Androgens and innate immunity in rehabilitated semi-captive orangutans (Pongo pygmaeus morio) from Malaysian Borneo.

Despite the implications for the development of life-history traits, endocrine-immune trade-offs in apes are not well studied. This is due, in part, to difficulty in sampling wild primates, and lack of methods available for immune measures using samples collected noninvasively. Evidence for androgen-mediated immune trade-offs in orangutans is virtually absent, and very little is known regarding their pattern of adrenal development and production of adrenal androgens. To remedy both of these deficiencies, sera were collected from orangutans (Pongo pygmaeus morio) (N = 38) at the Sepilok Orangutan Rehabilitation Centre, Sabah, Malaysia, during routine health screenings. Testosterone, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone-sulfate (DHEA-S) were assayed, along with two measures of functional innate immunity. DHEA-S concentrations, but not DHEA, increased with age in this sample of 1-18 year old animals. DHEA concentrations were higher in animals with higher levels of serum bacteria killing ability, while DHEA-S and testosterone concentrations were higher in animals with reduced complement protein activity. Patterns of DHEA-S concentration in this sample are consistent with patterns of adrenarche observed in other apes. Results from this study suggest that in addition to testosterone, DHEA and DHEA-S may have potent effects on immunological activity in this species.

Evolution of killer cell Ig-like receptor (KIR) genes: definition of an orangutan KIR haplotype reveals expansion of lineage III KIR associated with the emergence of MHC-C.

Orangutan (Pongo pygmaeus) MHC-C appears less evolved than human HLA-C: Popy-C is not fixed and its alleles encode only one (C1) of the two motifs for killer cell Ig-like receptor (KIR) ligands. To assess the structure and complexity of the orangutan KIR locus, the complete nucleotide sequence of an orangutan KIR haplotype was determined. The PopyKIR locus is flanked by LILR and FCAR and consists of seven genes and pseudogenes, two novel and five corresponding to known cDNA. Distinguishing all KIRs in this rapidly evolving KIR locus from the KIR3DX1 gene is an LTR33A/MLT1D element in intron 3. These two forms of KIR represent lineages that originated by duplication of a common ancestor. The conserved, framework regions of primate KIR loci comprise the 5' part of a lineage V KIR, the 3' part of a pseudogene, the complete 2DL4 gene, and the 3' part of a lineage II KIR. Although previously defined PopyKIR2DL4 alleles contain premature termination codons, the sequenced haplotype's PopyKIR2DL4 allele encodes a full-length protein. A model for KIR evolution is proposed. Distinguishing the orangutan KIR haplotype from the proposed common ancestor of primate KIR haplotypes is an increased number to give three lineage III KIR genes in the centromeric part of the locus, the site for most human lineage III genes encoding HLA-C specific KIR. Thus, expansion of lineage III KIR is associated with emergence of MHC-C.

NK cell receptors of the orangutan (Pongo pygmaeus): a pivotal species for tracking the coevolution of killer cell Ig-like receptors with MHC-C.

CD94, NKG2, Ly49, and killer cell Ig-like receptor (KIR) expressed by orangutan peripheral blood cells were examined by cloning and sequencing cDNA from a panel of individuals. Orthologs of human CD94, NKG2A, D, and F were defined. NKG2C and E are represented by one gene, Popy-NKG2CE, that is equidistant from the two human genes. Several Popy-CD94, NKG2A, and NKG2CE alleles were defined. Popy-Ly49L is expressed in cultured NK cells and has a sequence consistent with it encoding a functional receptor. Orangutan KIR corresponding to the three KIR lineages expressed in humans and chimpanzees were defined. Popy-KIR2DL4 of lineage I is the only ortholog of a human or chimpanzee KIR, but in all individuals examined, the transcripts of this gene produced premature termination, either in the D2 domain or at the beginning of the cytoplasmic domain. Ten Popy-KIR3DL and one Popy-KIR3DS of lineage II are all closely related, but represent the products of at least two genes. The two Popy-KIR2DL and four Popy-KIR2DS of lineage III also represent two genes, both being more related to KIR2DS4 than to other human and chimpanzee KIR of lineage III. The Popy-KIR2D include ones predicted to be specific for the C1 epitope of MHC-C, but none specific for C2. This correlates with the observation that all orangutan MHC-C allotypes examined have the C1 motif.

