The Journal of Immunology
Identification of a Major Susceptibility Locus for Lethal Graft-versus-Host Disease in MHC-Matched Mice1 Thai M. Cao,2* Laura C. Lazzeroni,† Schickwann Tsai,* Wendy W. Pang,‡ Amy Kao,‡ Nicola J. Camp,§ Alun Thomas,§ and Judith A. Shizuru‡ Graft-vs-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hemopoietic cell transplantation. From a genetic perspective, GVHD is a complex phenotypic trait. Although it is understood that susceptibility results from interacting polymorphisms of genes encoding histocompatibility Ags and immune regulatory molecules, a detailed and integrative understanding of the genetic background underlying GVHD remains lacking. To gain insight regarding these issues, we performed a forward genetic study. A MHC-matched mouse model was used in which irradiated recipient BALB.K and B10.BR mice demonstrate differential susceptibility to lethal GHVD when transplanted using AKR/J donors. Assessment of GVHD in (B10.BR ⴛ BALB.K)F1 mice revealed that susceptibility is a dominant trait and conferred by deleterious alleles from the BALB.K strain. To identify the alleles responsible for GVHD susceptibility, a genome-scanning approach was taken using (B10.BR ⴛ BALB.K)F1 ⴛ B10.BR backcross mice as recipients. A major susceptibility locus, termed the Gvh1 locus, was identified on chromosome 16 using linkage analysis (logarithm of the odds, 9.1). A second locus was found on chromosome 13, named Gvh2, which had additive but protective effects. Further identification of Gvh genes by positional cloning may yield new insight into genetic control mechanisms regulating GVHD and potentially reveal novel approaches for effective GVHD therapy. The Journal of Immunology, 2009, 183: 462– 469.
I
n a broadly accepted pathophysiological model, graft-vs-host disease (GVHD)3 occurs as a complication of allogeneic hemopoietic cell transplantation through a three-step process (1). First, recipient conditioning leads to tissue injury and inflammation. These events trigger, in the second phase, activation of tissue-resident host APCs that efficiently present alloantigen to donor T cells (2, 3). Ag-activated donor T cells consequently undergo differentiation and expansion and, in the final phase, recruit additional effector cells to cause tissue injury via a combination of cellular and humoral inflammatory mechanisms. This general framework of GVHD pathophysiology can be viewed, from a genetic perspective, as a complex phenotypic trait with a polygenic basis. Genetic susceptibility to GVHD is firstly conferred by deleterious alleles encoding histocompatibility Ags (HAg) that are disparate between donors and recipients. Allele effects of major HAg, encoded by classical MHC genes (4), and minor HAg, arising from endogenously expressed polymorphic au*Blood and Marrow Transplantation Program, Department of Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132; †Department of Psychiatry and Behavioral Sciences, Stanford University of School of Medicine, Stanford, CA 94305; ‡Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305; and §Department of Biomedical Informatics, University of Utah School of Medicine, Salt Lake City, UT 84132 Received for publication February 13, 2009. Accepted for publication April 22, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
This work was supported in part by Grants P01CA049605 and R01HL087240 (to J.A.S.) and K08HL067847 (to T.M.C.) from the National Institutes of Health.
2
Address correspondence and reprint requests to Dr. Thai M. Cao, Blood and Marrow Transplant and Myeloma Program, Division of Hematology, University of Utah School of Medicine, 30 North 1900 East, Room SOM 5C402, Salt Lake City, UT 84132. E-mail address:
[email protected]
tosomal gene products (5, 6), are the antigenic basis for allogeneic donor T cell activation. Whether or not mismatched HAg becomes functionally expressed as GVHD is contingent on further interaction with polymorphisms of immune regulatory modifier genes. Examples of modifier gene effects include allelic variants of cytokines and cytokine receptors, the protein products of which either dampen or augment graft-vs-host alloimmune responses (7). Although many of these genes and gene effects are well characterized, a detailed understanding of genetic control mechanisms underlying GVHD remains lacking. Further, novel molecular targets for effective GVHD therapy are still needed. To address these challenges, we initiated a forward genetic screen in a murine model of severe GVHD. In this model, irradiated BALB.K mice are susceptible whereas B10.BR mice are resistant to lethal GVHD when transplanted using hemopoietic cells from MHC-identical AKR/J donors (8). Using (B10.BR ⫻ BALB.K)F1 mice as transplant recipients, we found that lethal GVHD in these mice is a dominant gene effect with susceptibility conferred by deleterious alleles from the BALB.K strain. A backcross (BC) generated between the B10.BR parental strain and (B10.BR ⫻ BALB.K)F1 was then bred for genome scanning using linkage analysis. A major susceptibility locus on chromosome 16 was identified, which we have termed the Gvh1 locus. Gvh1 appears to regulate the development of lethal as opposed to mild and clinically insignificant GVHD. Underscoring the complex regulatory mechanisms controlling GVHD, a second locus with additive but protective BALB.K allele effects was identified on chromosome 13, termed Gvh2. These results lay the groundwork for positional cloning of Gvh genes and gene discovery as a prerequisite to developing new methods for predicting, preventing, or treating GVHD.
