Richard L. Grendell

A soluble isoform of the rhesus monkey nonclassical MHC class I molecule Mamu-AG is expressed in the placenta and the testis.

The nonclassical MHC class I locus HLA-G is expressed primarily in the placenta, although other sites of expression have been noted in normal and pathological situations. In addition, soluble HLA-G isoforms have been detected in the serum of pregnant and nonpregnant women as well as men. The rhesus monkey placenta expresses a novel nonclassical MHC class I molecule Mamu-AG, which has features remarkably similar to those of HLA-G. We determined that the rhesus placenta expresses Mamu-AG mRNA (Mamu-AG5), retaining intron 4 as previously noted in HLA-G5. Immunostaining experiments with Ab 16G1 against the soluble HLA-G5 intron 4 peptide demonstrated that an immunoreactive protein(s) was present in the syncytiotrophoblasts of the chorionic villi of the rhesus placenta, within villous cytotrophoblasts, and occasionally within cells of the villous stroma. The Mamu-AG5 mRNA was readily detected in rhesus testis (although not in ejaculated sperm). Whereas an Ab against membrane-bound Mamu-AG stained few cells, primarily in the interstitium of the testis, there was consistent immunostaining for Mamu-AG5 in cells within the seminiferous tubules, which was corroborated by localization of Mamu-AG mRNA by in situ hybridization. While primary spermatocytes were negative, Sertoli cells, spermatocytes, and spermatids were consistently positive for 16G1 immunostaining. The specific recognition of the soluble Mamu-AG isoform was confirmed by Western blotting of Mamu-AG5 expressed in heterologous cells. The results demonstrate that a soluble nonclassical MHC class I molecule is expressed in the rhesus monkey placenta and testis, and confirm and extend the unique homology between HLA-G and the rhesus nonclassical molecule Mamu-AG.

Passive Immunization against the MHC Class I Molecule Mamu-AG Disrupts Rhesus Placental Development and Endometrial Responses

The unique MHC phenotype of the human and nonhuman primate placenta has suggested a potential role in maternal-fetal immune tolerance, pregnancy success, and maternal as well as fetal well-being. In the rhesus monkey (Macaca mulatta) a nonclassical MHC class I molecule, Mamu-AG, is a putative homologue of HLA-G and is hypothesized to play a role in maternal-fetal immune interactions during pregnancy. Rhesus monkeys were passively immunized during the second week after implantation with a mAb against Mamu-AG. Passive immunization altered the growth and vascularization of the fetal placenta, the placental modification of maternal endometrial vessels, the maternal leukocyte response to implantation, and the differentiation of epithelial and stromal cells in the endometrium. These data are the first to demonstrate in vivo the importance of MHC class I molecules expressed on primate trophoblasts in establishing an important environment for pregnancy success through coordinated interactions between endometrial and fetal tissues.

Expression of indoleamine 2,3-dioxygenase in the rhesus monkey and common marmoset

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal-fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal-fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal-fetal immune dialogue.

Characterization of cynomolgus and vervet monkey placental MHC class I expression: diversity of the nonhuman primate AG locus

Nonhuman primates are important animal models for the study of the maternal immune response to implantation within the decidua. The objective of this study was to define the placental expression of major histocompatibility complex (MHC) class I molecules in the cynomolgus (Macaca fascicularis) and vervet (African green) (Chlorocebus aethiops) monkeys. Early pregnancy (d36-42) cynomolgus and vervet placentas were obtained by fetectomy and prepared for histological evaluation. A pan-MHC class I monoclonal antibody demonstrated MHC class I expression in both vervet and cynomolgus placental trophoblasts, with particularly high expression in the villous syncytium, as previously shown in the rhesus and baboon. Placental cytotrophoblasts were isolated by enzymatic dispersion and gradient centrifugation and cultured, and multicolor flow cytometry was used to phenotype cell populations. Culture of isolated villous cytotrophoblasts demonstrated that MHC class I expression was linked to syncytiotrophoblast differentiation. A monoclonal antibody against Mamu-AG, the nonclassical MHC class I homolog of HLA-G in the rhesus monkey, demonstrated intense immunostaining and cell surface expression in cynomolgus placental trophoblasts; however, staining with vervet placenta and cells was low and inconsistent. Reverse transcriptase polymerase chain reaction was used to clone MHC class I molecules expressed in cynomolgus and vervet placentas. While Mafa-AG messenger RNA (mRNA) was readily detectable in cynomolgus placental RNA and was >99% identical at the amino acid level with Mamu-AG, 7/8 Chae-AG complementary DNAs had an unusual 16 amino acid repeat in the α1 domain, and all clones had an unexpected absence of the early stop codon at the 3′-end of the mRNA diagnostic for rhesus, cynomolgus, and baboon AG mRNAs, as well as HLA-G. We conclude that while the vervet monkey has retained the placental expression of a primate-specific nonclassical MHC class I locus, diversity is also revealed in this locus expressed at the maternal–fetal interface, thought to participate in placental regulation of the maternal immune response to embryo implantation and pregnancy.

Suppression of Mamu-AG by RNA interference.

Problem  The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu‐AG, the rhesus monkey homology of human leukocyte antigen (HLA)‐G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function.

ORIGINAL ARTICLE: Suppression of Mamu-AG by RNA Interference: SUPPRESSION OF MAMU-AG BY RNA INTERFERENCE

Problem  The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu‐AG, the rhesus monkey homology of human leukocyte antigen (HLA)‐G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function.

ORIGINAL ARTICLE: Suppression of Mamu-AG by RNA Interference

Problem  The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu‐AG, the rhesus monkey homology of human leukocyte antigen (HLA)‐G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function.