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The Journal of Immunology, 1999, 162: 4502-4510.
Copyright © 1999 by The American Association of Immunologists

Biochemical and Immunogenetic Analysis of an Immunodominant Peptide (B6dom1) Encoded by the Classical H7 Minor Histocompatibility Locus1

Peter A. Eden2,*, Gregory J. Christianson*, Pierre Fontaine{dagger}, Peter J. Wettstein{ddagger}, Claude Perreault{dagger} and Derry C. Roopenian*

* The Jackson Laboratory, Bar Harbor, ME 04609; {dagger} Research Center, Maisonneuve-Rosemont Hospital, Montreal, Canada; and {ddagger} Department of Surgery and Immunology, Mayo Foundation, Rochester, MN 55901


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Of the many minor histocompatibility (H) Ags that have been detected in mice, the ability to induce graft vs host disease (GVHD) after bone marrow transplantation is restricted to a limited number of immunodominant Ags. One such murine Ag, B6dom1, is presented by the H2-Db MHC class I molecule. We present biochemical evidence that the natural B6dom1 peptide is indistinguishable from AAPDNRETF, and we show that this peptide can be isolated from a wide array of tissues, with highest levels from the lymphoid organs and lung. Moreover, we employ a novel, somatic cell selection technique involving CTL-mediated immunoselection coupled with classical genetics, to show that B6dom1 is encoded by the H7 minor H locus originally discovered ~40 years ago. These studies provide a molecular genetic framework for understanding B6dom1, and exemplify the fact that mouse minor H loci that encode immunodominant CTL epitopes can correspond to classical H loci originally identified by their ability to confer strong resistance to tumor transplantation. Additionally, these studies demonstrate the utility of somatic cell selection approaches toward resolving H Ag immunogenetics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Minor histocompatibility (H)3 Ags are polymorphic peptides derived from intracellular processing of cellular proteins and presented in the Ag groove of MHC molecules for recognition by T cells (1, 2, 3). CD8+ CTLs recognize on APCs the complex formed by minor H peptides and specific class I molecules. Clinically, such CTLs are implicated in chronic graft rejection and graft vs host disease (GVHD) in MHC-matched recipients (reviewed in 4). In addition, antileukemic activity seen after bone marrow transplantation has been ascribed to CTL cell recognition of cancer cell-associated minor H Ags (4, 5).

CTL responses generated after immunization with allogeneic cells presenting multiple incompatible minor H Ag epitopes vary from strong (immunodominant) to weak (dominated) (reviewed in 6 and 7). Indeed, in all mouse strain combinations, and despite a complex array of alloantigens present on APCs, a limited number of minor H Ags is preferentially recognized as foreign by CTLs (8, 9, 10, 11). Based on evidence in human (4, 12) and mouse GVHD (13, 14), it is reasonable to suggest that epitopes dominating the immune response are most likely to be of inherent clinical significance.

B6dom1 is a class I (H2-Db)-restricted immunodominant minor H Ag epitope originally detected when C3H.SW mice were immunized with C57BL/6 (B6) spleen cells (15, 16). Edman degradation analysis of the reverse-phase HPLC-separated natural ligand, coupled with testing of synthetic peptide analogues, suggested that the peptide AAPDNRETF is antigenically cross-reactive and biochemically similar to the natural ligand (15). CTLs directed against this peptide out-compete CTLs responding to a number of minor H Ags, including male-specific HY peptides, when presented on the same APC (16). This finding may be because the H2-Db/B6dom1 peptide complex displays an unusually high cell surface density (1012 copies per cell) compared with dominated H2-Db-restricted HY peptides (10 copies per cell), and that this complex engages the TCR with an optimal avidity and thus triggers rapid expansion of cognate CTLs (17). Moreover, the B6dom1 peptide is highly antigenic. C3H.SW mice primed with the B6dom1 peptide in CFA generate cytotoxic activity and T lymphocytes from B6dom1 peptide-primed mice invoke manifestations of GVHD (skin lesions and thymic hypoplasia) when transplanted into irradiated C57BL/6 recipients (15). However, the tissue distribution of the B6dom1 precursor protein is poorly understood and nothing is known regarding the genetic control of this immunodominant Ag.

The purpose of this study was to elucidate the biochemical and immunogenetic basis of the B6dom1 Ag. We show that the natural peptide has biochemical properties indistinguishable from AAPDNRETF, that this Ag dominates the CTL response among several inbred mouse strains, and that the peptide can be extracted from an array of organs but most abundantly from lymphoid organs and lung. Moreover, we employ a novel somatic cell selection technique coupled with classical genetics to show that the B6dom1 peptide is encoded by the H7 minor H gene originally discovered by Snell ~40 years ago (18). These studies provide a molecular genetic framework for understanding B6dom1, and highlight the fact that mouse minor H loci encoding immunodominant CTL epitopes often correspond to classical H loci originally identified by their ability to confer strong resistance to tumor transplantation.


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

All of the mouse strains used in this study were obtained from The Jackson Laboratory (Bar Harbor, ME) in accordance with accepted husbandry practices. Mice used for CTL generation were generally 6–12 wk of age.

