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The Journal of Immunology, 2003, 170: 1019-1026.
Copyright © 2003 by The American Association of Immunologists

Novel Quantitative Trait Loci Controlling Development of Experimental Autoimmune Encephalomyelitis and Proportion of Lymphocyte Subpopulations1

Jenny Karlsson, Xiangshan Zhao2, Irina Lonskaya, Malin Neptin, Rikard Holmdahl and Åsa Andersson3

Department for Cell and Molecular Biology, Section for Medical Inflammation Research, University of Lund, Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The B10.RIII mouse strain (H-2r) develops chronic experimental autoimmune encephalomyelitis (EAE) upon immunization with the myelin basic protein 89–101 peptide. EAE was induced and studied in a backcross between B10.RIII and the EAE-resistant RIIIS/J strain (H-2r), and a complete genome scan with microsatellite markers was performed. Five loci were significantly linked to different traits and clinical subtypes of EAE on chromosomes 1, 5, 11, 15, and 16, three of the loci having sex specificity. The quantitative trait locus on chromosome 15 partly overlapped with the Eae2 locus, previously identified in crosses between the B10.RIII and RIIIS/J mouse strains. The loci on chromosomes 11 and 16 overlapped with Eae loci identified in other mouse crosses. By analyzing the backcross animals for lymphocyte phenotypes, the proportion of B and T cells in addition to the levels of CD4+CD8- and CD4-CD8+ T cells and the CD4+/CD8+ ratio in spleen were linked to different loci on chromosomes 1, 2, 3, 5, 6, 11, and 15. On chromosome 16, we found significant linkage to spleen cell proliferation. Several linkages overlapped with the quantitative trait loci for disease phenotypes. The identification of subphenotypes that are linked to the same loci as disease traits could be most useful in the search for candidate genes and biological pathways involved in the pathological process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Multiple sclerosis (MS)4 is an inflammatory disease in the CNS characterized by demyelination and axon damage leading to severe neurological dysfunction. Leukocytes are present in the CNS lesion, and CD4+ T cells and macrophages are considered the primary cell types in the initiation stage of the disease process (1). The triggering event for the pathogenesis in MS is not known, but both genetic and environmental factors contribute to disease. Association studies have demonstrated that susceptibility is associated with genes in the MHC (2, 3, 4, 5). A number of additional, potentially important genetic regions have been suggested (6, 7, 8, 9, 10), but to date no major susceptibility gene has been found.

Animal models, resembling human disease, are important tools for studying complex genetic traits, and recent comparable analysis of the mouse and human genomes has highlighted the striking similarity in their genes and genetic organization (11). Experimental autoimmune encephalomyelitis (EAE), a model for MS, is induced in genetically susceptible rodents by immunization with myelin proteins or peptides. The myelin basic protein (MBP) peptide 84–102 was demonstrated to bind to the MHC class II molecule of the HLA-DR2 haplotype (12), which is strongly associated with susceptibility to MS. In a humanized mouse model for MS, this peptide, when bound to HLA-DR2, confirmed susceptibility to disease (13). Moreover, the MBP epitope 85–99, bound to DR2, was detected in human MS lesions (14), emphasizing the importance of this MBP epitope in MS pathogenesis. The B10.RIII mouse strain develops chronic EAE after immunization with the MBP peptide 89–101 (15). This strain expresses the MHC haplotype H-2r also expressed in the EAE-resistant RIIIS/J mouse strain, suggesting a role for non-MHC genes in susceptibility to EAE. This was demonstrated in an F2 intercross between the B10.RIII and RIIIS/J strains, in which EAE development was linked to one locus on chromosome 15 (Eae2) and an additional locus on chromosome 3 (Eae3) (16). In addition to the Eae1 (MHC) (17), Eae2, and Eae3 loci, genetic studies in crosses between the mouse strains SJL/J and B10.S have revealed 23 loci linked to different traits of EAE (18, 19, 20) (http://www.informatics.jax.org).

