Molecular evidence that the H-2D and H-2L genes arose by duplication. Differences between the evolution of the class I genes in mice and humans.

To resolve issues regarding the evolution of D region class I MHC genes and their relationship to other class I-encoding regions of the mouse, as well as man, we characterized the class I genes from the Dq region of the B10.AKM mouse strain. The Dq region was selected because it was known to express multiple gene products, yet two of the products previously characterized have structural features in common with the Ld molecule. Since DNA hybridization data defined similarities between the Dd and Dq regions, we used low-copy genomic or oligonucleotide probes derived from the Dd region of BALB/c (H-2d) to screen a B10.AKM cosmid library. Cosmid clones containing Dq, D2q, D3q, D4q, Lq, and Q1q genes have been isolated and aligned with the corresponding genes of the BALB/c MHC, thus demonstrating a similar gene organization. The two classical transplantation genes, Dq and Lq were found to be strikingly similar to each other such that exons 1-3 of Dq and Lq, are approximately 97% homologous, and exons 4-8 are identical. Furthermore, the implied amino acid sequences of both Lq and Dq molecules show considerable homology to Ld, particularly in regions presumed to be involved in ligand binding. These comparisons suggest not only that the Dq and Lq genes arose from the duplication of an Ld-like progenitor, but also that there is a selective advantage for the maintenance of an Ld-like structure. In addition, the 5' portion of the D4q gene was sequenced and found to have a 13-bp deletion and a 4-bp insertion within the alpha 2 exon. These result in a frame shift that creates a premature termination codon and potential polyadenylation site, respectively. Thus, D4q does not encode a typical class I molecule. Sequence comparisons suggest that the D4q gene did not arise from a duplication event involving an Ld-like gene such as Dq and Lq. Interestingly, the D4q molecule, if produced, would have amino acid residues in common with K and/or Q molecules that differ from those observed in D/L molecules. These findings, in conjunction with hybridization data, provide evidence that the D2, D3, and D4 genes were derived from Q genes by an unequal crossover event. Additional hybridization data using low-copy D region probes suggest that several different D region gene organizations exist among mice of different haplotypes. These and other recent molecular studies provide multiple examples of expansion and contraction of the class I genes in the D region.(ABSTRACT TRUNCATED AT 400 WORDS)

The murine MHC class I genes, H-2Dq and H-2Lq, are strikingly homologous to each other, H-2Ld, and two genes reported to encode tumor-specific antigens.

Two phenomena appear to distinguish the D region class I genes from those in the K region in the murine MHC: (a) haplotype disparity in the number of expressed D region class I molecules has been observed; and (b) clines of closely related D region class I molecules among and within mice of different H-2 haplotypes can be defined. Both of these observations have been based on serological and peptide mapping analyses of these molecules. Recent reports using molecular biological approaches have corroborated these findings. Since the mouse strain B10.AKM expresses multiple D region class I antigens, all of which are closely related to the prototypic Ld molecule, we investigated the Dq region of B10.AKM using molecular approaches. Three D region class I genes were isolated from genomic B10.AKM bacteriophage and cosmid libraries. Based on alignment of those genes with the BALB/c D region class I genes by analogous restriction endonuclease sites and by hybridization of one of those genes with a D4d gene-derived oligonucleotide probe, we have designated these genes as Dq, Lq, and D4q. As determined by DNA-mediated gene transfer to mouse L cells followed by serological analyses, the Dq and Lq genes encode previously characterized Dq region class I antigens. The nucleic acid sequence comparisons of the Dq and Lq genes demonstrated a higher level of homology with the Ld and Db genes than with other D region class I genes. In addition, CTL stimulated with a Dq, Lq, or Ld gene transfectant showed strong crossreactions with the other transfectants as targets, suggesting that the products of these genes are also functionally related. Thus, these studies suggest that the L molecule represents a prototypic structure shared by several D region gene products, and furthermore, the duplication of an Ld-like progenitor gene resulted in two Dq region class I genes, Dq and Lq. Unexpectedly, the sequences determined for the Dq and Lq genes are nearly identical to the sequences of two genes, A166 and A149, respectively, which were reported to encode the tumor-specific antigens; these novel class I genes were isolated from an H-2k fibrosarcoma, 1591. This raises the distinct possibility that these purported tumor-specific class I genes were introduced into this tumor by contamination.