Materials and Methods Mouse strains and crosses
3
Abbreviations used in this paper: GVHD, graft-vs-host disease; HAg, histocompatibility Ag; BC, backcross; BM, bone marrow; TBI, total-body irradiation; LOD, logarithm of the odds. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900454
Mice were bred and maintained at the Stanford University Research Animal Facility (Stanford, CA). Hemopoietic cell donor AKR/J (H2k, Thy1.1) mice were 6 –10 wk of age at bone marrow (BM) harvest. Recipient MHC congenic BALB.K (H2k, Thy1.2) and B10.BR (H2k, Thy1.2) mice were
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⬎8 wk of age at the time of transplantation. Male BALB.K and female B10.BR mice were mated to generate a (B10.BR ⫻ BALB.K)F1 population. F1 mice were subsequently mated with females of the B10.BR parental strain to produce (B10.BR ⫻ (B10.BR ⫻ BALB.K)F1) BC mice for linkage analysis. All work using animals was reviewed and approved by the Administrative Panel on Laboratory Animal Care at the Stanford University School of Medicine.
nome-wide p values to define suggestive ( p ⬍ 0.63), significant ( p ⬍ 0.05), and highly significant ( p ⬍ 0.001) threshold levels of LOD scores for linkage were applied (15). For our backcross, this threshold corresponded to LOD scores of 1.4, 2.7, and 4.4, respectively. Approximate confidence intervals for the locations of linked loci were obtained using the 2.0-LOD dropoff method (16).
T cell isolation
Results
Donor T cells were isolated from the spleen of AKR/J mice by micromagnetic bead separation using methods modified from procedures previously described (9). For pan T cell isolation, splenocytes were labeled with PEconjugated anti-B220 (6B2), anti-Mac1 (M1/70), anti-DX5, and antiTer119 mAbs (BD Biosciences). This was followed by second-stage labeling with anti-PE MicroBeads for negative selection on a MidiMACS column (Miltenyi Biotech). The resulting unbound CD3⫹B220⫺ Mac1⫺DX5⫺Ter119⫺ T cells were ⬎99% pure as determined by FACS. For isolation of T cell subsets from spleen, PE-conjugated anti-CD4 (GK1.5) and anti-CD8 (53-6.7) were added, respectively, to the Ab labeling mixture for isolating CD8 (CD4⫺CD8⫹) and CD4 (CD4⫹CD8⫺) T cell populations, again by negative selection.
GVHD phenotype analysis BM transplantation and GVHD analyses were performed as previously described (8). Recipient mice were conditioned with a lethal dose of totalbody irradiation (TBI) delivered in two split fractions on day 0. A shieldedroom Phillips Unit Irradiator providing x-ray dose outputs of 250 kV and 15 mA was used for irradiation. The TBI dose was 800 cGy for BALB.K and 900 cGy for B10.BR, (B10.BR ⫻ BALB.K)F1, and BC mice. After irradiation, mice were injected with 2.5 ⫻ 106 BM cells plus defined doses of purified splenic T cells or T cell subsets. Survival, weight loss, and clinical signs of GVHD were monitored daily for 60 –100 days after transplantation. T cell chimerism in surviving mice was determined by peripheral blood FACS for Thy1.1 (OX-7) and Thy1.2 (53.21) expression. Selected mice were euthanized before death or at the end of experiments for histopathological analysis of liver, intestine, and skin where indicated.