Cell lines and bulk cultures

Cell lines were maintained in DMEM medium (Life Technologies, Gaithersburg, MD) supplemented with 2 mM glutamine, 1 mM pyruvate, 50 µM ß-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS (HyClone, Logan, UT) as described (19). Abelson virus-transformed R8 cells (clone 1.3) were a kind gift of T. V. Rajan (University of Connecticut, Storrs). TAP-deficient human T2 cells expressing H-2Db (T2-Db) and H2-Kb (T2-Kb) (20) were generously provided from P. Cresswell (Yale University, New Haven, CT). The H3aa-specific CTL line LPa/2R-1 (alternative name C1) has been described (21). All CTL lines were maintained by weekly stimulation with C57BL/10 (B10) spleen cells and 30–50 U/ml rIL-2 using established conditions (22). To generate bulk effector populations, mice were primed i.p. with two weekly injections of 2 x 107 splenocytes (200 Gy-irradiated) and 7 days later 2 x 107 primed responder cells were harvested and stimulated in MLC with 3 x 107 200 Gy-irradiated B6 splenocytes, in modified DMEM medium supplemented on day 3 of culture with 50 U/ml rIL-2, using established conditions (21). For restimulation in vitro, cells from the MLCs were cocultured with 5 x 106 200 Gy-irradiated spleen cells pulsed with 1 nM peptide for 30' at 37°C and then washed 1x in DMEM medium. The B6dom1-specific cell line SW10/B was generated from such an MLC in which the cells were restimulated by consecutive rounds of in vitro passage using C3H.SW stimulator cells pulsed with 1 nM AAPDNRETF peptide.

Synthetic peptides

B6dom1 (AAPDNRETF) peptide was synthesized using standard fluorenylmethionylleucyl phenylalanine chemistry by Chiron (Victoria, Australia). Purity, as determined by reverse-phase HPLC, was >97%. The H2-Db-binding HY peptide WMHHNMDLI was kindly provided by D. Scott (Hammersmith Hospital, London). The B6dom1 peptide was stored as 1 mM stock solution in PBS and the HY peptide was stored in dimethyl sulfoxide.

Extraction and HPLC fractionation of natural minor H peptides

Two methods were used for extraction of natural B6dom1 peptide from C57BL/6 cells and organs: 1) acid elution in citrate-phosphate buffer (0.131 M citric acid/0.066 M Na2HPO4, pH 3.3) performed on live cell suspensions (see Fig. 2Go, B and C), and 2) extraction from tissue homogenates in 0.1% trifluoroacetic acid (TFA) (Fig. 2GoD). Both methods were performed in the presence of protease inhibitors (25 mM iodoacetamide, 1 mM aprotinine, 1 mM PMSF) as described previously (16, 23, 24). After prepurification on a C18 Sep-Pak column (Waters) extracts were fractionated on an HPLC system using a Superpac Pep-S C18 column (5 µm, 4 x 250 mm, Pharmacia, Uppsala, Sweden) (16). Solvents used were 99.9% water/0.1% TFA (solvent A) and 99.9% acetonitrile/0.1% TFA (solvent B). The gradient consisted of the following linear step intervals: 0% solvent B (0–5 min), up to 20% solvent B at 10 min, up to 55% solvent B at 55 min, plateau at 55% solvent B from 55 to 60 min, and up to 100% solvent B at 70 min. Flow rate was 1 ml/min; 1 ml fractions were collected and lyophilized.



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  		FIGURE 2. Biochemical and expression analysis of the natural B6dom1 antigen. A, HPLC elution profile of synthetic AAPDNRETF peptide. B, Natural peptides were extracted from 5 x 108 C57BL/6 spleen cells by acid elution and HPLC-fractionated. Individual fractions were incubated with C3H.SW Con A blasts, and the Con A blasts were tested for lysis by SW10/B effectors. C, Same as for B except that targets were T2-Db and T2-Kb cells. D, TFA extracts (each containing 10 mg protein) from 10 different organs were fractionated by HPLC. Fraction 19 was incubated with T2Db targets, and these were tested for lysis by SW10/B effectors. In control experiments, no lysis was observed when target cells were incubated with peptides extracted from C3H.SW organs (data not shown). Results are presented as the mean ± SD of three to five experiments. E:T ratio was 5:1.

 
Immunoselection and H2 alloantigen screening of growth-positive cultures

Immunoselection was conducted after procedures described by Zuberi et al. (25). To generate cultures containing independent mutational events, the B6dom1 heterozygous cell line R8 was seeded at 200 cells per well into 10 96-well round bottom plates. Six days later, the number of cells had increased to about 1 x 105 R8 cells per well. B6dom1-specific SW10/B CTLs were added at 1 x 105 per well to achieve an approximate 1:1 effector to R8 cell ratio. Growth-positive microwells were examined for Kb and Db expression by conventional immunofluorescence methods using anti-Kb mAb 28–13.3 and the anti-Db mAb MA62. Analysis was performed on viable cells gated by propidium iodide exclusion, and analyzed using LYSIS II software on a FACScan (Becton Dickinson, Mountain View, CA). H2b-positive cells were then assayed for B6dom1 expression in a growth inhibition assay (GIA) (25 and M. E. Dudley, P. R. Chandler, G. J. C., and D. C. R., unpublished data). The GIA consisted of coincubating either SW10/B or LPa/2R-1 CTLs washed free of IL-2 in 96-well round-bottom plates at 1 x 105 per well with 1 x 104 variant cells. After 48 h, cells were labeled with 1 µCi per well of [3H]thymidine for 12 h. The level of [3H]thymidine incorporation is represented as a cpm of wells containing variants and CTLs divided by cpm from wells with only variant cells.