Restricted VH gene usage by murine hybridomas directed against the human N, but not M, blood group antigen.

The M and N human blood group antigens are complex glycopeptide determinants at the amino terminus of the red blood cell membrane glycoprotein, glycophorin A. The heavy and light chain variable region cDNA sequences were determined for seven murine monoclonal antibodies recognizing glycophorin A. Three of the antibodies were anti-M and four were anti-N. Each of the anti-M antibodies was composed of VH and VL regions derived from distinct germline gene families (VH1 (J558), VH4 (X24), VH5 (7183), VK5, VK8, and VK19). In contrast, all four anti-N heavy chains were composed of VH regions derived from the VH2 (Q52) germline gene family and all used the same J4 gene segment. In addition, two of the anti-N light chains were composed of VK regions from the VK8 germline gene family and used the J1 gene segment. Since each anti-N hybridoma was derived from different mice immunized by different protocols, these results suggest that the murine immune response to the N, but not the M, human blood group antigen is restricted.

Use of gene therapy to suppress the antigen-specific immune responses in mice to an HLA antigen.

Hematopoietic chimerism has been used in the laboratory to induce life-long immunologic tolerance to donor antigens. The present study demonstrates that mice transplanted with autologous bone marrow cells retrovirally transduced to express HLA-A2.1 develop a significantly depressed immune response to this antigen while retaining normal reactivity to HLA-B7. Retrovirus-mediated transduction was performed using whole bone marrow-producer cell coculture. This approach did not result in significant gene transfer into hematopoietic progenitor cells. Despite this, the antibody response to HLA-A2.1 in mice reconstituted with genetically modified BMC was completely suppressed three months following bone marrow transplantation. Cell-mediated immunity to HLA-A2.1 was partially suppressed in three-fourths of animals tested three months later, although one animal had a CTL profile similar to that an of HLA-A2.1 transgenic mouse. Complete suppression of the antibody-mediated immune response occurred when only one-third of mice had evidence of the introduced genes in their spleen and one-tenth had the introduced sequences in their circulating WBCs by PCR. In conclusion, engineering of BMC to express donor MHC genes may be an alternative to xenogeneic BMT to induce chimerism and tolerance. More efficient transduction of bone marrow progenitor cells may result in more persistent gene expression and long-lasting transplantation tolerance in recipients of genetically modified bone marrow. Successful application of this technology may also be useful in altering immune responses to other external and self antigens.

Transfusion-induced graft-versus-host disease after liver transplantation. Documentation using polymerase chain reaction with HLA-DR sequence-specific primers.

Graft-versus-host disease (GVHD) occurring after liver transplantation can pose a difficult diagnostic dilemma. Similar clinical and pathologic skin and gastrointestinal manifestations can result from other causes (i.e., drugs, infections). Treatment for each of these entities differs, and the high mortality associated with GVHD makes this distinction critical. GVHD has been assumed to result from the cotransplantation of donor lymphoid tissue along with the allograft. In most instances, the patient also receives blood products during the operation, and occasionally during the postoperative period, and the lymphoid cells in these products are also a potential source of concern. In this report, we describe a patient who developed GVHD after liver transplantation. Using molecular diagnostic techniques, we determined that the source for this GVHD was not the organ donor, but was most likely nonirradiated blood products received during the hospital course. Our results suggest that transplant recipients with concomitant hematopoietic dysfunction would benefit from irradiated blood products to reduce the likelihood of this complication.

Biochemical and functional characterization of soluble multivalent MHC L(d)/Fc gamma 1 and L(d)/Fc mu chimeric proteins loaded with specific peptides.

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.

Loss of heterozygosity detected in a short tandem repeat (STR) locus commonly used for human DNA identification.