In the present study, the aim was to find additional genetic loci linked to development of EAE in a cross between B10.RIII and RIIIS/J. In the previous F2 intercross, two loci were significantly linked to incidence of EAE. The genetic context of a backcross (BC) reduces the genetic variance compared with an F2 intercross and may also neutralize or reduce the impact of the previously identified loci, Eae1, Eae2, and Eae3. We therefore expected to find novel quantitative trait loci (QTLs) in this experiment. EAE was induced in a (B10.RIII x RIIIS/J)F1 x B10.RIII BC, disease development and clinical EAE subtypes were studied, and a complete genome scan with microsatellite markers was performed. In addition, we investigated the lymphocyte compartment for levels of different subpopulations and for function in terms of proliferative response. The statistical analysis revealed significant linkages for EAE development to chromosomes 1, 5, 11, 15, and 16, and suggestive linkage to chromosome 18, with a sex influence on four of the linkages. The analyses of subphenotypes demonstrated that some of the lymphocyte phenotypes were linked to the same genetic regions as disease phenotypes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

B10.RIII animals were originally provided by J. Klein (Tübingen, Germany), and kept in our breeding colony. RIIIS/J animals were purchased from The Jackson Laboratory (Bar Harbor, ME). (B10.RIII x RIIIS/J)F1 hybrids and (B10.RIII x RIIIS/J)F1 x B10.RIII animals were produced in our colony and kept in the animal facility at the Section for Medical Inflammation Research, University of Lund. The mice, ranging in age between 14 and 24 wk, were all immunized at the same day.

Induction of EAE and evaluation of disease

The MBP 89–101 peptide was synthesized on an Applied Biosystems (Foster City, CA) peptide synthesizer model ABI430A. The animals were immunized at the root of the tail with 0.1 ml of an emulsion containing 50 µg MBP 89–101 in PBS mixed with 100 µg Mycobacterium tuberculosis H37RA (Difco Labs, Detroit, MI) in IFA (Difco Labs). The animals were given injections with 400 ng of Bordetella pertussis toxin in 100 µl PBS i.p. on days 0 and 2. From day 8, the mice were monitored for clinical signs. The scores were graded from 0 to 8 as follows: 0, no clinical signs of disease; 1, tail weakness; 2, tail paralysis; 3, tail paralysis and mild waddle; 4, tail paralysis and severe waddle; 5, tail paralysis and paralysis of one limb; 6, tail paralysis and paralysis of two limbs; 7, tetraparesis; 8, moribund or deceased.

Incidence of EAE was considered as positive if the disease score was higher than 1. Severity of disease was analyzed as the highest score recorded for each individual. The onset trait is the day when clinical signs were first observed. Area under the curve (auc) was calculated by adding together the scores for each day with clinical symptoms. Mice were assigned to different clinical subtypes according to Fig. 1 and Table II, and were scored for 63 days.



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  		FIGURE 1. Clinical subtypes of EAE observed in the (B10.RIII x RIIIS/J)F1 x B10.RIII BC. The EAE subtypes are demonstrated by one representative animal from each group: a, chronic disease; b, acute disease; c, remitting-relapsing disease; and d, acute progressive disease.

 

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  		Table II. Incidence of EAE in (B10.RIII x RIIIS/J)F1 x B10.RIII mice

 
Cell preparations and stimulation in vitro

Spleens were excised in DMEM (Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 5% FCS. Single cell suspensions were prepared, and RBCs were depleted using 0.84% NH4Cl. The number of splenocytes were counted in a cell counter (Sysmex CDA 500; Toa Medical Electronics, Kobe, Japan), and cells were cultured in DMEM supplemented with 10% FCS, antibiotics, HEPES, and 2-ME (cell culture medium). For proliferation assays, total spleen cells (7 x 105/well) were cultured in medium only, or in the presence of ConA at a final concentration of 5 µg/ml. After 48 h, 1 µCi 3H-labeled thymidine was added to each well and 3H-labeled thymidine incorporation was measured after 16 h.

