Removal of Qa-2 antigen alters the Ped gene phenotype of preimplantation mouse embryos.

Embryo survival is influenced by both genetic and environmental factors. Previous research in our laboratory has identified one gene associated with embryonic survival, the Ped gene, a gene that is linked to the major histocompatibility complex (MHC) of the mouse. The Ped gene has been shown to influence the rate of preimplantation embryonic cleavage division, as well as litter size, birth weight, and weaning weight. Genetic mapping of the Ped gene has located it in the Q region of the MHC and has suggested that possible Q region genes encoding the Ped gene are Q3, Q5, Q6, Q7, Q8, and/or Q9. Whereas the protein products of the Q3 and Q5 genes are unknown, the protein product of the very similar Q6, Q7, Q8, and Q9 genes is the Qa-2 antigen. Two forms of membrane-bound Qa-2 antigen are known: glycosylphosphatidylinositol (GPI)-linked and transmembrane bound. Only the GPI-linked form is sensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC). The first purpose of the present study was to determine the nature of the linkage of the Qa-2 antigen to the cell surface of preimplantation mouse embryos. It was found that all detectable Qa-2 antigen on the embryonic cell surface is sensitive to cleavage by PI-PLC and is therefore bound to the cell membrane by a GPI linkage. Furthermore, removal of Qa-2 antigen from the embryonic cell surface slows down the rate of development of preimplantation mouse embryos. These results suggest the likelihood that the Qa-2 antigen is the Ped gene product.     

Preimplantation embryo development (<Emphasis Type="Italic">Ped</Emphasis>) gene copy number varies from 0 to 85 in a population of wild mice identified as <Emphasis Type="Italic">Mus musculus domesticus</Emphasis>

The preimplantation embryo development (Ped) gene regulates the rate of preimplantation embryonic cleavage division and subsequent embryo survival. In the mouse, the Ped gene product is Qa-2 protein, a nonclassical MHC class I molecule encoded by four tandem genes, Q6/Q7/Q8/Q9. Most inbred strains of mice have all four genes on each allelic chromosome, making a total of eight Qa-2 encoding genes, but there are a few strains that are missing all eight genes, defining a null allele. Mouse strains with the presence of the Qa-2 encoding genes express Qa-2 protein and produce embryos with a faster rate of preimplantation embryonic development and a greater chance of embryo survival compared to mouse strains with the null allele. There is extensive evidence that the human homolog of Qa-2 is HLA-G. HLA-G in humans, like Qa-2 in mice, is associated with enhanced reproductive success. The human population is an outbred population. Therefore, for a better comparison to the human population, we undertook an investigation of the presence of the genes encoding Qa-2 in an outbred population of mice. We used Real-Time Quantitative PCR to quantify the number of Qa-2 encoding genes in a population of 32 wild mice identified as Mus musculus domesticus both by morphologic assessment and by PCR analysis of their DNA. We found great variability in the number of Qa-2 encoding genes in the wild mice tested. The wild mouse with the highest number of Qa-2 encoding genes had 85 such genes, whereas we discovered one wild mouse without any Qa-2 encoding genes. Evolutionary implications of a range of Qa-2 encoding gene numbers in the wild mouse population are discussed, as well as the relevance of our findings to humans.     

Analysis of Qa-2 antigen expression by preimplantation mouse embryos: possible relationship to the preimplantation-embryo-development (Ped) gene product

The preimplantation-embryo-development (Ped) gene, a gene that controls the cleavage rate of preimplantation mouse embryos, maps to the Qa-2 subregion of the mouse major histocompatibility complex (MHC). A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure was used to detect Qa-2 antigens on mouse embryos. The use of a monoclonal antibody specific for Qa-2 antigens showed that Qa-2 antigens were present on oocytes, 2-cell, 8-cell, and blastocyst-stage embryos, with the greatest expression found on blastocysts. Expression of Qa-2 antigens by the embryos correlated completely with Ped gene phenotype. Those embryos expressing the fast Ped allele showed the presence of Qa-2 antigens (Qa-2a mice), whereas those embryos expressing the slow Ped allele showed the absence of Qa-2 antigens (Qa-2b mice). It is hypothesized that the Qa-2 antigen may be the Ped gene product.     

