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Some pygmy chimpanzees (also called Bonobos) give much simpler patterns of hybridization on Southern blotting with killer cell immunoglobulin-like receptor (KIR) cDNA probes than do either humans or common chimpanzees. Characterization of KIRs from pygmy chimpanzees having simple and complex banding patterns identified nine different KIRs, representing seven genes. Five of these genes have orthologs in the common chimpanzee, and three of them (KIRCI, KIR2DL4, and KIR2DL5) also have human orthologs. The remaining two genes are KIR3D paralogous to the human and common chimpanzee major histocompatibility complex A- and/or -B-specific KIRs. Within a pygmy chimpanzee family, KIR haplotypes were defined. Simple patterns on Southern blot were due to inheritance of "short" KIR haplotypes containing only three KIR genes, KIRCI, KIR2DL4, and KIR3D, each of which represents one of the three major KIR lineages. These three genes in pygmy chimpanzees or their corresponding genes in humans and common chimpanzees form the conserved "framework" common to all KIR haplotypes in these species and upon which haplotypic diversity is built. The fecundity and health of individual pygmy chimpanzees who are homozygotes for short KIR haplotypes attest to the viability of short KIR haplotypes, indicating that they can provide minimal, essential KIRs for the natural killer and T cells of the hominoid immune system.

Purpose: We generated a humanized antibody, HuLuc63, which specifically targets CS1 (CCND3 subset 1, CRACC, and SLAMF7), a cell surface glycoprotein not previously associated with multiple myeloma. To explore the therapeutic potential of HuLuc63 in multiple myeloma, we examined in detail the expression profile of CS1, the binding properties of HuLuc63 to normal and malignant cells, and the antimyeloma activity of HuLuc63 in preclinical models., Experimental Design: CS1 was analyzed by gene expression profiling and immunohistochemistry of multiple myeloma samples and numerous normal tissues. HuLuc63-mediated antimyeloma activity was tested in vitro in antibody-dependent cellular cytotoxicity (ADCC) assays and in vivo using the human OPM2 xenograft model in mice., Results: CS1 mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1 antibody staining of tissues showed strong staining of myeloma cells in all plasmacytomas and bone marrow biopsies. Flow cytometric analysis of patient samples using HuLuc63 showed specific staining of CD138+ myeloma cells, natural killer (NK), NK-like T cells, and CD8+ T cells, with no binding detected on hematopoietic CD34+ stem cells. HuLuc63 exhibited significant in vitro ADCC using primary myeloma cells as targets and both allogeneic and autologous NK cells as effectors. HuLuc63 exerted significant in vivo antitumor activity, which depended on efficient Fc-CD16 interaction as well as the presence of NK cells in the mice., Conclusions: These results suggest that HuLuc63 eliminates myeloma cells, at least in part, via NK-mediated ADCC and shows the therapeutic potential of targeting CS1 with HuLuc63 for the treatment of multiple myeloma.

Beta2-microglobulin (beta2m) associates with MHC and related class I H chains to form cell surface glycoproteins that mediate a variety of functions in defense. In humans, monomorphism of a single beta2m gene contrasts with the diversity and polymorphism of the class I H chain genes, and a similar picture was seen in almost all other species examined. In this regard, rainbow trout (Oncorhynchus mykiss) appeared unusual: trout beta2m genes gave a complicated and polymorphic pattern in Southern blots, and a minimum of 10 different mRNA encoding two distinct types of beta2m were expressed by a single fish. Characterization of genomic clones from the same fish now shows that the rainbow trout beta2m locus consists of two expressed genes and one partial gene that are closely linked. Four copies of the locus were identified and allelic variants of each gene defined, largely through comparison of the noncoding regions. A dramatic variation in the lengths of introns is caused by variable repetitive elements and accounts for the complex pattern seen in Southern blots. By comparison to noncoding sequences, the coding regions are conserved but the three loci differ within a cluster of codons that encode residues of beta2m that do not interact with class I H chains. Additional diversity in the trout beta2m genes appears to be due to somatic mutation that might be facilitated by the abundance of repetitive DNA elements within the 12 beta2m genes of an individual rainbow trout.