Short tandem repeat (STR) markers are commonly used in basic genetic research and in human identification testing. Clinically, STRs can be used to study genetic alterations in tumors. A genetic deletion common to many types of cancer is referred to as the loss of heterozygosity (LOH). Numerous examples of LOH in cancer have been described and some have been mapped to areas located in close proximity to markers employed in human identity testing. Despite this fact, LOH has rarely been observed for STR loci commonly employed in forensic testing. Recently, for medico-legal purposes, we were asked to determine whether a tissue biopsy originated from a particular individual. For a reference source we assessed two specimens, one from normal tissue and one from cancerous tissue. When both reference specimens were used to generate DNA profiles, we observed LOH at one STR locus, D13S317. As demonstrated in other cancers only the cancerous biopsy demonstrated LOH. The forensic community should be cognizant of these unusual circumstances because, as identification of human DNA continues to be used more extensively, certain instances will arise in which reference material will not be readily available. In these situations, archived specimens may be employed as a reference source. Clinical specimens such as tissue biopsies should be used with caution if they have not been confirmed to contain normal tissue.

Natural DNA mixtures generated in fraternal twins in utero.

Analysis of multiple genetic loci using short tandem repeats (STR) is widely used in human identity testing because the extensive polymorphism at these loci allows for a high degree of discrimination among individuals. We recently received a forensic case that included several pieces of evidence and reference blood samples. Upon initial testing, one of the suspects had a DNA profile that included three alleles at four of the nine loci tested (vWA, FGA, TH01, and D5S818). At each locus, two of the alleles appeared to be "major" alleles with a third "minor" allele present. The profile appeared to be a mixture of two people. Contamination of this first reference sample was suspected and a second, unopened blood specimen was requested from this individual. The DNA profile from this second reference specimen was identical to that of the original specimen at each locus. One of the evidence samples also displayed an identical mixed DNA profile matching that of the reference specimens mentioned above. The relative peak heights of the two "major" and one "minor" allele remained constant in all three samples. Additional background information revealed that the suspect had not received a bone marrow transplant or blood transfusion. However, it was disclosed that this individual is a fraternal (dizygotic) twin. We hypothesize that an exchange of blood cells between the fetuses occurred in utero and that the additional alleles present in these reference samples are derived from cells contributed by his twin sibling. No additional specimens from the suspect or his twin could be obtained for confirmation, and our hypothesis remains untested. Forensic scientists should be aware of this possibility when faced with a DNA profile in which extra alleles at multiple loci are detected.

Molecular studies of murine mutant BALB/c-H-2dm2 define a deletion of several class I genes including the entire H-2Ld gene.

Inbred mouse strains carrying spontaneous mutations in class I genes have been extremely informative in studies of the genetic mechanisms generating polymorphism in the major histocompatibility gene complex. In this report, we determine the molecular basis of the spontaneous loss mutation in BALB/c-H-2dm2 mice, which were previously shown not to express Ld antigens while maintaining normal expression of two other class I antigens, Kd and Dd. We show BALB/c-H-2dm2 mice do not transcribe detectable levels of Ld mRNA, indicating they do not produce a truncated Ld molecule as previously reported. Furthermore, in Southern blot comparisons using a series of low-copy genomic probes, the deletion was found to be approximately 140 kilobases and include the entire Ld gene along with three or more other class I genes mapping between Dd and Ld. These data represent direct genetic evidence for a spontaneous contraction in the genes encoding class I histocompatibility antigens, which in this case probably resulted from the misalignment of the 3' flanking regions of the Dd and Ld genes.

Responses of B6.C-H-2bm12 to heterologous insulins show no correlation with the putative gene conversion but define Iabm12 as functionally unique.

The IA mutant mouse strain, B6.C-H-2bm12 (bm12) has been used to address several important questions for the role of Ia molecules in immune responses to foreign antigens. Numerous publications using bm12 mice have led to conclusions concerning (1) the number and relative importance of functional sites on Ia molecules; (2) the effects of qualitative versus quantitative differences in Ia; (3) whether T cells recognize Ia sequence or conformation; and (4) if gene conversion events, such as the one that putatively occurred in bm12, transfer functional Ir gene epitopes. Because of the importance of these conclusions, as well as their controversial nature, we have undertaken a comprehensive and systematic analysis of the aberrant immune response of bm12 mice to heterologous insulin. Responses to beef, horse, and sheep insulin were compared in B6 and bm12 mice by T cell proliferation, enumeration of plaque-forming cells, and quantitation of serum antibody levels. Various doses of antigen were administered and the kinetics of each response was monitored at various times. The findings of these studies suggest (1) B6 and bm12 mice both mount comparably high levels of response to sheep and horse insulins; (2) in contrast to the good response of B6 mice to beef insulin, bm12 mice showed dramatically impaired responses, as evident from both the lower magnitude of the response in all three assays as well as the difference in the kinetics of the response in B6 and bm12 mice; and (3) the response to sheep insulin is controlled by IA and IE encoded genes. These new findings differ from and extend previously published reports using bm12 mice, and therefore have substantive implications on the above stated conclusions regarding recognition of Ia. One such implication is that the bm12 gene conversion did not result in the transfer of a functional epitope for sheep insulin, but rather resulted in the creation of a functionally unique Ia molecule. Furthermore, this critical definition of the Ir gene lesion in bm12 permits us to address mechanistic questions regarding the nature of its Ir gene defect to beef insulin.