Genotyping Genomic DNA was isolated from tail tips of BC and control mice using the DNeasy Tissue Kit according to the vendor’s instructions (Qiagen). Genotypes were performed by PCR amplification of 90 informative microsatellite markers using standard PCR conditions and cycling parameters (10). The markers spanned all 19 autosomal chromosomes and a list of microsatellite markers and genetic map position data is provided as supplemental Table I.4 The average internal spacing between markers was 13.0 cM. Proximal and distal markers for each chromosome were anchored within an average of 5.7 cM from chromosome ends. Marker position and order assignment were based on the Mouse Genome Database and accessed via The Jackson Laboratory Mouse Genome Informatics online resource (http://www.informatics.jax.org; Ref. 11). PCR amplicons were stained with ethidium bromide for size separation and allele determination on 4% agarose gels. Where necessary, markers were evaluated in duplicate until genotypes at all loci were determined for all BC mice to allow linkage analysis with no missing marker data.
Statistical and linkage analysis A 90-marker genome scan was completed by genotyping 180 BC mice that were phenotyped for lethal GVHD. Linkage analysis was performed using R/qtl version 1.05-2, an add-on package to the R general statistical package (12). GVHD susceptibility was analyzed as a binary trait: surviving mice were scored as 0; and nonsurviving mice were scored as 1. Simple interval mapping was performed using the expectation-maximization algorithm to test for maximum likelihood of single locus effects on a 1-cM grid along the genome (13). Single-locus effects were further evaluated by composite interval mapping implemented in R/qtl by performing the genome scan with the inclusion of significantly linked background loci as additive and interactive covariates. Background markers were chosen at the location of the maximum logarithm of the odds (LOD) score calculated by simple interval mapping. Hazard ratios for the GVHD phenotype according to genotype at linked loci was calculated using Cox proportional hazards regression with survival time as the dependent variable. Genome-wide significance thresholds were determined by empirical permutation testing using 1000 permutation replicates (14). Standard ge4
The online version of this article contains supplemental material.
Variable GVHD in H2k-matched mice We previously reported a mouse model of allogeneic hemopoietic cell transplantation that uses a single donor mouse strain, AKR/J, and two MHC-congenic recipients, BALB.K and B10.BR (8). In the prior studies, GVHD was induced by cotransferring purified hemopoietic stem cells and unseparated donor splenocytes into irradiated recipients. Because mapping susceptibility to a small genomic interval requires a large number of mice, this experimental protocol was modified to permit high-throughput GVHD phenotypic evaluation for linkage analysis. Thus, hemopoietic stem cells, which require a rigorous two-step isolation procedure for purification, was replaced with BM. In addition, splenic T cells isolated by micromagnetic bead separation were used in place of whole splenocytes. As shown in Fig. 1A, BALB.K mice conditioned with a lethal dose of whole-body irradiation and injected with AKR/J BM along with either of two doses of T cells developed aggressive and lethal GVHD, consistent with our prior studies. Median survival time after transplantation was 9 days. Before death, all BALB.K mice displayed clinical features of GVHD including bloody diarrhea, weight loss, ruffled fur, and hunched posture. In contrast, similar AKR/J 3 B10.BR transplants resulted in no mortality as survival of B10.BR mice given donor BM plus T cells did not differ from control mice given BM alone (Fig. 1B). Further consistent with our previous results was the observation that, although not associated with lethality, AKR/J 3 B10.BR transplants using BM and splenic T cells resulted in detectable mild GVHD. Clinically, this GVHD syndrome was manifested by minimal chronic weight loss in almost all recipients (data not shown). No overt skin lesions, diarrhea, or dysmotility was observed. Further evidence of mild GVHD was the finding that B10.BR mice given BM plus splenic T cells engrafted with full donor T cell chimerism, rather than mixed T cell chimerism as was observed when mice were given BM alone (Fig. 1C). Lastly, histological examination of B10.BR mice