, respectively. However, in this system, more exten- sive treatments were required to effectively deplete the T cell system. The requirement for T cells was not apparent at first because TxIrBM treatment was ineffective in altering the course of L. danovani infection in C57Bl mice. Similarly, North (36) observed that resistance to Listeria was not altered in TxIrBM mice even though resistance to Listeria in mice had previously been shown to be T cell dependent (25). The same treatment was effective in suppressing immune resistance to tuberculosis. However, the mycobacterial growth rate prior to the onset of immunity was greater' than the rate of growth in TxIrBM host after the onset of immunity in simultaneously infected controls. In this study, the pattern of L. donovani growth in deprived mice mirrored this trend suggesting that the immune response was incompletely suppressed. Preston et al. (37) also observed that the course of L. trap- ica infection in some TxIrBM mice was prolonged and less acute. There is 37 no evidence to implicate that humoral mechanisms could account for the apparent residual resistance, however, several lines of evidence have shown that T cells exist both in bone marrow of normal mice and in the spleens of irradiated mice (17,23,41). It is possible that resistance to different organisms may require different types or different quantities of T cells for the induction of immunity. It was concluded that the incomplete removal of T cells was the major factor contributing to the failure of the TxIrBM treatment to sup- press the immune response to L. danavani. Additional treatments were there— fore implemented to eliminate the potential sources of T cells. Bone mar- row donors were thymectomized and marrow cells were treated in vitro with aBAe serum prior to injection into TxIr recipients. In addition, recip— ient mice were treated with A2 on the day of infection. Az has been shown to have a relatively selective affect in inhibiting the immune response to Listeria (44), thymidine incorporation in the mixed lymphocyte reaction (1), and T-rosette forming cells (3,4). Splenectomy was employed because the spleen was presumed to be the most likely source of residual T cells as well as the possible source of a putative primary-lymphoid-organ function (6,13,41). Splenectomy appar- ently removed residual T cells, the putative primary-lymphoid—organ func— tion, or the procedure produced an environment in which Az was more effective. An immunosuppressive protocol which extensively removed T cells from both, donor and recipient mice, was required to eliminate the immune response of C57Bl mice to L. donovani. The effects of TxIrBM, aBAO serum, and Az treatments are well established, but the effect of Splenectomy on cellular immunity has not been elucidated. Splenectomy is known to increase susceptibility of some animals to malaria, but only delays the early 38 antibody response to soluble antigens given i.v. (38). Several lines of evidence suggest that the spleen may play several important roles in cell-mediated immunity. Battisto et a1. (6) and Bucsi et al. (13) have observed a deficiency in the T-B cooperative capacity in neonatauy splenectomized and congenitally spleenless mice. Ito et al. (24) have suggested that B cells require both a thymic influence and the splenic environment for differentiation into antibody producing cells. It has been postulated that the spleen in rabbits plays an important role in the gen- esis of effector cells or secretes a hormone which allows progenitor cells to develop (7). Bach et al. (2) found that Splenectomy did not modify serum thymic activity in normal mice, but when followed by thymectomy, the effects of thymectomy became apparent much sooner. Relative to cellular immunity, splenectomized animals have been observed to have a more effective response to tumor grafts. Gershan et al. (20) have advanced the premise that Splenectomy soon after the transplantation of thymocytes increases graft— vs-host reactivity in the splenectomized mouse by removal of spleen seek- ing suppressor cells. In the second article of this series (39), we re— ported that immunity to L. donavani was reconstituted by thymocytes and/or lymph node cells; thus we concluded that in this study, the major effect of Splenectomy was the removal of residual T cells in TxIrBM mice. The greater suppression of IgG than IgM in deprived mice parallels the findings by Preston et al. (37) that in L. trapica infected TxIrBM mice, immunofluorescent antibody titers were reduced while agglutination titers were equal in control and TxIrBM mice. The superimposition or absence of one of the IgG bands in the radial immunodiffusion of sera from deprived mice suggests that one subclass of IgG was reduced more than the other. If so, these observations agree with evidence reported by Torrigiani (43) that mouse IgGl is relatively more T cell dependent than IgG2 and IgM. 39 Studies of the adoptive transfer of protection against infection with Listeria (31) and mycobacteria (26) indicate that the protective cell populations vary depending on the nature of the infective agent and the time cells are taken during the course of infection. During an acute in- fection or early phaSe of a chronic infection, large short-lived noncircu— lating lymphoid cells passively transfer resistance. In contrast, small long-lived circulating lymphocytes were responsible for protection after three weeks in chronic infections. These Observations raise the question as to whether these cells represent distinct T cell populations. 