The conformation of Ld induced by beta 2-microglobulin is fixed during de novo synthesis and irreversible by exchange or dissociation.

Data are presented that support the hypothesis that beta 2m controls the folding of the ligand binding site of newly synthesized class I molecules. This conclusion was indicated by comparisons of two antigenic forms of the Ld molecule separated by sequential immunoprecipitation. Whereas, mAb 30-5-7+ Ld molecules were found to exist either as free H chains or associated with beta 2m, 30-5-7- Ld molecules showed no beta 2m association. Chemical comparisons showed 30-5-7- Ld molecules to be highly sensitive to proteolysis relative to 30-5-7+ Ld molecules. Experiments employing a construct with the Ld gene juxtaposed to the inducible metallothionein promoter indicated that the ratio of the antigenic forms of Ld was determined by the relative synthesis of beta 2m vs class I proteins. Pulse-chase experiments demonstrated that the two antigenic forms of Ld do not share a precursor-product relationship, but do display disparate rates of intracellular transport. beta 2m dissociation or exchange at the cell surface was found not to affect the ratio of the two antigenic forms of Ld. In contrast to these findings with Ld, the Dd and Ddml molecules were not detected in alternative conformations, thus mapping this property to the N-terminus of the class I molecule. These findings support the notion that beta 2m induces conformation on the alpha 1/alpha 2 domains of Ld molecules during de novo synthesis and once beta 2m-conformed, the class I structure is fixed and irreversible.

Peptide ligand-induced conformation and surface expression of the Ld class I MHC molecule.

Newly synthesized major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum are thought to bind peptides of foreign and endogenous antigens. Several lines of evidence indicate that beta-2 microglobulin (beta 2m) and/or peptide ligand participate in the intracellular transport and surface expression of class I molecules, but the nature of their involvement is still unclear. Here we present evidence that culturing non-mutant cells (fibroblast, thymoma or mastocytoma) with a peptide ligand specific for the Ld class I molecule of the mouse leads to a dramatic (fourfold) and specific induction of Ld surface expression. Surprisingly, this peptide ligand-induced expression of Ld does not result in an increased intracellular association of Ld with beta 2m. These findings demonstrate that the previously reported decrease in surface expression of Ld results from its failure to be saturated with endogenous self-peptide ligands. This unique feature of Ld could also contribute to the fact that several virus-specific cytotoxic T cell responses have been found to be Ld-restricted.

Histocompatibility screening by molecular techniques: use of polymerase chain reaction products and heteroduplex formation.

Novel molecular approaches have recently become available allowing improved major histocompatibility complex (MHC) matching of potential allogeneic bone marrow donors and recipients. Current cellular and serological assays are hindered by aberrant cell populations and limited reagents which only detect an individuals' phenotype. Therefore, a molecular screening protocol which discriminates at the genotypic level would be advantageous. Here we describe a two-step DNA-based approach that can be applied to large-scale screening of potential donors. A primary screen, utilizing polymerase chain reaction (PCR), reduces the potential donor population, whereas a secondary or fine resolution screen uses DNA heteroduplex analysis to determine identity or non-identity at specific loci. Heteroduplex analysis generates a DNA migration pattern that is unique for alleles at a given locus, and is more sensitive than serology in discriminating among individuals. Here we demonstrate the potential feasibility of this approach by analyzing results at one MHC locus, HLA-DQ. Since this method does not rely on typing sera or viable lymphocytes, it is not subject to the variability found in the traditional methods. In contrast to traditional methods, these molecular techniques can provide the critical information needed to select a potential bone marrow donor.

