RESISTANCE AND ABROGATION OE RESISTANCE TO ... - Nature

AJEBAK 59 (Pt. 5) 539-554 (1981)

© RESISTANCE AND ABROGATION OE RESISTANCE TO CUTANEOUS LEISHMANIASIS IN RECONSTITUTED BALB/c NUDE MICE by GRAHAM F. MITCHELL, JOAN M. CURTIS, ROLAND G. SCOLLAY AND EMANUELA HANDMAN (From the Laboratory of Immunoparasitology, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia.) (Accepted for publication May 25. 1981.) Summary. Normal BALB/c mice and hypothymlc BALB/c nude mice are highly susceptible to infection with an isolate of the intramacrophage protozoan parasite, Leishmania tropica. developing severe cutaneous disease. Nude mice reconstituted with large numbers of BALB/c lymphoid cells are also susceptible but are resistant when injected with low numbers of cells (e.g. 5 X lO" spleen plus mesenteric lymph node cells). Susceptibility lo chronic cutaneous disease is thus quile different in minimally reconstituted HALB/c nude mice than In BALB/c mice with a normal or ne«r-normal comptemcnt of T cells. Recovered minimally reconstituted nudes are susceptible lo reinfection but. again. lesions heal. The minimally reconstituted, resistant nude mouse has been used as a screen for cellular and serum components which may contribute to chronicity of cutaneous disease in infected BALB/c mice. Low numbers of T cell-enriched spleen cells from chronically infected BALB/c mice are not protective in nude mice and. in fact, abrogate the protective effect of low numbers of syngeneic normal mouse spleen cells in a mix experiment. Treulment of chronically infected mouse spleen cells with an[i-Ly2 reagents does not affect the capacity of the cells to abrogate resistance. A proportion of minimally reconstituted nude mice were susceptible to cutaneous disease after multiple injections of serum from chronically infected mice. However, this abrogation of resistance differed from that mediated by ceils of chronically infected mice in being inconsistent. The results suggest that T cell-dependent immune responses (but perhaps not Ly2+ TM cell-dependent effects) contribute to chronicity of infection in genetically susceptible BALB/c mice and inhibit healing of lesions in this mouse strain. Although the possibility is raised by the data, no compelling evidence was obtained in [his series of experiments that Tii cell-dependent 'blocking antibodies', or other serum components, are involved in perpetuating chronic disea.se in BALB/c mice. Abbreviations tised in this paper: B cell, bone marrow-derived precursor of antibodysecreting cells: CIM. chronically infected mouse: FCS, foetal c:ilf serum; HEM, Hepesbuffered Eagle's medium: H-2. major hislocompatibility complex (MHC) in the mouse; Lyl, Ly2, locus designalions for Iwo T cell markers; NM. normal mouse: NNN medium, medium of Novy. McNeal and Nicolle (Nicolle. 1908); SEM. standard error of the arithmetic mean; T cell, thymus-derived (-influenced) cell; Tn, helper T cell for antibody production; T«A, postulated macrophage-activating T cell; Ts, suppressor T cell.

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INTRODUCTION BALB/c mice differ from numerous other common inbred strains in being highly susceptible to development of cutaneous lesions and disease caused by the intramacrophage protozoan parasite, Leishmania tropica (Kellina, 1973; other literature cited in Mitchell et ai, 1980; Howard, Hale and Liew, 1981; Perez, Arredondo and Gonzalez, 1978). A cutaneous injection of in vitroderived L. tropica promastigotes (of various isolates) to BALB/c mice results in an expanding superficial lesion, visceralization of infection and death of the host. With the reasonable assumption that infected macrophages are the targets of immune aggression, one proposed mechanism to account for increased susceptibility of BALB/c mice is that L. tropica-Meded macrophages are recognized inefficiently by sensitized Lyl"^ T cells of the short-range, macrophageactivating type (TM.\) in the early stages of infection (Mitchell et ai, 1980). Parasite antigens are present on the surface of infected macrophages (at least in vitro), and the reduced efficiency of recognition by TMA may result from reduced expression of H-2 coded associative (restrictive) recognition molecules on the surface of infected macrophages (Handman, Ceredig and Mitchell. 1979) in the skin. A consequence of T cell recognition of parasite antigens not in association with H-2 molecules on the infected macrophage surface may be the generation of suppressor T cells (Ts). Indeed, Howard, Hale and Liew (1980) have shown that heavily infected BALB/c mice contain T cells in spleen (not yet proven to be Ly2"'' cells) which adoptively suppress delayedtype hypersensitivity to an L. tropica promastigote antigen mixture. The presence of Ts reactive to antigens of infected macrophages may perpetuate a situation of non-resolving infection in chronically infected BALB/c mice perhaps by simply competing with, and thus further reducing the efficiency of recognition by, Lyl"^TMA cells. A surprising observation made during the course of studies on the susceptibility of nude mice of various genotypes was that the majority of minimally reconstituted BALB/c nude mice were resistant to cutaneous disease caused by L. tropica infection. In this respect they differed markedly from either intact BALB/c mice or BALB/c nude mice. In addition, some evidence was obtained that infected macrophages in BALB^^c nude skin, grafted on intact BALB/c mice, were more vulnerable to immune attack (Mitchell et ai, 1980). If this is so, then infected macrophages of BALB/c genotype are not entirely refractory to host anti-parasite effector mechanisms which presumably operate via intracellular parasitostatic or parasitocidal effects subsequent to appropriate T cell-dependent macrophage activation. Supporting evidence for the notion that infected BALB/c macrophages are not refractory to immune attack in vivo has come from studies on sublethally irradiated and T cell-depleted BALB/c mice (Howard et ai, 1980; 1981) and cyclophosphamide-injected mice (Modabber et ai. 1980). Such manipulated BALB/c mice are more resistant than intact mice to disease. No information is currently available on the relative contributions of anti-parasite antibodies, immune complexes, circulating and tissue parasite antigens, TM.V and T.-^ cells in the causa-