Influence of the preimplantation-embryo-development (ped) gene on mouse blastocyst differentiation

Mouse preimplantation embryonic cleavage rate is dependent upon the presence or absence of the Preimplantation-embryo-development (Ped) gene; which is linked to the Qa-2 subregion of the H-2 complex. Expression of Qa-2 antigens by fast developing mouse embryos correlates with Ped gene pheno-type: Qa-2(a). It is not known if the Ped gene (Qa-2(a)) participates in cell differentiation in the preimplantation mouse blastocyst. Therefore, the study objective was to determine the differentiation of cells to the inner cell mass (ICM) and trophectoderm (TE) in Qa-2(a) positive (Ped +) and Qa-2(a) negative (Ped -) mouse blastocysts. One-cell stage embryos were recovered from the excised oviducts of PMSG (5 IU) and hCG (5 IU) primed virgin female (3-4 weeks) BALB/cByJ (Qa-2(a): Ped -) and BALB/cJ (Qa-2(a): Ped +) mice mated to fertile males (12+ weeks). Embryos were collected, 14 hr after hCG, and cultured in modified alpha-MEM, to the hatched blastocyst stage in an atmosphere of 5% CO2 in air, 95% relative humidity at 37 degrees C. Cell differentiation was determined by differential staining (bis-benzimide and propidium iodide) and fluorescence microscopy. Data were analyzed by Students t-test. There was no significant difference in total cell number between BALB/cJ (mean 139) and BALB/cByJ (mean 143) embryos. A significant difference (p < 0.001) was found in the number of cells differentiating to the ICM between BALB/cJ (mean 59.0) and BALB/cByJ (mean 29.0) mouse embryos. The number of cells differentiating to the TE, between BALB/cJ (mean 80.0) and BALB/cByJ (mean 114) embryos, approached significance (p = 0.062). The results suggest that the Ped gene (Qa-2(a)) may have an influential role in preimplantation blastocyst cell differentiation. Additional studies are warranted to further elucidate the role of the Ped gene in preimplantation embryo development and blastocyst formation.     

Promiscuous antigen presentation by the nonclassical MHC Ib Qa-2 is enabled by a shallow, hydrophobic groove and self-stabilized peptide conformation.

BACKGROUND: Qa-2 is a nonclassical MHC Ib antigen, which has been implicated in both innate and adaptive immune responses, as well as embryonic development. Qa-2 has an unusual peptide binding specificity in that it requires two dominant C-terminal anchor residues and is capable of associating with a substantially more diverse array of peptide sequences than other nonclassical MHC. RESULTS: We have determined the crystal structure, to 2.3 A, of the Q9 gene of murine Qa-2 complexed with a self-peptide derived from the L19 ribosomal protein, which is abundant in the pool of peptides eluted from the Q9 groove. The 9 amino acid peptide is bound high in a shallow, hydrophobic binding groove of Q9, which is missing a C pocket. The peptide makes few specific contacts and exhibits extremely poor shape complementarity to the MHC groove, which facilitates the presentation of a degenerate array of sequences. The L19 peptide is in a centrally bulged conformation that is stabilized by intramolecular interactions from the invariant P7 histidine anchor residue and by a hydrophobic core of preferred secondary anchor residues that have minimal interaction with the MHC. CONCLUSIONS: Unexpectedly, the preferred secondary peptide residues that exhibit tenuous contact with Q9 contribute significantly to the overall stability of the peptide-MHC complex. The structure of this complex implies a "conformational" selection by Q9 for peptide residues that optimally stabilize the large bulge rather than making intimate contact with the MHC pockets.     

