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

描述(申请人提供)：这项研究的长期目标是阐明避免母体免疫系统排斥半异基因胎儿的机制。妊娠期间，胎儿通过在滋养层细胞上表达Ib类人类白细胞抗原-G(人类白细胞抗原-G)，积极参与自身的保护。人类白细胞抗原-G被认为是半异基因胎儿存活的关键；它将母胎交界处的免疫细胞编程为免疫抑制表型。人类白细胞抗原G功能的确切证据仍然难以捉摸，因为出于伦理方面的考虑，在人体上进行实验是不可能的。我们一直在评估使用橄榄狒狒(Papio Anubis)作为体内研究的模型的适宜性。为了实现这一目标，我们在狒狒中发现了一种独特的类似于HLAG的MHC类Ib基因，称为Paan-AG。HL A-G和PAAN-AG在RNA加工方面有显著的相似之处，包括mRNA的选择性剪接，导致编码膜锚定的转录本(HLA-G1-4或PAAN-AG1-4)和可溶性蛋白(分别为HLA-G5-7或PAAN-AG5)。在人类中，人类白细胞抗原-G5正在成为怀孕、移植和癌症进展中重要的关键亚型之一。人类胎盘中的绒毛细胞和绒毛外细胞滋养层细胞分别高表达人类和狒狒胎盘中的HLA-G5和其对应的PAAN-AG5[1，4，6]。基于这些观察结果，我们的假设是，与人类胎盘组织中的人类白细胞抗原-G5一样，狒狒胎盘组织中的PAAN-AG5会将母体免疫反应推向有利于妊娠的途径。为了验证这一假说，有必要首先确定PAAN-AG5蛋白对免疫细胞的作用，以便与人类白细胞抗原G5的作用进行比较。人类白细胞抗原G5可与所有主要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起到类似的作用。这项建议的具体目的是(I)制备和鉴定重组PAAN-AG5蛋白，以及(Ii)评估PAAN-AG5作为免疫抑制分子的能力，以利于妊娠。本实验室建立的技术将用于在细菌中产生重组原核PAAN-AG5和在人胚胎肾细胞中产生真核标记的PAAN-AG5(HEK293)。我们将评估纯化的蛋白对自然杀伤细胞(NK细胞)、CD4+和CD8+T细胞、单核细胞、巨噬细胞和树突状细胞的影响。这项研究的结果可能为PAAN-AG5(由此推断，人类白细胞抗原-G5)在妊娠中的潜在功能提供关键数据。阐明人类白细胞抗原-G在妊娠中的作用是评估重组人类白细胞抗原-G蛋白在妊娠相关病理和移植中的治疗潜力的先决条件。这些研究也可能为在体内评价重组人类白细胞抗原G5的治疗潜力的研究提供重要的信息。 公共卫生相关性：这项拟议的研究与公共健康相关，因为人类不适当的HLA-G5和/或HLA-G6表达与早产、先兆子痫或其他妊娠病理导致的受孕困难或妊娠失败有关。这表明，这些蛋白的重组异构体在设计治疗策略以解决这些妊娠问题方面可能有价值。拥有与人类相似的生殖系统的动物模型，如橄榄狒狒，对于进行临床前测试以评估人类白细胞抗原G的治疗应用是必不可少的。这项建议旨在评估以橄榄狒狒为模型进行体内实验的可行性。

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.