Class I-restricted cytotoxic T cell recognition of split peptide ligands.

We developed a novel approach to probe the molecular basis of TCR recognition of the MHC class I-peptide complex and to determine how constraints placed on peptide binding by the class I molecule influence T cell recognition. We synthesized peptide pairs derived from the N- and C-terminal regions of class I peptide ligands in which the TCR contacts and dominant binding residues were placed together or were separated. Complementary peptide pairs derived from two well-characterized Ld peptide ligands, tum- (QNHRALDL) and p2Ca (LSPFPFDL), were tested for the ability to sensitize targets for recognition by peptide-specific cytotoxic T lymphocytes (CTL). The tum-derived tetramer QNHR, containing both primary TCR contact residues (H17 and R18), is recognized only when used in combination with ALDL which contains the primary binding residues (A19, D21 and L22). This suggests that both peptides of the pair contribute to positioning of the TCR contacts. Remarkably, CTL clone P24 recognized target cells sensitized with a trimer (QNH) combined with a pentamer (RALDL), demonstrating that TCR recognition can occur when the TCR contacts are separated (placed on separate peptide subunits). For the p2Ca peptide LSPFPFDL, the C-terminal tetramer PFDL, which contains both the primary TCR contact residue (P) and the dominant binding residue (L), is sufficient for recognition. In addition, PFDL was able to bind effectively to Ld and to activate naive antigen-specific T cells. These data suggest that peptide subunits and complementary peptide pairs composed of trimeric, tetrameric or pentameric peptides can bind independently to the Ld molecule in the same register and orientation as they do when contained within the parent peptide.

Human leukocyte antigen class II associations in patients with idiopathic dilated cardiomyopathy. Myocarditis Treatment Trial Investigators.

BACKGROUND: Idiopathic dilated cardiomyopathy (IDC) is a disease of unknown etiology for which immune abnormalities, possibly related to viral infections, are suspected but unproven. Previous serologic studies have reported associations between human leukocyte antigen DR4 and IDC. A molecular study of human leukocyte antigen associations was undertaken in patients with IDC to further explore the possibility of susceptibility markers of genetically determined disease. METHODS AND RESULTS: In this study, 36 patients from the Myocarditis Treatment Trial (32 IDC and 4 myocarditis patients) were examined using restriction fragment length polymorphism analysis and polymerase chain reaction amplification with sequence-specific primers to perform class II typing. All 4 myocarditis patients were DQ5 positive and 3 possessed the allele DQB1*0501. In the IDC group, the frequency of human leukocyte antigen DR4 was similar to that reported in the normal population. In addition, there was no excess prevalence of any molecularly defined DR4 alleles (0401-0419). There was an increase in the frequency of DR12 in IDC patients. The frequencies of the alleles DQB1 *0503 and DQB1*0301 and/or *0304 were also increased in IDC patients versus the normal population. CONCLUSION: The molecular studies point to a relationship between the DQ locus and IDC.

Mutation at amino acid position 133 of H-2Dd prevents beta 2m association and immune recognition but not surface expression.

The histocompatibility loss mutation H-2dm6 was derived from a mouse treated with the chemical mutagen ethylnitrosourea, and previous mapping studies implicated Dd as the affected locus. Southern blot analyses of DNA from H-2dm6 cells did not detect major deletions in the Ddm6 gene, suggesting that H-2dm6 was different from the previously characterized D region mutants H-2dm1 and H-2dm2. RNA blot analysis identified Ddm6 transcripts of appropriate size and a Ddm6 protein was immunoprecipitated from biosynthetically labeled H-2dm6 cells. Interestingly, the Ddm6 protein showed no beta 2m association and was only precipitated by a mAb to the alpha 3 domain. Furthermore, oligosaccharide maturation and low levels of surface expression of Ddm6 molecules were detected. However, the surface Ddm6 was nonfunctional as a target Ag in in vitro cytotoxicity assays, consistent with its original in vivo detection as a loss mutation. Sequence analyses of Ddm6 cDNA identified a single nucleotide base difference from wild-type, resulting in the substitution of a Trp to Arg at position 133. The significance of this substitution is discussed in the context of other class I expression variants.

Full hematopoietic engraftment after allogeneic bone marrow transplantation without cytoreduction in a child with severe combined immunodeficiency.

Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.