Ped gene deletion polymorphism frequency in wild mice

The Ped gene influences the rate of cleavage of preimplantation embryos and their subsequent survival. Embryos that express the product of the Ped gene, Qa-2 protein, cleave at a faster rate than embryos with an absence of Qa-2 protein. In addition, the Ped gene has pleiotropic effects on reproduction. Thus, there is a reproductive advantage to those mouse strains that are Qa-2 positive. The presence or absence of Qa-2 is reflected at the DNA level by the presence or absence (deletion polymorphism) of the gene(s) encoding Qa-2 protein. Many inbred and wild-derived mouse strains have been characterized as Qa-2 positive or negative, but no previous studies have looked at the distribution of the Ped gene in a population of free-living wild mice. The purpose of this study was to determine the Ped gene deletion polymorphism frequency in a sample of free-living wild mice. Twenty-nine mice were collected and identified as Mus musculus. Genomic DNA extraction was performed on tail tips, and PCR was used to amplify a region from the Ped gene. Known Qa-2 positive and negative mice were used as controls. Results showed that all 29 wild mice were positive for the Ped gene. Since the Ped gene is dominant and provides a reproductive advantage, it is not surprising that all of the wild mice were Qa-2 positive. However, our assay could not distinguish homozygous from heterozygous mice. It is possible that the Qa-2 deletion polymorphism is segregating in the population, and a larger sample size would identify some Qa-2 negative mice.     

Selection in favor of the Ped fast haplotype occurs between mid-gestation and birth

 The preimplantation embryo development (Ped) gene that encodes the class Ib major histocompatibility complex protein Qa-2 influences the rate of embryonic cleavage during the preimplantation stages of development. Embryos from strains of mice that lack the Ped gene cleave slowly, while embryos that have a functional Ped gene cleave more rapidly. This effect is observed both in vivo and in vitro with the Ped fast haplotype showing dominance over the Ped slow haplotype. The Ped gene is associated with pleiotropic effects on reproduction. Certain strains of mice lacking the Ped gene (Ped slow) have smaller litters and the pups weigh less at birth and at weaning. Previously our laboratory reported that in litters derived from Ped fast/slow F1 mice backcrossed to the slow/slow parent, there were significantly more Ped fast pups than the 50% expected, at two months of age. This implies that there is selection in favor of the Ped fast haplotype at some point during development. The present study was designed to determine at what point during development selection occurs. Using a polymerase chain reaction assay, we determined that selection does not occur by days post coitus 14.5. However, our results show that there are significantly more Ped fast pups than Ped slow pups remaining in backcross litters just after birth, indicating that selection in favor of the Ped fast haplotype occurs between day 14.5 and birth. Received: 5 June 1998 / Revised: 20 January 1999     

Molecular basis of Qa-11 antigen and paradoxical Qa-gene expression in an H-2 recombinant

The Qa-11 Ag expressed in certain strains with the B2-microglobulin-b allele, apparently maps into the Tla region as well as into the Qa-2 region. Moreover Qa-11 has been shown to be biochemically indistinguishable from Qa-2. Genetic complementation studies combining the right Qa and Tla regions failed to lead to Qa-11 expression. To elucidate the molecular basis of this apparent paradox, we examined the expression of Qa-11 on products of transfected Q-region class I genes. Immunochemical analysis has shown that the Qa-11 Ag is expressed on class I molecules encoded by the Q7 gene from both C57BL/10 (Q7b) and BALB/c (Q7d), but not on the protein product of the Q9 gene isolated from the C57BL/10 strain (Q9b). Inasmuch as the predicted protein products of the Q7b and Q9b genes would differ at a single amino acid, a residue critical for Qa-11 expression has been identified. Based on these results it is proposed that among the beta-2-mb strains, the Qa-11+/Qa-2+ mice are likely to express at least the Q7 gene, whereas Qa-11-/Qa-2+ mice express only Q9. In support of this model, the Qa-2+/Q-11- recombinant B6.K2, essential for the apparent mapping of Qa-11 into the Tla region, expresses only Q9 but not Q7 encoded molecules on the cell surface, and only Q9 and no processed Q7 mRNA is detected in the cytoplasm. This expression pattern in B6.K2 cannot be explained on the basis of a single crossing-over event.     

