Anti-human red cell monoclonal antibodies produced by macaque-mouse heterohybridomas: their reactivity with human and nonhuman primate erythrocytes.

Eighteen monoclonal antibodies (Mabs) against human red blood cells (RBCs) produced by macaque mouse heterobybridomas gave uniformly positive reactions with all human samples except for some with particular null phenotypes. Based on reactions with latter cells, the human antigenic targets of 11 antibodies could be identified: six were specific for glycophorin-related antigens (Wr(b), En(a), Ge4), and each of the live remaining antibodies showed one of the following specificities: CD55, CD44, CD59, Kell, and Rh proteins. Four Mabs recognized the Vc antigen of the chimpanzee V-A-B-D system. Six macaque Mabs detected polymorphisms in chimpanzee, gorilla, orangutan, and gibbon that did not correspond to any known blood group in these animals, while other Mabs gave monomorphic reactions with ape RBCs. The reagents produced by macaque hybridomas are useful tools not only for human blood grouping tests, but also for tracing the relationships among blood group antigens of man and anthropoid apes.

Characterization of a macaque anti-Rh17-like monoclonal antibody.

In order to produce macaque monoclonal antibodies (mAbs) against human red blood cell (RBC) antigens, macaques were immunized with human and gorilla RBCs and their spleen lymphocytes were fused with man-mouse heteromyeloma cells. One macaque-mouse heterohybridoma produced a macaque IgGx (Cvn2-4D5) which agglutinated all human RBCs but not rare human variants Dc-,D-, and Rhnull. Thus, Cyn2-4D5 exhibited RH17-like reactivity. The specificity of Cyn2-4D5 for RHCE-encoded polypeptides was confirmed by specific immunoprecipitation of RhcE and RhCe polypeptides from human RBCs and the absence of immunoprecipitation of the RhD polypeptides extracted from D-RBCs. This study demonstrates that it is possible to produce macaque mAbs against human RBC blood group antigens.

Reactivity of anti-glycophorin monoclonal antibodies (Mabs) in tests with red cells of non-human primates.

Seventy Mabs against human glycophorins (GP) and band 3 were tested with red blood cells (RBCs) of various non-human primates, from anthropoid apes to monkeys. Differences among Mabs reactivity in tests with non-human primate RBCs reflect the complexity of the immune reactions to human GPs and provide insights into aspects of evolution and a tool to epitope map.

Monoclonal antibodies directed against human Rh antigens in tests with red cells of non-human primates.

Human anti-D (Rho) monoclonal antibodies (Mabs) of the IgG (70) and IgM (27) classes were tested with red blood cells (RBCs) of various non-human primates, from anthropoid apes to New World monkeys. Significant differences in reactivity were observed among antibodies of two classes depending on taxonomic position of primate animals. Only IgM Mabs gave positive reactions (9 out of 18 Mabs) with blood of Old World monkeys. Allotypic reactions with RBCs of African apes were produced by a majority of IgG Mabs but by very few IgM reagents, most of the latter reacting with RBCs of all chimpanzees and all gorillas tested. Eight out of 70 IgG anti-D defined chimpanzee polymorphisms related to chimpanzee Rc antigen which is the chimpanzee counterpart of human D antigen. Most of IgG anti-D Mabs (61/70) were found specific of Dgor antigen (gorilla counterpart of human antigen D). Most of anti-D which were found negative with all chimpanzee RBCs were also negative with human DIVb RBCs and most of anti-D which agglutinated human DIVb RBCs were positive with some or all chimpanzee blood samples. Differences among Mabs evidenced in tests with non-human primate RBCs reflect the complexity of the immune reactions to the human D antigen. The results obtained with anti-Rh Mabs of specificities other than D confirmed that chimpanzee, gorilla and gibbon express c-like epitopes and that antigens C, E, e are absent in non-human primates.

Red cell polymorphisms in nonhuman primates: a review.

Development as well as current status of the knowledge of nonhuman primate blood groups are discussed together with some practical implications of the red cell antigen polymorphisms in anthropoid apes, Old and New World monkeys and prosimians. Recent data on molecular biology and genetics throw light on the relationships among simian and human red cell antigens and their evolutionary pathways.

Structural analysis of the RH-like blood group gene products in nonhuman primates.

Rh-related transcripts present in bone marrow samples from several species of nonhuman primates (chimpanzee, gorilla, gibbon, crab-eating macaque) have been amplified by RT-polymerase chain reaction using primers deduced from the sequence of human RH genes. Nucleotide sequence analysis of the nonhuman transcripts revealed a high degree of similarity to human blood group Rh sequences, suggesting a great conservation of the RH genes throughout evolution. Full-length transcripts, potentially encoding 417 amino acid long proteins homologous to Rh polypeptides, were characterized, as well as mRNA isoforms which harbored nucleotide deletions or insertions and potentially encode truncated proteins. Proteins of 30-40,000 M(r), immunologically related to human Rh proteins, were detected by western blot analysis with antipeptide antibodies, indicating that Rh-like transcripts are translated into membrane proteins. Comparison of human and nonhuman protein sequences was pivotal in clarifying the molecular basis of the blood group C/c polymorphism, showing that only the Pro103Ser substitution was correlated with C/c polymorphism. In addition, it was shown that a proline residue at position 102 was critical in the expression of C and c epitopes, most likely by providing an appropriate conformation of Rh polypeptides. From these data a phylogenetic reconstruction of the RH locus evolution has been calculated from which an unrooted phylogenetic tree could be proposed, indicating that African ape Rh-like genes would be closer to the human RhD gene than to the human RhCE gene.

