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Passive immunization protects guinea pigs from lethal toxoplasma infection
FEMS MicrobiologyImmunology89 (1992)97-104 © 1992Federation of European MicrobiologicalSocieties0920-8534/92/$05.00 Published by Elsevier
97
FEMSIM 00193
Passive immunization protects guinea pigs from lethal toxoplasma infection Charles S. Pavia 1, Susan J. Bittker 1 and K at hi e E. Curnick 2 1 Department of Medicine, Division of Infectious Diseases and Department of Microbiology and Immunology, New York Medical College, Valhalla, New York, and 2 Trudeau Institute, Saranac Lake, New York, U.S.A.
Received2 October 1991 Accepted 9 October 1991 Key words: Transfer of immunity to lethal toxoplasmosis; anti-Toxoplasma immunity; Toxoplasma gondii; Immunotherapy to toxoplasmosis
1. SUMMARY The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti-Toxoplasma immunoglobulins or immune ceils that had been enriched for B ceils prior to transfer. Cells residing in the spleen,
Correspondence to: C.S. Pavia, Divisionof InfectiousDiseases,
Room 209SE, Macy Pavilion, Westchester Medical Center, Valhalla, NY 10595 U.S.A.
lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 x 10 7 sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cellmediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.
2. INTRODUCTION With the use of appropriate animal models and passive transfer systems, past studies [1-4] have shown that acquired resistance to infection with the RH strain of Toxoplasma gondii depends upon activation of both humoral and cellular components of the host's immune system. In this
98 regard, while there is convincing evidence for the T cell-mediated nature of anti-Toxoplasma immunity [2,3,5,6] there is now mounting evidence that antibodies or other humorat factors [1,2,7] are also protective against toxoplasmosis. However, the manner in which T cells interact with each other as well as with B cells and macrophages in providing protection or in limiting effective immune mechanisms throughout various stages of toxoplasmosis is not fully known. Recent experiments from our laboratory [2,3] have demonstrated the feasibility of using immunocompetent and T cell-deficient inbred guinea pigs for delineating the nature of the immune mechanisms that may be operative during a local, intradermally introduced Toxoplasma infection. As an extension of this experimental system, we report here the results of passive transfer experiments designed to determine the role of humoral and cell-mediated immunity in affording protection against the usually lethal (intraperitoneally induced) infection [2] caused by the RH strain of T. gondii. Our results also demonstrate that whole serum or partially purified immunoglobulins, as well as both T cells and B cells, from guinea pigs resistant to Toxoplasma can confer complete protection against lethal T. gondii infection.
3. MATERIALS AND METHODS
3.1. Animals Strain 2 guinea pigs were bred under barriersustained conditions at the Animal Breeding Facility at the Trudeau Institute from animals originally obtained from the Division of Research Services, National Institutes of Health, Bethesda, MD. Adult (3-20-month-old) animals weighing 400-1100 g and having negative reactions in the indirect hemagglutination (IHA) assay (TPMTest; Wampole Laboratories, Division of CarterWallace, Ind., Cranbury, N J) were used in these experiments. Male and female 12-20-week-old B6D2 and AB6 mice were used for the routine maintenance of T. gondii parasites by serial peritoneal passage [2,3].
3.2. Detection of antibodies Whole blood was collected from guinea pigs before infection and at various intervals postinoculation. Serum was assayed for the presence of anti-Toxoplasma antibodies by using the IHA test performed in accordance with the instructions supplied by the manufacturer.
3.3. Parasites and infections The virulent RH strain of T. gondii was obtained from Dr. Jack Remington (Palo Alto Medical Research Foundation, Palo Alto, CA). In passive transfer experiments (described below), guinea pigs were injected i.p. with 10 4 tachyzoites. The latter parasites were derived from peritoneal exudates of infected passage animals and were prepared as detailed elsewhere [2,3].
3.4. Preparation of lymphocytes Spleens and draining lymph nodes (popliteal, inguinal, and iliac-lumbar) from normal and resistant donors were removed aseptically, and single cell suspensions were prepared in RPMI 1640 medium by teasing apart the excised organs with forceps and gently pressing the tissue through a 60-mesh stainless steel screen. After washing once, harvested cells were resuspended in RPMI 1640. Viable nucleated ceils were enumerated after staining with 0.4% trypan blue. Peritoneal exudate cells (PEC) were obtained by injecting 25 ml of sterile light mineral or paraffin oil (Fisher Scientific, Springfield, N J) into the peritoneal cavity of normal or immune donor guinea pigs. Three to four days later, the guinea pigs were injected with 100 ml of heparinized (20 units/ml) RPMI 1640. After washing twice, the harvested cells were resuspended in 2-3 ml RPMI 1640 and were counted after staining with 0.4% trypan blue. Differential staining of thin smears with Giesma stain and phagocytosis of latex particles revealed that 75-80% of the PEC were macrophages or monocytes, 10-15% were lymphocytes and the remainder were granulocytes (neutrophils, eosinophils and basophils).
3.5. Enrichment for T cells and B cells In some experiments, spleen and lymph node cell suspensions were enriched for T cells by a
99 modification [2,8] of the technique of Mage et al. [9] which depletes B ceils by adhering them to anti-IgG-coated plates. For T cell depletion, cell suspensions were treated with anti-T cell 8BE6 monoclonal antibody [10] plus complement [2] and were prepared for inoculation as described previously [2]. Spleen cell suspensions were depleted of macrophages by allowing them to adhere to plastic dishes (No. 4030; Lab-Tek Division, Miles Laboratories, Elkhart, IN) for 1-2 h at 37°C. The high degree of enrichment of these preparations for either T cells or B cells was verified in cytotoxic, immunofluorescent and functional assays as described recently by us [2,3,8] and as shown in Table 1. In all cell transfer experiments (described below), cell preparations used for adoptive immunization were parasitefree based on subinoculation of aliquots of donor cells into test mice as detailed elsewhere [2].
