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Selection of the host for resistance: Genetic control of protective immunity to schistosomes
SELECTION
OF THE HOST FOR RESISTANCE: GENETIC CONTROL OF PROTECTIVE IMMUNITY TO SCHISTOSOMES
ALAN SHER,RODRIGO CORREA-OLIVE~~,PAULBRINDLEY
AND *STEPHANIEL.JAMES
Laboratory of Parasitic Disease, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892. USA and *Departments of Medicine and Microbiology, George Washington University School of Medicine, Washington, DC 20037, USA
INTRODUCTION The influence of host genotype on resistance to parasitic infection is a major area of investigation for immunoparasitologists. That host genetic differences can play a crucial role in governing the nature and outcome of parasitism has been well documented in both protozoan and helminth infections (Wakelin, 1978; Sher & Scott, 1982). In outbred populations, these genetically determined variations in host susceptibility may actually be exploited by parasites in promoting their own survival (Wakelin, 1984). From the point of view of the veterinary parasitologist, knowledge of genetically based polymorphisms in host susceptibility and protective immunity has facilitated the selected breeding of livestock resistant to or readily immunized against wasting parasitic infections (Dineen & Windon, 1980; Dargie, 1982). Although poorly documented at present, the influence of host genotype on human parasitic disease remains an important concern for clinical parasitologists attempting to understand the variable pathogenesis of parasitic infection in man (Sher & Scott, 1982). At a more fundament~ level, immunogenetics has become an impo~ant and highly effective tool for characterizing immune mechanisms responsible for innate as well as acquired resistance to parasitic infection (Mitchell, Anders, Brown, Handman, Roberts-Thompson, Chapman, Forsyth, Kahl & Cruise, 1982). For example, knowledge of the genetics of visceral and cutaneous leishmania infections in inbred mice has led to the identification of genes controlling survival of the parasite in macrophages as well as immunoregulatory mechanisms governing healing and chronicity of infection (Howard, 1985). Similarly, elegant studies by Wassom and colleagues (1984) have identified and elucidated the role of H-2 and non-H-2 linked genes in governing lymphocyte interactions involved in innate and acquired resistance to Trichinella spiratis infection in mice. In the work summarized in this paper, we have employed an immunogenetic approach to characterize the effector mechanism of protective immunity against the important human parasite Schistosoma mansoni. We were led to this approach by the failure of in vitro studies to define a relevant immune mechanism responsible for parasite killing as well as by the limited success of adoptive transfer experiments is dissecting this mechanism irr vivo. Previous work (Murrell, Clark, Dean & Vannier, 1979) had indicated that inbred mice vaccinated with highly irradiated cercariae develop reproducibly consistent levels of immunity against challenge infection. We decided to study the genetic control of protective immunity in this model in depth. The goals of this work were (1) to study the effects of previously characterized congenital immunodeficiencies on vaccine induced resistance, and (2) to identify genes controlling vaccine induced immunity and study their mode of action. RESULTS E$ects
of known
congenital
immunodeficiencies
on protective
immunity
By surveying inbred mouse strains with known immune response defects, certain features of the effector mechanism of vaccine induced resistance were rapidly established. Thus, the observation that athymic nuinu mice failed to develop protection against challenge infection established the T lymphocyte dependency of parasite rejection in the irradiated vaccine model @her, Hieny, James & Asofsky, 1982). In contrast, a variety of other congenital defects in humoral and cellular immune response functions failed to significantly effect vaccine induced resistance. These included the beige (b&g) defect which effects
53
54
A. Sher, R. Correa-Oliveira,
P. Brindley and S. James
natural killer cell and eosinophil activity (James & Sher, 1983), tissue mast cell deficiency controlled by the w” gene and defective IgE anti-schistosome antibody responses observed in SJLiJ mice @her, CorreaOliveira, Hieny & Hussain, 1982), the CS deficiency displayed by the BlO.D2/osn strain @her, Hieny, James & Asofsky, 1982) as well as defective anti-schistosome IgM responses controlled by the xid gene (Correa-Oliveira & Sher, 1985). The possible involvement of humoral antibody in parasite rejection was suggested by the failure of k-suppressed mice to develop significant immunity to challenge infection (Sher, Hieny, James & Asofsky, 1982). Inbred mice belonging to the P strain were found to be unique in failing to develop significant vaccine induced immunity (James & Sher, 1983). These mice had previously been characterized as possessing cellular defects in lymphokine production and macrophage activation in a tumoricidal assay (Boraschi & Meltzer, 1980). Similar defects were apparent in S. munsoni vaccinated P strain animals which were found to develop only minimal delayed-type hypersensitivity responses to schistosome antigens, produced only low levels of lymphokine (gamma interferon) when their lymphocytes were stimulated with parasite