RI strain distribution analysis

To establish the strain distribution pattern (SDP) of B6dom1, H2b-positive CXB, BXH, and AXB recombinant inbred (RI) strains were used in a GIA (M. E. Dudley, P. R. Chandler, G. J. C., and D. C. R., unpublished data). PBMCs from mice were isolated from 200 µl of blood as described (26) and a concentration of ~105 cells per round-bottom microwell were activated with 50 µg/ml LPS. The cells were either cultured alone or with 5 x 105 CTLs washed free of IL-2. After 48 h, cells were labeled with 1 µCi [3H]thymidine per well for 12 h. The level of [3H]thymidine incorporation is represented as cpm from wells containing LPS-stimulated spleen cells and CTLs divided by cpm from wells containing only LPS-stimulated spleen cells.

Cell mediated lysis assay

A standard 51Cr release assay was used (21). For peptide-loaded targets, 51Cr-labeled T2-Db cells were incubated with 1 nM concentrations of synthetic peptides for 30 min at 37°C, washed 2x to remove unbound peptide, and then coincubated at 37°C with effector cells in V-bottom plates at various E:T ratios. Lysis of target cells was measured as specific cytolysis, based on the level of 51Cr released into the supernatate relative to spontaneous and maximal 51Cr release, and is shown as mean of triplicate cultures. Normal splenocyte target cells were Con A-stimulated lymphoblasts.

Genetic mapping of the B6dom1 locus

Genomic DNA was isolated from variant cells and R8 tumor cells by standard methods or purchased from The Jackson Laboratory DNA resource (http://www.jax.org). Genomic DNA isolated from the immunoselected variant cells was pooled, and along with R8 DNA as well as DNA isolated from H7 congenic strains B6.C-H7c (HW23) and B10.C-H7c (47N), was used as template for PCR DNA amplification. PCR conditions included initial denaturation at 95°C for 5 min, followed by 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, for 35 cycles with a 10 min 72°C extension step at the end of the program. Oligonucleotide PCR primers designed to amplify a telomeric region on each chromosome were obtained from Research Genetics (Huntsville. AL) and from B. Taylor (The Jackson Laboratory), and were chosen based on the reported chromosomal map position described in the Whitehead Institute/MIT mouse simple sequence length polymorphism (SSLP) database (www-genome.wi.mit.edu). PCR products were run on 3% agarose gels and visualized with a UV transilluminator, after ethidium bromide staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bulk and clonal analysis of CTLs responding to the B6dom1 peptide AAPDNRETF

To clarify the immunogenetic basis of the B6dom1 Ag, we examined the specificity of CTLs generated in bulk MLC by female C3H.SW mice and BALB.B mice after immunization and in vitro restimulation with male B6 spleen cells and B10 spleen cells, respectively. We then tested the ability of the CTLs from these MLCs to lyse H2-Db-transfected T2 target cells that were loaded with either the synthetic B6dom1 peptide (AAPDNRETF) or the H2-Db-restricted HY peptide (WMHHNMDLI; 27). High levels of specific lysis were routinely observed against B6dom1-loaded target cells and no specific lysis was apparent against HY peptide-loaded target cells (Fig. 1Go, A and B). These results indicate that the Ag defined by CTL activity against AAPDNRETF is immunodominant over the HY peptide in both of these strain combinations.



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  		FIGURE 1. Bulk and clonal analysis of the CTLs responding to the B6dom1 peptide AAPDNRETF. A and B, Female C3H.SW and BALB/B mice, respectively, were immunized in vivo and restimulated in MLC with cells from male B10 mice and tested in a conventional CML assay at specified effector to target cell ratios. C, Long-term CTL lines SW9/B and SW10/B were tested against target cells loaded with various concentrations of synthetic peptides loaded onto target cells. T2-Db target cells were loaded with the indicated concentrations of B6dom1 peptide AAPDNRETF (•), HY peptide WMHHNMDLI ({circ}), or the H13a peptide SSVVGVWYL ({square}). HY and H13a loaded targets were lysed to high levels by control CTLs (data not shown). Data from individual mice tested against the indicated target cells.

 
We then generated two AAPDNRETF-specific CTL lines from C3H.SW anti-B6 MLCs through repeated cycles of in vitro stimulation of the C3H.SW effector cells with syngeneic spleen cells coated with synthetic AAPDNRETF peptide (described in Materials and Methods). These CTLs were tested for their ability to lyse T2-Db target cells that were loaded with either the B6dom1 peptide or a negative control Db-binding H13a minor H Ag peptide, SSVVGVWYL (Fig. 1GoC). Both lines were AAPDNRETF-specific in that they lysed T2-Db cells loaded with <10 pM of synthetic AAPDNRETF peptide, while these CTLs did not lyse control cells loaded with the H13 peptide at any concentration.

