Functional and genetic analysis of two CD8 T cell subsets defined by the level of CD45RC expression in the rat

Differential cytokine production by T cells plays an important role in the outcome of the immune response. We show that the level of CD45RC expression differentiates rat CD8 T cells in two subpopulations, CD45RC(high) and CD45RC(low), that have different cytokine profiles and functions. Upon in vitro stimulation, in an Ag-presenting cell-independent system, CD45RC(high) CD8 T cells produce IL-2 and IFN-gamma while CD45RC(low) CD8 T cells produce IL-4, IL-10, and IL-13. In vitro, these subsets also exhibit different cytotoxic and suppressive functions. The CD45RC(high)/CD45RC(low) CD8 T cell ratio was determined in Lewis (LEW) and Brown-Norway (BN) rats. These two rat strains differ with respect to the Th1/Th2 polarization of their immune responses and to their susceptibility to develop distinct immune diseases. The CD45RC(high)/CD45RC(low) CD8 T cell ratio is higher in LEW than in BN rats, and this difference is dependent on hemopoietic cells. Linkage analysis in a F(2)(LEW x BN) intercross identified two quantitative trait loci on chromosomes 9 and 20 controlling the CD45RC(high)/CD45RC(low) CD8 T cell ratio. This genetic control was confirmed in congenic rats. The region on chromosome 9 was narrowed down to a 1.2-cM interval that was found to also control the IgE response in a model of Th2-mediated disorder. Identification of genes that control the CD45RC(high)/CD45RC(low) CD8 T cell subsets in these regions could be of great interest for the understanding of the pathophysiology of immune-mediated diseases.     

Genetic isolation of quantitative trait loci for blood pressure development and renal mass on chromosome 5 in the spontaneously hypertensive rat

Total genome scans of genetically segregating populations derived from spontaneously hypertensive rats (SHR) and other rat models of essential hypertension suggested a presence of quantitative trait loci (QTL) regulating blood pressure on multiple chromosomes, including chromosome 5. The objective of the current study was to test directly a hypothesis that chromosome 5 of the SHR carries a blood pressure regulatory QTL. A new congenic strain was derived by replacing a segment of chromosome 5 in the SHR/Ola between the D5Wox20 and D5Rat63 markers with the corresponding chromosome segment from the normotensive Brown Norway (BN/Crl) rat. Arterial pressures were directly monitored in conscious, unrestrained rats by radiotelemetry. The transfer of a segment of chromosome 5 from the BN strain onto the SHR genetic background was associated with a significant decrease of systolic blood pressure, that was accompanied by amelioration of renal hypertrophy. The heart rates were not significantly different in the SHR compared to SHR chromosome 5 congenic strain. The findings of the current study demonstrate that gene(s) with major effects on blood pressure and renal mass exist in the differential segment of chromosome 5 trapped within the new SHR.BN congenic strain.     

Localization of a blood pressure QTL to a 2.4-cM interval on rat chromosome 9 using congenic strains

A blood pressure (BP) quantitative trait locus (QTL) was previously found on rat chromosome 9 using Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. A congenic strain, S.R(chr9), constructed by introgressing an R chromosomal segment into the S background, previously proved the existence of a BP QTL in a large 34.2-cM segment of chromosome 9. In the current work congenic substrains were constructed from the progenitor congenic strain, S.R(chr9). BP and heart weight comparisons between these congenic substrains and their S control localized the BP QTL to a 4.6-cM interval. Two solute carrier (Na(+)/H(+) exchanger) genes, Nhe2 and Nhe4, were excluded as candidates based on their map locations. A second iteration of congenic substrains was used to localize the QTL further to a 2.4-cM interval. Another solute carrier (Cl(-)/HCO3- exchanger) gene, Ae3, is in this reduced interval and was sequenced for both S and R strains, but no coding sequence variations were found. Ae3 mRNA was not differentially expressed in the kidney of congenic compared to S rats. Although the identity of the QTL remains unknown its map location has been reduced from an interval of 34.2 to 2.4 cM.     