Sequence-based typing of a breeding population (G1) consisting of 84 Atlantic salmon individuals revealed the presence of 7 Sasa-DAA and 7 Sasa-DAB expressed alleles. Subsequent typing of 1,182 individuals belonging to 33 families showed that Sasa-DAA and Sasa-DAB segregate as haplotypes. In total seven unique haplotypes were established, with frequencies in the population studied ranging from 0.01 to 0.49. Each haplotype is characterized by a unique minisatellite marker size embedded in the 3' untranslated region of the Sasa-DAA gene. These data corroborate the fact that Atlantic salmon express a single class II locus, consisting of tightly linked class II A and class B genes. The seven haplotypes give rise to 15 genotypes with frequencies varying between 0.01 and 0.23; 21 class II homozygous individuals were present in the G1 population. We also studied the frequency distribution in another breeding population (G4, n=374) using the minisatellite marker. Only one new marker size was present, suggesting the presence of one new class II haplotype. The marker frequency distribution in the G4 population differed markedly from the G1 population. The genomic organizations of two Sasa-DAA and Sasa-DAB alleles were determined, and supported the notion that these alleles belong to the same locus. In contrast to other studies of salmonid class II sequences, phylogenetic analyses of brown trout and Atlantic class II A and class II B sequences provided support for trans-species polymorphism.

Here we describe two rainbow trout major histocompatibility complex (MHC) class I genes characterized from lambda phage genomic clones prepared from a single fish. Clone GC71 contains all exons except a leader peptide-encoding exon. An open reading frame is maintained, and thus the gene MhcOnmy-U71 could be expressed in this individual. The class I gene found on clone GC41 lacks exons encoding the leader peptide and cytoplasmic domain. This gene, MhcOnmy-U41p, is a pseudogene due to a deletion in the alpha(2) domain-encoding exon causing premature termination. Both the Onmy-U71 and Onmy-U41p genes are distinguished by long introns between the exons encoding the alpha(1) and alpha(2) domains. Clone GC41 also contains the 3' exons of the LMP7/ PSMB8 gene encoding the gamma-interferon-induced proteosome subunit of rainbow trout.

To assess polymorphism and variation in human and chimpanzee NK complex genes, we determined the coding-region sequences for CD94 and NKG2A, C, D, E, and F from several human (Homo sapiens) donors and common chimpanzees (Pan troglodytes). CD94 is highly conserved, while the NKG2 genes exhibit some polymorphism. For all the genes, alternative mRNA splicing variants were frequent among the clones obtained by RT-PCR. Alternative splicing acts similarly in human and chimpanzee to produce the CD94B variant from the CD94 gene and the NKG2B variant from the NKG2A gene. Whereas single chimpanzee orthologs for CD94, NKG2A, NKG2E, and NKG2F were identified, two chimpanzee paralogs of the human NKG2C gene were defined. The chimpanzee Pt-NKG2CI gene encodes a protein similar to human NKG2C, whereas in the chimpanzee Pt-NKG2CII gene the translation frame changes near the beginning of the carbohydrate recognition domain, causing premature termination. Analysis of a panel of chimpanzee NK cell clones showed that Pt-NKG2CI and Pt-NKG2CII are independently and clonally expressed. Pt-NKG2CI and Pt-NKG2CII are equally diverged from human NKG2C, indicating that they arose by gene duplication subsequent to the divergence of chimpanzee and human ancestors. Genomic DNA from 80 individuals representing six primate species were typed for the presence of CD94 and NKG2. Each species gave distinctive typing patterns, with NKG2A and CD94 being most conserved. Seven different NK complex genotypes within the panel of 48 common chimpanzees were due to differences in Pt-NKG2C and Pt-NKG2D genes.

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family.

The human leukocyte receptor complex (LRC) at Chromosome 19q13.4 encodes Ig superfamily proteins which regulate the function of various hematopoietic cell types. We investigated characteristics of the Ig-like transcript (ILT)/leukocyte Ig-like receptor (LIR) group of LRC genes in comparison with the other major LRC loci encoding the killer cell Ig-like receptors (KIRs). In direct contrast to KIR genes, the ILT/LIR loci of ethnically diverse individuals did not display haplotypic variations in gene number. Investigation of gene expression identified novel cDNA sequences related to the ILT2/LIR1, ILT4/LIR2, ILT3/LIR5, and ILT7 loci, while phylogenetic analysis revealed two distinct lineages of ILT/LIR genes. These two lineages differ in both the nature and extent of their sequence polymorphism. The presence of certain transcription factor-related motifs in the 5' untranslated region of ILT/LIR cDNAs correlates with the specific cell types in which particular ILT/LIR genes are expressed. Although extensive gene duplications and conversion events have apparently forged the LRC, our results indicate striking conservation in the organization of the ILT/LIR genes when compared with the related and closely linked KIR genes. This suggests the evolutionary maintenance of a significant function consistent with the cellular distribution of the ILT/LIR proteins.

