The long-term goal of this research is to elucidate the mechanisms by which rejection of the semiallogeneic fetus by the maternal immune system is avoided. The fetus actively participates in its own protection by expression of the class Ib human leukocyte antigen-G (HLA-G), on trophoblast cells during pregnancy. HLA-G is thought to be critical in survival of the semi-allogenic fetus;it programs immune cells at the maternal-fetal interface into immunosuppressive phenotypes. Definitive proof of HLA-G function remains elusive since in vivo experiments in humans are not possible due to ethical concerns. We have been assessing the suitability of using the olive baboon (Papio anubis) as a model for HLA-G in vivo studies. In pursuit of this goal, we identified a unique HLA-G-like MHC class Ib gene termed Paan-AG in the baboon. HLA-G and Paan-AG display remarkable similarity in the RNA processing, including alternative splicing of the mRNA, resulting in transcripts that encode membrane-anchored (HLA-G1-4 or Paan-AG1-4) and soluble proteins (HLA-G5-7 or Paan-AG5 respectively). In humans, HLA-G5 is emerging as one of the critical isoforms important in pregnancy, transplantation and cancer progression. Both HLA-G5 and the baboon counterpart, Paan-AG5, are highly expressed by villous and extravillous cytotrophoblast cells in the human and baboon placenta respectively [1,4,6]. Based on these observations, our hypothesis is that baboon placental Paan-AG5, as with human placental HLA-G5, drives the maternal immune response into pathways beneficial to pregnancy. To test this hypothesis, it is necessary to first establish the effects of Paan-AG5 protein on immune cells for comparison with HLA-G5 effects. HLA-G5 interacts with all major subsets of immune cells, including natural killer (NK) cells, CD4+ and CD8+ T-lymphocytes, B-lymphocytes, macrophages and dentritic cells. The planned experiments are designed to assess whether recombinant Paan-AG5 acts similarly on sub- sets of immune cells purified from baboon peripheral blood leukocytes.
The specific aims of this proposal are to (i) generate and characterize recombinant Paan-AG5 protein, and (ii) assess the ability of Paan- AG5 to act as an immune suppressor molecule for the benefit of pregnancy. Techniques established in our laboratory will be used to generate recombinant prokaryotic Paan-AG5 in bacteria and eukaryotic FLAG- tagged Paan-AG5 in human embryonic kidney cells (HEK293). We will assess effects of the purified proteins on natural killer (NK) cells, CD4+ and CD8+ T-cells, monocytes, macrophages and dentritic cells. The results of this study may provide critical data on the potential function of Paan-AG5 (and by inference, HLA-G5) in pregnancy. Elucidation of the role of HLA-G in pregnancy is essential as a prerequisite to assessing the therapeutic potential of recombinant HLA-G proteins in pregnancy-related pathologies and transplantation. These studies may also provide important information regarding the suitability of the olive baboon as a model for in vivo HLA-G functional studies to assess the therapeutic potential of recombinant HLA-G5.
The proposed study is relevant to public health because inappropriate expression of HLA-G5 and/or HLA-G6 in humans has been associated with difficulties in conception or pregnancy failure due to preterm labor, preeclampsia or other pregnancy pathologies. This suggests that recombinant isoforms of these proteins may be of value in designing therapeutic strategies to address these pregnancy problems. An animal model with a reproductive system similar to that of humans, such as the olive baboon, is essential in order to perform pre- clinical tests to assess therapeutic applications of HLA-G. This proposal is aimed at assessing the feasibility of using the olive baboon as a model for HLA-G in vivo experiments.