3.6. Preparation of immune and normal serum Normal strain 2 guinea pigs were infected i.d. with a total of 2 × 106 T. gondii followed by an i.d. challenge infection with 106 tachyzoites 5 weeks later [2]. After another week, guinea pigs were bled out by intracardiac puncture to obtain sera. Titers of anti-T, gondii antibody in pooled immune sera, measured by the I H A test, ranged from 1024-2048. A group of uninfected syngeneic guinea pigs with negative I H A titers were similarly bled to obtain normal serum. The pooled immune serum samples and the pooled normal serum were filter-sterilized (pore size, 0.45 Izm)
and stored at - 7 0 ° C until used. In some experiments, high titer ( > 1024) anti-Toxoplasma immune guinea pig serum and normal guinea pig serum were enriched for immunoglobulin by ammonium sulfate precipitation [11]. After extensive dialysis in phosphate buffered saline, pH 7.2, each crude Ig fraction was sterilized by filtration, analysed for reactivity with rabbit anti-guinea pig IgG (Miles Laboratories) based on immunodiffusion in gels [12], and was tested for antibody activity in the I H A assay. Antibody titers in these crude Ig fractions for the Toxoplasma samples usually increased two- to three-fold following ammonium sulfate treatment, whereas supernatant fractions resulting from ammonium sulfatetreated immune serum were almost totally depleted of antibody (see RESULTS).
3. 7. Passive transfer experiments in guinea pigs Recipient guinea pigs were injected i.v. with 8 - 1 0 ml of immune or normal serum per kilogram of body weight or with 3 - 4 ml of the relevant ammonium sulfate-treated fraction. For cell transfer experiments, age- and sex-matched syngeneic recipients were infused i.v. with 8 × 10 8 unfractionated spleen and lymph node cells, or 2 x 10 8 PEC, or with 4 × 10 8 fractionated (T-enriched or T-depleted) cells suspended in 2 - 3 ml of R P M I 1640; serum, serum fractions, or cells were transferred within 2 h after i.p. challenge with 1 x 10 4 tachyzoites (obtained from freshly harvested, infected peritoneal exudates of syngeneic guinea pigs or AB6 mice).
Table 1 Evidence for enrichment of T cells by the panning technique and of B cells by treatment with 8BE6 plus complement (C) Experiment
Cell group a
Proliferative response to: No mitogen Phytohemagglutinin
1 2 3 4
Unfractionated cells Nonadherent (panned) cells Cells treated with C alone Cells treated with 8BE6+ C
1028 c 757 1231 949
72177 95 545 83 708 4451
Lipopolysaccharide 57221 8937 51684 77660
% Fluorescent positivecells t~ 23-31 4- 9 21-30 92-97
a Cells were derived from the draining lymph nodes of normal or immune guinea pigs. b % of viable cells (range) reacting with a fluorescent anti-guinea pig IgG reagent. c Data are reported as mean counts per minute of tritiated thymidine incorporation of triplicate cultures from six separate experiments. The standard deviations were + 15% or less and are not shown. Culture conditions were identical to those described in references 18 and 19.
100 --A--A--Za O O
6
4. R E S U L T S
•- - O - - O - -
4.1. Passive transfer of anti-T, gondii immunity An earlier study conducted from this laboratory [2] revealed that i.v. infusion of T. gondiisensitized T cells or immune anti-Toxoplasma serum conferred partial protection against local (i.d.) and disseminated T. gondii infection in recipient guinea pigs. It also showed that, in guinea pigs, the R H strain of T. gondii causes a dose-dependent fatal infection when parasites are inoculated i.p. whereas a self-healing infection occurs and long-lasting immunity is generated and sustained when parasites are introduced by the i.d. route. It was, therefore, of interest to determine whether immunity to lethal infection ever becomes expressed in animals surviving an i.d. infection. Three groups of guinea pigs (previously infected) were challenged i.p. with 10 4 T. gondii tachyzoites at 5, 10, or 15 weeks after a primary cutaneous infection with 2 million parasites. Five weeks was the earliest time-point to be tested since prior experiments [2,3] had indicated that by 5 weeks of a primary T. gondii infection, parasites had been cleared from all organ sites and maximum resistance to cutaneous (secondary) challenge occurs in such previously infected guinea pigs. Table 2 shows that all previously infected guinea pigs survived an i.p. challenge with the R H strain of T. gondii, irrespective of the duration of the primary immunizing infection. In sharp contrast, all normal, previously uninfected guinea pigs died within nine days after i.p. parasite challenge. In the next series of experiments, groups of 5 - 6 guinea pigs were infused i.v. with either T.
~--A----.-~
O
O~---O--
\ 0
L
--~ I m m u n e cell recipients --~ I m m u n e T-cell r e c i p i e n t s
O--O
",%_°
L I I 4 8 Days of t o x o p l o s m o
t
-4,- N o r m a l cell recipients
112
//
218
in~ection
Fig. 1. Evidence that guinea pigs receiving unfractionated, immune spleen and lymph node cells (zx), or immune T cells (e) enriched by the panning technique, are fully protected against an i.p. challenge with 1 × 10 4 T. gondii relative to challenged recipients of normal spleen/lymph node cells or normal T cells (©).
gondii-immune lymphocytes, normal lymphocytes, immune serum or normal serum 2 h before i.p. infection with 104 T. gondii tachyzoites. It was found that all recipients of immune material survived T. gondii infection whereas 83-100% of the control guinea pigs had died by day 10 of infection (Figs. 1 and 2). These studies were subsequently repeated using fractionated immune cells. All guinea pigs receiving immune T cells or immune B cells survived lethal parasite challenge but recipients of non-immune cells died shortly after challenge infection (Figs. 1 and 2). Additional experiments revealed that this transfer of protection against lethal i.p. infection can be accomplished using ceils taken from either the spleen, lymph nodes or peritoneal exudate, but not from the thymus, of T. gondii-immune donor animals (Table 3). The minimum cell requirement
Table 2 Temporal development of resistance to lethal Toxoplasmainfection Status of guinea pigs Normal, previouslyuninfected Infected i.d. 5 to 15 weeks earlier (immunized)
Number of survivors at week 1 5a 10 a
15 a
Mean survival time in days_+SD
2/4 b Not done
0/4 4/4
7.8 +_0.96 >_28 c
0/4 4/4
0/4 4/4
a Week of primary i.d. infection with 2 million parasites for the immunized group. b Number of survivors/number challenged with 10 4 T. gondii i.p. c All survivors were healthy at 28 days and selected members of each group were observed for well beyond 2 months after challenge.