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tion of different disease patterns in manipulated versus intact BALB/c mice. No evidence for the existence of host-protective antibodies has been obtained in the L. tropica/mouse system (cf. the L. e/ir/Vfrr/guinea-pig system, Poulter, 1980a), and the possibility that 'blocking antibodies' may perpetuate cutaneous disease has been mentioned (Bryceson et ai. 1972; Garnham and Humphrey, 1969). The availability of cell-injected BALB/c nude mice which are resistant to disease has enabled experiments to be performed on the 'protection-inhibiting' activity of cells and sera from chronically infected BALB/c mice. The data reported in this paper demonstrate that the otherwise resistant minimally reconstituted BALB/c nude provides a convenient readout system for inhibition of protection mediated by spleen cell suspensions and sera from long-term infected, diseased BALB/c mice. MATERIALS AND METHODS Mice Male and female BALB/c and female hypothymic BALB/c.nu/nu (nude) mice were used throughout. Intact mice were 7 to 10 weeks of age at the commencement of experiments, whereas nude mice were 6 to 9 weeks old. All mice were derived from a specific pathogenfree facility at the Hall Institute but. during experimentation, were maintained under conventional mouse house conditions in a small isolation facility. Cells and sera Spleen cell suspensions (or spleen plus mesenteric lymph nodes) were prepared in Eisen's balanced salt solution In the cold by aspirating cut pieces of organs through a I ml disposable syringe and allowing large fragments to settle under unit gravity. Cell suspensions were washed once prior to counting nucleated cells in a haemocyiometer and intraperitoneal injection to nude recipients. In Ihe case of the markedly enlarged spleens from chronically infected mice, a prolonged period of unit gravity sedimentation was employed and the top 5 ml of a 10 ml tube taken for washing and subsequent treatment. For preparation of T ceii-enriched spleen cells from chronically infected mice, approximately lO'* cells per 9 cm petri dish (Costar, Cambridge, Massachusetts. U.S.A.) were incubated for 30 to 45 min at 37° in 10% foetal calf serum (FCS) (Commonwealth Serum Laboratories. Melbourne, Australia) in Hepes-buffered Eagle's medium (HEM), the non-adherent cells removed and approximately lO** in 2 ml warm 10% FCS: HEM loaded on to washed, sterile nylon wool columns (0-6 g per 10 ml disposable plastic syringe barrel) and incubated for 4560 min at 37°. Warm FCS:HEM was added (20 ml) and the wash-through collected dropwise. Celts were injected intraperitoneally to mice immediately after collection and centrifugation or were further treated as follows: cells were Incubated with a monoclonal rat anti-mouse Lyt-2 (Ly2) reagent (Ledbetter and Herzenberg. 1979) followed by affinity-purified rabbit anti-rat immunoglobulin then complement from young rabbits (selected for low mouse T cell cytotoxicity). Treatment with each antibody source was performed at 0° for 30 min and complement treatment was performed at 37° for 20 min. Cells were washed between steps. This treatment, in control experiments, killed 85-90% of thymocytes, approximately 30% of nylon wool purified lymph node cells, and reduced the cytotoxic activity of alloreactive T cells by > 90%. The second stage (i.e. rabbit anti-rat immunoglobulin) killed 30-50% of immunoglobulin positive cells. Unanaesthetized mice were bled from the orbital plexus, or, at kill bleeding, from the axilla of anaesthetized mice, the blood allowed to clot at room temperature and serum removed after overnight storage at 4° and centrifugation (1000 g for 10 min). Sera were