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15--20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II beta-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles alpha 1 and alpha 2 domains into different combinations.

That NK cell receptors engage fast-evolving MHC class I ligands suggests that they, too, evolve rapidly. To test this hypothesis, the structure and class I specificity of chimpanzee KIR and CD94:NKG2 receptors were determined and compared to their human counterparts. The KIR families are divergent, with only three KIR conserved between chimpanzees and humans. By contrast, CD94:NKG2 receptors are conserved. Whereas receptors for polymorphic class I are divergent, those for nonpolymorphic class I are conserved. Although chimpanzee and human NK cells exhibit identical receptor specificities for MHC-C, they are mediated by nonorthologous KIR. These results demonstrate the rapid evolution of NK cell receptor systems and imply that "catching up" with class I is not the only force driving this evolution.

Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UAC32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the alpha 3 domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2-6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14-16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene.

Previous studies of class I MHC molecules have shown that the owl monkey (Aotus) possesses at least two variants of the beta2-microglobulin (beta2m) protein. These two variants have different isoelectric points, and exhibit differential reactivity with the monoclonal antibody W6/32. We report cDNA sequences of the B2m gene, from W6/32-positive and W6/32-negative Aotus cell lines. The two beta2m variants we identified exhibit a single amino acid difference at position three. An arginine residue at position 3 was correlated with W6/32 reactivity, whereas histidine was associated with non-reactivity. W6/32 reactivity was conferred to a W6/32-negative Aotus cell line when it was transfected with the B2m from the W6/32-positive cell line. Residue 3 of beta2m is located at the surface of the class I molecule. It is also close to position 121 of the MHC class I heavy chain, which has previously been shown to influence W6/32 antibody binding. We conclude that W6/32 binds a compact epitope on the class I molecule that includes both residue 3 of beta2m and residue 121 of the heavy chain. We examined the distribution of the two beta2m motifs in a sample Aotus population using an allele-specific polymerase chain reaction assay. The pattern of beta2m segregation we observed matches that which was defined previously by serology. Additionally, we identified laboratory-born hybrid animals who possess both variants of beta2m.

The presence and expression of killer inhibitory receptor (KIR) and CD94:NKG2 genes from 68 donors were analyzed using molecular typing techniques. The genes encoding CD94:NKG2 receptors were present in each person, but KIR gene possession varied. Most individuals expressed inhibitory KIR for the three well-defined HLA-B and -C ligands, but noninhibitory KIR genes were more variable. Twenty different KIR phenotypes were defined. Two groups of KIR haplotypes were distinguished and occurred at relatively even frequency. Group A KIR haplotypes consist of six genes: the main inhibitory KIR, one noninhibitory KIR, and a structurally divergent KIR. Allelic polymorphism within five KIR genes was detected. Group B comprises more noninhibitory KIR genes and contains at least one additional gene not represented in group A. The KIR locus therefore appears to be polygenic and polymorphic within the human population.

For mammals beta2-microglobulin (beta2m), the light chain of major histocompatibility complex (MHC) class I molecules, is invariant (or highly conserved) and is encoded by a single gene unlinked to the MHC. We find that beta2m of a salmonid fish, the rainbow trout (Oncorhynchus mykiss), does not conform to the mammalian paradigm. Ten of 12 randomly selected beta2m cDNA clones from an individual fish have different nucleotide sequences. A complex restriction fragment length polymorphism pattern is observed with rainbow trout, suggesting multiple beta2m genes in the genome, in excess of the two genes expected from the ancestral salmonid tetraploidy. Additional duplication and diversification of the beta2m genes might have occurred subsequently. Variation in the beta2m cDNA sequences is mainly at sites that do not perturb the structure of the mature beta2m protein, showing that the observed diversity of the trout beta2m genes is not primarily a result of pathogen selection.