Flow cytometry

Cells were washed with staining buffer (PBS, 3% FCS, 0.01% NaN3) and incubated on ice with supernatant from the 2.4G2 hybridoma to block non-Ag-specific binding of Igs to FcRs. Cells were stained with FITC-, PE-, or biotin-conjugated primary Abs for 15 min on ice. Following two washes, biotin-conjugated Abs were revealed by incubating the cells with streptavidin-PE for 15 min, followed by two washes. Ab-labeled cells were analyzed on a FACScan (BD Biosciences, San Jose, CA). The following dye- or biotin-coupled Abs were obtained from BD PharMingen (San Diego, CA): anti-B220 (RA3-6B2), anti-CD3 (145-2C11), anti-CD4 (H129.19), anti-CD8 (53-6.7).

Genotyping and linkage analysis

DNA was prepared from tail biopsies according to a previously reported protocol (21). One hundred and fifty fluorescence-labeled microsatellite primers (INTERACTIVA, Ulm, Germany), covering the autosomal chromosomes and the X chromosome, were used for genotyping. A complete list of the microsatellite markers used and the result from the parental screening with genomic DNA from B10.RIII and RIIIS/J will be found on http://net.inflam.lu.se. PCRs were performed with 5 ng of DNA in a reaction volume of 10 µl containing: 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 1.5 pmol of the respective forward and reversed primer, 0.4 mM dNTPs (Advanced Biotechnologies, Surrey, U.K.), and 0.25 U Taq DNA polymerase (Amersham Pharmacia Biotech, Uppsala, Sweden). The following conditions were used for amplification of DNA: denaturation at 94°C for 3 min, annealing at 56°C for 45 s, polymerization at 72°C for 1 min, followed by 31 cycles of 94°C for 30 s, 56°C for 45 s, and 72°C for 1 min. The final cycle ended by elongation at 72°C for 7 min. The PCR products were analyzed on an ABI 377 (PE Applied Biosystems) or a Megabace 1000 (Amersham Pharmacia Biotech), according to the manufacturers’ protocols.

Linkage analysis and permutation tests were conducted using the Map Manager QTXb11 software (http://mapmgr.roswellpark.org/mmQTX.html). The marker map was generated using the Kosambi map function. Permutation tests (1000 permutations) were conducted to establish the empirical significance thresholds on a genome-wide level (p < 0.05) for each phenotype. Interval mapping was conducted at 1-cM increments under the additive regression model to calculate the test statistics. The Map Manager QTXb11 software generates likelihood ratio statistics that can be converted to conventional likelihood of odds score by dividing it by log10. Results from the calculations were exported from the Map Manager software to the QGene software (http://www.qgene.org) to create the linkage curves in Fig. 2. The marker map covered >95% of the genome at a 20-cM intermarker distance (disregarding the Y chromosome). Arithmetic mean and maximum distance to the next marker or chromosome end were 11.4 and 32.6 cM, respectively.



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  		FIGURE 2. Logarithm of odds ratio score curves for genetic linkage to traits of EAE development on chromosomes 11, 15, and 16. The dotted lines indicate the empirical significance levels.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EAE development in a (B10.RIII x RIIIS/J)F1 x B10.RIII BC

The B10.RIII mouse strain is susceptible to induction of EAE with 250 µg of the MBP peptide 89–101 (15, 16, 22). The RIIIS/J strain, which expresses the same MHC haplotype (H-2r), is resistant to induction of disease with this protocol (16). In an attempt to find an immunization protocol that resulted in a milder disease course in the B10.RIII strain and might reveal the most penetrant genes in a gene segregation cross, we compared EAE development in B10.RIII and (B10.RIII x RIIIS/J)F1 mice immunized with 250 or 50 µg of MBP 89–101. Immunization with the lower dose of peptide resulted in lower incidence and severity in the B10.RIII strain (Table I). Subsequently, 402 (B10.RIII x RIIIS/J)F1 x B10.RIII mice were immunized with 50 µg of the MBP peptide 89–101, and clinical scores were recorded for 63 days (Table I). Similar to what has been reported by Butterfield et al. (19), we could distinguish four different clinical subtypes of EAE (Table II and Fig. 1). A later day of onset in mice developing chronic disease compared with animals with acute or remitting-relapsing EAE (p = 0.039 and p = 0.0022, respectively) was observed. Severity of disease was higher in mice with chronic EAE compared with the two other groups (p < 0.0001) (Table III).