To determine whether the naturally presented B6dom1 Ag is indeed AAPDNRETF, we eluted HPLC-fractionated natural peptides from C57BL/6 splenocytes. We then determined in CTL sensitization assays whether the SW10/B CTLs lyse C3H.SW Con A-stimulated lymphoblasts loaded with these HPLC fractions. SW10/B effectors recognized a single peak (compare Fig. 2Go, B and C). Furthermore, the natural peptide eluted at the same time as the synthetic AAPDNRETF peptide (Fig. 2GoA) and was recognized when loaded on T2-Db but not on T2-Kb targets (Fig. 2GoC). These results suggest that the naturally processed peptide presented by H2-Db and recognized by the SW10/B cell line is biochemically similar to AAPDNRETF.

Tissue distribution of the B6dom1 Ag

The B6dom1 Ag has been detected on lymphoid cells, but otherwise little is known regarding its distribution. To better understand B6dom1 tissue distribution, we determined whether T2-Db cells loaded with HPLC-fractionated peptides extracted from various organs of C57BL/6 mice were lysed by SW10/B CTLs. Results pooled from 3–5 such experiments show that B6dom1 has a wide organ distribution, but was most abundant in the spleen, thymus, and lung (Fig. 2GoD).

CTL-mediated immunoselection produces B6dom1-loss variant cells

Nothing is known about the genetic basis of B6dom1. To map the gene encoding this Ag, we employed CTL immunoselection (25, 28, 29, 30). During CTL-mediated immunoselection, cell lines heterozygous (or hemizygous) for the recognized epitope are killed, and rare epitope-loss variants survive; these cells often exhibit loss of a chromosomal segment containing the immunoselected allele, manifested as a loss of heterozygosity (LOH). Immunoselected variant cells obtained in this manner can be evaluated for LOH using allele-specific molecular markers. We exploited the fact that BALB-background mice lack the B6dom1 Ag (Fig. 1GoB) while B6 mice express it. The Abelson virus-transformed R8 lymphoblastoid cell line was produced from (BALB/c x B6) F1 hybrid mice, and thus expresses B6dom1 heterozygously. We imposed B6dom1-specific negative selection on the R8 line through coculturing with a 1:1 ratio of SW10/B CTLs. In doing so, almost all R8 cells were killed. However, rare R8 cells survived, as evidenced by presumably clonal outgrowths of viable cells detected in 42% of the R8 microwell cultures after 10 days of coculture with SW10/B CTLs.

Microcultures containing outgrowths were screened for Ag expression. Because the anti-B6dom1 SW10/B CTLs recognize peptide in the context of H2-Db, either a mutational loss of H2b or loss of the B6-derived allele of B6dom1 would allow survival of R8 cells. To distinguish R8 cultures that survived immunoselection caused by loss of H2b from cells that survived by virtue of B6dom1 loss, we screened cells from growth-positive microwells by FACS analysis, using H2-Kb- and H2-Db-specific Abs to monitor for H2-Kb and H2-Db alloantigens. These results are summarized in Table IGo. Approximately 68% of the cells from growth-positive wells showed complete H2b loss, whereas cells from two microwells showed Db but not Kb loss; these cells were excluded from further analyses. Approximately 21% expressed both Kb and Db; these potential B6dom1-loss mutant R8 cultures were selected for further analyses.


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  		Table I. Summary of FACS analysis of growth-positive immunoselected microwells1

 
The H2-Db-positive, immunoselected R8 cultures were screened for B6dom1 expression using the GIA (25). In this assay, CTLs inhibit proliferation of transformed target cells that express the Ag against which the CTLs are directed. Ag loss is indicated by unimpaired proliferation of the immunoselected R8 cells in the presence of excess SW10/B CTLs. Such conditions failed to inhibit proliferation of the immunoselected, H2-Db-positive R8 microcultures, but inhibited proliferation of parental R8 cells (Fig. 3GoA). A positive control CTL line, LPa/2R-1 (specific for the H2-Db-restricted H3aa minor H Ag), inhibited growth of all immunoselected cells, indicating that the LOH variants had selectively lost expression of the B6dom1 Ag and functionally confirming H2-Db expression (Fig. 3GoB).



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  		FIGURE 3. GIA screen of CTL-immunoselected variant cultures identifies B6dom1-loss variants. Data are presented as a ratio of mean cpm of [3H]thymidine incorporation of variant microcultures and parental R8 cells cultured in the presence of anti-B6dom1 CTL SW10/B or control H3aa-specific CTL Lpa/2R-1 divided by cpm from the replicate microcultures cultured without CTLs. CTL cultures alone did not proliferate appreciably (data not shown). Variant no. 32 is a known Db-loss variant and shown for comparative purposes.

 
Mapping the gene encoding the B6dom1 Ag

To determine whether we could detect a genetic loss accompanying B6dom1 Ag loss, the panel of B6dom1-loss variant cell cultures that retained H2-Db was screened for LOH of B6-derived alleles. In previous studies, the most common genetic mutation that led to Ag loss as a consequence of immunoselection was a single mitotic crossover event leading to LOH, starting from a site proximal to the Ag-encoding locus and extending to the end of the chromosome (25). We therefore reasoned that the majority of B6dom1-negative R8 variant cells would likely exhibit LOH that includes a locus at the end of the chromosome encoding B6dom1. Loss of a B6 allele at such a telomeric locus would thus identify the chromosome that encodes B6dom1.