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Thymic dependence of the different classes. J. Immunol. 108:161. Tripathy, S. P. and G. B. Mackaness. 1969. The effect of cytotoxic agents on the primary immune response to Listeria monacytagenes. J. Exp. Med. 30:1. Visher, W. A. and E. Suter. 1954. Intracellular multiplication of Taxoplasma gondii in adult mammalian macrophages cultivated in vitro. Proc. Soc. Exp. Biol. Med. 86:413. Wagner, H. 1973. Synergy during in vitro cytotoxic allograft re- sponses. 1. Evidence for cell interaction between thymocytes and peripheral T cells. J. Exp. Med. 138:1379. 43 TABLE 1 Relative amounts of IgG and IgM in pooled sera from deprived and control mice during the convalescent stage of infection. Percentage of IgG and IgM in normal mouse serum8 Days post infection 24 40 50 IgG IgM IgG IgM IgG IgM % t % % t % Deprived 78 96 71 95 83 88 Control 106 107 116 105 127 116 aAmounts of immunoglobulin were quantitated by the radial immunodif- fusion method and expressed as the percentage of IgG or IgM found in pooled sera from uninfected normal mice. Serum from 5 to 10 mice was pooled for each group; mice with incomplete thymectomy were not excluded from the deprived group. Each serum sample was tested in duplicate. 44 Figure 1. Growth curve of L. danavani in the livers and spleens of control, irradiated (Ir), and thymectomized-irradiated-bone marrow reconstituted (TxIrBM) mice infected i.v. with 20 x 106 LD bodies. Each point represents the mean parasite count from 5 mice. 45 1000 \ . \JVGI’ \ \ \ 0 1 00 .00.. x. coaco\oa._ coas— Control \ Spleen 60 4O 20 Days Past Infection Figure l 46 Figure 2. Growth curve of L. danavani from 24 to 60 days post infection in livers of untreated controls compared to mice receiving various treatments including thymectomy (Tx), irradiation (Ir), re- constitution with theta-negative bone marrow cells (e’BM), and aza- thioprine (A2). The mice were infected i.v. with 20 x 106 LD bodies. Each point represents the mean number of parasites in livers from 5-8 mice except for group-A on day 40 which represent 3 mice. 47 100 Mean LDs/Liver (x 10“) [TX Ir GEM A2 + +++ 20 4O 60 Days Past Infection Figure 2 48 Figure 3. Growth curve of L. donovani from day 24 to 40 in con- trol, splenectomized (Sx), and splenectomized-irradiated—bone marrow reconstituted (SxIrBM) mice injected i.v. with 20 x 106 LD bodies. Each point represents the mean number of liver-parasite from 5 mice. Mean LDs/Liver (X 106) 49 20 40 Days Past Infection Figure 3 .5 50 Figure 4. Growth curve of L. danavani from day 24 to 60 in con— trol and deprived mice infected i.v. with 20 x 106 LD bodies. Mice were deprived by thymectomy—Splenectomy-irradiation-reconstitution with theta-negative bone marrow, and azathioprine (TxSxIre’BMAz). A second group received the same treatment except that Splenectomy was omitted (TxIrO'BMAz). Each point represents the mean number of liver- parasites from 5-8 mice. 1000 10 Mean LDS/ Liver (x 106) 51 TxlereEMAz TxIrBBMAz Control 4O 60 Days Past Infection Figure 4 52 Figure 5. Liver section from a deprived mouse about 4 wk after infection i.v. with 10 x 106 LD bodies. Note the absence of pathological manifestations around macrophages (M) which are filled with amastigotes (LD). H and E. (X 1000 and 2400). 53 Figure 5 ARTICLE 2 Cellular Immunity to Leishmania danavani. 11. Evidence for synergy between thymocytes and lymph node cells (T-T) in reconstitution of acquired resistance to L. danavani in mice. Clare B. Skov and Donald W. Twahy Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 (To be submitted to J. Immunol.) This is Journal Article No. 6791 from the Michigan Agricultural Experiment Station. 55 1This work was supported by Grant No. DADA 17-69C-9135 from the U.S. Army Medical Research and Development Command under sponsorship of the Commission on Parasitic Diseases of the Armed Forces Epidemiology Board, and with support from the Michigan Agricultural Experiment Sta- tion. Journal Article No. 6791 from the Michigan Agricultural Experi— ment Station. 2Abbreviations used in this paper: T, thymus-dependent; LNC, lymph node cell; Thym, thymocyte; GVH, gfaft-vs-host; Az, azathioprine; aBAO, anti-brain associated theta serum; PEC, peritoneal exudate cells; EDTA, sodium ethylene diaminetetraacetate; i.p., intraperitoneally; i.v., intravenously. 