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injected or were frozen at —20" until used (within 6 weeks of collection). For intraperitoneal injection of cells or serum, volumes of 0' 1 to 0 5 ml were given by under-running the dermis on the ventral surface of the taut abdominal wall with a 26G needle prior to deposition intraperitoneally after puncturing the muscle layers. In this way, no backflushing of injected material occurred. Parasites were injected within i-2 h of cell injection on day 0. Parasites and assessment of lesions The derivation and in vitro maintenance in NNN medium of t^. tropica isolate LRC-L137 has been described (Handman et al.. 1979). Washed promastigotes (tO"' to 2 X 10" per mouse) were injected using a 26G needle in a volume of 50 f\ intradermally and subcutaneously on the dorsum of the mouse close to the base of the tail. The injection area in BALB/c mice was plucked free of hair immediately prior to injection of promastigotes. Unless otherwise stated, an injection of 1 X 10" washed in vi'rro-derived promastigotes was roulinely used for challenge. Lesion scores were determined at regular Intervals according to the following scoring system; 0 = no lesion or healed sc;ir; I = small swelling (0 5 = questionable score 1); 2 ^ large swelling or lesion less than 5 mm in average diameter ( 1 5 ^ questionable score 2 and consisted of a relatively large area of minor swelling at the injection site with no lesion apparent): 3 = lesion 5-10 mm in diameter; 4 -- lesion greater than 10 mm in diameter and/or obvious cutaneous metastases. Data are expressed as the arithmetic mean ( ± standard error) of the lesion scores for the groups of 4 to 6 mice, the number of infected mice per group being indicated in some graphs at certain time points.

RESULTS Protective effect of syngeneic normal mouse (NM) lymphoid celts in infected BALB/c nudes BALB/c nude mice, like intact BALB/c mice, are highly susceptible to cutaneous leishmaniasis (Handman et ai. 1979) but most can be protected by injection of 10" to lO'^ NM spleen cells (Mitchell et ai, 1980). A dose response study was performed in nude mice using various numbers of syngeneic spleen plus mesenteric lymph node cells. These mice were injected cutaneously with I X 10" promastigotes of L. tropica. As seen in Fig. 1, injections of 2 or 20 X 10" NM cells were highly effective at mediating resolution of cutaneous disease in nude mice with 0 2 X lO" being marginally less effective. A large number of cells (360 X 10") was not effective at protecting nude mice against cutaneous disease, lesions being controlled initially but increasing rapidly in size later (Fig. 1). This particularly interesting result with low versus high numbers of cells used for reconstitution raises the possibility that a fine balance between T cell-dependent host-protective and T cell-dependent parasite-protective immune responses exists in mice and that the parasiteprotective immune responses may dominate in chronically infected intact BALB/c mice. Spleen cells from chronically infected mice (CIM) inhibit protection mediated by NM spleen cells in BALB/c nudes In the first experiment in a series aimed at detecting inhibitory (suppressor) activities in lymphoid cell populations of CIM, large inocula of cells were used. Approximately lO"" nucleated cells from the grossly enlarged spleen of BALB/c mice infected for 4 months (and with lesion scores of 4) did not

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DAYS AFTER INJECTION OF 10^ PROMASTIGOTES OF L. tropica Fig. 1. Titrytion of cells in nude mice. Cutaneous lesions in BALB/c.nu/nu mice given 1 X 10" promastigotes of L. tropica and no cells (O O)* 0-2 X 10'* spleen plus mesenteric lymph node cells from syngeneic mice ( • • ) , 2 X 10" cells (A A ) , 20 X 10" cells (A A) and 360 X 10" cells (X X ). As in succeeding Figures, ariihmelic mean lesion scores and SEM are indicated as well as deaths ( t ) , and the number of mice with lesions relative to the total number of mice in each group is indicated at certain time points.

protect nude mice. Moreover, this inoculum totally abrogated the protection afforded by 10 X 10" NM spleen cells. The susceptibility of BALB/c nude mice given a mixture of CIM and NM spleen cells was no different from that of BALB/c nude mice given no cells. Thus, suppression of the protective effect of NM spleen cells was clearly demonstrated. However, in this experiment, lesions were noted in all recipients of CIM spleen cells on the abdomen (from

