Functional Analysis of PAAN-AG5, the Baboon Counterpart of HLA-G5

描述（由申请人提供）：本研究的长期目标是阐明避免母体免疫系统对半异体胎儿排斥的机制。胎儿在怀孕期间通过在滋养细胞上表达b类人白细胞抗原g （HLA-G）积极参与自身保护。HLA-G被认为对半同种异体胎儿的存活至关重要；它将母胎界面的免疫细胞编程为免疫抑制表型。HLA-G功能的明确证据仍然难以捉摸，因为由于伦理问题，不可能在人体进行体内实验。我们一直在评估使用橄榄狒狒（Papio anubis）作为HLA-G体内研究模型的适用性。为了实现这一目标，我们在狒狒中发现了一种独特的hla - g样MHC类Ib基因，称为Paan-AG。HLA-G和Paan-AG在RNA加工中表现出显著的相似性，包括mRNA的选择性剪接，导致转录本编码膜锚定蛋白（HLA-G1-4或Paan-AG1-4）和可溶性蛋白（HLA-G5-7或Paan-AG5）。在人类中，HLA-G5正在成为妊娠、移植和癌症进展中重要的关键亚型之一。HLA-G5和狒狒对应的Paan-AG5分别在人和狒狒胎盘中的绒毛和外绒毛细胞滋养细胞中高度表达[1,4,6]。基于这些观察，我们的假设是狒狒胎盘Paan-AG5，与人类胎盘HLA-G5一样，驱动母体免疫反应进入有利于怀孕的途径。为了验证这一假设，有必要首先建立Paan-AG5蛋白对免疫细胞的作用，并与HLA-G5的作用进行比较。HLA-G5与所有主要的免疫细胞亚群相互作用，包括自然杀伤（NK）细胞、CD4+和CD8+ t淋巴细胞、b淋巴细胞、巨噬细胞和树突状细胞。计划中的实验旨在评估重组Paan-AG5是否对从狒狒外周血白细胞中纯化的免疫细胞亚群有类似的作用。本提案的具体目的是：(i)生成和表征重组Paan-AG5蛋白，以及（ii）评估Paan-AG5作为免疫抑制分子对妊娠有益的能力。我们实验室建立的技术将用于在细菌中产生重组原核Paan-AG5，并在人胚胎肾细胞（HEK293）中产生真核FLAG标记的Paan-AG5。我们将评估纯化蛋白对自然杀伤细胞（NK）、CD4+和CD8+ t细胞、单核细胞、巨噬细胞和树突状细胞的影响。本研究的结果可能为Paan-AG5（以及由此推断的HLA-G5）在妊娠中的潜在功能提供关键数据。阐明HLA-G在妊娠中的作用是评估重组HLA-G蛋白在妊娠相关病理和移植中的治疗潜力的必要前提。这些研究也可能为橄榄狒狒作为体内HLA-G功能研究模型的适用性提供重要信息，以评估重组HLA-G5的治疗潜力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.