56 ABSTRACT C57Bl/6J mice were infected with Leishmania danavani after a series of treatments that were designed to deplete thymus-dependent (T) cell populations. Deprived mice did not acquire immune resistance to the parasite. The capacity to resist infection was restored by either lymph node cells (LNC) or thymocytes, but 20-25 times more thymocytes than LNC were required. Reconstitution of immunocompetence by both cell types was inhibited by in viva treatment with azathioprine or anti-brain as- sociated theta serum. Limiting dilution experiments showed that con- stant minimal numbers of LNC combined with graded numbers of thymocytes, or vice versa, conferred greater protection than expected from the addi- tive effect of both cell types acting independently. Cultured peritoneal macrophages, harvested from normal and reconstituted mice 44 days after infection, suppressed parasite multiplication. Macr0phages from infec- ted deprived mice were unable to prevent parasite multiplication in vitro. These results provide strong evidence that acquired resistance of mice to L. danavani involves T-T cell synergy and relies principally on cellular immunity. INTRODUCTION Cell-mediated immunity represents a wide range of immune phenomena including inductiOn mechanisms of humoral responses to many antigens as well as the more classically defined cellular immunity. Thymus- dependent (T) cell interactions with other lymphoid cells may be a basic hallmark of the development of the immune response since induction and adaptive transfer of cell-mediated immunity can be reduced or abrogated by T cell depletion. 57 Cantor and Asofsky (6) recently presented evidence far Tl—T2 cell synergy in the graft-versus-host (GVH) response, a classical model of cellular immunity. Several lines of evidence support the concept of T cell heterogeneity and T-T cell interaction. Subpopulations of T cells reportedly differ with regard to surface antigens (11), sensitivity to corticosteroids (8) and azathioprine (2), "homing" patterns (24), re- sponsiveness to mitogens (17,22) and their immune function (6,7,22,24,27). A substantial body of evidence has shown that acquired immunity to many intracellular parasites is initiated and maintained by T cells and activated macrophages. The nature or stage of differentiation of cells capable of passively transferring protection to Listeria (12) and myco- bacteria (14) varies depending on the infective agent employed and the time during the course of the immune response when donor cells are col- lected. Corticosteroid treatment reportedly differentiates between cells which confer immunity in Listeria infections (18) and graft rejection (8). These differences in cell pr0perties may also be a result of T cell heterogeneity. Since T cell interaction in cellular immune reactions to infectious intracellular parasites has not been reported, this study was designed to test for T-T cell interaction in cellular immunity against Leishmania dbnavani, an intracellular protozoan parasite. Acquired immunity to leishmaniasis has been reported to be dependent on T cells (4,19) and on activated macr0phages (15). This paper reports the results of limiting dilution experiments in which infected deprived mice were reconstituted with thymocytes and/or lymph node cells (LNC). The results are consis- tent with the evidence of Tl-T2 cell synergy found by Cantor and Asofsky (6) and Wagner (27). 58 MATERIALS AND METHODS Experimental design, The general techniques and procedures for treatment and maintenance of mice and parasites was previously described (20). Fig. 1 summarizes the basic protocol employed for preparing and reconstituting T cell deprived C57BL/6J mice (Jackson Laboratory, Bar Harbor). In brief, thymectomized, splenectomized, and lethally irradia- ted mice were reconstituted intravenously (i.v.) with T cell depleted bone marrow cells. Recipient mice were infected with 10 x 106 L. danavani (3S) amastigotes (LD bodies) and injected intraperitoneally (i.p.) with azathiprine (Az) about 18 hr later. Five days after Az treatment, recipi- ent mice were reconstituted i.p. with graded numbers of thymocytes and/or LNC. To test far synergistic effects, one cell type was injected in graded numbers together with a constant number of the second cell type which was supplied at a level that provided minimal or undetectable re- constitution alone. As previously described (20), liver impression smears were used to assess the number of parasites in control mice at the peak of infection (day 24) and in all groups 16-26 days later. In viva effect of A2 and anti-brain associated theta (aBAe) serum. A2 and aBAe serum were prepared as previously described (20) and used to test the T cell dependency of reconstitution. One group of deprived mice was reconstituted with 106 thymocytes and a second group was reconstitu— ted with 10S LNC. Bath major groups were divided into 3 subgroups: the first subgroup in each major group served as untreated controls; the second and third subgroups were injected once with 1 m1 of a 1/2 dilu- tion of aBAe serum or 150 mg/kg body weight/m1 of A2, respectively. Both treatments were administered about 20 hr after reconstitution. The number of liver-parasites in treated, untreated reconstitution controls, and deprived mice was compared on day 45. 59 In vitro correlates of resistance. Peritoneal exudate cells (PEC) were collected from infected mice immediately prior to liver harvests in one experiment. Six mice per group were stimulated by i.p. injections of S x 106 L0 bodies. After 48 hr the PEG donors were killed by cervi- cal dislocation and injected i.p. with 2.5 ml (5 ml to each deprived animal) of a 20 mg% solution of EDTA (sodium ethylene diaminetetraacetate) in 0.85% NaCl, neutralized to pH 7.0 with NaHCOs. The PEC were with- drawn 10 to 20 min later with a syringe and perforated needle. The exu- date from each mouse was transferred to a centringe tube containing 5 ml of cold NCTC 135 medium, centrifuged at 63g for 10 min, resuspended in 2 m1 of complete culture medium (NCTC 135 medium supplemented with 2% bovine embryo extract, 40% horse serum, 400 