101 Table 3
Table 4
Ability of lymphocytes from the spleen, lymph nodes, peritoneal cavity or thymus to transfer anti-Toxoplasma immunity
Titration of transferred protective spleen and lymph node cells in challenged recipients
Status of donors Normal, uninfected Previously infected b (immunized) a
Source of guinea pig lymphoid cells Spleen
Lymph Peritoneal nodes cavity
Thymus
Number of transferred immune cells ~
0/4 a
0/4
0/4
0/4
2x108 1 x 108 5 X 107 2 X 107
4/4
4/4
4/4
0/4
Number of survivors/number challenged i.p. with 104 T,
gondii; all non-survivors died between 7 to 9 days after challenge. b Guinea pigs from this group had undergone a primary i.d. infection of at least 5 weeks duration and each of the various cell preparations from these donors were found to be parasite-free based on subinoculation into test mice as previously described [2].
for the t r a n s f e r of p r o t e c t i o n against lethal toxoplasmosis is illustrated by the results of a subseq u e n t e x p e r i m e n t that tested the effect, o n adoptive i m m u n i z a t i o n , of infusing g r a d e d n u m b e r s of sensitized cells. T a b l e 4 shows that the i n f u s i o n of at least 2 x 10 7 sensitized T cells o r B ceils c o n f e r r e d p r o t e c t i o n in T. gondii-challenged recipients. It was also f o u n d that g u i n e a pigs given B ceils a l o n e or B + T ceils, but n o t T cells alone, p r o d u c e d relatively high levels of a n t i b o d y ( T a b l e 4).
1 X 10 7
0b
Cell type B cell
T cell
B + T cell
3/3c (256) 3/3 (256) 3/3 (128) 2/3 (128) 0/3 ( < 64) 0/3 (-)
3/3 3/3 3/3 3/3 0/3 0/3
3/3 3/3 3/3 3/3 0/3 0/3
( < 64) ( < 64) ( < 64) ( < 64) ( < 64) (-)
(512) (256) (256) (256) ( < 64) (-)
Number of B or T cells transferred to recipients challenged i.p. with 104 T. gondii. This group received 2 x 10s normal cells instead of immune cells, prior to parasite challenge. Number of survivors remaining at 28 days after challenge infection/total number infected guinea pigs. Values in parentheses are mean IHA titers of recipients on day 7 of challenge; a minimum titer of 64 is considered the lowest positive value in the IHA test.
4.2. Antibody-mediated transfer of anti-Toxoplasma immunity Since c o m p l e t e p r o t e c t i o n against toxoplasmosis was achieved by a d m i n i s t r a t i o n of i m m u n e g u i n e a pig serum, passive t r a n s f e r e x p e r i m e n t s were d e s i g n e d in o r d e r to d e t e r m i n e w h e t h e r a c o m p a r a b l e degree of p r o t e c t i o n could be conferred to recipient g u i n e a pigs i n f u s e d with fractions of i m m u n e s e r u m e n r i c h e d for i m m u n o g l o b ulins or with i m m u n e s e r u m lacking high levels of antibodies. A f t e r i.p. challenge with 10 4 T. gondii,
Table 5 Immune properties and activities of various fractions of anti-Toxoplasma guinea pig serum Experimental groups
No. of survivors a No. infected
Mean IHA titer of donor fraction
1. Guinea pigs given normal Ig fraction after ammonium sulfate precipitation
0/6
negative
2. Guinea pigs given anti-T, gondii Ig fraction after ammonium sulfate precipitation
6/6 b
4096
3. Guinea pigs given anti-T, gondii non-Ig supernatant after ammonium sulfate precipitation
0/6
_<64
a All guinea pigs were infected i.p. with 104 T. gondii; all non-survivors died between 7 to 9 days after challenge. b All survivorswere healthy at 28 days and selected members of this group were observed for well beyond 2 months after challenge.
102 ! 6 L - - A - -o~-----~- -o- ~ - - A _
oL
--~
--~ immune serum recip,ents
\o
~ IN. . . . I/serumo~cel~ recipients 4 8 12 28 Days of toxoplasma infection
Fig. 2. Evidence that guinea pigs receiving immune serum (zx), immune B cells (o) enriched by treatment with 8BE6 anti-T cell monoclonal antibody plus complement, are fully protected against an i.p. challenge with 1 x 104 T. gondii when compared to recipients of normal serum or cells (o).
6 of 6 guinea pigs receiving crude Ig fractions of immune serum (mean I l i a titer of greater than or equal to 4096) survived during a 28-day observation period (Table 5) while all challenged guinea pigs receiving normal immunoglobulins, or the non-Ig-containing, ammonium sulfate fraction of immune serum, rapidly succumbed to Toxoplasma infection.