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  		Table I. EAE development in B10.RIII, F1, and BC micea

 

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  		Table III. Onset and severity of EAE in (B10.RIII x RIIIS/J)F1 x B10.RIII mice

 
Subphenotypes

The genetic contribution to the disease phenotype is complex, and many genes are involved to control each trait. To divide the disease phenotypes into smaller entities, subphenotypes can be studied. To identify informative subphenotypes, we screened the parental strains and selected a number of phenotypes from analyses of spleen cell populations to be included in the BC study (Table IV).


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  		Table IV. Lymphocyte subpopulations in spleena

 
In support of a previous study, we observed that the RIIIS/J mouse strain has reduced spleen size (23) (Table IV). The proportion of T and B cells in spleen differed significantly between RIIIS/J and F1 mice as compared with B10.RIII. In RIIIS/J and F1 mice, the T cells comprise a larger proportion of the spleen lymphocytes, whereas the B cells are in majority in the B10.RIII spleen. The relative numbers of CD4+ T cells were significantly higher in spleens of RIIIS/J mice, while there was no difference in the percentage of CD8+ T lymphocytes, giving a considerably higher ratio between CD4+ and CD8+ T lymphocytes in the RIIIS/J strain compared with B10.RIII (Table IV).

The proliferative response to Con A was tested on spleen cells from the parental and F1 mice. The proliferation was significantly higher with cells from the RIIIS/J and F1 strains compared with spleen cells from the B10.RIII strain (data not shown). Interestingly, we observed a difference in the background proliferation of spleen cells from different animals in the BC, which we did not find in the parental strains. The level of background proliferation was not correlated to incidence of EAE nor to the level of proliferation after stimulation in vitro, and was included as a subphenotype in the genetic analyses.

Linkage analysis

A complete genome scan with 150 microsatellite markers was performed for all animals. Informative microsatellite markers were chosen from a screen on B10.RIII and RIIIS/J genomic DNA. As previously reported, we observed that in addition to the MHC region, the B10.RIII strain has a large RIIIS/J genomic fragment on chromosome 10 (24). Incidence, onset, severity, auc, and different clinical subtypes were investigated in the linkage analysis. In addition, 200 of the BC animals were investigated for different subphenotypes, and the results were included in the linkage analysis. Genome-wide interval mapping revealed linkages to different traits of EAE on chromosomes 1, 5, 11, 16, and 18 that have not previously been reported in crosses between the B10.RIII and RIIIS/J strains. Similar to a previous report (19), we found that the different clinical subtypes of EAE were linked to different genetic regions (Table V). Development of acute disease in males was linked to a region on chromosome 5. Genetic linkage to development of remitting EAE in female mice, including mice with both acute and remitting-relapsing disease, was revealed toward the distal end of chromosome 1 (Table V).


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  		Table V. Genotype distribution of affected and unaffected animals at different loci linked to clinical subtypes of EAEa

 
In Table V, the genotypes for the loci linked to different disease subtypes are shown for affected and nonaffected mice. For the locus on chromosome 5, linked to acute disease in males, the inheritance pattern suggested that the RIIIS/J allele protects from disease development. The same inheritance pattern, a dominant protective RIIIS/J effect on disease, was found for the female specific linkage to remitting disease on chromosome 1 (Table V). No Eae loci have to date been reported on chromosomes 1 and 5 in mice, and we consider the two regions as novel QTLs for EAE that should be designated Eae26 (chromosome 5) and Eae27 (chromosome 1). Significant and suggestive linkage to the relative numbers of CD8+ and CD4+ T cells in spleen, respectively, peaked in the same region as the linkage to acute disease in male mice on chromosome 5 (Table VI). Linkage to CD8+ T cells was, in addition, found at the distal end of chromosome 1, close to the Eae27 locus (Table VI).


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  		Table VI. Inheritance patterns of lymphocyte phenotypesa

 
The central part of chromosome 11 harbors several Eae loci (Eae6b, Eae7, Eae23) (18, 19, 25, 26). A region between 33 and 44 cM on chromosome 11 was linked to incidence, development of chronic disease, day of onset, and auc in the BC (Table V and Fig. 2). As shown in Table V, susceptibility was associated with two B10.RIII alleles at this locus. The linkage to the relative proportions of B and T cells in the spleen overlapped with the QTLs for the different disease traits on chromosome 11 (Tables V and VI, and Fig. 2).