units of penicillin, and 400 mg of streptomycin per m1), and duplicate 0.2 ml aliquots of each cell suspension were transferred to apprOpriate chambers of 8-chamber Lab Tek tissue culture slides. After the PEC were incubated at 37 C far 2 hr in a humidified 95% air-5% CO2 atmosphere, the medium was replaced with fresh complete culture medium containing 1.5 x 106 LD bodies per m1 and the cultures were reincubated. Culture-chamber slides containing in- fected adherent macrophages were periodically removed from culture, fixed with 5% glutaraldehyde in phosphate buffer (pH 7), and stained in May- Gruenwald Giemsa. The numbers of macrophages and intracellular LD bodies were counted in 5 microscoPe fields per chamber to compare the relative potential of macr0phages from experimental and control mice to prevent amastigote proliferation in vitro. ReconStitution with cells from immunized danors. In this experi- ment we compared the potentials of cells from non-immune and immunized donors to reconstitute resistance. Two groups of donors were immunized: 60 the first group was infected i.v. with 20 x 106 LD bodies 30 days be- fore cell transfers; 8 days later, the second group was injected i.v. with 20 x 106 LD bodies combined with S x 107 thymocytes followed by an i.p. injection of 20 x 107 thymocytes ("T-boosted"). A third group of normal mice served as donors of non—immune cells. Deprived recipients were divided into 6 groups which were reconstituted with graded numbers of one of the following cell types: 1) normal thymocytes, 2) normal LNC, 3) a mixture of normal thymocytes and LNC, 4) spleen cells from donors immunized 30 days earlier; 5) spleen cells from "T-boosted" donors im- munized 22 days earlier, and 6) a mixture of normal thymocytes with LNC from the "T-boosted" immune donors. Liver-parasites were counted 45 days later. Cell suspensions. Thymuses were excised from 3-4 wk old syngeneic donors, freed of fascia, rinsed in cold MEM (minimal essential medium), and teased into suspension. Cell viability was determined by the trypan blue exclusion method. After the cells were counted in a hemocytometer they were diluted with MEM to the desired concentration. Lymph nodes and spleens were excised from adult mice and handled in a like manner. Cells used for reconstitution were injected i.p. 5 days after recipients were treated with A2 as described above. Statistical methods. Regression lines of the log number of para- sites as a function of the number of inoculated lymphoid cells were calculated by the least squares method. The slopes and individual data points were compared by the Student's t test. Extremely variant data, such as data associated with mice which were runted or when complete thymectomy was in doubt, were excluded on the basis of an outlier test. 61 The parallel line assay was used to compare relative immunocompetency of thymocytes and LNC. The statistical significance of reconstitutive po— tency of different papulations of graded cells or cell combinations, as depicted by regression lines, was evaluated by analysis of covariance. RESULTS Reconstitution with thymocytes and LNC. The effect of reconstitu- tion with different numbers of thymocytes or LNC from syngeneic mice was determined using groups of 8-10 deprived mice as recipients for each con- centration and cell type employed. The geometric means of the number of parasites found in the livers of each group at 45 days post infection were plotted against the corresponding log number of inoculated cells (Fig. 2). Because of deaths and the exclusion of some data due to incomplete thymec- tomy, final parasite counts were based on 3-9 mice per group; however, most points represent the means derived from 5 or more mice. The dif- ference between the number of parasitesin deprived and control mice was highly significant (P<0.001). On the basis of the parallel line assay, the relative reconstitu- tive potency of LNC was calculated to be 20-25 times greater than thymo- cytes. An analysis of covariance showed that the immunocompetence of thymocytes and LNC were significantly different (P<0.001). The number of parasites in deprived mice and mice reconstituted by 104 LNC was not significantly different, but extrapolation of the regression line shows that equal levels of infection could be expected in deprived mice and mice reconstituted with about 6 x 103 LNC. Reconstitution with 10S thymocytes also did not confer measurable immmity by 40-50 days post infection. 62 Inhibition with A2 and aBAG serum. Approximately 18-22 hr after 5 LNC, either Az or aBAe i.p. injections of either 106 thymocytes or 10 serum.was administered i.p. The results of this experiment are summa- rized in Table 1. Treatment of LNC reconstituted mice with A2 or aBAe serum eliminated the protection conferred as compared with untreated mice (P<0.001 and P<0.02, respectively). A2 and aBAe serum treatment of thymocyte reconstituted mice only partially inhibited the protective effect since both groups had fewer parasites than the deprived group (P<0.05 and P<0.001, respectively) but more parasites than the untreated mice (P<0.02 and P<0.05, respectively). The moderate level of reconsti- tution in untreated mice was not significantly different from the levels of reconstitution by the same number of cells in other experiments. Synergy among thymocytes and LNC. Fig.3 shows