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day 30 or so) presumably at the site of intraperitoneal injection of the CIM cells. Parasites must be contained in these whole spleen cell suspensions. Thus, the suppressive effects of CIM spleen cells on NM spleen cell-mediated protection against persistence of cutaneous lesions may not be due to lymphocyte components of the inoculum but to systemically-iocated parasites, parasitized niacrophages or parasite antigens. Also, an injection of lO'^ + 10" cells is getting close to the 360 X 10^ normal cells used above (Fig. 1) and which were shown to be supra-optimal and unable to induce resistance to cutaneous disease. T cell-enriched CIM spleen cells inhibit protection mediated by NM spleen cells in BALB/c nudes Incubation of CIM spleen cells on plastic dishes and nylon wool was performed in an attempt to reduce cells other than T cells in CIM spleen cell suspensions for injection to minimally reconstituted (NM spleen cell-injected)

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Fig. 2. Abrogation of protection in nude mice. Cutaneous lesions in BALB/c.nu/nu mice (• • ) , BALB/c.nu/nu given 25 X 10" T cell-enriched spleen cells from chronically infected BALB/c mice (X X ) . BALB/c.nu/nu given 10 X 10" normal BALB/c spleen cells (O O) • 3nd BALB/c.nu/nu mice given both types of spleen cells (A A). Promastigoies (1 X 10'') were injected cutaneously at day 0 (the day of cell injection) and, in the recovered nude mice given normal mouse spleen cells (O O ) . a' day ^^ as well.

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DAYS AFTER INJECTION OF 10^ PROMASTIGOTES OF L. tropica Fig. 3. Abrogation of protection in nude mice. Cutaneous lesions in groups of 5 BALB/c.nu/nu mice given 1 X 10" promastigotes and no cells ( • • ) . 2 5 X 10" normal BALB/c spleen cells (O O) or normal cells plus 10 X 10'' T cell-enriched spleen cells from chronically infected BALB/c mice and treated with either complement alone (X X) or anti-Ly2 antibodies plus complement (A A ) . Anti-Ly2 treatmenl resulted in a 30% loss of nucleated cells and each mouse in the group of nudes receiving treated cells was therefore injected with 7 X 10" cells. In a repeat experiment nude mice injected with 2 X 10" normal BALB/c spleen cells had fully recovered by day 58. whereas cutaneous disease was severe and comparable at this time point in recipients of normal ceils plus approximately 5 X 10" T cell-enriched spleen cells from chronically infected mice and treated with anti-Ly2 plus complement or left untreated (mean lesion scores of approximately 2 in both groups).

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syngeneic BALB/c nude mice. The data in Fig. 2 indicate that 25 X 10'' T celJ-enriched CIM spleen cells did not protect nudes, that 10 X IO" NM spleen cells were highly effective at inducing resi.stance to persistent cutaneous disease, and that a mixture of T cell-enriched CTM plus NM spleen cells was not protective. The high susceptibility of recipients of the cell mixture may reflect the operation of Ts cells or Tn cells which facilitate 'blocking antibody' production by host (nude) B cells or contaminant primed donor B cells. It is most unlikely to reflect the presence of infected macrophages and no lesions were seen at the abdominal cell injection site in this experiment. An experiment in which different numbers of CIM spleen cells were injected into minimally reconstituted nude mice demonstrated that 12 X 10" CIM cells were only partially effective, whereas 42 X 10*' CIM cells were highly effective at abrogating protection mediated by 3 X 10" NM spleen cells. In an attempt to determine the partial Ly phenotype of CIM spleen cells responsible for suppression of resistance to disease in minimally reconstituted nude mice, T cell-enriched CIM spleen cells were depleted of Ly2+ cells by treatment with hybridoma-derived anti-Ly2 antibodies and complement. It can be seen in Fig. 3 that depletion of Ly2 • cells had no effect on suppression of resistance to persistent disease; injection of 7 X 10*"' Ly2- T cell-enriched CIM spleen cells abrogated the protective effect of 2-5 X 10" NM spleen cells. In this experiment (and a repeat, see caption to Fig. 3). the number of eells treated with anti-Ly2 reagents and injected to nudes was kept to a minimum. This was done to increase the sensitivity of the readout by decreasing the probability that any residual Ly2^ cells in the treated cell population would eontribute to the outeome. Resistance to reinfection in recovered cell-injected nude mice The recovered positive control group of mice in Fig. 2 (i.e. nude mice given NM spleen cells) was reinjected with 1 X 10" promastigotes at a time when no lesions were apparent (i.e. day 48). Such mice developed definite lesions (to a greater extent than after primary infection) but they recovered soon after (Fig. 2). A repeat experiment in BALB/c nude mice injected with NM spleen cells also demonstrated that recovered mice are susceptible to reinfection when injected at day 55 after primary injection of promastigotes, but that lesions heal. Nude mice injected with NM spleen cells treated with anti-Ly2 antibodies plus complement were injected with promastigotes and the course of primary infection recorded (Fig. 4). Recovery was demonstrated in recipients of Ly2- NM spleen cells as it was in nude reeipients of control treated NM spleen cells. Moreover, reinfection and recovery were identical in the two groups of mice. No increased resistance to reinfection was seen in nude recipients of Ly2- NM spleen cells, suggesting that the susceptibility to reinfection after recovery and transient disease is not influenced by the presence or absence (?high or low numbers) of Ly2+ cells.