5. DISCUSSION The results of this study show that long-lasting immunity to lethal toxoplasmosis can be induced in guinea pigs by a prior sub-lethal, self-healing infection with homologous parasites. This finding, together with the evidence that the onset of acquired resistance was associated with generation in the peripheral lymphoid organs of T cells and B cells that were capable of adoptively immunizing normal guinea pigs against lethal T. gondii infection, correlates well with the hypothesis that cell-mediated immunity is a crucial host-defense mechanism against toxoplasmosis [5,6]. While the involvement of T cell-mediated effector mechanisms in immunity to this disease has been reasonably well established [2,4], there is less con-
vincing evidence for ascribing an important role for B cells or their products (antibodies) in affording protection against T. gondii infection. In this regard, earlier studies [13,16] showed that passive immunization with homologous or heterologous serum containing polyclonal antiToxoplasma antibodies conferred little or no protection to recipient mice against virulent parasite challenge, nor does passive transfer of antibody to infected T cell-deficient mice promote their survival [16,17]. More recently, however, monoclonal antibodies to T. gondii have been shown to impart significant protection [1,7] in mice challenged with moderately virulent or highly virulent parasite strains. Consistent with these latter findings, the experiments presented here are supportive of an important role for antibodies by the demonstration that fractions of immune serum enriched for guinea pig immunoglobulins are fully protective against lethal toxoplasmosis. It is noteworthy, however, that, unlike the guinea pig, we have found (Pavia et al., unpublished observation) that mice could not be successfully immunized against the lethal RH strain of T. gondii nor could protection to toxoplasmosis be transferred to syngeneic recipients using spleen cells or serum derived from previously immunized donors. Such divergent results suggest that the choice of a suitable host species as well as selecting a particular parasite strain may be critical factors when performing passive protection studies and for drawing conclusions about the hostparasite relationship associated with toxoplasmosis. Interestingly, in this regard, the only other reported successful adoptive cell transfer systems for analyzing Toxoplasma immunity were found to occur in hamsters [18] or in rats [4]. An earlier study from this laboratory [2] had shown that immune guinea pig serum was partially protective against disseminated, non-lethal toxoplasmosis, yet B cells were not. Although this latter finding would seem to contradict the results reported here, showing that guinea pigs receiving immune B cells alone are completely resistant to lethal infection, the apparent discrepancies are probably related to differences associated with the route and dose of parasite inoculation (i.d. with 10 6 parasites vs. i.p. with 10 4 parasites), and
103
the parameters used to measure resistance (parasite dissemination: tissue immunity vs. survival:generalized immunity). It is also possible that immune donor guinea pigs generate sufficient numbers of B cells to promote host survival against lethal i.p. infection but not enough to prevent a significant reduction in parasite dissemination or growth resulting from a cutaneous infection. Another consideration is that B cells, under different experimental conditions, may express suppressor activity as they do in certain mouse strains infected with the Me49 strain of T. gondii [19]. The importance, however, of a specific T. gondii-induced suppressor mechanism in naturally occurring human infections is unclear and would seem to have little or no relevance under these circumstances when compared to other parasitic diseases [20]. This notion is based on the compelling fact that the predominant number of Toxoplasma infections are asymptomatic and that life-threatening disease usually arises following reactivation of encysted latent forms of the parasite in patients immunocompromised from transplant- or cancer-related chemotherapy [21] or after being stricken with the acquired immune deficiency syndrome [23] or following maternal transmission of parasites to the immunologically immature fetus during pregnancy [23]. In conclusion, the demonstration that passive transfer of protection to lethal toxoplasmosis can be accomplished with guinea pig T cells confirms their participation in the expression of antiToxoplasma immunity deduced from earlier studies in T cell-deficient [3,4,16] and antibody-depleted (/z-suppressed) animals [24], and in certain immunosuppressed patients [21,22]. Such findings have now been extended to include B cells as a relevant host defense mechanism against toxoplasmosis. Together with the evidence presented here that immune guinea pig serum and antibodies are equally protective, it is suggested that T lymphocytes function in this model as helper cells by interacting with B cells in the formation of specific anti-T, gondii antibodies and as effector cells or by activating macrophages for enhanced phagocytosis [6,25] and destruction of the parasite.
ACKNOWLEDGEMENT This work was supported in part by a grant (to C.S.P.) from the J.M. Foundation, New York, NY.
REFERENCES [ll Sharma, S.D., Araujo, F.G. and Remington, J.S. (1984) Toxoplasma antigen isolated by affinity chromatography with monoclonal antibody protects mice against lethal infection with Toxoplasma gondii. J. Immunol. 133, 28182820. [2] Pavia, C.S. (1986) Protection against experimental toxoplasmosis by adoptive immunotherapy. J. Immunol. 137, 2985-2990. [3] Pavia, C.S. (1987) Thymocyte-dependent immunity to toxoplasmosis in the normal and immunocompromised guinea pig host. Parasite Immunol. 9, 205-218. [4] Duquesne, V., Auriault, C., Darcy, F., Decavel, J.P. and Capron, A. (1990) Protection of nude rats against Toxoplasma infection by excreted-secreted antigen-specific helper T cells. Infect. Immun. 58, 2120-2126. [5] Frenkel, J.K. (1982) Experimental analysis of tissue immunity in toxoplasmosis. Lyon Med. 248 (Suppl. 17), 67-73. [6] Remington, J.S. and Krahenbuhl, J.L. (1982) Immunology of Toxoplasma gondii. In: Immunology of human infection, part I1. Viruses and parasites, (Nahmias, A.J. and Reilly, R.J., Eds), pp. 327-371. Plenum Publishing Co., New York. [7] Johnson, A.M., McDonald, P.J. and Neoh, S.H. (1983) Monoclonal antibodies to Toxoplasma cell membrane surface antigens protect mice from toxoplasmosis. J. Protozool. 30, 351-356. [8] Pavia, C.S. and Niederbuhl, C.J. (1985) Adoptive transfer of anti-syphilis immunity with lymphocytes from Treponema pallidum-infected guinea pigs. J. Immunol. 135, 2829-2834. [9] Mage, M.F., McHugh, L.L. and Rothstein, T.L (1977) Mouse lymphocytes with and without surface immunoglobulin: preparative scale separation in polystyrene tissue culture dishes coated with specifically purified anti-immunolobulin. J. Immunol. Methods 15, 47-56. [10] Chiba, J., Chused, T.M., Leiserson, W.M., Zweig, S.E. and Shevach, E.M. (1983) Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens. J. Immunol. Methods 63, 246261. [11] Campbell, D.H., Garvey, J.S., Cremer, N.E. and Sussdorf, D.H. (1970) Methods in Immunology, pp. 189-191. W.A. Benjamin Inc., New York. [12] Pavia, C.S., Niederbuhl, C.J. and Saunders, J. (1985) Antibody-mediated protection of guinea pigs against in-