Evidence for an HLA-C-like locus in the orangutan Pongo pygmaeus.

HLA-B and C are related class I genes which are believed to have arisen by duplication of a common ancestor. Previous study showed the presence of orthologues for both HLA-B and C in African apes but only for HLA-B in Asian apes. These observations suggested that the primate C locus evolved subsequent to the divergence of the Pongidae and Hominidae. From an analysis of orangutan Tengku two HLA-C-like alleles (Popy C*0101 and Popy C*0201) were defined as well as three HLA-B-like (Popy-B) alleles. By contrast, no Popy-C alleles were obtained from orangutan Hati, although three Popy-B alleles were defined. Thus an HLA-C-like locus exists in the orangutan (as well as a duplicated B locus), implying that the primate C locus evolved prior to the divergence of the Pongidae and Hominidae and is at least 12-13 million years old. Uncertain is whether all orangutan MHC haplotypes contain a C locus, as the failure to find C alleles in some individuals could be due to a mispairing of HLA-C-specific primers with certain Popy-C alleles. These results raise the possibilities that other primate species have a C locus and that the regulation of natural killer cells by C allotypes evolved earlier in primate evolution than has been thought.

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.

Characterization of a simian T-lymphotropic virus from a wild-caught orang-utan (Pongo pygmaeus) from Kalimantan, Indonesia.

In a recent serological survey among 143 ex-captive orang-utans two individuals were found that reacted positive in an ELISA detecting antibodies which cross-react with human T-lymphotropic virus type I (HTLV-I) antigens. Infection of both animals with an HTLV-I or simian T-lymphotropic virus (STLV)-like virus was confirmed by Western blot analysis. A third wild-caught animal, which was not part of the original serological survey, was also found to be infected with an HTLV-related virus in a diagnostic PCR assay and Western blot assay. Nucleotide sequence analysis of the 709 bp PCR fragment from the tax/rex region of the HTLV/STLV genome confirmed infection of orang-utans with an STLV similar to but clearly distinct from other Asian STLVs.

Sequence convergence in the peptide-binding region of primate and rodent MHC class Ib molecules.

In addition to the universally expressed and highly polymorphic class Ia genes, the major histocompatibility complex (MHC) of placental mammals includes class Ib genes that are characterized by restricted expression and low levels of sequence polymorphism. The functional importance of class Ib genes as well as their actual function has long been controversial. Phylogenetic analyses have suggested that there are no orthologous relationships among class Ib loci of mammals belonging to different orders, suggesting that these loci have evolved independently since the placental mammals diverged. Here, we present evidence of convergent evolution at the molecular sequence level in the putative peptide-binding regions (PBRs) of human and mouse class Ib genes. So far, there are few if any convincing examples of convergent evolution at the amino acid sequence level, and such evolution is believed to be likely to occur only as a result of strong positive selection. Because the present case involves the functionally important PBR and because the primate and rodent molecules are known to bind similar peptides, this study represents both a convincing case of molecular-level convergence and evidence that MHC class Ib molecules, although not orthologous, may evolve similar functions convergently.

Mhc-E polymorphism in Pongidae primates: the same allele is found in two different species.