The role of structurally conserved class I MHC in tumor rejection: contribution of the Q8 locus

The mouse multimember family of Qa-2 oligomorphic class I MHC genes is continuously undergoing duplications and deletions that alter the number of the two "prototype" Qa-2 sequences, Q8 and Q9. The frequent recombination events within the Q region lead to strain-specific modulation of the cumulative Qa-2 expression levels. Q9 protects C57BL/6 hosts from multiple disparate tumors and functions as a major CTL restriction element for shared tumor-associated Ags. We have now analyzed functional and structural properties of Q8, a class I MHC that differs significantly from Q9 in the peptide-binding, CTL-interacting alpha(1) and alpha(2) regions. Unexpectedly, we find that the extracellular domains of Q8 and Q9 act similarly during primary and secondary rejection of tumors, are recognized by cross-reactive antitumor CTL, have overlapping peptide-binding motifs, and are both assembled via the transporter associated with the Ag processing pathway. These findings suggest that shared Ag-presenting functions of the "odd" and "even" Qa-2 loci may contribute to the selective pressures shaping the haplotype-dependent quantitative variation of Qa-2 protein expression.     

Linkage of the preimplantation-embryo-development (Ped) gene to the mouse major histocompatibility complex (MHC)

The preimplantation-embryo-development (Ped) gene influences the rate of cleavage division of preimplantation mouse embryos. The location of the Ped gene in the mouse major histocompatibility complex (MHC), the H-2 complex, has been inferred from the analysis of cleavage rates of embryos from mouse strains congenic at the H-2 complex. In this paper, formal genetic linkage studies were undertaken to evaluate linkage of the Ped gene to the H-2 complex. Co-segregation of Ped gene phenotype and H-2 haplotype was found in back-cross embryos. These data support the hypothesis that the Ped gene is linked to the H-2 complex.     

Ped gene expression by embryos cultured in vitro

The rate of cleavage division of preimplantation mouse embryos has been shown to be influenced by the Ped gene, a gene linked to the H-2 complex, the major histocompatibility complex of the mouse. There are two functional alleles of the Ped gene, slow and fast. To examine Ped gene expression outside of the maternal uterine environment, embryos from inbred and congenic mouse strains were cultured in vitro, in chemically defined medium, for various lengths of time. The results of these studies show that the difference in the rate of cleavage division between slow-developing strains (Ped slow) and fast-developing strains (Ped fast) is maintained in vitro. Thus, the Ped gene phenotype of developing embryos is an intrinsic property of the embryos themselves.     

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.     

Antigenic Heterogeneity of Qa-2 Antigens in C57BL/6

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of¹²⁵I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.     

Expression of theQ8/9 d gene by T cells of the mouse

TheQ genes, specifying Qa antigens and situated in the extended part of the major histocompatibility complex (MHC) of the mouse, comprise a subgroup of MHC class I genes whose significance and function are still largely unknown. In screening a cDNA library made from the BALB/c inducer T-cell line Cl.Ly1-T1, we isolated 11 clones representingQ8/9, but none representingQ6 orQ7. Confirmatory evidence is given that theQ8/9 gene originated from fusion of the 5′ region of theQ8 gene with the 3′ region of theQ9 gene at a recombination site or hot spot in the vicinity of intron 4. Contrary to previous impressions thatQ8/9 is an inert pseudogene, we find that theQ8/9 gene can be functional and encode a Qa-2,3 antigen. One variety of the 11 Q8/9 clones isolated lacked exon 5, which encodes the transmembrane domain of class I glycoproteins, and thus may account for secretion of a soluble form of Qa-2,3 antigen thought to be released by activated T cells.