antigen and displayed defective macrophage mediated killing of schistosome larvae in an in vitro assay (James, Correa-Oliveira & Leonard, 1984). In addition, vaccinated P mice were found to develop approximately one third the level of IgM anti-larval antibodies displayed by mouse strains exhibiting normal levels of protective immunity (Correa-Oliveira, Sher & James, 1984). In contrast, extensive examination of vaccinated P mice failed to reveal significant abnormalities in IgG, IgA and IgE antischistosome antibody responses (Correa-Oliveira et ul., 1984), schistosome antigen triggered lymphocyte proliferation, IL-l, IL-2, IL-3 production as well as defects in macrophage mediated antigen presentation (James, DeBlois & Langehorne, submitted for publication). In summary, these studies on immunodeficient mice strongly suggested that T-lymphocyte dependent cell-mediated immunity plays an important role in vaccine induced immunity. Although an additional function for humoral antibody was suggested by the abrogation of resistance induced by k-suppression, this evidence was not conclusive because of the known effects of in vivo B lymphocyte depletion on certain T cell responses. Variation in vaccine induced resistance amongst inbred und H-2 congenic mouse strains
The analysis of vaccine induced resistance in a series of standard inbred mouse strains indicted that with the exception of the P strain (which as described above fails to develop significant immunity) all strains develop either high (5570% reduction in challenge recovery) or intermediate levels (30-55% reduction in challenge recovery) of protection in response to immunization with irradiated cercariae (James & Sher, 1983). The C57BL/6 (B6) strain was chosen as the prototype of the high resistance group whereas the A/J (A) strain was selected as the intermediate group prototype. Mice of the latter strain consistently develop levels of vaccine induced resistance ranging from 3040% and similar to animals of the P strain are defective in macrophage killing activity against schistosomula (James, Skamene & Meltzer, 1983). The strain survey suggested the possible influence of the murine major histocompatibility complex (MHC) on protective immunity in that all strains displaying high levels of resistance possessed either the b or d MHC haplotypes. This quantitative influence was confirmed by analysing a series of BlO, B6 and BALB H-2 congenic strains. When k or a haplotypes were substituted on these backgrounds lower levels of immunity were observed (Sher, 1984). Nevertheless, it was clear that the variation in resistance observed amongst mouse strains was not due entirely to the influence of the MHC. For example, BIO.P (H-2P) mice developed a high level of resistance comparable to that displayed by BlO (H-2b) animals (Correa-Oliveira, James, McCall & Sher, submitted for publication) yet clearly greater than that exhioited by the defective P (H-2*) strain. Genetic control of defects in vaccine induced immunity
In order to formally establish that the observed variations in vaccine induced immunity and immune responses are under genetic control and to test for possible association of resistance and immune response defects, a series of genetic crosses were performed. It was decided to focus on the inheritance of the P strain immunity, IgM antibody, and macrophage larvacidal activity defects and at the same time on the inheritance of the A strain immunity and macrophage larvacidal defects. FI and FZ crosses as well as reciprocal backcrosses were constructed between the P or A strains and the non-defective, highly resistance C57BL/6J (B6) strain. The progeny were then vaccinated and assayed for their resistance to challenge infection, 1gM anti-schistosomulum antibody levels, macrophage larvacidal activity as well as their MHC haplotypes and immunoglobulin heavy chain allotypes. The results of this study obtained to date are summarized in Table 1. The vaccine immunity, IgM and macrophage larvacidal defects of P or A mice were found to be inherited in the FL generation with B6 as fully recessive autosomal traits. Analysis of the FZ and reciprocal backcross generations indicated that the P mouse resistance and IgM defects segregate with a distribution consistent with control by single genetic loci. Since both traits are quantita-
Genetic control of parasite immunity
55
tive, continuously distributed variables, independent support for the single locus hypothesis of inheritance was obtained by means of a statistical procedure (MAXLIK) specifically designed for the analysis of genetic data with this property (Correa-Oliveira et al., submitted for publication). Similarly, partial analyses performed on either FZ or backcross progeny suggested that the P strain larvacidal defect as well as the A strain immunity and larvacidal defects are also controlled by single loci (Table 1). We have designated the genes controlling the P and A strain defects in protective immunity to S. mansonias ism-P and ism-A respectively. TABLE I-INHERITANCE %
Defect P strain P strain P strain A strain A strain
resistance IgM antibody larvacidal activity resistance larvacidal activity
Predicted distribution for recessively expressed single genetic locus**
* it **
OF VACCINE DEFECTS IN
Non-defective
PxB6 OR AxB6 GENETIC
progeny (observed)* Cross F,xP Fz or A
Ft
FlxB6
100 100 96 100 89
100 100 n.t. n.t. 89
43 48 n.t. n.t. 58
73 70 75 75 n.t.