The Eae11 locus at chromosome 16 was previously demonstrated to be associated with incidence of disease in male mice (19). In the present EAE experiment, the region on chromosome 16 was linked to the time for disease onset in male mice (Fig. 2). In contrast with the previous report on a cross between SJL/J and B10S/DvTe, in which heterozygosity at this locus was correlated with susceptibility to disease, homozygosity for the B allele was associated with disease development in this experiment. Lymphocyte proliferation upon Con A stimulation in vitro, in addition to background proliferation in male mice, showed linkage to the distal part of chromosome 16 (Table VI).

At the telomeric end of chromosome 18, we found a region with suggestive linkage to development of remitting disease in male mice (Table V). This region has previously been designated Eae18. In one investigation, the chromosome 18 locus was linked to the level of demyelination in the spinal cord in male mice (20), and another study revealed suggestive linkage to paralysis (27).

In an F2 intercross between the B10.RIII and RIIIS/J mouse strains, it was demonstrated that development of EAE is controlled by genes located on chromosomes 15 (Eae2) and 3 (Eae3) (16). In the present BC, a region that is believed to include the Eae2 locus showed linkage to the trait auc (Fig. 2). The peak area for this linkage extends down to the central part of chromosome 15, which should be compared with the previous experiment in which the confidence interval covered the distance from the centromeric region down to ~20 cM (16). Furthermore, the inheritance pattern for susceptibility to disease is different in the BC compared with the previous intercross in which the affected mice were heterozygous at the Eae2 locus. In the present BC, the mice with the highest auc values are homozygous for B10.RIII alleles. The data suggest a second Eae locus at chromosome 15, close to the Eae2, that may seclude the effect of Eae2 in the genetic context of a BC. We therefore denote this locus as Eae2b. Linkage analysis with the CD4+/CD8+ ratio phenotype revealed that this trait is linked to the Eae2 region in females. The Eae2 locus has been shown to interact with Eae3 on chromosome 3 (16). In the BC, we found a weak suggestive linkage to the trait auc in the Eae3 region (likelihood ratio statistics = 8.6) (data not shown). Linkage to the relative numbers of CD4+ and CD8+ T cells in spleen was found at the same location (Table VI).

In Table VI, the QTLs for various lymphocyte subphenotypes are shown. In addition to the already mentioned QTLs for subphenotypes in genetic regions linked to disease traits, we found QTLs for lymphocyte traits on chromosomes 2 and 6. On the middle part of chromosome 2, we identified a QTL for the percentage of CD4+ T cells in the spleen that confirms the recently published description of a similar locus in a cross between C57BL/6 and DBA/2 (28). The CD4+/CD8+ ratio was, in addition to the Eae2 region on chromosome 15, linked to chromosomes 2 and 6. On chromosomes 2 and 15, this association was female specific. In Table VI, the inheritance patterns of different QTLs for the subphenotypes are shown, and Fig. 3 shows a schematic summary of QTLs for disease and lymphocyte traits in crosses between the B10.RIII and RIIIS/J strains.