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[13]
[14]
[15] [16] [17]
[18] [19]
fection with Treponema pallidum. Immunology 56, 195202. Foster, B.G. and McCulloch, W.F. (1968) Studies of active and passive immunity in animals inoculated with Toxoplasma gondii. Can. J. Microbiol. 14, 103-110. Krahenbuhl, J.L., Ruskin, J. and Remington, J.S. (1972) The use of killed vaccines in immunization against an intracellular parasite: Toxoplasma gondii. J. Immunol. 108, 425-431. Nakayama, I. (1965) Effects of immunization procedures in experimental toxoplasmosis. Keio. J. Med. 14, 63-72. Lindberg, R.E. and Frenkel, J.K. (1977) Toxoplasmosis in nude mice. J. Parasitol. 63, 219-221. Strannegard, O. and Lycke, E. (1972) Effect of antithymocyte serum on experimental toxoplasmosis in mice. Infect. Immun. 5, 769-774. Frenkel, J.K. (1967) Adoptive immunity to intracellular infection. J. Immunol. 98, 1309-1319. Brinkman, V., Sharma, S.D. and Remington, J.S. (1986) Differential regulation of the L3T4-T cell subset by B
[20] [21] [22]
[23]
[24]
[25]
cells in different mouse strains bearing the H-2 k haplotype. J. Immunol. 137, 2991-2997. Bloom, B.R. (1979) Games parasites play: how parasites evade immune surveillance. Nature 279, 21-26. Ruskin, J. and Remington, J.S. (1976) Toxoplasmosis in the compromised host. Ann. Int. Med. 84, 193-199. Luft, B.J., Brooks, R.G., Conley, F.K., McCabe, R.E. and Remington, J.S. (1984) Toxoplasmic encephalitis in patients with acquired immune deficiency syndrome. J. Am. Med. Assoc. 252, 913-917. Remington, J.S. and Desmonts, G. (1990) Toxoplasmosis. In: Infectious diseases of the fetus and newborn infant. (Remington, J.S. and Klein, J.O., Eds.), pp. 89-195. W.B. Saunders Co., Philadelphia. Frenkel, J.K. and Taylor, D.W. (1982) Toxoplasmosis in immunoglobulin M-suppressed mice. Infect. Immun. 38, 360-367. Anderson, S.E. and Remington, J.S. (1974) Effect of normal and activated human macrophages on Toxoplasma gondii, J. Exp. Med. 1349, 1154-1174.
97
FEMSIM 00193
Passive immunization protects guinea pigs from lethal toxoplasma infection Charles S. Pavia 1, Susan J. Bittker 1 and K at hi e E. Curnick 2 1 Department of Medicine, Division of Infectious Diseases and Department of Microbiology and Immunology, New York Medical College, Valhalla, New York, and 2 Trudeau Institute, Saranac Lake, New York, U.S.A.
Received2 October 1991 Accepted 9 October 1991 Key words: Transfer of immunity to lethal toxoplasmosis; anti-Toxoplasma immunity; Toxoplasma gondii; Immunotherapy to toxoplasmosis
1. SUMMARY The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti-Toxoplasma immunoglobulins or immune ceils that had been enriched for B ceils prior to transfer. Cells residing in the spleen,
Correspondence to: C.S. Pavia, Divisionof InfectiousDiseases,
Room 209SE, Macy Pavilion, Westchester Medical Center, Valhalla, NY 10595 U.S.A.
lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 x 10 7 sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cellmediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.
2. INTRODUCTION With the use of appropriate animal models and passive transfer systems, past studies [1-4] have shown that acquired resistance to infection with the RH strain of Toxoplasma gondii depends upon activation of both humoral and cellular components of the host's immune system. In this
98 regard, while there is convincing evidence for the T cell-mediated nature of anti-Toxoplasma immunity [2,3,5,6] there is now mounting evidence that antibodies or other humorat factors [1,2,7] are also protective against toxoplasmosis. However, the manner in which T cells interact with each other as well as with B cells and macrophages in providing protection or in limiting effective immune mechanisms throughout various stages of toxoplasmosis is not fully known. Recent experiments from our laboratory [2,3] have demonstrated the feasibility of using immunocompetent and T cell-deficient inbred guinea pigs for delineating the nature of the immune mechanisms that may be operative during a local, intradermally introduced Toxoplasma infection. As an extension of this experimental system, we report here the results of passive transfer experiments designed to determine the role of humoral and cell-mediated immunity in affording protection against the usually lethal (intraperitoneally induced) infection [2] caused by the RH strain of T. gondii. Our results also demonstrate that whole serum or partially purified immunoglobulins, as well as both T cells and B cells, from guinea pigs resistant to Toxoplasma can confer complete protection against lethal T. gondii infection.
3. MATERIALS AND METHODS
3.1. Animals Strain 2 guinea pigs were bred under barriersustained conditions at the Animal Breeding Facility at the Trudeau Institute from animals originally obtained from the Division of Research Services, National Institutes of Health, Bethesda, MD. Adult (3-20-month-old) animals weighing 400-1100 g and having negative reactions in the indirect hemagglutination (IHA) assay (TPMTest; Wampole Laboratories, Division of CarterWallace, Ind., Cranbury, N J) were used in these experiments. Male and female 12-20-week-old B6D2 and AB6 mice were used for the routine maintenance of T. gondii parasites by serial peritoneal passage [2,3].
3.2. Detection of antibodies Whole blood was collected from guinea pigs before infection and at various intervals postinoculation. Serum was assayed for the presence of anti-Toxoplasma antibodies by using the IHA test performed in accordance with the instructions supplied by the manufacturer.
3.3. Parasites and infections The virulent RH strain of T. gondii was obtained from Dr. Jack Remington (Palo Alto Medical Research Foundation, Palo Alto, CA). In passive transfer experiments (described below), guinea pigs were injected i.p. with 10 4 tachyzoites. The latter parasites were derived from peritoneal exudates of infected passage animals and were prepared as detailed elsewhere [2,3].
3.4. Preparation of lymphocytes Spleens and draining lymph nodes (popliteal, inguinal, and iliac-lumbar) from normal and resistant donors were removed aseptically, and single cell suspensions were prepared in RPMI 1640 medium by teasing apart the excised organs with forceps and gently pressing the tissue through a 60-mesh stainless steel screen. After washing once, harvested cells were resuspended in RPMI 1640. Viable nucleated ceils were enumerated after staining with 0.4% trypan blue. Peritoneal exudate cells (PEC) were obtained by injecting 25 ml of sterile light mineral or paraffin oil (Fisher Scientific, Springfield, N J) into the peritoneal cavity of normal or immune donor guinea pigs. Three to four days later, the guinea pigs were injected with 100 ml of heparinized (20 units/ml) RPMI 1640. After washing twice, the harvested cells were resuspended in 2-3 ml RPMI 1640 and were counted after staining with 0.4% trypan blue. Differential staining of thin smears with Giesma stain and phagocytosis of latex particles revealed that 75-80% of the PEC were macrophages or monocytes, 10-15% were lymphocytes and the remainder were granulocytes (neutrophils, eosinophils and basophils).