100
100
SO
75
Phenotypes assigned on the basis of parental data range. Assessed by Spearman’s rank correlation analysis. In each instance the observed distribll?ions fit the predicted distributjons
CROSSES
Linkage to MHC or Igh loci
Quantitatively associated responses?
none none none none n.t.
larvacidal activity none resistance n-t. n.t.
as assessed by Chi square analysis.
Linkage and relationship of the genes controlling vaccine immune response defects
In agreement with our findings on xid mice (Correa-Oliveira & Sher, 1985), no linkage or quantitative association between ism-P and P mouse IgM anti-larval antibody defect was observed in the analysis of F2 progeny (Table 1). Similarly, no correlation or linkage was detected between the P mouse IgM antibody and macrophage larvacidal defects. However, a highly significant (p
OF P AND A STRAIN VACCINE DEFECTS IN
(PxA)Fl
HYBRIDS.
Mice Parameter Resistance to challenge infection (%)* Macrophage larvacidal activity?
* f
C57BLi6
P
A
(PxA)Fl
64 35
12 17
30 20
59 39
Percent reduction in challenge infection adult worm recovery in vaccinated vs age matched control mice. Percent of schistosomula killed by schistosome antigen elicited peritoneal macrophages obtained from immunized
mice at the time of challenge infection (4 weeks after vaccination). The chromosomal locations of the ism-P and ism-A loci have yet to be defined. No linkage with either the MHC (chromosome 17) or the ~mmuno~obulin heavy chain locus (chromosome 12) was detected in our genetic crosses nor were the P and A strain larvacidal defects and P strain IgM defect found to be linked to these loci. Recent data (Correa-Oliveira, Skamene & Sher, unpublished) have indicated a
56
A. Sher, R. Correa-Oliveira, P. Brindley and S. James
tentative linkage for the ism-A locus. Sixteen different AxB6 (AxB/BxA) recombinant inbred strains were assayed for their vaccine induced immunity. The resulting strain distribution pattern indicated a highly significant association of the ism-A defect with markers on chromosome 6. We are now attempting to formally confirm this linkage by means of genetic crosses. DISCUSSION The results of these studies provide one of the first formal demonstrations of single locus control of protective immunity to helminth infection. Of perhaps greater significance, is the observed striking association of defects in macrophage activation with defective vaccine immunity. Indeed, in our analyses of different inbred mouse strains, no correlation of other congenital humoral or cellular immune defects with decreased resistance has been observed. Although further work is required to confirm the genetic association of the ism-P and ism-A defects with defective macrophage function and to investigate the possible causality of this relationship, the hypothesis that T-lymphocyte dependent macrophage activation plays a critical role in the effector mechanism of schistosome immunity is supported by recent experiments (James, 1985) in which the induction of strong cell-mediated immunity against parasite antigens was found to result in highly significant protection of mice against challenge infection. The P and A strain immune defects delineated in our experiments are not uniquely associated with protection against schistosome infection. Thus, A strain mice show enhanced susceptibility to lethal rickettsial infection (Nacy & Meltzer, 1982). In addition, P strain mice have been shown to be unusually susceptible to infection with Leishmaniu major(Nacy, Fortier, Pappas & Henry, 1983). In both infections, the genetic defects in resistance are associated with defective macrophage microbicidal activity against these intracellular parasites. In the case of L. major, the defective resistance of the P strain appears to be controlled by a single locus (Fortier, Meltzer & Nacy, 1984). Thus, it is possible that the ism-P and ismA loci detected in our experiments on protective immunity against schistosomes may play a more general role in the genetic control of microbial and parasitic infection. Therefore, knowledge of the identity and precise immunologic function of these loci would appear to be highly relevant to our understanding of the genetic basis of host susceptibility. SUMMARY The genetic control of protective immunity to Schistosoma mansoni infection was studied in an attempt to characterize the effector