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  		FIGURE 3. Schematic overview of QTLs for disease traits and subphenotypes found in crosses between B10.RIII and RIIIS/J (16 39 47 ). Significant and suggestive linkages are shown in bold and plain text, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MS is a complex inflammatory disease, and susceptibility is dependent on both environment and genetic background. To date, only genes within the MHC have been shown to be associated with development of MS. In genetic studies on mouse models for MS, 26 Eae loci, linked to different traits of EAE development, have been reported (http://www.informatics.jax.org). The majority of the Eae loci have been described in crosses between the SJL/J and B10.S/DvTe strains. In this model, EAE is induced with spinal cord homogenate (18, 19, 20, 26). To date, two Eae loci, Eae2 and Eae3, were reported from an EAE experiment in a cross between the mouse strains B10.RIII and RIIIS/J (16). In addition, the Eae3 was confirmed in other crosses involving B10 strains (19, 27, 29). In the present study, regions on chromosomes 11, 16, and 18 were linked to different traits of EAE and could presumably be the same as Eae6b, Eae7, Eae23 (chromosome 11), Eae 11 (chromosome 16), and Eae18 (chromosome 18). In addition, homologous regions linked to disease traits have been identified in rat EAE experiment. The part of mouse chromosome 11, containing three Eae loci, is homologous to a locus on rat chromosome 10, which was demonstrated to correlate with development of EAE (30). Eae18 on mouse chromosome 18 is homologous to rat chromosome 18, in which suggestive linkage to severity and duration of disease was reported (31). Interestingly, this locus is close to the MBP gene (http://www.informatics.jax.org). The finding of Eae loci on the homologous genetic locations in different mouse and rat models strengthens the importance of the particular QTLs in disease development.

In the present study, one locus on chromosome 5, Eae26, was linked to development of acute EAE in males. No Eae locus has previously been reported on mouse chromosome 5, but three arthritis loci have recently been identified on the middle part of the chromosome: proteoglycan-induced arthritis (Pgia16) (32) and Borrelia burgdorferi-associated arthritis (Bbaa2 and Bbaa3) (33). In addition, a homologous region on chromosome 12 in the rat, linked to EAE, has been identified (31).

We identified a weak, but significant linkage to development of remitting disease in females on the distal part of chromosome 1 in which no Eae locus has previously been isolated in mouse. Several QTLs for inflammatory diseases are mapped to this location: the Cia9 (collagen-induced arthritis QTL 9) (34), Stia1 (serum transfer-induced arthritis) (35), Orch4 (autoimmune orchitis) (36), in addition to a number of susceptibility loci for models of systemic lupus erythematosus (37, 38). It has been suggested that genes clustered at this end on chromosome 1 might be important for various steps in pathways leading to autoimmune disease (34).

The Eae2 locus on chromosome 15 was previously described in an F2 intercross between B10.RIII and RIIIS/J (16). Importantly, this region is syntenic, with a locus on human chromosome 5 demonstrated to be associated to MS development in a Finnish population (7). In the previous F2 intercross between B10.RIII and RIIIS/J, the most severe disease was observed in mice with one B10.RIII and one RIIIS/J allele, while homozygosity for RIIIS/J genes at this genetic location was associated with the absence of disease. In the BC, Eae2, or a locus close to Eae2, was linked to the trait auc. The confidence interval in this linkage extended further distant from the centromeric region compared with the linkage in the previous report. Moreover, homozygosity for the B allele was associated with disease. This discrepancy could be explained by the contribution of several genes of importance for the disease trait in this region. In the previous intercross experiment, the Eae3 locus was demonstrated to act together with Eae2 in an additive fashion (16). In the present BC experiment, we found a weak suggestive linkage to Eae3. The Eae3 locus has recently been confirmed in Eae3 congenic animals (M. Johannesson et al., manuscript in preparation), demonstrating that disease development is decreased in mice with two RIIIS/J alleles at this locus on the B10.RIII background. This explains why we do not find significant linkage to Eae3 in the BC experiment in which there are no individuals with RIIIS/J homozygous loci. Similarly, in the present BC experiment, we did not observe an association to chromosome 7, in which we recently described a locus linked to acute disease in an F2 intercross between B10.RIII and the Eae2 congenic B10.RIII.Eae2RIIIS/J (39). Linkage to this locus was, however, not expected in the BC because it requires RIIIS/J homozygous genes at Eae2. These results emphasize that inflammatory diseases such as EAE are genetically complex, and point toward the importance of the genetic context in which the genes are operating. Thus, the different genetic segregation pattern, and possibly also the usage of a modified immunization protocol, containing a lower concentration of MBP 89–101, led to the identification of several novel Eae loci in the BC.