at day ⫹60 after transplant revealed low-grade GVHD pathology restricted to the liver and not present in the skin, ileum, or colon (Fig. 1D). By comparison, BALB.K mice sacrificed early in the transplant course at day ⫹5 before death exhibited severe GVHD pathology in both the colon and liver. No histopathological abnormalities were seen in B10.BR mice sacrificed at the day ⫹5 time point. Depending on the strain combination, GVHD mortality in MHC-identical, minor HAg-mismatched mice can be mediated by either CD4⫹ or CD8⫹ T cells alone, by both in combination with synergistic effects or not at all regardless of the graft cell composition (17). We characterized our GVHD model in this regard by depleting splenic T cell subsets to produce CD4 (CD4⫹CD8⫺) and CD8 (CD4⫺CD8⫹) T cell populations for transplant experiments. These studies showed that severe GVHD in AKR/J 3 BALB.K transplants was mediated primarily by the donor CD4⫹ T cell subset (Fig. 2A). Lethality following coinjection of BM and CD4⫹CD8⫺ T cells was rapid and uniform. Only a weak effect was seen with CD8⫹ T cells, reflected by a small proportion of mice dying after transplants of donor BM plus CD4⫺CD8⫹ T cells. Lethal GVHD could not be induced in AKR/J 3 B10.BR transplants with either CD4⫹ or CD8⫹ T cell subsets (Fig. 2B). Only late-onset liver GVHD histopathology was detected in all
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FIGURE 1. Variable graft-vs-host disease (GVHD) in MHC-matched AKR/J 3 BALB.K and AKR/J 3 B10.BR mice. Shown are survival for BALB.K (A) or B10.BR (B) recipient mice following lethal irradiation and injection of AKR/J BM and splenic T cells. C, FACS for peripheral blood T cell chimerism from representative recipients at day ⫹60 posttransplant in surviving B10.BR mice using Thy1.1 (donor) and Thy1.2 (recipient) markers. D, Histopathology of skin, ileum, colon, and liver from BALB.K and B10.BR recipient mice sacrificed at day ⫹5 or surviving B10.BR mice sacrificed at day ⫹60 posttransplant. Shown in the colon of BALB.K mice are areas of mononuclear cell infiltrate and crypt dropout (ⴱ), crypt destruction with sloughing of colonic mucosa (dashed arrow), and crypt abscesses with goblet cell depletion or total loss of crypt epithelia cells (solid arrows). In the liver of BALB.K mice are a periportal mononuclear cell infiltrate and extensive hepatocyte microvesicular steatosis and necrosis (outlined by solid arrowheads). A periportal mononuclear cell infiltrate is observed in B10.BR mice at day ⫹60 (outlined by open arrowheads) but not day ⫹5 posttransplant. No pathological abnormalities were seen in the skin and ileum of all mice.
B10.BR mice as well as surviving BALB.K mice given CD8⫹ T cells sacrificed before the end of each experiment (supplemental Fig. 1). Taken together, these results show that the GVHD phe-
notype in AKR/J ⫻ BALB.K and AKR/J ⫻ B10.BR mice closely resemble other established models of murine GVHD and was thus suitable for genetic studies. Lethal GVHD susceptibility is a dominant trait The most common strategy for performing a genome scan in mice involves breeding random genetic recombinants from parental strains that differ with regard to a trait of interest, followed by statistical analysis to identify chromosomal regions that segregate with the phenotype and are thus shared among affected individuals (18). To facilitate design of the experimental cross, we began by generating (B10.BR ⫻ BALB.K)F1 littermates to determine whether GVHD is the result of dominance or additive effects and to assess the directionality of the allele effect. As shown in Fig. 3, irradiated F1 mice transplanted with AKR/J BM and donor T cells developed rapidly aggressive GVHD with typical clinical features and uniform lethality. Only a transient latency in median survival time as compared with BALB.K parents was observed. These results show that inheritance of lethal GVHD susceptibility is governed by a dominant gene effect and that susceptibility is conferred by deleterious alleles from the BALB.K strain. GVHD susceptibility in a backcross
FIGURE 2. T cell subsets mediating GVHD in AKR/J 3 BALB.K and AKR/J a` B10.BR mice. Survival for BALB.K (A) or B10.BR (B) recipient mice following lethal irradiation and injection of AKR/J BM alone or with either unseparated T cells (CD4⫹CD8⫹) or CD4 (CD4⫹CD8⫺) and CD8 (CD4⫺CD8⫹) T cell subsets (f) compared with irradiation controls (䡺). Results are pooled from three independent experiments.