mechanism of attenuated vaccine induced resistance and to define genetic loci regulating this process. Of all inbred mouse strains surveyed, the P and A (A/J) strains developed the lowest levels of resistance to challenge infection after vaccination with irradiated cercariae. Genetic cross experiments indicated that the protective immunity defects of these two strains are controlled by single yet distinct loci unlinked to either the murine H-2 or immunoglobulin heavy chain loci. Vaccinated P and A strain mice were also found to be defective in their capacity to produce activated macrophages which kill schistosome larvae in vitro. In the case of the P strain, an association of this macrophage larvacidal defect with defective vaccine induced resistance was observed in the progeny of FZ genetic crosses. No other congenital defects in humoral or cellular immune response have been detected which genetically correlate with impaired protective immunity. These results support the hypothesis that T-lymphocyte dependent macrophage activation plays a crucial role in the effector mechanism of anti-schistosome immunity. Since P and A strain mice have previously been shown to be defective in their resistance to other infections, it is possible that the loci determining the impaired protective immunity of these strains to schistosomes may play a general role in the regulation of host susceptibility. REFERENCES BORASCHI D. & MELTZER M. 1980. Defective turmoricidal capacity of macorphages from P/J mice: Turmoricidal defect involves abnormalities in lumphokine activation stimuli and in mononuclear phagocyte responsiveness. Journal of Immunology 125: 777-782. CORREA-OLIVEIRA R., SHER A. & JAMES S.L. 1984. Defective vaccine-induced immunity to Schistosoma mansoniin P strain mice I. Analysis of antibody responses. Journal of Immunology133: 1581-1586. CORREA-OLIVEIRA R. & SHER A. 1985. Defective IgM responses to vaccination or infection with Schistosoma mansoni in xid mice. Infection and Immunity 50: 409-414. DARGIE J.D. 1982. The influence of genetic factors in the resistance of ruminants to gastrointestinal nematode and trypanosome infections. In: Animal Models in Parasitology, pp 17-51 (Edited by Owen D.G.) Macmillan, London. DINEEN J.K. & WINDON R.G. 1980. The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology 10: 189-196. FORTIER A., MELTZER M.S. & NACY C.A. 1984. Susceptibility of inbred mice to Leishmania tropicainfection: genetic control of the development of cutaneous lesions in P/J mice. Journal of Immunology 133: 454-459.
Genetic control of parasite immunity HOWARD J. 1985. Immunological regulation Experimental Pathology. In press.
and control of experimental
57
leishmaniasis.
International
Review of
JAMES S.L. & SHER A. 1985. Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae III. Identification of a mouse strain, P/N, that fails to respond to vaccination. Parasite Immunology 5: 567-576.
JAMES S., SKAMENE E., MELTZER M.S. 1983. Macrophages as effector cells of protective immunity in murine schistosomiasis. V. Variation in macrophage schistosomulacidal and tumoricidal activites among mouse strains and correlation with resistance to re-infection. Journal of Immunology131: 945-953. JAMES S.L., CORREA-OLIVEIRAR. & LEONARD E.J. 1984. Defective vaccine induced immunity to Schistosoma mansoniin P strain mice. II. Analysis of cellular responses. Journal of Immunology 133: 1587-1593. JAMES S.L., CORREA-OLIVIERAR., MCCALL D. & SHER A. 1985. Resistance to schistosomes. In: Genetic Control of Host Resistance to Infection and Malignancy (Edited by Skamene E.) Alan R. Liss Publishers, New York. In press. MITCHELLG.F., ANDERSR.F., BROWNG.V., HANDMANE., ROBERTS-THOMSON I.C., CHAPMANC.B., FORSYTHK., KAHL, L.P. & CRUISE K.M. 1982. Analysis of infection characteristics and anti-parasite immune responses in resistant compared with susceptible hosts. Immunological Reviews 61: 137-188. MURRELLK.D., CLARK D.S., DEAN D.A. & VANNIERW.E. 1979. Influence of mouse strain on induction of resistance with irradiated Schistosoma mansoni cercariae. Journal of Parasitology 65: 829-834. NACY C.A. & MELTZERM.S. 1982. Macrophages in resistance to rickettsial infection: Strains of mice susceptible to the lethal effects of Rickettsia akari show defective macrophage richettisicidal activity in vitro. Infection and Immunity 36: 1096-1101.