Mhc-E intron 1, exon 2, intron 2, and exon 3 from pygmy chimpanzee (Pan paniscus), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) have been sequenced; six new Mhc-E alleles have been obtained but sequence changes are only placed either in introns or in synonymous exonic bases. One pygmy chimpanzee Mhc-E DNA sequence is identical to another sequence from chimpanzee; the fact that no variation is found also at the intronic level suggests that these two species of chimpanzee may have recently separated and/or that both of them might only represent subspecies. Mhc-E phylogenetic trees separate two evolutionary groups: Pongidae, including humans, and Cercopithecinae; this is also found by studying another non-classical class I gene, Mhc-G. The Mhc-E alleles' invariance at the protein level supports that strong selective forces are operating at the Mhc-E locus, as has also been found in both Cercopithecinae and humans. These allelic and evolutionary data suggest an altogether different functionality for HLA-E (and also HLA-G) compared with classical class I proteins: i.e., sending negative (tolerogenic) signals to NK and T cells.

The synonymous substitution rate of the major histocompatibility complex loci in primates.

Because the divergence of many allelic lineages at the major histocompatibility complex (MHC) loci predates species divergence, standard methods of calculating synonymous substitution rates are not applicable to this system. We used three alternative methods of rate estimation: one based on the minimum number of substitutions (Dm), another on the nucleotide difference (Dxy), and the third on the net nucleotide difference (Dn). We applied these methods to the protein-encoding sequences of primate MHC class I (A, B, and C) and class II (DRB1) genes. To determine the reliability of the different estimates, we carried out computer simulation. The distribution of the estimates based on Dxy or Dn is generally much broader than that based on Dm. More importantly, the Dm-based method nearly always has the highest probability of recovering true rates, provided that Dm is not smaller than 5. Because of its desirable statistical properties, we used the Dm-based method to estimate the rate of synonymous substitutions. The rate is 1.37 +/- 0.61 for A, 1.84 +/- 0.40 for B, 3.87 +/- 1.05 for C, and 1.18 +/- 0.36 for DRB1 loci, always per site per 10(9) years. Hence despite the extraordinary polymorphism, the mutation rate at the primate MHC loci is no higher than that of other loci.

Immunoglobulin Gm allotypes in apes: comparison with man.

Serum samples from 245 apes (184 Pan troglodytes, five Pan paniscus, 28 Gorilla gorilla, 23 Pongo pygmaeus abelei, and five Pongo pygmaeus pygmaeus) were tested for G1m (1,2,3,17), G2m (23), and G3m (5,6,10,11,13,14,15,16,21,24,28) immunoglobulin allotypes by the classical method of inhibition of hemagglutination. Some phenotypes are species specific while a few are shared by man and African apes.

Molecular cloning of orangutan and gibbon MHC class I cDNA. The HLA-A and -B loci diverged over 30 million years ago.

To investigate whether the classical HLA MHC class I loci have been preserved during evolution of the primates, we have cloned, sequenced, and expressed eight MHC class I cDNA from orangutan and gibbon lymphocytes. Both the HLA-A and -B loci are present in both of these species. In fact, lymphocytes from the orangutan expressed three HLA-B-related gene products, suggesting that the ancestral homologue of the HLA-B locus had undergone a duplication in this species. Interestingly, several amino acid motifs thought to be important in the Ag-presenting function of MHC class I molecules were preserved in the Ag-recognition sites of the orangutan and gibbon MHC class I molecules. Finally, these findings suggest that the recombination event between the HLA-A and -E loci occurred over 38 million years ago. These data indicate that the HLA-A and -B loci are extremely stable and that recombination between them is rare. Furthermore, the data presented here argue against the role of concerted evolution in the evolution of primate MHC class I molecules.

Evolution of immunoglobulin allotypes and phylogeny of apes.

Serum samples from 72 Pan troglodytes, 5 Pan paniscus, 22 Gorilla gorilla, 23 Pongo pygmaeus abelii, 5 Pongo pygmaeus pygmaeus, 2 hybrids P.p. abelii X P.p. pygmaeus and 13 Hylobates lar were tested for Gm(1, 2, 3, 5, 6, 10, 11, 13, 14, 15, 16, 17, 21, 24, 28), Km(1) and Bm(1, 2, 3, 4, 5, 6, 7, 8) immunoglobulin allotypes by the classical hemagglutination inhibition method. The distribution of the various alleles and phenotypes makes it possible to distinguish each species or subspecies. Common chimpanzees have the richest polymorphism. Pygmy chimpanzees share common phenotypes with gorillas. Bornean and Sumatran orangutans have their own patterns of polymorphism, as do gibbons. Our principal component plot and dendrogram are compatible with the traditional classification of Hominoidea [e.g. Simpson, Bull. Am. Mus. nat. Hist. 85: 1-350, 1945] in 3 families: Hominidae, Pongidae and Hylobatidae.