To test this rationale, we scanned the end of all autosomal chromosomes and the X chromosome. We selected telomeric, PCR-amplifiable dinucleotide repeat MIT marker loci that yielded discernible SSLP between BALB/c and B6, for each chromosome-specific locus. To streamline this procedure, we combined genomic DNA isolated from each independent B6dom1 variant culture into a single, pooled DNA sample. Such pooling methods have proven successful in identifying recombinant chromosomes within DNA pools from F2 mice (31). Assuming the majority of B6dom1 -negative R8 variants carry LOH that extends to the telomere, we expected to observe a reduction in the B6-specific amplification product from the chromosome that encodes B6dom1. The results comparing the amplification products of pooled variant cell DNA with B6dom1-heterozygous R8 DNA are summarized in Fig. 4GoA. A LOH on chromosome 11 was present in the parental R8 cell line and in the pooled sample. This is likely because of a spontaneous mitotic recombination event that became fixed in the parental R8 line before immunoselection. Among the remaining autosomal chromosomes scanned, the pooled variant DNA sample demonstrated lack of only one B6-derived amplification product, D9 Mit18, which maps to the telomeric end of chromosome 9. This result suggested that the gene encoding B6dom1 maps to chromosome 9.



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  		FIGURE 4. LOH analysis of B6dom1-negative immunoselected variants maps the gene encoding B6dom1 to the distal end of mouse chromosome 9. A, Genome-wide scanning of telomeric markers for LOH. DNA from pooled variant cultures and the parental R8 cell line were screened for telomeric dinucleotide repeat markers. The chromosome 11 marker was uninformative, as the R8 cell line was homozygous for the BALB allele at this locus. The chromosome 13 marker showed a diminished B6-specific band, but in repeated testing (data not shown), this amplification product was confirmed. B, Chromosome 9-specific scanning of pooled variant DNAs. C, Analysis of individual, informative variant DNAs that carried the B6 allele at D2 Mit182, and DNAs from H7-congenic mouse strains HW23 and 47N.

 
Once linkage to chromosome 9 was indicated, more refined mapping was performed using chromosome 9 markers (D9 Mit67, D9 Mit130, D9 Mit182, D9 Mit214, and D9 Mit18) that span this chromosome and yield readily discernible B6 and BALB/c-specific amplification products. Fig. 4GoB shows that the B6-specific amplification products of the pooled variant DNA sample were diminished and ultimately absent as increasingly distal markers were examined. Indeed, D9 Mit182 showed a greatly diminished amplification product, and D9 Mit214 and D9 Mit18 yielded no detectable B6-specific amplification product. This result suggested that the great majority of the variants exhibit a LOH including D9 Mit182, D9 Mit214, and D9 Mit18, thus mapping B6dom1 near to or distal to D9 Mit182.

The diminished B6-specific D9 Mit182 product also suggested that a subset of the variants from the pool were heterozygous for this locus; if so, such variants could conceivably provide a more refined chromosomal position for B6dom1. To identify this subset, we screened DNAs from each of the individual variant cultures comprising the pool. Fig. 4GoC shows more refined allele typing of the three variants among this pool (V.61, V.78, and V.79) that retained the B6 D9 Mit182 allele, using markers distal to this locus. The LOH breakpoint for these three variants occurred between markers D9 Mit182 and D9 Mit214, suggesting that B6dom1 maps telomeric to D9 Mit182.

To confirm and refine this map position, we analyzed the SDP of B6dom1 using recombinant inbred strains. We selected informative strain backgrounds in which one parental strain (B6) expresses B6dom1 while the other fails to express this Ag. We then used a modified GIA to analyze B6dom1 expression of the subset of CXB, AXB, and BXH RI mice known to carry the H2b haplotype and for which one parental strain (B6) is B6dom1-positive and the other (C3H/HeJ, BALB/c, or A/J) parent is negative for this Ag (Fig. 1GoA and data not shown). This type of GIA measures Ag expression by the ability of CTLs to specifically inhibit LPS-induced proliferation of PBMCs (Dudley et al., unpublished work). Results are presented in Fig. 5GoA. In agreement with the immunoselection-based mapping, the SDP was most consistent with the telomeric region of chromosome 9 near D9 Mit 18. However, none of the SDPs of molecularly-defined chromosome 9 markers were completely concordant with that of B6dom1. It was notable, however, that the RI SDP of the CTT1 Ag, an immunodominant minor H Ag generated by B6 mice in response to BALB.B cells (32, 33), correlated inversely with the expression of B6dom1 (Fig. 5GoB).