Length polymorphism of a microsatellite in human and non human primates.

Microsatellites are tandem repeats of short sequences elements (most often CA repeats) interspersed in many genomes and which frequently show multiallele polymorphism. They have proved invaluable for genomic mapping in man and other species and may be used for evolutionary studies provided that the available primers can be used in different species. The dystrophin gene, which shows high sequence conservation between man, rodents and chicken contains such polymorphic CA repeats. Using the oligonucleotides primers developed for testing the polymorphic CA repeat of the 3'untranslated region of the dystrophin gene in man, we achieved the amplification by polymerase chain reaction (PCR) of the analogous region in five non human primates species (Pan troglodytes, Gorilla gorilla, Macaca tonkeana, Macaca fascicularis, Lemur fulvus). All were proved to possess the CA repeat while intraspecies variations of the microsatellite length was observed in chimpanzees, gorillas and tonkean macaques. As it was demonstrated by sequencing, these length variations depend on the number of CA repeats in the microsatellite.

Molecular genetics of chimpanzee Rh-related genes: their relationship with the R-C-E-F blood group system, the chimpanzee counterpart of the human rhesus system.

As the chimpanzee R-C-E-F blood group system appears to be the chimpanzee counterpart of the human Rhesus (RH) system, we have tried to determine whether chimpanzee Rh-like genes encode R-C-E-F-related proteins. Chimpanzee genomic DNA, digested by any of eight endonucleases and hybridized with three Rh exon-specific probes, exhibits a high degree of polymorphism. Analysis of DNA from unrelated individuals of different R-C-E-F types revealed that the presence of some restriction fragments is correlated with particular R-C-E-F types. The cosegregation of these fragments with R-C-E-F haplotypes was confirmed by family studies. Oligonucleotides complementary to regions flanking human exons were used as PCR primers on chimpanzee DNA; the resulting amplified fragments were identical in size to their human counterparts. Moreover, the nucleotide sequences of the fragments present a high degree of similarity to the corresponding human regions.

Comparative study of human monoclonal anti-D antibodies of IgG and IgM classes in tests with red cells of nonhuman primates.

Forty-nine human anti-D (Rho) monoclonal antibodies of the IgG and IgM classes were tested with red blood cells of various nonhuman primates, from anthropoid apes to Prosimians, and significant differences in reactivity were observed among antibodies of two classes depending on taxonomic position of primate animals. By and large, higher percentage of IgM mAbs gave positive reactions with nonhuman primate red cells and, particularly, with blood of lower monkeys: Old and New Worlds monkeys and Prosimians, than did those of IgG class. Allotypic reactions with red cells of African apes were produced by majority of IgG mAbs but by very few IgM reagents. Some of those reactions defined epitopes related to human D, such as chimpanzee Rc and gorilla Dgor. By contrast, individual differences among Old World monkey species were revealed almost exclusively in tests using anti-D mAbs of IgM class. Some of the epitopes detected by these antibodies on the red cells of macaques are related to human D alloantigen, as confirmed by absorption experiments. Differences among mAbs evidenced in tests with nonhuman primate red cells reflect the complexity of the immune reactions to the human D antigen.

Relationship between chimpanzee Rh-like genes and the R-C-E-F blood group system.

The antigenic closeness between the chimpanzee alloantigen Rc of the R-C-E-F system, and the human alloantigen Rho(D) suggests a phyloge-connection between their genes. To confirm at the molecular level the common origin of these genes, genomic DNA from 16 unrelated chimpanzees of various R-C-E-F phenotypes were digested by three restriction enzymes and analyzed by Southern blot using a human Rh cDNA probe and three exon-specific probes. Restrictions profiles displayed reach polymorphism. Correlations between some bands and certain R-C-E-F phenotypes demonstrate that the human Rh cDNA probe defines in chimpanzee genomic DNA some genes of the R-C-E-F system.

The R-C-E-F blood group system of chimpanzees: serology and genetics.

Six chimpanzee alloimmune antibodies define 20 phenotypes of the R-C-E-F blood group system, the counterpart of the human Rh system. Of the several specificities of this system, the Rc constitutes the crucial link with human Rh since the reactions of some chimpanzee alloimmune anti-Rc sera with human red cells parallel those obtained with human anti-Rho reagents. Reciprocally, properly absorbed human anti-Rho sera detect Rc specificity on chimpanzee red cells. Tests with large panels of human monoclonal anti-D antibodies confirm the notion of shared epitopes between human alloantigen Rho(D) and chimpanzee alloantigen Rc.