3.5. Enrichment for T cells and B cells In some experiments, spleen and lymph node cell suspensions were enriched for T cells by a
99 modification [2,8] of the technique of Mage et al. [9] which depletes B ceils by adhering them to anti-IgG-coated plates. For T cell depletion, cell suspensions were treated with anti-T cell 8BE6 monoclonal antibody [10] plus complement [2] and were prepared for inoculation as described previously [2]. Spleen cell suspensions were depleted of macrophages by allowing them to adhere to plastic dishes (No. 4030; Lab-Tek Division, Miles Laboratories, Elkhart, IN) for 1-2 h at 37°C. The high degree of enrichment of these preparations for either T cells or B cells was verified in cytotoxic, immunofluorescent and functional assays as described recently by us [2,3,8] and as shown in Table 1. In all cell transfer experiments (described below), cell preparations used for adoptive immunization were parasitefree based on subinoculation of aliquots of donor cells into test mice as detailed elsewhere [2].
3.6. Preparation of immune and normal serum Normal strain 2 guinea pigs were infected i.d. with a total of 2 × 106 T. gondii followed by an i.d. challenge infection with 106 tachyzoites 5 weeks later [2]. After another week, guinea pigs were bled out by intracardiac puncture to obtain sera. Titers of anti-T, gondii antibody in pooled immune sera, measured by the I H A test, ranged from 1024-2048. A group of uninfected syngeneic guinea pigs with negative I H A titers were similarly bled to obtain normal serum. The pooled immune serum samples and the pooled normal serum were filter-sterilized (pore size, 0.45 Izm)
and stored at - 7 0 ° C until used. In some experiments, high titer ( > 1024) anti-Toxoplasma immune guinea pig serum and normal guinea pig serum were enriched for immunoglobulin by ammonium sulfate precipitation [11]. After extensive dialysis in phosphate buffered saline, pH 7.2, each crude Ig fraction was sterilized by filtration, analysed for reactivity with rabbit anti-guinea pig IgG (Miles Laboratories) based on immunodiffusion in gels [12], and was tested for antibody activity in the I H A assay. Antibody titers in these crude Ig fractions for the Toxoplasma samples usually increased two- to three-fold following ammonium sulfate treatment, whereas supernatant fractions resulting from ammonium sulfatetreated immune serum were almost totally depleted of antibody (see RESULTS).
3. 7. Passive transfer experiments in guinea pigs Recipient guinea pigs were injected i.v. with 8 - 1 0 ml of immune or normal serum per kilogram of body weight or with 3 - 4 ml of the relevant ammonium sulfate-treated fraction. For cell transfer experiments, age- and sex-matched syngeneic recipients were infused i.v. with 8 × 10 8 unfractionated spleen and lymph node cells, or 2 x 10 8 PEC, or with 4 × 10 8 fractionated (T-enriched or T-depleted) cells suspended in 2 - 3 ml of R P M I 1640; serum, serum fractions, or cells were transferred within 2 h after i.p. challenge with 1 x 10 4 tachyzoites (obtained from freshly harvested, infected peritoneal exudates of syngeneic guinea pigs or AB6 mice).
Table 1 Evidence for enrichment of T cells by the panning technique and of B cells by treatment with 8BE6 plus complement (C) Experiment
Cell group a
Proliferative response to: No mitogen Phytohemagglutinin
1 2 3 4
Unfractionated cells Nonadherent (panned) cells Cells treated with C alone Cells treated with 8BE6+ C
1028 c 757 1231 949
72177 95 545 83 708 4451
Lipopolysaccharide 57221 8937 51684 77660
% Fluorescent positivecells t~ 23-31 4- 9 21-30 92-97
a Cells were derived from the draining lymph nodes of normal or immune guinea pigs. b % of viable cells (range) reacting with a fluorescent anti-guinea pig IgG reagent. c Data are reported as mean counts per minute of tritiated thymidine incorporation of triplicate cultures from six separate experiments. The standard deviations were + 15% or less and are not shown. Culture conditions were identical to those described in references 18 and 19.
100 --A--A--Za O O
6
4. R E S U L T S
•- - O - - O - -
4.1. Passive transfer of anti-T, gondii immunity An earlier study conducted from this laboratory [2] revealed that i.v. infusion of T. gondiisensitized T cells or immune anti-Toxoplasma serum conferred partial protection against local (i.d.) and disseminated T. gondii infection in recipient guinea pigs. It also showed that, in guinea pigs, the R H strain of T. gondii causes a dose-dependent fatal infection when parasites are inoculated i.p. whereas a self-healing infection occurs and long-lasting immunity is generated and sustained when parasites are introduced by the i.d. route. It was, therefore, of interest to determine whether immunity to lethal infection ever becomes expressed in animals surviving an i.d. infection. Three groups of guinea pigs (previously infected) were challenged i.p. with 10 4 T. gondii tachyzoites at 5, 10, or 15 weeks after a primary cutaneous infection with 2 million parasites. Five weeks was the earliest time-point to be tested since prior experiments [2,3] had indicated that by 5 weeks of a primary T. gondii infection, parasites had been cleared from all organ sites and maximum resistance to cutaneous (secondary) challenge occurs in such previously infected guinea pigs. Table 2 shows that all previously infected guinea pigs survived an i.p. challenge with the R H strain of T. gondii, irrespective of the duration of the primary immunizing infection. In sharp contrast, all normal, previously uninfected guinea pigs died within nine days after i.p. parasite challenge. In the next series of experiments, groups of 5 - 6 guinea pigs were infused i.v. with either T.