the results of re- constituting deprived mice with graded numbers of thymocytes mixed in each case with 104 LNC. By themselves, 104 LNC provide little or no pro- tection. The level of protection conferred by thymocyte-LNC combinations was approximately 10 times greater than the level of protection afforded by thymocytes alone and greatly exceeded the level of protection ex- pected from an additive effect of both cell types acting independently. Although the slopes were not significantly different, the differences in the levels of protection were highly significant (P<0.01). Fig. 4 shows that when a constant, non-protective number of thymo- cytes (105) was inoculated together with graded numbers of LNC, the pro- tection conferred by combinations of both cell types also exceeded that predicted by an additive effect. Reconstitution with 104 LNC combined with 10S thymocytes conferred significantly greater protection than 104 LNC alone (P<0.01). The synergistic effect was most evident when fewer than 5 x 104 LNC were inoculated with 10s thymocytes. Reconstitution 63 with higher numbers of LNC in either group resulted in levels of protec— tion which approached those observed in normal mice and differences be- tween groups became insignificant. The slope of the regression line representing reconstitfition with this synergistic cell combination was less than the slope of lines representing the effect of LNG alone or the reversed synergistic mixture of cells (Fig. 3). In vitro response to parasites. In one experiment, PEC were col- lected prior to liver harvest at 44 days post infection. This experi- ment was designed to compare parasite survival and/or proliferation in cultured macrophages from infected control, reconstituted, and deprived hosts. For each time interval, duplicate cultures of PECOfrom each mouse (6 mice per group) were infected with amastigotes at the beginning of the culture period. Fig. 5 shows that a smaller number of macr0phages were harvested from deprived and reconstituted animals than from normal controls, but that cells from all except one reconstituted group of mice followed similar survival patterns. The number of parasites in macro- phages from control and reconstituted mice decreased during the 44 hr culture period. In contrast, macrophages from deprived mice were unable to prevent parasite multiplication. Differences in the number of para- sites in control as compared to deprived cell cultures were highly sig- nificant after 24-44 hr in culture (P<0.001). The results of parasite death or survival in macrophages were consistent with the results from in viva tests shown in Figs. 2 and 4 where reconstitution with 5 x 104 LNC conferred a moderate level of protection and protection conferred by 5 x 106 thymocytes was equivalent to controls. The in vitro response of macrophages to parasites roughly reflected the level of immunocompetence in the hosts as determined from liver impression smears. 64 ReconStitution with immune'cells. This experiment was designed to compare the levels of protection conferred by reconstituting deprived mice with cells from immunized and non-immunized donors. The data.was incom- plete as a result of the unprecedented death toll which occurred either as a result of Pseudomonas aeruginosa infections and/or predisposing trauma and exposure experienced in transit for irradiation treatment. Spleen cells collected from convalescing donors 4 wk after infection were used as a source of sensitized cells because this source had been shown to have a maximum effect in adoptive transfer experiments (Twahy, unpublished). A second group of donors was "T-boosted" at the time of infection with thymocytes from 26 day old donors in an attempt to in— crease the capacity of donor cells to adoptively transfer protection. Im- mune Spleen cells (105) from donors immunized 4 wk (group-A) and 3 wk (group-C) prior to cell transfers conferred greater protection than 105 normal thymocytes, which afforded little or no protection (P<0.05 and P<0.01, respectively; Fig. 6). Resistance conferred by normal thymocytes (105) combined with 104 LNC from donors that were "T-boosted" and immu- nized 3 wk prior to cell transfer (group-D) was greater than the level of resistance given by the same cell combination from non-immune donors (group—B) or from 105 thymocytes alone (P<0.02 and P<9.01, respectively). Both groups of mice which were reconstituted with cells from "T-boosted" donors exhibited a higher level of protection than mice reconstituted with spleen cells from 4 wk immune donors (groups C and D vs A; P<0.02). The slopes and the relative levels of immunocompetence of non-immune thy- mocytes and LNC shown in Fig. 6 are consistent with those shown in Fig. 2. 