NACY CA., FORTIER A.H., PAPPAS, M.G. & HENRY R.R. 1983. Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibilty with in vitro defective macrophage microbicidal activities. Cellular Immunology 77: 298-307.
SHER A. & SCOTT P. 1982. Genetic factors influencing the interaction of parasites with the immune system. Clinics in Immunology
and Allergy 2: 489-510.
SHER A., HIENY S., JAMES S.L. & ASOFSKY R. 1982. Mechanisms of protective immunity against Schistosoma mansoni in mice vaccinated with irradiated cercariae. II. Analysis of immunity in hosts deficient in T lymphocytes, B-lymphocytes or complement. Journal ofImmunology 128: 1880-1884. SHER A., CORREA-OLIVEIRAR., HIENY S. & HUSSAIN R. 1983. Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. IV. Analysis of the role of IgE antibodies and mast cells. Journal of Immunology 131: 1460-1465. SHER A., HIENY S. & JAMES S.L. 1984. Mechanisms of protective immunity against Schistosoma mansoni in mice vaccinated with irradiated cercariae. VI. Influence of the major histocompatibility complex. Parasite Immunology 6: 319-328.
WAKELIN D. 1978. Genetic control of susceptibility
and resistance
to parasitic infection.
Advances
In Parasitology
16: 219-308.
WAKELIN D. 1984. Evasion of the immune response: survival within low responding individuals of the host population. Parasitology 88: 639-657.
WASSOMD., WAKELIN D., BROOKSB.O., KROO C.J. & DAVID C.S. 1984. Genetic control of immunity to Trichinella spiralis infections of mice. Hypothesis to explain the role of H-2 genes in primary and challenge infections. Immunology 51: 625-63 1.
OF THE HOST FOR RESISTANCE: GENETIC CONTROL OF PROTECTIVE IMMUNITY TO SCHISTOSOMES
ALAN SHER,RODRIGO CORREA-OLIVE~~,PAULBRINDLEY
AND *STEPHANIEL.JAMES
Laboratory of Parasitic Disease, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892. USA and *Departments of Medicine and Microbiology, George Washington University School of Medicine, Washington, DC 20037, USA
INTRODUCTION The influence of host genotype on resistance to parasitic infection is a major area of investigation for immunoparasitologists. That host genetic differences can play a crucial role in governing the nature and outcome of parasitism has been well documented in both protozoan and helminth infections (Wakelin, 1978; Sher & Scott, 1982). In outbred populations, these genetically determined variations in host susceptibility may actually be exploited by parasites in promoting their own survival (Wakelin, 1984). From the point of view of the veterinary parasitologist, knowledge of genetically based polymorphisms in host susceptibility and protective immunity has facilitated the selected breeding of livestock resistant to or readily immunized against wasting parasitic infections (Dineen & Windon, 1980; Dargie, 1982). Although poorly documented at present, the influence of host genotype on human parasitic disease remains an important concern for clinical parasitologists attempting to understand the variable pathogenesis of parasitic infection in man (Sher & Scott, 1982). At a more fundament~ level, immunogenetics has become an impo~ant and highly effective tool for characterizing immune mechanisms responsible for innate as well as acquired resistance to parasitic infection (Mitchell, Anders, Brown, Handman, Roberts-Thompson, Chapman, Forsyth, Kahl & Cruise, 1982). For example, knowledge of the genetics of visceral and cutaneous leishmania infections in inbred mice has led to the identification of genes controlling survival of the parasite in macrophages as well as immunoregulatory mechanisms governing healing and chronicity of infection (Howard, 1985). Similarly, elegant studies by Wassom and colleagues (1984) have identified and elucidated the role of H-2 and non-H-2 linked genes in governing lymphocyte interactions involved in innate and acquired resistance to Trichinella spiratis infection in mice. In the work summarized in this paper, we have employed an immunogenetic approach to characterize the effector mechanism of protective immunity against the important human parasite Schistosoma mansoni. We were led to this approach by the failure of in vitro studies to define a relevant immune mechanism responsible for parasite killing as well as by the limited success of adoptive transfer experiments is dissecting this mechanism irr vivo. Previous work (Murrell, Clark, Dean & Vannier, 1979) had indicated that inbred mice vaccinated with highly irradiated cercariae develop reproducibly consistent levels of immunity against challenge infection. We decided to study the genetic control of protective immunity in this model in depth. The goals of this work were (1) to study the effects of previously characterized congenital immunodeficiencies on vaccine induced resistance, and (2) to identify genes controlling vaccine induced immunity and study their mode of action. RESULTS E$ects