~--A----.-~
O
O~---O--
\ 0
L
--~ I m m u n e cell recipients --~ I m m u n e T-cell r e c i p i e n t s
O--O
",%_°
L I I 4 8 Days of t o x o p l o s m o
t
-4,- N o r m a l cell recipients
112
//
218
in~ection
Fig. 1. Evidence that guinea pigs receiving unfractionated, immune spleen and lymph node cells (zx), or immune T cells (e) enriched by the panning technique, are fully protected against an i.p. challenge with 1 × 10 4 T. gondii relative to challenged recipients of normal spleen/lymph node cells or normal T cells (©).
gondii-immune lymphocytes, normal lymphocytes, immune serum or normal serum 2 h before i.p. infection with 104 T. gondii tachyzoites. It was found that all recipients of immune material survived T. gondii infection whereas 83-100% of the control guinea pigs had died by day 10 of infection (Figs. 1 and 2). These studies were subsequently repeated using fractionated immune cells. All guinea pigs receiving immune T cells or immune B cells survived lethal parasite challenge but recipients of non-immune cells died shortly after challenge infection (Figs. 1 and 2). Additional experiments revealed that this transfer of protection against lethal i.p. infection can be accomplished using ceils taken from either the spleen, lymph nodes or peritoneal exudate, but not from the thymus, of T. gondii-immune donor animals (Table 3). The minimum cell requirement
Table 2 Temporal development of resistance to lethal Toxoplasmainfection Status of guinea pigs Normal, previouslyuninfected Infected i.d. 5 to 15 weeks earlier (immunized)
Number of survivors at week 1 5a 10 a
15 a
Mean survival time in days_+SD
2/4 b Not done
0/4 4/4
7.8 +_0.96 >_28 c
0/4 4/4
0/4 4/4
a Week of primary i.d. infection with 2 million parasites for the immunized group. b Number of survivors/number challenged with 10 4 T. gondii i.p. c All survivors were healthy at 28 days and selected members of each group were observed for well beyond 2 months after challenge.
101 Table 3
Table 4
Ability of lymphocytes from the spleen, lymph nodes, peritoneal cavity or thymus to transfer anti-Toxoplasma immunity
Titration of transferred protective spleen and lymph node cells in challenged recipients
Status of donors Normal, uninfected Previously infected b (immunized) a
Source of guinea pig lymphoid cells Spleen
Lymph Peritoneal nodes cavity
Thymus
Number of transferred immune cells ~
0/4 a
0/4
0/4
0/4
2x108 1 x 108 5 X 107 2 X 107
4/4
4/4
4/4
0/4
Number of survivors/number challenged i.p. with 104 T,
gondii; all non-survivors died between 7 to 9 days after challenge. b Guinea pigs from this group had undergone a primary i.d. infection of at least 5 weeks duration and each of the various cell preparations from these donors were found to be parasite-free based on subinoculation into test mice as previously described [2].
for the t r a n s f e r of p r o t e c t i o n against lethal toxoplasmosis is illustrated by the results of a subseq u e n t e x p e r i m e n t that tested the effect, o n adoptive i m m u n i z a t i o n , of infusing g r a d e d n u m b e r s of sensitized cells. T a b l e 4 shows that the i n f u s i o n of at least 2 x 10 7 sensitized T cells o r B ceils c o n f e r r e d p r o t e c t i o n in T. gondii-challenged recipients. It was also f o u n d that g u i n e a pigs given B ceils a l o n e or B + T ceils, but n o t T cells alone, p r o d u c e d relatively high levels of a n t i b o d y ( T a b l e 4).
1 X 10 7
0b
Cell type B cell
T cell
B + T cell
3/3c (256) 3/3 (256) 3/3 (128) 2/3 (128) 0/3 ( < 64) 0/3 (-)
3/3 3/3 3/3 3/3 0/3 0/3
3/3 3/3 3/3 3/3 0/3 0/3
( < 64) ( < 64) ( < 64) ( < 64) ( < 64) (-)
(512) (256) (256) (256) ( < 64) (-)
Number of B or T cells transferred to recipients challenged i.p. with 104 T. gondii. This group received 2 x 10s normal cells instead of immune cells, prior to parasite challenge. Number of survivors remaining at 28 days after challenge infection/total number infected guinea pigs. Values in parentheses are mean IHA titers of recipients on day 7 of challenge; a minimum titer of 64 is considered the lowest positive value in the IHA test.
4.2. Antibody-mediated transfer of anti-Toxoplasma immunity Since c o m p l e t e p r o t e c t i o n against toxoplasmosis was achieved by a d m i n i s t r a t i o n of i m m u n e g u i n e a pig serum, passive t r a n s f e r e x p e r i m e n t s were d e s i g n e d in o r d e r to d e t e r m i n e w h e t h e r a c o m p a r a b l e degree of p r o t e c t i o n could be conferred to recipient g u i n e a pigs i n f u s e d with fractions of i m m u n e s e r u m e n r i c h e d for i m m u n o g l o b ulins or with i m m u n e s e r u m lacking high levels of antibodies. A f t e r i.p. challenge with 10 4 T. gondii,
Table 5 Immune properties and activities of various fractions of anti-Toxoplasma guinea pig serum Experimental groups
No. of survivors a No. infected
Mean IHA titer of donor fraction
1. Guinea pigs given normal Ig fraction after ammonium sulfate precipitation
0/6
negative
2. Guinea pigs given anti-T, gondii Ig fraction after ammonium sulfate precipitation
6/6 b
4096
3. Guinea pigs given anti-T, gondii non-Ig supernatant after ammonium sulfate precipitation
0/6
_<64
a All guinea pigs were infected i.p. with 104 T. gondii; all non-survivors died between 7 to 9 days after challenge. b All survivorswere healthy at 28 days and selected members of this group were observed for well beyond 2 months after challenge.
102 ! 6 L - - A - -o~-----~- -o- ~ - - A _
oL
--~
--~ immune serum recip,ents
\o
~ IN. . . . I/serumo~cel~ recipients 4 8 12 28 Days of toxoplasma infection
Fig. 2. Evidence that guinea pigs receiving immune serum (zx), immune B cells (o) enriched by treatment with 8BE6 anti-T cell monoclonal antibody plus complement, are fully protected against an i.p. challenge with 1 x 104 T. gondii when compared to recipients of normal serum or cells (o).