Having determined the genetic model and allele effect we next generated a [B10.BR ⫻ (B10.BR ⫻ BALB.K)]F1 backcross. Detection of dominance effects is more efficient with a backcross, requiring about one-half the progeny size of an F2 intercross population because of lower background genetic variance (18). The
The Journal of Immunology
FIGURE 3. Susceptibility to lethal GVHD is a dominant trait in AKR/ J 3 BALB.K mice. Shown is survival for (B10.BR ⫻ BALB.K)F1 recipient mice following lethal irradiation and injection of AKR/J BM and splenic T cells. Results are pooled from two representative independent experiments.
backcross was made to B10.BR parents, as a segregating locus in a backcross to the BALB.K strain would not contribute to phenotypic variance. We generated 180 (F1 ⫻ B10.BR) BC littermates, and the GVHD phenotype for these mice is shown in Fig. 4. The mice were divided into seven groups for lethal irradiation and injections of sex-matched AKR/J BM and T cells, each performed as individual experiments. When survival outcome for all 180 BC mice were pooled, 28 mice (16%) were found to have a phenotype similar to the BALB.K strain and died before day 20 posttransplant with clinical signs characteristic of GVHD. When follow-up was extended to 100 days posttransplantation, an intermediate phenotype emerged whereby 12 additional BC mice died at time points indicated by the survival curve. The cumulative mortality before day 100 was thus 40 mice (22%). BC mice surviving for ⬎100 days were considered to have survived the transplant without lethal GVHD. Similar to the resistant B10.BR strain, complete donor peripheral blood T cell engraftment was seen in all surviving BC mice by FACS (supplemental Fig. 2A). Surviving BC mice further resembled B10.BR parents in that variable liver abnormalities consistent with GVHD was detected in representative mice evaluated by histology (supplemental Fig. 2B). Interval mapping for lethal GVHD Individual BC mice were numbered and genomic DNA was isolated from tail tip sections before irradiation for a 90-marker genome-wide scan involving all 180 BC mice. Genotyping for markers was performed in duplicate as necessary until 100% of mice were genotyped at all makers for a total of 16,200 genotypes. The genotype distribution was 49.4% B10.BR homozygous and 50.6%
FIGURE 4. Lethal GVHD susceptibility in [B10.BR ⫻ (B10.BR ⫻ BALB.K)F1] BC mice. Shown is survival for recipient BC mice (n ⫽ 180) following lethal irradiation and injection of AKR/J BM and splenic T cells. Dashed vertical bars indicate cumulative number of BC mice with survival ⬍28 and ⬍100 days, respectively.
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FIGURE 5. Genome-wide scan for lethal GVHD reveals a highly significant susceptibility locus on chromosome 16. Interval mapping was performed for BC mice following transplantation using survival values dichotomized as a binary trait. Shown are LOD scores for survival for ⬍20 days (dashed line) or ⬍100 days (solid line) plotted according to chromosome position. Vertical lines on x-axis indicate positions of microsatellite markers used for genotyping. Horizontal dashed lines indicate significant (p ⬍ 0.05) and highly significant (p ⬍ 0.001) LOD threshold levels determined by permutation testing.
B10.BR/BALB.K heterozygous, not significantly different from the expected 50:50 distribution. We first evaluated for genotyping errors according to the method of Lincoln and Lander (19). Using the Haldane map function to convert genetic distances into recombination fraction and an assumed genotyping error rate of 0.01, we found no markers with significant error scores to suggest genotyping error. As a further test of genotype data integrity, we performed mock linkage analysis for coat color. BALB.K mice are albino, and B10.BR mice have black coat color. BC mice are either black or brown. Linkage analysis for coat color in the 180 BC mice identified precise localization to the agouti locus on chromosome 2 (data not shown), which regulates coat color variation in these mice (20). We then proceeded with genome-wide linkage analysis for lethal GVHD by interval mapping. We considered but ultimately rejected evaluating for linkage using survival days as a quantitative trait because nearly 80% of BC mice survived for ⬎100 days, a clear departure from the standard assumption of normal distribution for interval mapping. As shown in supplemental Fig. 3, a log10 transformation of the survival time failed to resolve the skewed
FIGURE 6. Interval mapping for lethal GVHD on chromosome 16. LOD scores for survival for ⬍20 days (dashed line) or ⬍100 days (solid line) plotted according to chromosome 16 markers. Marker names and map positions are indicated on the x-axis. A GVHD susceptibility locus, designated Gvh1, is demarcated by a rectangle indicating the 2-LOD confidence interval for survival ⬍100 days.