of known
congenital
immunodeficiencies
on protective
immunity
By surveying inbred mouse strains with known immune response defects, certain features of the effector mechanism of vaccine induced resistance were rapidly established. Thus, the observation that athymic nuinu mice failed to develop protection against challenge infection established the T lymphocyte dependency of parasite rejection in the irradiated vaccine model @her, Hieny, James & Asofsky, 1982). In contrast, a variety of other congenital defects in humoral and cellular immune response functions failed to significantly effect vaccine induced resistance. These included the beige (b&g) defect which effects
53
54
A. Sher, R. Correa-Oliveira,
P. Brindley and S. James
natural killer cell and eosinophil activity (James & Sher, 1983), tissue mast cell deficiency controlled by the w” gene and defective IgE anti-schistosome antibody responses observed in SJLiJ mice @her, CorreaOliveira, Hieny & Hussain, 1982), the CS deficiency displayed by the BlO.D2/osn strain @her, Hieny, James & Asofsky, 1982) as well as defective anti-schistosome IgM responses controlled by the xid gene (Correa-Oliveira & Sher, 1985). The possible involvement of humoral antibody in parasite rejection was suggested by the failure of k-suppressed mice to develop significant immunity to challenge infection (Sher, Hieny, James & Asofsky, 1982). Inbred mice belonging to the P strain were found to be unique in failing to develop significant vaccine induced immunity (James & Sher, 1983). These mice had previously been characterized as possessing cellular defects in lymphokine production and macrophage activation in a tumoricidal assay (Boraschi & Meltzer, 1980). Similar defects were apparent in S. munsoni vaccinated P strain animals which were found to develop only minimal delayed-type hypersensitivity responses to schistosome antigens, produced only low levels of lymphokine (gamma interferon) when their lymphocytes were stimulated with parasite antigen and displayed defective macrophage mediated killing of schistosome larvae in an in vitro assay (James, Correa-Oliveira & Leonard, 1984). In addition, vaccinated P mice were found to develop approximately one third the level of IgM anti-larval antibodies displayed by mouse strains exhibiting normal levels of protective immunity (Correa-Oliveira, Sher & James, 1984). In contrast, extensive examination of vaccinated P mice failed to reveal significant abnormalities in IgG, IgA and IgE antischistosome antibody responses (Correa-Oliveira et ul., 1984), schistosome antigen triggered lymphocyte proliferation, IL-l, IL-2, IL-3 production as well as defects in macrophage mediated antigen presentation (James, DeBlois & Langehorne, submitted for publication). In summary, these studies on immunodeficient mice strongly suggested that T-lymphocyte dependent cell-mediated immunity plays an important role in vaccine induced immunity. Although an additional function for humoral antibody was suggested by the abrogation of resistance induced by k-suppression, this evidence was not conclusive because of the known effects of in vivo B lymphocyte depletion on certain T cell responses. Variation in vaccine induced resistance amongst inbred und H-2 congenic mouse strains
The analysis of vaccine induced resistance in a series of standard inbred mouse strains indicted that with the exception of the P strain (which as described above fails to develop significant immunity) all strains develop either high (5570% reduction in challenge recovery) or intermediate levels (30-55% reduction in challenge recovery) of protection in response to immunization with irradiated cercariae (James & Sher, 1983). The C57BL/6 (B6) strain was chosen as the prototype of the high resistance group whereas the A/J (A) strain was selected as the intermediate group prototype. Mice of the latter strain consistently develop levels of vaccine induced resistance ranging from 3040% and similar to animals