The importance of T lymphocytes in the pathogenesis of EAE and MS is well established. T cells are believed to take part in the initiation of the disease process, but why T cells in the peripheral immune system lose tolerance to CNS Ags remains an open question. In the present study, lymphocyte phenotypes were investigated in addition to the EAE traits. The studies included proportions of splenic T and B cells, CD4+ and CD8+ T cells, in addition to proliferation of spleen cells upon stimulation. The proportions of lymphocyte populations are genetically controlled, as demonstrated by the difference in lymphocyte levels between different mouse strains (40, 41). The lymphocyte phenotypes are, like the disease phenotypes, dependent of many genes and of a complex interplay between genes. It is reasonable to believe, however, that some genes controlling subphenotypes, in addition, are involved in the pathways leading to disease. For some of the QTLs associated with EAE development, we found linkage to lymphocyte phenotypes, which will now be further investigated in mice congenic for the different EAE loci. Recently, a human study reported a number of QTLs controlling variations in blood lymphocyte subpopulations (42). Interestingly, a locus at the distal end of chromosome 1, which is homologous to the same end of mouse chromosome 1, showed linkage to the levels of CD8+ T cells, similar to what was found in mouse in the present study. In another study, this part of chromosome 1 was found to be linked to the proportion of B lymphocytes in mouse peripheral blood (43).

The ratio between CD4+ and CD8+ in the peripheral immune system is a reflection of the proportion of T cell subsets established in the thymus. It has been suggested that the CD4+/CD8+ ratio is not a result of thymic selection processes, but rather of genetically controlled intrinsic properties of the differentiating thymocyte population (44). In other studies, however, a role for the MHC locus in the determination of the levels of CD4+ and CD8+ cells has been reported (45, 46). The observation of a significant difference in the splenic CD4+/CD8+ ratio in the two MHC congenic mouse strains, B10.RIII and RIIIS/J, as reported in this work, in addition to the definition of three non-MHC regions controlling this trait, would suggest that other genes are operating as well. The ratio between CD4+ and CD8+ T cells was linked to different chromosomes and chromosomal regions than the percentage of the different subpopulations. The reason for this might be that the ratio is not dependent on the total number of T cells in the spleen, but rather on genes controlling the differentiation and maintenance of the respective subpopulation. For several of the QTLs linked to relative levels of CD4+ and CD8+ T cells, the inheritance pattern is opposite to the levels in the parental strains. This could reflect the activity of modifier genes in the parental strain that are lost due to gene segregation in the BC.

In the BC, we found strong linkage of different disease traits to the middle part of chromosome 11 containing several Eae loci. The ratio of B and T lymphocytes in spleen was linked to this region, showing that a high percentage of B cells was associated with homozygosity for the B allele, which is concomitant with the inheritance pattern for development of EAE.

In summary, we have identified four new QTLs linked to different traits of EAE on chromosomes 1, 5, 11, 15, and 16, and one suggestive linkage on chromosome 18 not previously demonstrated in crosses between the B10.RIII and RIIIS/J mouse strains. Two of the loci have not previously been identified in EAE mouse models. In addition, we analyzed a large number of animals from the BC for levels of lymphocyte subpopulations and found significant linkages to these traits on loci associated with disease development.


   Acknowledgments
 
We thank C. Palestros and L. Lindström for help with animal care, A.-K. Lindqvist for expertise in linkage analyses, and A. Treschow for linguistic corrections.


   Footnotes
 
1 This work was supported by grants from the Swedish Research Council; the Swedish Association for Neurologically Disabled; the Swedish Rheumatism Association; and the Magnus Bergvall, Tore Nilsson, Crafoord, Greta and Johan Kock, Åke Wiberg, and Österlund Foundations. X.Z. received a fellowship from the foundation Wenner-Grenska Samfundet. Back

2 Current address: Department of Medicine, College of Medicine, University of Illinois at Chicago, and VA Chicago Healthcare System (West Side Division), Chicago, IL 60612. Back

3 Address correspondence and reprint requests to Dr. Åsa Andersson, Section for Medical Inflammation Research, I11, BMC, S-221 84 Lund, Sweden. E-mail address: asa.andersson{at}inflam.lu.se Back

4 Abbreviations used in this paper: MS, multiple sclerosis; auc, area under the curve; BC, backcross; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; QTL, quantitative trait locus. Back

Received for publication August 19, 2002. Accepted for publication November 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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