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FIGURE 7. Composite interval mapping for lethal GVHD. Composite interval mapping was performed by using D16Mit4 genotype as an additive covariate for genome-wide scanning. Shown are LOD scores on chromosomes reaching at least suggestive thresholds for survival ⬍20 days (dashed lines) or ⬍100 days (solid lines) plotted according to chromosome location.
phenotype distribution. Therefore, lethal GVHD was analyzed as a binary trait: surviving mice were scored as 0; and dying mice were scored as 1. Separate analyses were performed for mice with survival for ⬍20 days (n ⫽ 28 affected) or for ⬍100 days (n ⫽ 40 affected, cumulatively). Linkage analysis was performed by interval mapping using the expectation-maximization algorithm to test for maximum likelihood of single locus effects on a 1-cM grid along the genome. This procedure allows localization of genomic regions linked to the phenotype by analyzing coinheritance of genetic markers and phenotype. As shown in Fig. 5, a single highly significant ( p ⬍ 0.001) lethal GVHD susceptibility locus on chromosome 16 was found for both survival for ⬍20 days and survival for ⬍100 days. A minor peak associated with markers on chromosome 5 met suggestive but not significant thresholds for survival for ⬍20 days. Another minor peak was found at a locus on chromosome 13 that met suggestive, but again not significant, thresholds for survival ⬍100 days. No suggestive linkage was identified elsewhere throughout the genome. To obtain additional evidence for linkage, we applied a second mapping analysis using a different statistical test. We performed individual marker regression implemented by the Map Manager QTX package (21) and found an identical result, which was a single highly significantly linked locus on chromosome 16 (supplemental Table II). The additive statistic for this locus was a positive value (0.37), confirming that susceptibility was conferred by BALB.K alleles. All BC mice were genotyped for 10 additional markers along chromosome 16 and interval mapping was repeated. Results are shown in Fig. 6. For survival for ⬍100 days, a broad and complex linkage pattern was observed with an LOD peak of 9.1 at map position 29 cM, flanked by markers D16Mit4 and D16Mit138. This linkage was followed by a secondary peak with a LOD score of 8.1 at position 52.2 cM, proximal to D16Mit189. For survival
for ⬍20 days, the LOD peak was marginally offset in the centromeric direction at position 28.0 cM, and the peak LOD score was slightly higher at 9.4. The 2-LOD confidence interval for survival for ⬍100 days, extending from 17.0 cM to 57.6 cM, was used as boundaries for a new GVHD susceptibility locus which we term the Gvh1 locus. Numerous mouse histocompatibility H loci have been mapped to autosomal chromosomes, as shown in Fig. 8A. These gene regions include the H2 locus on chromosome 17and the well-characterized H60 locus on chromosome 10 (22). The Gvh1 locus is unique among these for being located on chromosome 16. The 2-LOD confidence interval for the Gvh1 locus extends from 23.6 to 86.1 Mb on the physical map, as shown in Fig. 8B. Composite interval mapping for lethal GVHD One explanation for the wide confidence interval for the Gvh1 locus is that more than one adjacent susceptibility locus was located on the same chromosome. To test for this possibility and to identify additional loci, a form of composite interval mapping was implemented by including D16Mit4 marker genotype as an additive cofactor for linkage analysis. This model assumes that the effect of a putative trait locus is not dependent on D16Mit4 marker genotype and allows detection of background loci with weak main effects. D16Mit4 at map position 27.3 cM on chromosome 16 was the individual marker with highest LOD score. As shown in Fig. 7, composite interval mapping controlling for additive D16Mit4 effects revealed that distal markers on chromosome 16 had a peak LOD score of 1.9 at position 55.2 cM. This finding indicates suggestive linkage for a second GVHD susceptibility locus on chromosome 16, but the significance threshold was not reached. Thus, whether or not more than one susceptibility locus is present on chromosome 16 could not be resolved with the current backcross. The chromosome 13 locus became significantly linked when all nonsurviving mice were scored as affected, including those with death between days 20 and 100, suggesting that this locus may have contributed to the intermediate phenotype. The peak LOD score was 3.4 and, in contrast to the Gvh1 locus, protective rather than deleterious effects were conferred by BALB.K alleles at the chromosome 13 locus. That is, BC mice homozygous for B10.BR alleles at this locus had higher susceptibility than mice that were heterozygous with both B10.BR and BALB.K alleles. This locus is designated Gvh2. No evidence for epistatic interactions between this chromosome 13 locus and Gvh1 was observed when composite interval mapping included D16Mit4 marker genotype as an interactive covariate, or when BC mice were partitioned into two groups according to D16Mit4 genotype for simple interval mapping (supplemental Fig. 4). Regression analysis of Gvh1 and Gvh2 Cox regression was used to calculate hazard ratios for lethal GVHD in BC mice according to their genotypes at D16Mit4 and D13Mit248. These markers were, respectively, the individual
Table I. Regression analysis of Gvh genotype and lethal GVHD Survival for ⬍100 Days
Genotype Group
No. of BC Mice
No. of Deaths
Gvh1 (D16Mit4)
Gvh2 (D13Mit248)
Hazard ratio
p
1 2 3 4
51 38 49 42
2 1 28 9
B10/B10 B10/B10 B10/BALB B10/BALB
B10/B10 B10/BALB B10/B10 B10/BALB
1 0.7 (0.06–7.4)a 20.3 (4.8–85.3) 6.3 (1.4–29.3)
0.75 ⬍0.001 0.02
a
Numbers in parentheses, 95% confidence intervals.