of the P strain are defective in macrophage killing activity against schistosomula (James, Skamene & Meltzer, 1983). The strain survey suggested the possible influence of the murine major histocompatibility complex (MHC) on protective immunity in that all strains displaying high levels of resistance possessed either the b or d MHC haplotypes. This quantitative influence was confirmed by analysing a series of BlO, B6 and BALB H-2 congenic strains. When k or a haplotypes were substituted on these backgrounds lower levels of immunity were observed (Sher, 1984). Nevertheless, it was clear that the variation in resistance observed amongst mouse strains was not due entirely to the influence of the MHC. For example, BIO.P (H-2P) mice developed a high level of resistance comparable to that displayed by BlO (H-2b) animals (Correa-Oliveira, James, McCall & Sher, submitted for publication) yet clearly greater than that exhioited by the defective P (H-2*) strain. Genetic control of defects in vaccine induced immunity
In order to formally establish that the observed variations in vaccine induced immunity and immune responses are under genetic control and to test for possible association of resistance and immune response defects, a series of genetic crosses were performed. It was decided to focus on the inheritance of the P strain immunity, IgM antibody, and macrophage larvacidal activity defects and at the same time on the inheritance of the A strain immunity and macrophage larvacidal defects. FI and FZ crosses as well as reciprocal backcrosses were constructed between the P or A strains and the non-defective, highly resistance C57BL/6J (B6) strain. The progeny were then vaccinated and assayed for their resistance to challenge infection, 1gM anti-schistosomulum antibody levels, macrophage larvacidal activity as well as their MHC haplotypes and immunoglobulin heavy chain allotypes. The results of this study obtained to date are summarized in Table 1. The vaccine immunity, IgM and macrophage larvacidal defects of P or A mice were found to be inherited in the FL generation with B6 as fully recessive autosomal traits. Analysis of the FZ and reciprocal backcross generations indicated that the P mouse resistance and IgM defects segregate with a distribution consistent with control by single genetic loci. Since both traits are quantita-
Genetic control of parasite immunity
55
tive, continuously distributed variables, independent support for the single locus hypothesis of inheritance was obtained by means of a statistical procedure (MAXLIK) specifically designed for the analysis of genetic data with this property (Correa-Oliveira et al., submitted for publication). Similarly, partial analyses performed on either FZ or backcross progeny suggested that the P strain larvacidal defect as well as the A strain immunity and larvacidal defects are also controlled by single loci (Table 1). We have designated the genes controlling the P and A strain defects in protective immunity to S. mansonias ism-P and ism-A respectively. TABLE I-INHERITANCE %
Defect P strain P strain P strain A strain A strain
resistance IgM antibody larvacidal activity resistance larvacidal activity
Predicted distribution for recessively expressed single genetic locus**
* it **
OF VACCINE DEFECTS IN
Non-defective
PxB6 OR AxB6 GENETIC
progeny (observed)* Cross F,xP Fz or A
Ft
FlxB6
100 100 96 100 89
100 100 n.t. n.t. 89
43 48 n.t. n.t. 58
73 70 75 75 n.t.
100
100
SO
75
Phenotypes assigned on the basis of parental data range. Assessed by Spearman’s rank correlation analysis. In each instance the observed distribll?ions fit the predicted distributjons
CROSSES
Linkage to MHC or Igh loci
Quantitatively associated responses?
none none none none n.t.
larvacidal activity none resistance n-t. n.t.
as assessed by Chi square analysis.
Linkage and relationship of the genes controlling vaccine immune response defects
In agreement with our findings on xid mice (Correa-Oliveira & Sher, 1985), no linkage or quantitative association between ism-P and P mouse IgM anti-larval antibody defect was observed in the analysis of F2 progeny (Table 1). Similarly, no correlation or linkage was detected between the P mouse IgM antibody and macrophage larvacidal defects. However, a highly significant (p
OF P AND A STRAIN VACCINE DEFECTS IN
(PxA)Fl
HYBRIDS.
Mice Parameter Resistance to challenge infection (%)* Macrophage larvacidal activity?