65 DISCUSSION The mechanism of acquired resistance to several intracellular proto- zoan parasites has been shown to be similar to that described for intra- cellular bacterial parasites. Like resistance to Listeria (13) and Myco- bacterium (23), protection against Tbxoplasma (25), Besnoitia (9), and Leishmania (15) are dependent on cellular immunity as determined by the requirement of cells rather than serum for passive transfer of delayed hypersensitivity or acquired resistance, and by the demonstration of para- siticidal behavior of "immune" macr0phages in vitro. Evidence that re- sistance to Leishmania is T cell dependent was found by Bryceson et al. (4) and Preston et al. (19) who demonstrated that T cell depletion re- sulted in increased susceptibility to cutaneous leishmaniasis in guinea pigs and mice. Increased susceptibility to visceral leishmaniasis in T cell deprived mice was reported in the preceeding paper (20). This paper describes the reconstitution of deprived mice with thymo- cytes and LNC as well as synergistic activity by combinations of the two cell types. Both cell types conferred protection against infection with L. donovani. Equivalent levels of protection required approximately 20- 25 times more thymocytes than LNC. The levels of protection which resulted when both cell types were injected tOgether greatly exceeded the levels of protection that were predicted from the additive effects of independently acting populations. It was hypothesized that as the varied population was given in greater numbers, it would account for a greater proportion of the resistance con- ferred by a mixed population of cells. Thus, if synergistic activity oc- curred, the level of resistance given by mixed cells was expected to pro- gressively approach the level of resistance which resulted when the varied cell population was administered alone. A flatter dose-response 66 (regression) curve did result when graded numbers of LNC were injected with 10S thymocytes (Fig. 3). However, the slope of the lines represent- ing graded numbers of thymocytes mixed with 104 LNC or graded numbers of thymocytes alone were not significantly different (Fig. 2). Perhaps a flatter regression line would have resulted in the second case had vary- ing numbers of thymocytes been mixed with a more limiting number of LNC. Alternatively, if the mechanism of interaction involves two-way communi- cation such that the reactivity of LNC depends on stimulation from thymo- cytes, which in turn causes LNC to enhance the activity of thymocytes, then the level of enhancement of thymocytes by a constant number of LNC could vary depending on the number of responding thymocytes. Under these conditions, reconstitution with graded numbers of thymocytes, with and without 104 LNC, could be expected to result in nearly parallel regres- sion lines as shown in Fig. 3. The flatter dose-response curve observed for reconstitution with graded numbers of LNC mixed with 105 thymocytes (Fig. 4) could also be expected under these circumstances since the re- quired signal, or stimulus from thymocytes, would be constant while the enhancing effect would vary with the number of LNC. Regarding the above hypothetical explanation of the differences in slopes, it is important to remember that neither population of cells used in these experiments were pure. Perhaps spleen-seeking and lymph node-seeking cells represent relatively pure subpopulations of T cells. Tigelaar (24) observed relatively flat dose-response curves when graded numbers of lymph node-seeking or spleen-seeking T cells were tested alone for their ability to provide a GVH reaction in irradiated mice. However, when graded numbers of lymph node-seeking cells and constant numbers of spleen-seeking cells were injected together, the dose-response slopes and the levels of GVH reactivity were much higher and resembled dose-response 67 curves resulting from injections of normal LNC or spleen cells. A wide range of host responses are characteristic for L. donovani. Human and hamster hosts usually permit unrestricted parasite prolifera— tion without developing immunity. In man, chemotherapy prevents death and is followed by strong but poorly understood immunity. Cotton rats also permit unrestricted multiplication of parasites but apparently suf- fer no ill effects from high levels of infection. Mice show a wide range of susceptibility, some strains possess complete innate resistance while other strains experience a long course of infection (3). When infected intravenously, CS7BL/6J female mice support high levels of infection which are followed by strong immunity (Twohy, unpublished). Deprived mice have been used in a variety of studies designed to elucidate lymphocyte roles and interactions in the immune response. The immunosuppressed CS7BL mouse system used in this investigation proved to be a useful model for demonstrating T-T cell interaction in the rejection of intracellular parasites. However, the interpretation of results in these experiments was complicated by several factors. As has been ob- served by other investigators (10), a great deal of variability may be inherent with reconstitution of deprived mice with thymocytes. This is especially true with adult thymocyte donors or donors of different ages (17). In these experiments, thymocyte donors were always between 23 and 28 days old. Reconstitution with adult thymocytes was poor and inconsis- tent. Another variable, runting, occurred occassionally among the groups of reconstituted and deprived mice. In most cases, the runted mice ex- hibited higher numbers of parasites in their livers than did others in the same group even though their body weight was much lower. Electron micrographs of intracellular Leishmania amastigotes show that two complete membranes surround LD bodies. Putatively, the inner 6. I membrane is the cytoplasmic membrane of the LD body while the outer mem= brane may be a vacuole-like membrane belonging to the host. Such