The amphibian Xenopus laevis is the most primitive vertebrate in which the major histocompatibility complex (MHC) has been defined at the biochemical, functional, and molecular genetic levels. We previously described the isolation and characterization of cDNA clones encoding X. laevis MHC class II beta chains. In the present study, genomic clones encoding class II beta chains were isolated from X. laevis homozygous for the MHC f haplotype. Three class II beta chain genes, designated Xela-DAB, Xela-DBB, and Xela-DCB, were identified. Sequence analysis of these genes showed that Xela-DBB and Xela-DCB correspond to the previously characterized cDNA clones F3 and F8, respectively, whereas Xela-DAB encodes a third, hitherto unidentified class II beta chain of the MHC f haplotype. As a representative of X. laevis class II beta chain genes, the Xela-DAB gene underwent detailed structural analysis. In addition, the nucleotide sequence of Xela-DABf cDNA clones was determined. The Xela-DAB gene is made up of at least six exons, with an exon-intron organization similar to that of a typical mammalian class II beta chain gene. The 5'-flanking region of the Xela-DAB gene contains transcriptional control elements known as X1, X2, and Y, but lacks typical TATA or CCAAT boxes. A notable feature of the X. laevis class II beta chain genes is that the sizes of the introns are larger than those of their mammalian counterparts. As assessed by northern blot analysis, the three class II beta chain genes had similar expression patterns, with the highest level of transcription detected in the intestine. Identification of the Xela-DAB, -DBB, and -DCB genes is consistent with our previous observations, which suggested that the MHC of the tetraploid frog X. laevis is diploidized at the genomic level and contains three class II beta chain genes per haplotype that cross-hybridize to one another under reduced stringency conditions.

The amphibian Xenopus is an ectothermic vertebrate in which the MHC has been studied extensively at the functional, biochemical, and genetic levels. A cDNA clone corresponding to the MHC class la gene (Xela-UAA1f) of Xenopus laevis was isolated by screening a cDNA phage library with oligonucleotides based on NH2-terminal protein sequence. Three pieces of evidence support its status as a class la gene: 1) Previous biochemical data suggested that only one polymorphic class la molecule is expressed per MHC haplotype in X. laevis. NH2-terminal sequencing of the class I protein encoded by the f haplotype showed a single unambiguous sequence of the first 22 amino acids; the deduced protein sequence of the cDNA clone matches precisely to this peptide sequence; 2) Genes that hybridized to the cDNA clone segregated perfectly with the serologically typed MHC in two family studies; and 3) There is a strong conservation of amino acids in the peptide-binding region that have been shown in mammals to dock peptides at their NH2- and COOH-termini. In contrast to all other species that have been examined, there appears to be only one class I locus present in the MHC of X. laevis. Xenopus speciates by allopolyploidization, and there are Xenopus species with different levels of ploidy (2n-12n). Functionally, the MHC has been shown to be "diploidized" in most Xenopus species. As in previous studies with MHC class II and HSP70 probes, there is a trend toward maintaining a diploid number of class la genes in all Xenopus species regardless of their chromosome number, probably accomplished through a deletional mechanism. Thus, there is a strong pressure in Xenopus to maintain very few MHC-linked class I genes, exemplified both by the number of class I genes per MHC haplotype and by the number of class la genes per organism.

A Xenopus class I cDNA clone, isolated from a cDNA expression library using antisera, is a member of a large family of non-classical class I genes (class Ib) composed of at least nine subfamilies, all of which are expressed at the RNA level. The subfamilies are well conserved in their immunoglobulin-like alpha 3 domains, but their peptide-binding regions (PBRs) and cytoplasmic domains are very divergent. In contrast to the great allelic diversity found in the PBR of classical class I genes, the alleles of one of the Xenopus non-classical subfamilies are extremely well conserved in all regions. Several of the invariant amino acids essential for the anchoring of peptides in the classical class I groove are not conserved in some subfamilies, but the class Ib genes are nevertheless more closely related in the PBR to classical and non-classical genes linked to the MHC in mammals and birds than to any other described class I genes like CD1 and the neonatal rat intestinal Fc receptor. Comparison with the Xenopus MHC-linked class Ia protein indicate that amino acids presumed to interact with beta 2-microglobulin are identical or conservatively changed in the two major class I families. Genomic analyses of Xenopus species suggest that the classical and non-classical families diverged from a common ancestor before the emergence of the genus Xenopus over 100 million years ago; all of the non-classical genes appear to be linked on a chromosome distinct from the one harboring the MHC. We hypothesize that this class Ib gene family is under very different selection pressures from the classical MHC genes, and that each subfamily may have evolved for a particular function.