Pnlip encoding pancreatic lipase is possible candidate for obesity QTL in the OLETF rat

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat exhibits polygenic obesity, and one of quantitative trait loci (QTLs) responsible for a susceptibility to obesity in the OLETF, Nidd6/of, has been mapped to the approximately 10-cM genomic region between D1Rat166 and D1Rat90 on chromosome 1 in (OLETF x normal) F2 intercross. In this study, we have attempted to identify the causal gene for the Nidd6/of QTL. A Nidd6/of congenic strain, constructed by introgressing the OLETF allele on the mapped Nidd6/of region in the normal F344 rat strain, confirmed the existence of the Nidd6/of as obesity QTL. The Nidd6/of region was refined to a approximately 2.3-cM genomic region between D1Rat225 and D1Rat90, using informative recombinants selected from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. Among 46 genes located within the approximately 2.3-cM region, pancreatic lipase gene, Pnlip, was regarded as the most prominent and physiologically relevant positional candidate for the Nidd6/of QTL. We found that Pnlip possesses an OLETF allele-specific increase of mRNA levels in the pancreas, and that the OLETF allele is longer in variable number of tandem repeat (VNTR) within the 5'-flanking region than normal alleles. We further showed that the Nidd6/of QTL completely cosegregates with Pnlip VNTR in the informative recombinants from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. These results suggest that Pnlip is possible candidate for the Nidd6/of QTL.     

Mapping diabetes QTL in an intercross derived from a congenic strain of the Brown Norway and Goto-Kakizaki rats

Genetic studies in experimental crosses derived from the inbred Goto-Kakizaki (GK) rat model of spontaneous diabetes mellitus have identified quantitative trait loci (QTL) for diabetes phenotypes in a large region of rat Chromosome (RNO) 1. To test the impact of GK variants on QTL statistical and biological features, we combined genetic and physiologic studies in a cohort of F₂ hybrids derived from a QTL substitution congenic strain (QTLSCS) carrying a 110-cM GK haplotype of RNO1 introgressed onto the genetic background of the Brown Norway (BN) strain. Glucose intolerance and altered insulin secretion in QTLSCS rats when compared with BN controls were consistent with original QTL features in a GK × BN F₂ cross. Segregating GK alleles in the QTLSCS F₂ cross account for most of these phenotypic differences between QTLSCS and BN rats. However, significant QTL for diabetes traits in both the QTLSCS and GK × BN F₂ cohorts account for a similar small proportion of their variance. Comparing results from these experimental systems provides indirect estimates of the contribution of genetic interactions and environmental factors to QTL architecture as well as locus and biological targets for future post-QTL mapping studies in congenic substrains. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Stephan C. Collins and Robert H. Wallis contributed equally to this work.     

Mapping of rat Chromosome 2 markers generated from chromosome-sorted DNA

A quantitative trait locus (QTL) for blood pressure has recently been mapped to a region of roughly 30 cM on rat Chromosome (Chr) 2 by linkage and by the use of congenic strains. For further fine mapping of the QTL, however, closely linked chromosome markers residing in this 30-cM region are required. In the current work, 36 new markers were generated by screening rat Chr 2-sorted DNA libraries and subsequently mapped using five F₂ populations. Combining new and existing markers, the marker density for the 30-cM region approaches, on average, one marker per 1.1 cM. Received: 11 April 1997 / Accepted: 12 May 1997     

Two loci, Tmevp2 and Tmevp3, located on the telomeric region of chromosome 10, control the persistence of Theiler's virus in the central nervous system of mice

Theiler's virus persistently infects the white matter of the spinal cord in susceptible strains of mice. This infection is associated with inflammation and primary demyelination and is studied as a model of multiple sclerosis. The H-2D gene is the major gene controlling viral persistence. However, the SJL/J strain is more susceptible than predicted by its H-2(s) haplotype. An (SJL/J x B10. S)F1 x B10.S backcross was analyzed, and one quantitative trait locus (QTL) was located in the telomeric region of chromosome 10 close to the Ifng locus. Another one was tentatively mapped to the telomeric region of chromosome 18, close to the Mbp locus. We now report the study of 14 congenic lines that carry different segments of these two chromosomes. Although the presence of a QTL on chromosome 18 was not confirmed, two loci controlling viral persistence were identified on chromosome 10 and named Tmevp2 and Tmevp3. Furthermore, the Ifng gene was excluded from the regions containing Tmevp2 and Tmevp3. Analysis of the mode of inheritance of Tmevp2 and Tmevp3 identified an effect of sex, with males being more infected than females.     