a mem- brane may in effect protect the parasite from recognition by the host. Alternatively, the amastigote, either by serial passage in the hamster or by the presence of a remnant outer coat, may acquire xenogeneic histo- compatibility antigens which would stimulate a graft rejection type of im- mune response against the parasite. It seems unlikely that the expres- sion of immunity toward Leishmania reported here was a result of graft rejection mechanisms since the xenogeneic antigens would have been signif- icantly diluted by the massive proliferation of the parasites and the im- mune rejection would have been expected to have occurred much earlier. Furthermore, macrophages from mice which were immunized with hamster spleen were unable to suppress parasite growth in vitro while the para— sites were killed by macrophages from donors which were immunized with amastigotes from mouse or hamster spleens (Twohy, unpublished). The relative number and behavior of macrophages harvested from the peritoneum of infected controls was similar to that reported by Miller and Twohy (15). More macrophages were harvested from control than from deprived animals. Parasites did not survive in macr0phages from the im- mune controls and macr0phages from deprived mice supported parasite pro- liferation similar to that seen in macrophages from normal animals (15). In general, the in vitro tests correlated well with the pattern of re- sistance seen in vivo. The Youmans hypothesized that cellular immunity can be completely dissociated from delayed hypersensitivity and that non- specific killing of intracellular parasites by activated macr0phages may be an expression of delayed hypersensitivity (26). The observations re- ported here do not clarify this question. However, the correlation of 69 the in vivo and in vitro expressions of immunity indicates that the in vitro assessment of immunity in this system was valid. Like the recall of passively transferred delayed hypersensitivity in other systems, spe- cific antigenic stimulation approximately 48 hr prior to PEC harvest was necessary to obtain macr0phages with parasiticidal properties. In the experiment in which the cells used for reconstitution were taken from infected donors, the injection of the donors simultaneously with parasites and thymocytes appeared to enhance the transfer of immu- nity when compared to donors which were immunized without "T-boosting". In previous passive transfer experiments, in vitro criteria were used to assess immunity. In those experiments, spleen cells from donors immu- nized 3 wk before cells were transferred, were inconsistent in conveying adoptive immunity (Twohy, unbuplished). This experiment was of a pre- liminary nature, and did not discern whether the advantage of "T—boosted" cells was a result of the cells having previous contact with.the para- site antigens, or whether the enhanced immunity was a result of the en- richment of immunocompetent cells via a selective migration of immuno- competent cells to lymph nodes and spleens. The passive transfer of the anamestic response may more readily be demonstrated in this system by methods designed to detect an earlier onset of immunity rather than by comparing levels of infection later in the course of infection. Synergy between central and peripheral T cell populations has been previously reported by Cantor and Asofsky (6) and Wagner (27) using the graft-versus-host system and the in vitro cytotoxic allograft response, respectively. Cantor and Asofsky found combinations of peripheral blood lymphocytes and thymocytes to be 2-3 times more potent than predicted from an additive effect while Wagner found a 20 fold increase in reactivi- ty. In contrast, we found about a 10 fold increase in the level of 70 protection given by combination of thymocytes and LNC. Like the results of the previous workers, our results show that peripheral T cells were approximately 20-25 times more immunocompetent than thymocytes. The dif- ferences in the degree of synergy, as reported by the above investigators, may have resulted because the levels of immunocompetence present in the limiting or constant cell population were different. Alternatively, each of the experimental systems may be different with respect to its sensitivity or its lymphocyte interactions and ef- fector mechanisms Nevertheless, evidence that T-T cell synergy may play a major role in immune responses against histocompatibility antigens and intracellular parasites unifies the concept of cellular immunity and may more clearly distinguish it from other cell-mediated immune phenomena, i.e., T-T cell synergy may be a criterion of cellular immunity. It is evident that T cells are required for protection of mice against L. donovani. Anti-theta serum and A2 have been shown to effective- ly abrogate T cell function (1,5,20,24). The differences observed be- tween the inhibitory effects of A2 and aBAe serum are consistent with the concept that T2 cells recirculate more than Tl cells and are consequently more susceptible to the inhibitory effects of both agents. Treatment of thymocytes with aBAe serum in vitro killed 100% of the cells while in vivo treatments l8-22 hrs after reconstitution only moderately inhibited the reconstitutive effects of thymocytes. The results of this investigation provide convincing evidence that acquired resistance to L. donovani in miCe relies principally on T cell mediated cellular immunity. Furthermore, the suppression—reconstitution model described provides a usefu1 system for studying the cellular com- ponents which are responsible for protective immunity against infectious 71 intracellular parasites. In this regard the data suggest that the mechanisms of cellular immunity to intracellular parasites are at least fundamentally analogous to those described fOr the GVH system (6) and the in vitro cytotoxic allograft response (27). 