Quantitative study of chimpanzee and gorilla counterparts of the human D antigen.

The reactivities of three human anti-D monoclonal antibodies (mAbs) with human, chimpanzee, and gorilla red blood cells (RBCs) were compared by quantitative radioimmunology and indirect immunofluorescence methods. The number of antigenic sites varies widely in gorillas (from 48,000-283,000), while in chimpanzees this number is very close to that observed in human R1R2 RBCs. The affinity of the anti-D antibodies was slightly lower with ape RBCs than with D-positive human RBCs. In chimpanzee, the D-like epitopes recognition is enhanced by papain while the gorilla D-like epitopes are destroyed by enzyme treatment.

The M-N--V-A-B-D blood group system of chimpanzee and other apes: serology and genetics.

Poly- and monoclonal anti-M and anti-N reagents detect on the red cells of anthropoid apes the M and/or N antigens which are similar to, but not identical with human M and N. A series of V-A-B-D specificities, closely related to the M-N system, are recognized on ape red blood cells by chimpanzee immune sera. To account for the distributions of the M-N--V-A-B-D types in man and in various apes, a genetic model is proposed that assumes the existence of two independent pairs of alleles: M/m, and N/n. In the processes of speciation, some of the alleles were lost or replaced by multiple mutations, resulting in chimpanzee in a series of codominant alleles responsible for as many as 16 M-N--V-A-B-D phenotypes.

Application of maximum likelihood statistics for the population and family studies of inheritance of the chimpanzee R-C-E-F and V-A-B-D blood groups.

Maximum likelihood statistics were applied to the analysis of serological data to confirm the originally proposed genetic models of the chimpanzee R-C-E-F and V-A-B-D systems. Five hundred ninety-nine chimpanzees, including 81 parents of 114 offspring, were tested for R-C-E-F, and 60 parents of 80 offspring were tested for V-A-B-D blood groups. An estimation-maximization procedure was used to obtain maximum likelihood estimates and support intervals of the haplotype frequencies. For each haplotype, the null hypothesis of nonexistence was evaluated. The frequencies obtained by this method do not differ significantly from those calculated by the square root formula, but put these estimates on a statistically more rigorous footing.

Human monoclonal anti-D-induced clearance of human D-positive red cells in a chimpanzee model.

Two human monoclonal anti-Rh0(D) antibodies, one IgG1, and one IgG3, were tested for their ability to clear human D-positive red blood cells (RBCs) from chimpanzee circulation. Human RBCs (phenotype A1, R1r) from 1 donor were radiolabelled with chromium 51 and injected into 4 chimpanzees. One day later the control animal received isotonic saline whereas 2 animals received 400 micrograms of purified human monoclonal anti-D, either IgG1 or IgG3. The remaining animal received both antibodies together (200 micrograms of IgG1 and 200 micrograms of IgG3). Both individual antibody-mediated clearance of human D-positive cells and synergy was not observed when both antibodies were used in combination. IgG1 was slightly more effective than IgG3. This animal model is a suitable alternative for conducting in vivo experiments in human beings, especially at the preclinical study phase of monoclonal anti-D antibodies.

Survival of human monoclonal anti-Rho (D) antibodies in the rhesus monkey.

In vivo half-life of a 125I-labeled human anti-D monoclonal antibody (mAb) and that of 131I-labeled Rho-GAM was assessed in a rhesus monkey injected simultaneously with both reagents. The half-life of the mAb was 7.9 days, compared to 17 days of Rho-GAM. Survival of the second dose of mAb, given 34 days after the first injection, was identical to that of the first dose, thus showing that the human mAb did not elicit an immune response. The in vitro produced human mAbs appear to be an alternative, unlimited source of anti-D antibodies for possible use in prevention of feto-maternal Rh immunization.

Diversity of human anti-D monoclonal antibodies revealed by reactions with chimpanzee red blood cells.

Fifty-three human anti-D monoclonal antibodies (mAbs) revealed a striking diversity of reactions in tests with panels of chimpanzee red blood cells (RBCs) of various R-C-E-F blood group phenotypes (counterparts of the human Rh-Hr groups). The reactivities of these antibodies, which depended on the agglutination technique used, could be classified into four main types. These patterns of reactivity of anti-D mAbs with chimpanzee RBCs showed only limited correlation with types of reactions observed with human D variant RBCs. Primate red cells may, therefore, constitute an independent test system for subclassification of human monoclonal antibodies. Comparison of reactivities of human anti-D mAbs with chimpanzee and human D variant RBCs confirms the homology between the chimpanzee Rc, and the human D antigens. The chimpanzee Rc shares with human D the epitopes epD5, epD6/7 and epD8, but lacks epitopes epD1, epD2, epD3 and epD4 of the Rh mosaic, thus resembling the human D variants IVb and Vc.