Studies on the use of nonhuman primates to determine the DR status of the human hematopoietic stem cell.

Monoclonal antibodies that recognize monomorphic determinants of human DR are potentially useful for the in vitro elimination of malignant cells from marrow for use in autologous transplantation. While DR is expressed on normal hematopoietic progenitor cells and the cells of the majority of the hematologic and lymphoid malignancies, there is the possibility that DR may not be expressed on the hematopoietic stem cells responsible for marrow regeneration after transplantation. To resolve the uncertainty regarding the DR status of the human stem cell, we determined whether antihuman DR monoclonal antibodies recognized analogous antigens on nonhuman primate hematopoietic progenitor cells to determine an appropriate animal transplant model. We used antihuman DR plus C'-mediated lysis of marrow progenitor cells as an indicator of whether the analogous nonhuman primate cells express similar antigens. Using two potent C'-fixing anti-DR monoclonal antibodies separately (5F3, AMG-12), human progenitor cells are reduced by 90%-100%. The range of progenitor cell depletion varied more widely with the nonhuman primates studied: 80%-99% with cells from the chimpanzee, 48%-100% with cells from the orangutan, and 62%-100% with cells from the rhesus monkey. Despite this, the majority of animals yielded results identical to that seen with human cells. We concluded that autologous transplantation with DR-depleted rhesus bone marrow into a lethally irradiated animal would be a practical and expeditious means to determine the DR status of the cell responsible for marrow regeneration, and by inference the DR status of the human hematopoietic stem cell.

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin.

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.

Antistreptococcal antibodies, rheumatoid factors and haptoglobin in apes and monkeys.

Sera of 157 baboons (Papio hamadryas and P. anubis) 21 chimpanzees (Pan troglodytes) five orang utans (Pongo pygmaeus) three mountain gorillas (Gorilla gorilla beringei) and three gibbons (Hylobates lar lar) were examined for the content of antistreptolysin O, antidesoxyribonuclease B, for the presence of rheumatoid factors as well as for the level and type of haptoglobin. The mean antistreptolysin O titer (AST) in baboons was 106 ASU +/- 18 in dextransulfate absorbed sera ("real" AST) and 182 ASU +/- 34 in non-absorbed sera. The mean decrease after absorption was 20% (i.e., 20% inhibitor + 80% antistreptolysin O), a value that is lower than previously found in rhesus monkeys (55%) or in man (40%). Raised values of anti-DNase B were found in two baboons only, and none of the sera of that species displayed presence of rheumatoid factors. In chimpanzees, the mean AST was 68 +/- 29 in absorbed and 87 ASU +/- 57 in non-absorbed sera. Anti-DNase B was raised in three animals, and in two cases the increase was correlated with raised AST. Of 19 chimpanzee sera examined, 13 were found to contain antigammaglobulins ("rheumatoid factors") the titers of which reached 1:64 or more. All primate sera tested so far showed haptoglobin type 1-1 or Hp 1-1-like patterns. The haptoglobin level in chimpanzees and baboons was comparable to that established in man; in rhesus monkey, on the other hand, much lower values (40-62 mg/100 ml) or ahaptoglobinemia were observed. The sera of all monkeys and apes tested so far showed a very low (undiluted, or up to 1:10 titer at most) agglutinating activity against T4-antigen-carrying streptococci. This is in agreement with our observations made previously which indicated that human or animal sera of haptoglobin type 1-1 agglutinated streptococci to a much lower degree than type Hp 2-2 or type Hp 2-1 sera.