10. REFERENCES Bach, J. F. and M. Dardenne. 1972. Antigen recognition by'T lympho- cytes. II. 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Evidence for cell interaction between thymocytes and peripheral T cells. J. Exp. Med. 138:1379. 74 TABLE I Inhibition of protection conferred by transplanted LNC or thymocytes by in viva treatment of recipients with A2 or aBAG serum. Cells used for Treatment Log LDs/liver Significance reconstitution in viva mean : sd untreateda deprivedb None* None 9.22 i 0.172 P<0.001 LNC (105) Untreated 8.05 i 0.370 p<0.001 LNC (105) A2 9.11 i 0.183 P<0.001 ns LNC (105) aBAG 8.92 i 0.392 P<0.02 ns Thymocytes (106) Untreated 7.63 i 0.386 P<0.001 Thymocytes (106) A2 8.64 i 0.394 P<0.02 P<0.05 Thymocytes (106) aBAe 8.14 i 0.154 P<0.05 P<0.001 * Designates deprived group. a Treated group compared to untreated group; b group compared to deprived group; ns, not significant. 75 Figure 1. Experimental design. Bone marrow cells from thymecto- mized syngeneic donors were treated with aBAe serum plus complement and used to restore the hemopoietic stem cell population in thymectomized, splenectomized, and irradiated (deprived) recipients. Following the in- fection of deprived recipients, they were treated with A2 after about 20 hr, and reconstituted 5 days later with various numbers of thymocytes and/or LNC. The level of protection conferred by reconstitution was assessed 40 to 50 days post infection from the number of parasites counted in liver impression smears. 76 H onsmfim may.» 5335.... Log. Ea: 3333 5:085 ._Hom-wv next 835 u. .5: BE 8220 uni/g 02.. 95:8: .co:2__~wcoo.om cocoa. .9502 2.93.00: A >3 77 Figure 2. Relative immunocompetence of thymocytes and LNC. The log number of parasites/liver 41 days post infection were plotted against the number of thymocytes (A) and LNC (O) that were injected for reconsti- tution. Each point represents the mean number of parasites/liver from 6 to 9 mice. SlOpes of regression lines were calculated by the method of least squares. The range between the mean numbers of parasites in livers from control mice 24 and 41 days post infection are depicted by the vertical bar while the horizontal bar represents the mean parasite count in deprived mice on day 41. log LDs/ Liver 78 l LNC Control 104 105 106 Number of Cells Figure 2 79 Figure 3. Synergistic effect of reconstitution with graded numbers 4 LNC. Each point represents the mean number of thymocytes mixed with 10 of liver-parasites from 4 to 8 mice at 44 days post infection. Regression lines were calculated by the least squares method and represent the rela- tive immunocompetence of thymocytes and LNC combined ((3) and thymocytes alone (0). Control and deprived groups are represented by vertical and horizontal bars (see Fig. 2). 80 ’6' II m oasmfla «:00 .o 89:52 or + Eb; 02.. v am>taoo I 6, ”.ue.~fi~“.....n.n.~m «a». ......U.//.n..a as??? (I) Jam/901 60: 81 Figure 4. Synergistic effect of reconstitution with graded numbers of LNG mixed with 10S thymocytes. Each point represents the mean nunber of liver-parasites in 5 to 7 mice at 47 days post infection. Regression lines (calculated by the least squares method) represent the relative immunocompetence restored by graded numbers of LNC conbined with 10S thymocytes (O) and LNC alone (0) . Control and deprived groups are repre- sented by vertical and horizontal bars (see Fig. 2). log L Ds/ Liver C. 3 I. 0:. a? -m 103 82 ~. Dep rived ~~ LNC + 105 Thyrh~‘4l Control 104 Number of Cells Figure 4 ~~3~ 105 83 Figure 5. Survival of parasites in peritoneal macr0phages from control, reconstituted, and deprived mice 44 days after infection and harvested 2 days after i.p. injection of 5 x 106 LD bodies. Each point represents the mean number of cells in duplicate cultures from 6 mice. 84 0 Control ‘ o Deprived a 5x104 LNC 6 t 104 LNC+ 5x10 Thy 6 ‘ MacrOphages A 10 [P“\“‘ (‘8 8 3'74 at " a? X ’8‘ * a F I, / g 35 6 ,’ 1k .5 ' o I (I) I, 23 .2 , 2 cu U 4 ’ \ 2 ° 2‘ LBS m .‘\“‘ Q *~\‘\““\ _I 2 ‘\“:\“ \*\“\“““‘* \ \ “ .‘ \. a. 85 Figure 6. Protection conferred by cells from non-immune and immune donors. Each point represents 4 to 6 mice at 50 days post infection. Solid regression lines (calculated by the least squares method) represent standards for protection by gradients of normal thymocytes (0) or LNC (O ) . Relative levels of protection were compared far group-A, 10S spleen cells from 4 wk immune donors ((3); group-B, 105 normal thymocytes mixed with 104 normal LNC (()); group-C, 105 spleen cells from 3 wk immune, "T-boosted" donors (it); and group-D, 105 normal thymocytes mixed with 104 immune LNC from "T-boosted" donors (i&). Control and deprived groups are represented by vertical and horizontal bars (see Fig. 2). 86 50-. a ouswam a... 0 .o .o as. a 2 e2 no. 10A l'l/ 801 so I llHIWNWlllHllWlllUllllllrlllHlllllNIHIIHIHHHI