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  		FIGURE 5. GIA analysis of RI strains for B6dom1 expression, and summary of LOH, RI, and congenic strain mapping of B6dom1. A, RI mice from strain backgrounds in which one parental strain (B6) expresses B6dom1 while the other fails to express this Ag, and known to be H2b, were tested. The subsets of CXB, AXB, and BXH RI mice known to carry the H2b haplotype were tested for the ability of SW10/B cells to specifically inhibit LPS-induced proliferation of PBMCs. Data are presented as a ratio of the mean [3H]thymidine cpm of triplicate cultures of LPS-induced PBMCs cultured in the presence of anti-B6dom1 CTL SW10/B divided by the cpm from the same PBMCs cultured without CTLs. Any value >=1 is considered Ag negative. It should be noted that cloned T cells enhance the proliferation of LPS-stimulated cells; this explains why the values of B6dom1-negative LPS-stimulated cells are always severalfold greater in the presence of added CTLs. B, Fine detail mapping of the B6dom1-encoding locus on distal chromosome 9. Markers and marker positions are from http://www-genome.wi.mit.edu. LOH data from molecular typing of immunoselected cells derived from the R8 parental cell line is presented. Typing data for the CXB, BXH, and AXB RI strains recombinant within this interval is shown, as well as typing data for the HW23 and 47N congenic strains. At the right is presented the inferred positions of the H61 and B6dom1-encoding (H7) loci. C, B, H, and A represent alleles of BALB/c, C57BL/6, C3H/HeJ, and A/J, respectively. An asterisk (*) denotes not tested.

 
Snell identified the H7 minor H locus by the production of the B10.C-H7c (47N) strain (18). This congenic strain carries a BALB-derived congenic segment that maps to the distal end of chromosome 9 (32, 33). A second, putatively H7 congenic mouse strain, B6.C-H7c (HW23), also carries a donor BALB/c-derived segment that maps to this region (32, 33). To test whether these congenic segments overlap with the region to which B6dom1 maps, genomic DNA from HW23 and 47N strains was allele-typed for markers on distal chromosome 9 (Fig. 4GoC). Results show that the region predicted to contain the B6dom1 locus is of BALB origin in strain 47N but of B6 origin in HW23 mice, agreeing with earlier studies (33) that the H7 congenic segment of 47N extends farther toward the telomere compared with the HW23 congenic segment.

Functional evidence that B6dom1 may be encoded by Snell’s classical H7 locus

If the gene encoding B6dom1 maps within the donor segment defined by 47N but not HW23 mice, 47N mice should not express B6dom1 while HW23 mice should express this Ag as its BALB-derived segment does not extend as far toward the telomere. To address this possibility, we first determined whether 47N- and HW23-derived target cells express B6dom1. The SW10/B CTL line failed to lyse cells from 47N mice but did lyse cells from HW23 mice (Fig. 6Goa), indicating that the congenic strain expression of B6dom1 correlates with the preceding molecular genetic data.



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  		FIGURE 6. Functional evidence that B6dom1 is encoded by Snell’s classical H7 locus. A, Anti-B6dom1 CTL SW10/B fails to kill cells from 47N female mice but does lyse cells from B10 and from HW23 female mice. B, Female 47N mice immunized and restimulated with male B10 cells generate B10-specific CTLs. C, CTLs from B are specific for T2-Db loaded with the B6dom1 peptide AAPDNRETF and show no reactivity toward the HY peptide WMHHNMDLI. D, HW23 mice immunized and restimulated with B6 cells fail to generate CTLs that lyse B6dom1 peptide-loaded cells but generate CTLs specific for T2-Db loaded with the HY peptide. Target cells: ({blacksquare}), female B10 Con A blasts; ({blacktriangleup}), female HW23 Con A blasts; ({square}), female 47N Con A blasts; (•), T2-Db cells loaded with 1 nM B6dom1 peptide AAPDNRETF; and ({circ}), T2-Db cells loaded with 1 nM HY peptide WMHHNMDLI. Data from cells from individual mice tested against the two indicated target cells.

 
One prediction, if B6dom1 is encoded by Snell’s H7 locus, is that CTLs generated by 47N mice immunized with B10 cells should kill target cells loaded with the B6dom1 peptide, while HW23 mice immunized with B6 cells should not be capable of generating such CTLs. To test this possibility, female 47N mice and female HW23 mice were immunized and restimulated in vitro with male-derived B10 and B6 cells, respectively, and tested for cytotoxic activity. (Male-specific HY Ags were included to ensure a helper stimulus from CD4+ T cells responding to HY.) As shown in Fig. 6Go, B and C, 47N mice generated high levels of specific lysis activity against B10 target cells as well as T2-Db cells loaded with B6dom1, but failed to demonstrate activity against cells from female 47N cells or T2-Db cells loaded with the H2-Db-binding HY peptide. Moreover, the B6dom1 peptide concentration required to sensitize T2-Db cells for lysis was equivalent for the bulk culture and the SW10/B line (data not shown). In contrast, HW23 mice failed to generate CTLs capable of lysing female B10 cells or B6dom1-pulsed syngeneic cells, and only showed a detectable CTL response against T2-Db cells loaded with the H2-Db-binding HY peptide (Fig. 6GoD). These results suggest that 1) the congenic segment by which the 47N congenic strain differs from the HW23 strain encodes the H7 locus and the B6dom1 Ag, and 2) in contrast to B6dom1/H7, the H Ag that is differentially expressed in HW23 mice does not elicit the generation of CTLs detectable by the conventional CML assay.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of this study was to elucidate the biochemical and immunogenetic basis of the B6dom1 Ag. The fact that this murine minor H Ag appears to play a role in GVHD (15) underscores the need to better understand this immunodominant epitope. B6dom1 dominates the CTL response not only of C3H.SW mice but also of BALB.B mice responding to B10 and/or B6 cells. This antigenic disparity is thus not unique to C3H.SW mice responding to B6 Ags but is instead common among inbred strains, and is consistent with the notion that autosomally encoded mouse minor H Ags arose as a consequence of rare genetic changes that became fixed as common alleles among mouse strains (26). Such common alleles of minor H Ag epitopes have also been reported in humans (34, 35).