Experimental autoimmune myasthenia gravis may occur in the context of a polarized Th1- or Th2-type immune response in rats.

Experimental autoimmune myasthenia gravis (EAMG) is a T cell-dependent, Ab-mediated autoimmune disease induced in rats by a single immunization with acetylcholine receptor (AChR). Although polarized Th1 responses have been shown to be crucial for the development of mouse EAMG, the role of Th cell subsets in rat EAMG is not well established. In the present work we show that while the incidence and severity of EAMG are similar in Lewis (LEW) and Brown-Norway (BN) rats, strong differences are revealed in the immune response generated. Ag-specific lymph node cells from LEW rats produced higher amounts of IL-2 and IFN-gamma than BN lymph node cells, but expressed less IL-4 mRNA. IgG1 and IgG2b anti-AChR isotype predominated in BN and LEW rats, respectively, confirming the dichotomy of the immune response observed between the two strains. Furthermore, although IL-12 administration or IFN-gamma neutralization strongly influenced the Th1/Th2 balance in BN rats, it did not affect the disease outcome. These data demonstrate that a Th1-dominated immune response is not necessarily associated with disease severity in EAMG, not only in rats with disparate MHC haplotype but also in the same rat strain, and suggest that in a situation where complement-fixing Ab can be generated as a consequence of either Th1- or Th2-mediated T cell help, deviation of the immune response will not be an adequate strategy to prevent this Ab-mediated autoimmune disease.     

An interspecific backcross linkage map of the proximal half of mouse chromosome 14

We have generated a 30-cM molecular genetic linkage map of the proximal half of mouse chromosome 14 by interspecific backcross analysis. Loci that were mapped in this study include Bmp-1, Ctla-1, Hap, hr, Plau, Psp-2, Rib-1, and Tcra. A region of homology between mouse chromosome 14 and human chromosome 10 was identified by the localization of Plau to chromosome 14. This interspecific backcross map will be valuable for establishing linkage relationships of additional loci to mouse chromosome 14.     

Genetic mapping of QTL controlling tissue-culture response on chromosome 2B of wheat (Triticum aestivum L.) in relation to major genes and RFLP markers

 Three quantitative trait loci (QTL) for tissue- culture response (Tcr) were mapped on chromosome 2B of hexaploid wheat (Triticum aestivum L.) using single-chromosome recombinant lines. Tcr-B1 and Tcr-B2, affecting both green spots initiation and shoot regeneration, were mapped in relation to RFLP markers in the centromere region and on the short arm of chromosome 2B, linked to the photoperiod-response gene Ppd2. A third QTL (Tcr-B3), influencing regeneration only, was closely related to the disease resistance locus Yr7/Sr9g on the long arm of chromosome 2B. The homoeologous relationships to the tissue-culture response loci Qsr, Qcg and Shd of barley are discussed. A possible influence of the earliness per se genes of wheat and barley is suggested. Received: 30 August 1996 / Accepted: 15 November 1996     

Comparative analysis and development of microsatellite markers on swine (Sus scrofa) chromosome 1qter

Several quantitative trait loci (QTL) have been detected on SSC1qter (Sus scrofa chromosome 1qter), including QTL for the number of vertebrae, as reported in our previous study. To provide the tools for analysis of QTLs on SSC1qter, we constructed a comparative map of swine and human. In addition, we identified 26 swine STSs and mapped 16 of them on SSC1qter using the INRA - University of Minnesota porcine radiation hybrid (IMpRH) panel. We screened a BAC library using these swine STSs and developed 35 new polymorphic microsatellite markers from the BAC clones, of which 26 were informative in our reference family. We also mapped nine microsatellite markers we had isolated previously. Consequently a total of 44 new polymorphic microsatellite markers were located within a 60-cM region of SSC1qter, spanning from SW1092 to the telomere.     

