Cardiac Xenotransplantation

心脏异种移植

• 批准号: 7594429
• 负责人: Keith A Horvath
• 金额: $ 69.83万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594429
• 关键词: Acute; Animal Model; Biological Assay; CD4 Positive T Lymphocytes; Cardiac; Cell Separation; Cells; Clinical; Coculture Techniques; Effectiveness; Family suidae; Heart; IL2 gene; IL2RA gene; Immune response; Immunologics; Immunosuppression; In Vitro; Interleukin-2; Measures; Methods; Modeling; Numbers; Organ Donor; Papio; Performance; Play; Production; Role; Solutions; Sorting - Cell Movement; Spleen; Surface; Sus scrofa; Techniques; Testing; Therapeutic Uses; Therapeutic immunosuppression; Transplantation; Week; Xenograft procedure; base; cytokine; immunoregulation; lymph nodes; magnetic beads; peripheral blood; research study; response; success

Xenotransplantation presents an effective solution to donor organ shortage but vigorous immune response across specie barrier limits its success. The level of current immunosuppression used to prolong xenograft survival is lethal and also impractical for clinical use. Therefore, there is a strong need to develop methods to limit immunosuppression and induce immunological tolerance or accommodation. Recently characterized CD4+CD25+ T regulatory cells (Tregs) offer some hope of inducing donor specific immune modulation without much lethality. One of the limitations of their therapeutic use is their presence in very low numbers. In this study we have tried to isolate and expand baboon Tregs and measured their effectiveness in suppressing pig to baboon xenogeneic response. Tregs were isolated from peripheral blood, spleen and lymph nodes of healthy baboons first by CD4+ cells enrichment by magnetic beads and then by sorting CD4+ CD25+ cells via FACS. These sorted cells were tested for their suppressive potential by addition to the co culture of irradiated pig PBMCs and CD4+ CD25- cells. The isolated Tregs were also expanded in culture for 4 weeks in presence of IL2 and irradiated pig PBMCs and were evaluated for their inhibitory effect. Further, flow cytometric assays for intracellular cytokines and surface expression of various activation markers were also performed to study the mechanism of action of these Tregs in this xenotransplant model. The Treg isolation technique was very effective and 97% pure Tregs (1-2% of CD4+ T cells) were isolated. These isolated cells effectively suppressed the vigorous CD4+CD25- cell proliferative response to irradiated pig PBMCs (85 % suppression). These cells also expressed high levels of FoxP3, a potent marker of Tregs. Isolated Tregs expanded 150-200 folds in culture and were also able to suppress CD4+CD25- cells in a similar manner as naove Tregs. Tregs also suppressed the cytokine production by the CD25- cells in response to pig PBMCs, suggesting a possible mechanism of Treg function. With the above experiments it is clear that Tregs are potent suppressors of pig to baboon xenogenic response and they can also be expanded in vitro to achieve sufficient quantity without losing their suppressive potential. Thus, Tregs offer a potential non lethal alternative to non specific immunosuppression currently used to overcome xenograft rejection. In addition to these cellular studies we have commenced performance of pig to baboon orthotopic cardiac xenotransplantation. As a result we are verifying the stability of this animal model and establishing a set of baseline control results from which to compare additional transplants treated with pharmacologic and cell based immunosuppression.

异种移植为供体器官短缺提供了有效的解决方案，但在特定屏障中有剧烈的免疫反应限制了其成功。用于延长异种移植生存的电流免疫抑制水平是致命的，对于临床使用也不十分。因此，迫切需要开发限制免疫抑制并诱导免疫耐受性或适应性的方法。最近表征的CD4+ CD25+ T调节细胞（TREG）提供了一些希望诱导供体特异性免疫调节而没有太多致命性的希望。他们的治疗用途的局限性之一是它们的存在数量很少。在这项研究中，我们试图隔离和扩大狒狒Treg，并测量其在抑制猪对狒狒异类反应方面的有效性。 首先，通过磁珠富集CD4+细胞，然后通过FACS对CD4+ CD25+细胞进行排序，从外周血，脾和健康狒狒的淋巴结中分离出Treg。通过在辐照的PIG PBMC和CD4+ CD25-细胞的CO培养物中，对这些排序细胞的抑制潜力进行了测试。在IL2存在下，在培养物中也将分离的Treg扩展了4周，并被辐照的Pig PBMC膨胀，并评估其抑制作用。此外，还进行了细胞内细胞因子的流式细胞仪测定和各种激活标记的表面表达，以研究这些异种移植模型中这些Tregs的作用机理。 Treg隔离技术非常有效，分离出97％的纯Treg（1-2％的CD4+ T细胞）。这些分离的细胞有效地抑制了对辐射的猪PBMC的剧烈CD4+CD25-细胞增殖反应（85％抑制）。 这些细胞还表达了高水平的FOXP3，这是Tregs的有效标记。孤立的Treg在培养物中膨胀了150-200倍，也能够以与Naove Treg相似的方式抑制CD4+CD25-细胞。 Treg还抑制了CD25细胞对PIG PBMC的细胞因子产生，这表明可能是Treg功能的机制。 通过上述实验，很明显，Treg是猪的有效抑制剂到狒狒的异种反应，也可以在体外扩展，以实现足够的数量而不会失去其抑制潜力。因此，Tregs提供了目前用于克服异种移植排斥的非特异性免疫抑制的潜在非致命替代方法。 除了这些细胞研究外，我们还开始了猪到狒狒骨骨质心异种移植的性能。 结果，我们正在验证该动物模型的稳定性，并建立一组基线控制结果，以比较用药物和基于细胞的免疫抑制处理的其他移植物。