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markers with peak LOD value for Gvh1 and Gvh2. As shown in Table I, we considered a proportional hazard model where baseline risk was conferred by the presence of homozygous B10.BR alleles at both loci. Compared with this reference group, BC mice with a deleterious BALB.K allele at Gvh1 had a 20-fold risk of death before day ⫹100 posttransplantation (hazard ratio, 20.3; 95% confidence interval, 4.8 – 85.3; p ⬍ 0.001) if Gvh2 remained B10.BR homozygous. The risk of lethal GVHD was reduced to 6-fold, however, if a BALB.K allele at Gvh1 was also accompanied by heterozygosity at Gvh2 (hazard ratio, 6.3; 95% confidence interval, 1.4 –29.3; p ⫽ 0.02), where the presence of a BALB.K allele was protective.
Discussion The overall picture of susceptibility that emerged from our genome scan is consistent with the idea that severe GVHD is a complex trait, because no one-to-one gene to phenotype relationship was identified in our mice. That said, major susceptibility was conferred by deleterious alleles from the BALB.K background with strong effect at a single locus on chromosome 16, the Gvh1 locus. Genetic control of GVHD was influenced by a second locus on chromosome 13, the Gvh2 locus, which acted independently of Gvh1 and contributed an additive but opposite allele effect. The Gvh2 locus was not significant when mice dying between days 20 and 100 were excluded from analysis, suggesting a contribution to the intermediate phenotype of delayed GVHD lethality. Lastly, the fully susceptible BALB.K parental strain by itself expresses both deleterious and protective alleles from Gvh1 and Gvh2, respectively. This is likely indicative of additional background gene effects not detected with the current genome scan. In this study, we performed a classical linkage analysis for lethal GVHD involving an experimental backcross in a mouse model. Data from linkage analyses and recent advances in mouse genome informatics, congenic and in silico haplotype mapping techniques, and high-throughput methodologies, such as gene expression profiling, have allowed accelerated positional cloning of many genes underlying susceptibility loci that are responsible for important physiological traits in mice. These include the cloning of latexin as a regulator of hemopoietic stem cell size, histamine receptor H1 as a mediator of T cell responses in autoimmune disease, complement factor 5 as a modifier of liver fibrosis, and the serine-threonine kinase ROP18 as a key host effector mechanism controlling Toxoplasma gondii virulence (23–26). It is our anticipation that similar strategies may allow identification of Gvh genes that will be clinically useful in the management of GVHD complications. Forward genetic approaches for finding GVHD susceptibility genes in mouse models have otherwise been sparingly used. As of this writing, the only previously published series of genetic linkage analyses investigated the trait variance in GVHD that results from alternatively using C57BL/6 vs DBA/2 parents as donors for transplants into (C57BL/6 ⫻ DBA/2)F1 recipients (27–30). In this model, transfer of C57BL/6 lymphoid cells results in development of acute GVHD, whereas transfer of DBA/2 lymphoid cells results in chronic GVHD with features of weight loss, autoantibody production, and nephritis. Several quantitative trait loci linked to chronic, rather than acute, GVHD were identified that mapped to chromosomes 1, 2, 4, and 17. The current report adds to these efforts by identifying novel loci on chromosomes 13 and 16 with major effects in conferring susceptibility to severe acute GVHD. Further, the mice used in our studies are MHC matched and minor Hag mismatched, a genetic combination more similar to clinical allogeneic hemopoietic cell transplantation. This major susceptibility locus, Gvh1, was associated with a complex linkage pattern and a wide confidence interval that
FIGURE 8. Physical location of the Gvh1 locus. A, Diagram of previously mapped autosomal histocompatibility H loci. B, Mouse chromosome 16 physical map illustrating genes and quantitative trait loci (QTL) previously mapped within the 2 LOD confidence interval denoted by horizontal bar.