Identification of two quantitative trait loci involved in antibody production on mouse chromosome 8

 Several quantitative trait loci (QTLs) contributing to the extreme phenotypes of the selected high (H) and low (L) antibody-responder lines of mice were mapped on distinct chromosomes. Successive backcrosses were bred to reduce the length of the QTL-bearing segment detected on chromosome 8 and to produce congenic lines to test gene effect independently of the other QTLs. An increase in antibody responses was repeatedly found to be associated with inheritance of the H-line allele at two markers separated by 30 cM on that chromosome. In the successive backcrosses, background and unlinked involved genes of H-line origin were progressively eliminated; however, unexpected within-progeny variations persisted in the third and even fourth backcross. Nevertheless, the presence of two QTLs within the considered interval was definitely demonstrated in distinct progenies of the fourth backcross which separately inherited one of the two gene-marker H-line alleles. The previously identified chromosome 8 segment therefore contains at least two QTLs involved in antibody responsiveness. Received: 19 August 1997 / Revised: 9 October 1997     

Alignment of VIM, MRC1, GAD2, and IL2RA genes on swine chromosome 10q by in situ hybridization and RH mapping

Since the distal half of swine chromosome (SSC) 10q was shown to contain a quantitative trait locus (QTL) influencing swine growth, the precise correspondence between this chromosome region and the orthologous human chromosome region (HSA10p) was investigated using chromosomal fluorescence in situ hybridization and RH mapping of type I loci spanning the growth QTL. The goal was to align this critical region of swine with the corresponding region in human for the purpose of identifying candidate genes. The HSA10p type I loci mapped in swine were VIM, MRC1, GAD2, and IL2RA. Locus order on SSC10q was shown to be centromere-VIM-MRC1-GAD2-IL2RA, while in human the order is centromere-GAD2-MRC1-VIM--IL2RA, indicating that the chromosome segment marked by VIM, MRC1 and GAD2 has been inverted relative to the centromere and IL2RA.     

Genome-wide linkage analysis of chronic relapsing experimental autoimmune encephalomyelitis in the rat identifies a major susceptibility locus on chromosome 9

The immunization of inbred Dark Agouti (DA) rats with an emulsion containing homogenized spinal cord and CFA induces chronic relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis. We report here the first genome-wide search for quantitative trait loci regulating EAE in the rat using this model. We identified one quantitative trait locus on chromosome 9, Eae4, in a [DA(RT1av1) x BN(RT1n)]F2 intercross showing linkage to disease susceptibility and expression of mRNA for the proinflammatory cytokine IFN-gamma in the spinal cord. Eae4 had a larger influence on disease incidence among rats that were homozygous for the RT1av1 MHC haplotype (RT1av1 rats) compared with RT1n/av1 rats, suggesting an interaction between Eae4 and the MHC. Homozygosity for the DA allele at markers in Eae4 and in the MHC was sufficient for EAE. Thus, Eae4 is a major genetic factor determining susceptibility to EAE in this cross of DA rats. In addition, there was support for linkage to phenotypes of EAE on chromosomes 1, 2, 5, 7, 8, 12, and 15. The chromosome 12 region has been shown previously to predispose DA rats to arthritis, and the chromosome 2 region is syntenic to Eae3 in mice. We conclude that Eae4 and probably the other identified genome regions harbor genes regulating susceptibility to neuroinflammatory disease. The identification and functional characterization of these genes may disclose critical events in the pathogenesis of multiple sclerosis; understanding these events could be essential for the development of new therapies against the disease.     

A murine locus on chromosome 18 controls NKT cell homeostasis and Th cell differentiation

Th cell differentiation is a critical event in the adaptive immune response. C57BL strains develop predominant Th1 responses while BALB/c develops a predominant Th2 response. To identify quantitative trait loci controlling this variation, we performed Th1/Th2 differentiation assays of F(1) x BALB/c progeny. A single strong quantitative trait locus was identified on chromosome 18, with weaker effects detectable on chromosomes 5, 12, and 14. By preparing a congenic BALB.B10.D2c18 strain, we were able to demonstrate that this single locus was sufficient to "repolarize" spleen cell cultures. This difference was not due to intrinsic differences in CD4(+) T cells. Rather, introgression of the chromosome 18 locus into BALB/c disrupted Va14Ja18 NKT cell homeostasis resulting in the almost complete absence of this T cell subset. Taken together, these data indicate that genes within chromosome 18 control strain-dependent development of Va14Ja18 NKT cells.     

Fine mapping of quantitative trait loci for improved fruit characteristics from Lycopersicon chmielewskii chromosome 1.