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Fig. 4. Resistance to reinfection in nude mice. CiUaneous lesions in groups of 5 BALB/c,nu/ nu mice given no spleen cells (X X) or normal BALB/c spleen cells treated with an[i-Ly2 plus complement ( • • ) or untreated (Q O) and injected on two occasions with 1 X 10" promastigotes of L. tropica on day 0 (the day of cell injection) and day 48. Recipients of antibody-treated cells received 7 X lOi^ cells each and recipients of untreated spleen cells received 18 X 10" cells each.

Effect of CIM serum on NM spleen cell-mediated protection of BALB/c nudes The data in Fig. 5 indicate that an injection of a total of 0-7 ml serum from 13-week CIM given over the first 8 days after injection of cells to recipient nudes partially abrogates the protection mediated by NM spleen cells. CIM serum alone is without protective eflfect and 0 7 ml serum from uninfected agematched normal mice (i.e. out of the specific pathogen-free facility for a comparable length of time as the chronically infected mice) does not influence the protective effect of NM spleen cells in nude recipients. Results of a repeat experiment are shown in Fig. 6 in which serum for injection was obtained from 5-14 week infected BALB/c mice (with lesion scores of 3 or 4 ) , and injection was continued over 10 weeks from the time of cell (and parasite) injection to recipient nudes. Again, only a proportion of recipients of CIM serum showed increased susceptibility compared with recipients of NM serum. In a third experiment CIM serum injected in the first week did not alter the time course of recovery in NM spleen cell-injected nudes. Clearly, more experiments with serum are required in which the number of NM spleen cells is kept either very low or is raised to a number somewhere between 20 X 10" and 320 X 10*' (Fig. 1)—i.e. at a level which is not optimal for promoting resistance in the nude recipients of serum. Currently, the system is not reliable enough to en-

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DAYS AFTER INJECTION OF 10^ PROMASTIGOTES OF L. tropica Fig. 5. Attempted abrogation of protection in nude mice. Cutaneous lesions In groups of 5 BALB/c.nL/nii mice given 1 X IC' promasligotes and no cells ( • • ). 20 X 10" normal BALB/c spleen cells (O O*. cells plus a total of 0 7 ml serum from 13-week infected BALB/c mice (**) or cells plus serum from age-malched normal mice (V-v) in the first 8 days after injection of parasites and cells. Other mice ) were injected with serum from chronically infected mice but nol with normal mouse spleen cells.

courage fractionation of sera on protein A columns or absorption of the effect with parasite antigens. Moreover, since the serum from chronically infected mice will be hypergammagiobulinaemic (Chapman et ai, 1979), any reproducible positive effect which is ultimately obtained will need to be controlled by injection of hypergammagiobulinaemic sera from mice infected with another unrelated parasite.


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0 10 20 30 40 50 60 70 BO 90 100 DAYS AFTER INJECTION OF 10^ PROMASTIGOTES OF L tropica Fig. 6. Attempted abrogation of protection in nude mice. Cutaneous lesions in groups of 5 BALB/c.nu/nu mice given no cells (X X ) . 5 X 10" normal BALB/c spleen (A A ) , cells plus a total of 3 5 ml serum from 5 to 14 week infected BALB/c (• • ) or cells plus serum from age-matched normal mice (Q O ) ' Serum injected on 12 occasions between days 0 and 68.

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DISCUSSION Genetically based variations in the manifestations of cutaneous leishmaniasis in L. tropica-'miccXtd mice (Handman et at., 1979; Behin. Mauel and Sordat, 1979; Preston, Behbehani and Dumonde, 1978; Nasseri and Modabber, 1979) are of particular interest to the protozoan immunoparasitologist. Analyses of such variations are providing new insights into host anti-parasite effector mechani.sms and 'immune evasion' by these intracellular protozoan parasites (Handman