spanned a large segment of chromosome 16. Although it is possible that closely linked allelic genes on the same chromosome account for this broad linkage pattern, we found that Gvh1 could not be fractionated into more than one detectable sublocus with the current mouse cross. In this regard, prior efforts describing congenic mapping of quantitative trait loci are insightful for the diverse genetic control mechanisms that may be uncovered. A single trait locus can in fact map to a single chromosomal segment and provide significant refinement of a confidence interval defined by standard linkage analysis (31). More often, however, two or even three separately linked subloci are found to underlie the single linkage-derived trait locus (32–34). Alternatively, a trait locus may be revealed to encompass multiple discrete gene effects with additive as well as epistatic elements, or two separate loci with additive but negating opposite allelic effects (35, 36). These and other possible outcomes can be envisioned when the Gvh1 locus is further interrogated by fine mapping. On the basis of current understanding of GVHD pathophysiology, we hypothesize that strain-specific polymorphisms of Gvh genes encoding immunodominant minor HAg and/or immune regulatory molecules are responsible for causing lethal GVHD in our model. We may eventually find a minor HAg-mediated basis underlying Gvh gene effects through positional cloning of susceptibility loci linked to lethal GVHD in our model. Because MHC restriction for murine minor HAg had no direct human MHC peptide binding equivalent, more clinically relevant may be discovering Gvh genes encoding novel immune regulatory molecules with a human homolog. The significance of important modifier genes that interact with mismatched major or minor HAg to influence GVHD is best illustrated by studies of cytokine polymorphisms. In humans, numerous studies have now implicated significant associations between GVHD and polymorphisms of genes for the IL-1, IL-2, IL-4, IL-6, IL-10, IL-18, IFN-␥, TGF-, and TNF-␣ cytokines and, in some instances, for their respective receptors as well
468 (7). Tissue injury resulting from recipient conditioning, particularly in the gastrointestinal tract, is a major source of inflammatory cytokines in the immediate posttransplant period (37). Mouse strains differ in their sensitivity to irradiation (38); thus, it is possible that variable response to TBI conditioning may contribute to GVHD susceptibility as well. In the context of these potential Gvh gene characteristics, we note that the 2 LOD confidence interval for the Gvh1 locus extends from 23.6 to 86.1 Mb on the physical map, as shown in Fig. 8B. A discussion of genes of interest is premature but the T cell costimulatory molecules CD80 and CD86 merit highlighting. Polymorphisms of the CD86 gene in humans have been characterized and emerging data suggests a functional effect by these variants on transplantation tolerance and allergic and autoimmune disease (39 – 41). The ␣v5 integrin is expressed in human dendritic cells and is important for Ag cross-presentation (42). Stefins A1, A2, and A3 (stfa1, stfa2, and stfa3) inhibit cysteine endo- and exopeptidases important in Ag processing, such as cathepsins L and S and may influence mHAg processing (43). Specific cathepsins have been shown to be important for initiating autoimmune disease, such as diabetes in the nonobese diabetic mouse model (44), and pharmacological inhibitors may be therapeutically promising in those settings (45). The CD200 surface Ag and receptor system are active in regulating immune responses related to allergic and autoimmunity conditions (46). The GA-binding protein is essential for the regulation of IL-7 receptor expression in T cells (47). Previously mapped within this region are quantitative trait loci for autoimmune ovarian dysgenesis (Aod1), collagen-induced arthritis (Lp1), Leishmania susceptibility (Lmr12), IL-4 and IL-10 production (Cypr1), and experimental allergic encephalomyelitis (Eae11; Refs. 48 –52). We present here a unique mouse model of GVHD where the genetic basis may be dissected to the level of gene identification by positional cloning of linked loci found in this study. Successful identification of a Gvh minor HAg would yield new insight into the biology of GVHD and unveil a snapshot of the genetic architecture underlying this complex trait. Alternatively, identification of a novel Gvh immune regulatory molecule with a human homolog could directly lead to testing of clinical hypotheses and possibly new therapies.
Acknowledgments We gratefully acknowledge Lucino Hidalgo for excellent care of our mouse colony and Dr. Guidot Tricot for a critique of the manuscript.
Disclosures The authors have no financial conflict of interest.
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