The suggestion that the natural B6dom1 peptide is AAPDNRETF was originally suggested from Edman degradation analysis in combination with empirically tested synthetic peptides (15). We show herein that the HPLC elution pattern of the natural Db-presented B6dom1 peptide is indistinguishable from the synthetic ligand. These findings are consistent with the natural peptide being either biochemically similar to or identical with the synthetic AAPDNRETF peptide. It is notable that B6dom1 is the only minor H Ag peptide that has been elucidated solely by such techniques; this is likely because of the abundance of the B6dom1 peptide on the cell surface (~103 copies on resting cells and ~105 copies per Con A blast) (17). Indeed, other natural minor H Ag peptides have tended to be of lower abundance (often <50 copies) on the cell surface (17, 36, 37) and therefore not as readily amenable to such analytical techniques.

Little is known regarding which tissues process class I-presented minor H Ags, especially those involved in GVHD. Goulmy and collaborators have performed limited tissue distribution analysis of human minor H Ags, and have suggested that immunodominant human GVHD-associated Ags, such as the HA1 and HA2 minor H Ags, are limited in their distribution to hemopoietic tissue (38). This idea was difficult to reconcile with the work of Korngold and Sprent, who showed in recipients that were hemopoietic chimeras that to trigger GVHD minor H Ags must be expressed by both hemopoietic and nonhemopoietic cells (39). In mice, Griem et al. (23) studied the tissue distribution of three minor H peptides, two of which were presented by Kb (H4b and mapki), and one by Db (H-Y). These three peptides were most abundant in extracts of lymphoid organs, the lung in the case of H-Y and H4b, the kidney and small intestine for mapki, and the skin for mapki and H4b (23). Our results for B6dom1 also show a wide organ distribution, which argues against a requirement for hemopoietically restricted tissue distribution when considering minor H Ag determinants potentially active in GVHD. That an increased amount of Ag was extracted from the spleen, thymus, and lungs (which is known to contain abundant leukocytes) compared with the other organs could be explained by levels of MHC class I expression on hemopoietic cells rather than differential expression of the minor H precursor protein (23). The high abundance of B6dom1 found in lung (if it is a characteristic shared with other dominant minor H Ags) could explain why this particular organ has been reported to be the most reactive site for donor T lymphocytes after allogeneic bone marrow transplantation (40). Indeed, while it was previously thought that, in bone marrow transplant recipients, minor H-specific donor T cells were sequestered and proliferated in the host’s spleen (41, 42), it was recently shown that these T cells migrated preferentially into the lung rather than the spleen (40). This may contribute to the frequent occurrence of lung injury reported in mouse and human models of GVHD (43, 44, 45). Finally, in this study, peptide extracts used to assess the tissue expression of B6dom1 were obtained from whole organ homogenates; detection of B6dom1 or other minor H Ags in such preparations should not be taken as conclusive evidence of expression by various organ-specific cells. Minor H Ags detected in some organs may derive from nonparenchymatous cells such as macrophages, dendritic cells, or endothelial cells. The best strategy to approach this issue and to evaluate heterogeneity in the distribution of minor H Ag/MHC complexes among cells from tissues and organs, might be to generate Abs that recognize specific peptide/MHC complexes (46).

Efficient chromosomal localization of the B6dom1-encoding locus by CTL immunoselection

Rapid advances in genomic analyses provide increasingly useful genetic information for biological problems such as tissue rejection. However, to approach such problems efficiently, it is important to possess the tools needed to connect the rejection phenotype to a genetic basis. The studies described here show that CTL immunoselection can be used with remarkable efficiency to derive accurate genetic information. Before this analysis, the genetics of B6dom1 was not understood, aside from the fact that the epitope was a CTL-defined immunodominant peptide Ag lacking in C3H.SW but present in B6 mice. The finding that BALB.B mice also lack the Ag made it possible to use (BALB/c x B6)F1-derived R8 cells as the target of immunoselection by B6dom1-specific SW10/B CTLs. B6dom1-negative variant cells selected from this procedure and subjected to molecular typing for SSLPs provided evidence that the B6dom1 Ag maps to the telomeric end of mouse chromosome 9, distal to D9 Mit182. These findings were confirmed by RI SDP and analysis of the H7-congenic mouse strains 47N and HW23. We have also successfully applied CTL immunoselection to the fine genetic analysis of the chromosome 2 H3a minor H Ag (25, 47, 48), and to male-specific HY Ags (28), and in both cases, the LOH observed led to the identification of the encoding genes (27, 30). In humans, it is quite difficult to use conventional genetic linkage analysis to derive accurate chromosomal position (e.g., 49). CTL immunoselection, followed by screening for LOH, offers a most promising means of localizing the genes encoding minor H Ags of potential clinical relevance.