The near-isogenic line (NIL) TA1150 contains a 56-cM introgression from Lycopersicon chmielewskii chromosome 1 and has several interesting phenotypic characteristics including fruit with orange color, high levels of soluble solids, thick pericarp, small stem scars, and good firmness. A set of overlapping recombinant lines (subNILs) was developed and field tested to fine map the quantitative trait loci (QTL) controlling these traits. The results indicated that the solids, pericarp thickness, and firmness QTL are distinct from the color locus. Several of the QTL mapped in this study, including the soluble-solids QTL, probably correspond to QTL mapped in other wild species of tomato. However, analysis of a set of TA523 subNILs containing complementary introgressions from Lycopesicon hirsutum chromosome 1 suggests that this wild species may contain a different locus for improved soluble solids. Thus, it might be possible to combine the L. chmielewskii and L. hirsutum alleles for these loci in a single line with the potential for extremely highly soluble solids. The TA1150 subNIL TA1688 contains the smallest introgression of the solids locus (approximately 19 cM), as well as the pericarp thickness and firmness QTL, with a yield that was equivalent to two of the three control lines. Isolation of recombinant subNILs from TA1688 should break the linkage between orange color and high solids and provide a small introgressed segment for marker-assisted breeding and genetic improvement of processing tomato.     

A high-density genetic map of the chromosome 13q14 atopy locus

Atopy describes a syndrome of immunoglobulin E (IgE)-mediated allergy that underlies asthma and infantile eczema. We have previously identified a locus on chromosome 13q14 that is linked to atopy and to the total serum immunoglobulin A concentration. We have therefore made a saturation genetic map of the region by typing 59 polymorphic microsatellite loci on chromosome 13q. Multipoint linkage analysis identified a 1-LOD support unit for the location of the atopy locus with a 7.5-cM region flanked by the loci D13S328 and D13S1269. The peak of linkage was at locus D13S161 with a nonparametric -log of P score of approximately 4.5. Parent of origin effects were present, with linkage primarily observed to paternally derived alleles. The genetic map of this region provides a basis for the effective identification of the chromosome 13 atopy gene.     

Polyclonal IgE increase after H9CI2 injections in BN and LEW rats: A genetic analysis

An autoimmune disease and a dramatic increase in total serum IgE concentration are observed in BN rats that are chronically injected with HgCl₂. In contrast, LEW rats do not develop the characteristic glomerulonephritis and are very low IgE responders. In this study, we examined the genetic control of total serum IgE increase after HgCl₂ injection in F₁ and F₂ hybrids, in both backcrosses between LEW and BN rats, and in LEW.1N congenic rats. Genetic analysis was performed using peak IgE concentrations expressed as log g/ml. A high IgE phenotype was found to be dominant. Eighty-five percent of F₂ variance was due to genetic factors (VG) while only 15% of this variance was caused by environmental factors (VE). From observations in F₂ hybrids and backcrosses, estimations of additive variance (VA) and dominance variance (VD) were made following three different methods. Genetic control by about four loci is demonstrated. One of these genes is RT1-linked. This gene contributes to 25% of the phenotypic difference observed between BN and LEW rats. No correlation was found between the peak total IgE level and autoimmune disease based on IgG deposition in spleen and/or kidney.     

A new locus for autosomal dominant stargardt-like disease maps to chromosome 4

Stargardt disease (STGD) is the most common hereditary macular dystrophy and is characterized by decreased central vision, atrophy of the macula and underlying retinal-pigment epithelium, and frequent presence of prominent flecks in the posterior pole of the retina. STGD is most commonly inherited as an autosomal recessive trait, but many families have been described in which features of the disease are transmitted in an autosomal dominant manner. A recessive locus has been identified on chromosome 1p (STGD1), and dominant loci have been mapped to both chromosome 13q (STGD2) and chromosome 6q (STGD3). In this study, we describe a kindred with an autosomal dominant Stargardt-like phenotype. A genomewide search demonstrated linkage to a locus on chromosome 4p, with a maximum LOD score of 5.12 at a recombination fraction of.00, for marker D4S403. Analysis of extended haplotypes localized the disease gene to an approximately 12-cM interval between loci D4S1582 and D4S2397. Therefore, this kindred establishes a new dominant Stargardt-like locus, STGD4.