* f
C57BLi6
P
A
(PxA)Fl
64 35
12 17
30 20
59 39
Percent reduction in challenge infection adult worm recovery in vaccinated vs age matched control mice. Percent of schistosomula killed by schistosome antigen elicited peritoneal macrophages obtained from immunized
mice at the time of challenge infection (4 weeks after vaccination). The chromosomal locations of the ism-P and ism-A loci have yet to be defined. No linkage with either the MHC (chromosome 17) or the ~mmuno~obulin heavy chain locus (chromosome 12) was detected in our genetic crosses nor were the P and A strain larvacidal defects and P strain IgM defect found to be linked to these loci. Recent data (Correa-Oliveira, Skamene & Sher, unpublished) have indicated a
56
A. Sher, R. Correa-Oliveira, P. Brindley and S. James
tentative linkage for the ism-A locus. Sixteen different AxB6 (AxB/BxA) recombinant inbred strains were assayed for their vaccine induced immunity. The resulting strain distribution pattern indicated a highly significant association of the ism-A defect with markers on chromosome 6. We are now attempting to formally confirm this linkage by means of genetic crosses. DISCUSSION The results of these studies provide one of the first formal demonstrations of single locus control of protective immunity to helminth infection. Of perhaps greater significance, is the observed striking association of defects in macrophage activation with defective vaccine immunity. Indeed, in our analyses of different inbred mouse strains, no correlation of other congenital humoral or cellular immune defects with decreased resistance has been observed. Although further work is required to confirm the genetic association of the ism-P and ism-A defects with defective macrophage function and to investigate the possible causality of this relationship, the hypothesis that T-lymphocyte dependent macrophage activation plays a critical role in the effector mechanism of schistosome immunity is supported by recent experiments (James, 1985) in which the induction of strong cell-mediated immunity against parasite antigens was found to result in highly significant protection of mice against challenge infection. The P and A strain immune defects delineated in our experiments are not uniquely associated with protection against schistosome infection. Thus, A strain mice show enhanced susceptibility to lethal rickettsial infection (Nacy & Meltzer, 1982). In addition, P strain mice have been shown to be unusually susceptible to infection with Leishmaniu major(Nacy, Fortier, Pappas & Henry, 1983). In both infections, the genetic defects in resistance are associated with defective macrophage microbicidal activity against these intracellular parasites. In the case of L. major, the defective resistance of the P strain appears to be controlled by a single locus (Fortier, Meltzer & Nacy, 1984). Thus, it is possible that the ism-P and ismA loci detected in our experiments on protective immunity against schistosomes may play a more general role in the genetic control of microbial and parasitic infection. Therefore, knowledge of the identity and precise immunologic function of these loci would appear to be highly relevant to our understanding of the genetic basis of host susceptibility. SUMMARY The genetic control of protective immunity to Schistosoma mansoni infection was studied in an attempt to characterize the effector mechanism of attenuated vaccine induced resistance and to define genetic loci regulating this process. Of all inbred mouse strains surveyed, the P and A (A/J) strains developed the lowest levels of resistance to challenge infection after vaccination with irradiated cercariae. Genetic cross experiments indicated that the protective immunity defects of these two strains are controlled by single yet distinct loci unlinked to either the murine H-2 or immunoglobulin heavy chain loci. Vaccinated P and A strain mice were also found to be defective in their capacity to produce activated macrophages which kill schistosome larvae in vitro. In the case of the P strain, an association of this macrophage larvacidal defect with defective vaccine induced resistance was observed in the progeny of FZ genetic crosses. No other congenital defects in humoral or cellular immune response have been detected which genetically correlate with impaired protective immunity. These results support the hypothesis that T-lymphocyte dependent macrophage activation plays a crucial role in the effector mechanism of anti-schistosome immunity. Since P and A strain mice have previously been shown to be defective in their resistance to other infections, it is possible that the loci determining the impaired protective immunity of these strains to schistosomes may play a general role in the regulation of host susceptibility. REFERENCES BORASCHI D. & MELTZER M. 1980. Defective turmoricidal capacity of macorphages from P/J mice: Turmoricidal defect involves abnormalities in lumphokine activation stimuli and in mononuclear phagocyte responsiveness. Journal of Immunology 125: 777-782. CORREA-OLIVEIRA R., SHER A. & JAMES S.L. 1984. Defective vaccine-induced immunity to Schistosoma mansoniin P strain mice I. Analysis of antibody responses. Journal of Immunology133: 1581-1586. CORREA-OLIVEIRA R. & SHER A. 1985. 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