6 of 6 guinea pigs receiving crude Ig fractions of immune serum (mean I l i a titer of greater than or equal to 4096) survived during a 28-day observation period (Table 5) while all challenged guinea pigs receiving normal immunoglobulins, or the non-Ig-containing, ammonium sulfate fraction of immune serum, rapidly succumbed to Toxoplasma infection.
5. DISCUSSION The results of this study show that long-lasting immunity to lethal toxoplasmosis can be induced in guinea pigs by a prior sub-lethal, self-healing infection with homologous parasites. This finding, together with the evidence that the onset of acquired resistance was associated with generation in the peripheral lymphoid organs of T cells and B cells that were capable of adoptively immunizing normal guinea pigs against lethal T. gondii infection, correlates well with the hypothesis that cell-mediated immunity is a crucial host-defense mechanism against toxoplasmosis [5,6]. While the involvement of T cell-mediated effector mechanisms in immunity to this disease has been reasonably well established [2,4], there is less con-
vincing evidence for ascribing an important role for B cells or their products (antibodies) in affording protection against T. gondii infection. In this regard, earlier studies [13,16] showed that passive immunization with homologous or heterologous serum containing polyclonal antiToxoplasma antibodies conferred little or no protection to recipient mice against virulent parasite challenge, nor does passive transfer of antibody to infected T cell-deficient mice promote their survival [16,17]. More recently, however, monoclonal antibodies to T. gondii have been shown to impart significant protection [1,7] in mice challenged with moderately virulent or highly virulent parasite strains. Consistent with these latter findings, the experiments presented here are supportive of an important role for antibodies by the demonstration that fractions of immune serum enriched for guinea pig immunoglobulins are fully protective against lethal toxoplasmosis. It is noteworthy, however, that, unlike the guinea pig, we have found (Pavia et al., unpublished observation) that mice could not be successfully immunized against the lethal RH strain of T. gondii nor could protection to toxoplasmosis be transferred to syngeneic recipients using spleen cells or serum derived from previously immunized donors. Such divergent results suggest that the choice of a suitable host species as well as selecting a particular parasite strain may be critical factors when performing passive protection studies and for drawing conclusions about the hostparasite relationship associated with toxoplasmosis. Interestingly, in this regard, the only other reported successful adoptive cell transfer systems for analyzing Toxoplasma immunity were found to occur in hamsters [18] or in rats [4]. An earlier study from this laboratory [2] had shown that immune guinea pig serum was partially protective against disseminated, non-lethal toxoplasmosis, yet B cells were not. Although this latter finding would seem to contradict the results reported here, showing that guinea pigs receiving immune B cells alone are completely resistant to lethal infection, the apparent discrepancies are probably related to differences associated with the route and dose of parasite inoculation (i.d. with 10 6 parasites vs. i.p. with 10 4 parasites), and
103
the parameters used to measure resistance (parasite dissemination: tissue immunity vs. survival:generalized immunity). It is also possible that immune donor guinea pigs generate sufficient numbers of B cells to promote host survival against lethal i.p. infection but not enough to prevent a significant reduction in parasite dissemination or growth resulting from a cutaneous infection. Another consideration is that B cells, under different experimental conditions, may express suppressor activity as they do in certain mouse strains infected with the Me49 strain of T. gondii [19]. The importance, however, of a specific T. gondii-induced suppressor mechanism in naturally occurring human infections is unclear and would seem to have little or no relevance under these circumstances when compared to other parasitic diseases [20]. This notion is based on the compelling fact that the predominant number of Toxoplasma infections are asymptomatic and that life-threatening disease usually arises following reactivation of encysted latent forms of the parasite in patients immunocompromised from transplant- or cancer-related chemotherapy [21] or after being stricken with the acquired immune deficiency syndrome [23] or following maternal transmission of parasites to the immunologically immature fetus during pregnancy [23]. In conclusion, the demonstration that passive transfer of protection to lethal toxoplasmosis can be accomplished with guinea pig T cells confirms their participation in the expression of antiToxoplasma immunity deduced from earlier studies in T cell-deficient [3,4,16] and antibody-depleted (/z-suppressed) animals [24], and in certain immunosuppressed patients [21,22]. Such findings have now been extended to include B cells as a relevant host defense mechanism against toxoplasmosis. Together with the evidence presented here that immune guinea pig serum and antibodies are equally protective, it is suggested that T lymphocytes function in this model as helper cells by interacting with B cells in the formation of specific anti-T, gondii antibodies and as effector cells or by activating macrophages for enhanced phagocytosis [6,25] and destruction of the parasite.
ACKNOWLEDGEMENT This work was supported in part by a grant (to C.S.P.) from the J.M. Foundation, New York, NY.
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[18] [19]
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[20] [21] [22]
[23]
[24]
[25]
cells in different mouse strains bearing the H-2 k haplotype. J. Immunol. 137, 2991-2997. Bloom, B.R. (1979) Games parasites play: how parasites evade immune surveillance. Nature 279, 21-26. Ruskin, J. and Remington, J.S. (1976) Toxoplasmosis in the compromised host. Ann. Int. Med. 84, 193-199. Luft, B.J., Brooks, R.G., Conley, F.K., McCabe, R.E. and Remington, J.S. (1984) Toxoplasmic encephalitis in patients with acquired immune deficiency syndrome. J. Am. Med. Assoc. 252, 913-917. Remington, J.S. and Desmonts, G. (1990) Toxoplasmosis. In: Infectious diseases of the fetus and newborn infant. (Remington, J.S. and Klein, J.O., Eds.), pp. 89-195. W.B. Saunders Co., Philadelphia. Frenkel, J.K. and Taylor, D.W. (1982) Toxoplasmosis in immunoglobulin M-suppressed mice. Infect. Immun. 38, 360-367. Anderson, S.E. and Remington, J.S. (1974) Effect of normal and activated human macrophages on Toxoplasma gondii, J. Exp. Med. 1349, 1154-1174.