B6dom1 is encoded by Snell’s H7 locus

Our results suggest that the B6dom1 Ag is encoded by Snell’s H7 locus. Supportive evidence includes: 1) LOH mapping, RI SDPs, and congenic strain analysis all are consistent with mapping of B6dom1 within the region defined by Snell’s 47N congenic strain; and 2) 47N mice generate a vigorous B6dom1-specific CTL response after immunization with B10 or B6 cells. Snell’s H7-congenic 47N mouse strain was produced by backcross-intercross cycles, each time selecting for ability of the mice to resist a B10-derived leukemia (18). This was a demanding phenotype, as it required eradication of the tumor in order for the mouse to survive and breed. The fact that B6dom1 is immunodominant and potently immunogenic in BALB-background (as well as C3H.SW) mice challenged with B10 cells strongly suggests that this Ag played a crucial role in the selection of the 47N congenic strain. The cytotoxic determinants of several congenically defined minor H loci have recently been attributed to single genes that result in single differential peptides rather than more antigenically complex settings (30, 36, 37). In keeping with these findings, the most parsimonious explanation is that B6dom1 is the sole cytotoxic determinant encoded by the H7 locus. It remains to be determined whether the H7 locus also encodes an MHC class II-restricted determinant that stimulates the CD4+ helper T cell arm of immunity.

The relationship between Snell’s 47N and Bailey’s HW23 congenic strains

As indicated in Fig. 5GoB, the gene(s) encoding the B6dom1 and the CTT1 Ags map within the congenic segment carried by 47N mice but distal to the congenic segment carried by HW23 mice. Bailey’s congenic strains were selected to carry BALB/c chromosome segments that encode HAgs that were rejection barriers to B6 mice (50). One of the congenic strains produced in this manner was HW23, and the congenic segment defined by this strain was shown to be a strong skin transplantation barrier in the B6 anti-HW23 direction and in the HW23 anti-B6 direction (50).

Bailey suggested that the BALB-derived HW23 congenic strain included H7 (50). This conclusion is inconsistent with our findings in which we map H7 more telomeric than the congenic segment defined by HW23 (Fig. 5GoB). The basis for Bailey’s assignment was the classical F1 complementation test (51); he found that mice from a B6 x 47N cross failed to reject HW23 skin grafts, and thus concluded that both strains carried overlapping BALB-derived H7 congenic segments (unpublished observations). Given the genetic information (Fig. 5GoB), we suggest an alternative interpretation: the HW23 strain defines a second minor H locus (now referred to as H61), which is distinct from and maps proximal to H7. We have been unable to detect conventional CTL activity in responses between HW23 and B6 mice (Fig. 6Go and data not shown), and thus suggest that H61 might not encode Ags that stimulate CTLs directed against lymphoid target cells.

The immunogenetic relationship between B6dom1 and CTT1

It is of interest that the genetic mapping of B6dom1 is completely concordant with that of the immunodominant CTT1 Ag described by Vagliani and collaborators (32, 33). Moreover, there is reciprocal concordance in expression of these two Ags in common inbred strains, RI strains, and in the 47N and B10 strains. Reciprocal Ags have also been observed for other minor H loci of both mitochondrial and autosomal origin, and the recent elucidation of the molecular basis of several minor H Ags has revealed a pattern in which such reciprocal Ags arise from amino acid polymorphisms in TCR contact sites within the same core MHC class I-bound peptide (2, 30, 36). It is thus plausible that B6dom1 and CTT1 are allelic peptides. If so, it should be possible to use the B6dom1 peptide core sequence as a foundation to design analogues that act as a ligand for CTT1-specific CTLs. More generally, the finding of a potentially reciprocal relationship between B6dom1 and CTT1 provides increasing support for the possibility that many minor H genes encode reciprocally antigenic peptides.


   Acknowledgments
 
We are grateful to Shari Roopenian and Thomas J. Sproule for excellent technical assistance, Ben Taylor for helpful discussions and for generously providing dinucleotide repeat markers, and to Alexander Chervonsky and Len Schultz for critical review. This work was supported by The Jackson Laboratory Core Grant CA34196.


   Footnotes
 
1 This research was supported by National Institutes of Health Grant RO1 AI28802 (to D.C.R.), National Institutes of Health National Research Service Award Postdoctoral Fellowship 1 F32 CA 80777-01 to (P.A.E.), and National Cancer Institute Grant 008079 to (C.P.). Back

2 Address correspondence and reprint requests to Dr. Peter Eden, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609. E-mail address: Back

3 Abbreviations used in this paper: H, histocompatibility; GVHD, graft-versus-host disease; GIA, growth inhibition assay; SDP, strain distribution pattern; LOH, loss of heterozygosity; SSLP, simple sequence length polymorphism; RI, recombinant inbred strain; TFA, trifluoroacetic acid. Back

Received for publication November 24, 1998. Accepted for publication January 28, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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