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.

异种器官移植是解决供体器官短缺的有效方法，但强烈的跨种属免疫反应限制了异种器官移植的成功。目前用于延长异种移植物存活的免疫抑制水平是致命的，并且对于临床使用也是不切实际的。因此，迫切需要开发限制免疫抑制和诱导免疫耐受或调节的方法。最近鉴定的CD 4 + CD 25 + T调节细胞（TCRs）提供了诱导供体特异性免疫调节而没有太多致命性的一些希望。其治疗用途的局限性之一是其存在的数量非常低。在这项研究中，我们试图分离和扩大狒狒Tribunal和测量其有效性，抑制猪狒狒异种反应。 从健康狒狒的外周血、脾脏和淋巴结中分离T细胞，首先通过磁珠富集CD 4+细胞，然后通过流式细胞仪分选CD 4 + CD 25+细胞。通过将这些分选的细胞添加到经辐照的猪PBMC和CD 4 + CD 25-细胞的共培养物中来测试它们的抑制潜力。还将分离的T细胞在IL 2和经照射的猪PBMC存在下在培养物中扩增4周，并评价其抑制作用。此外，还进行了细胞内细胞因子和各种活化标志物的表面表达的流式细胞术测定，以研究这些T细胞因子在该异种移植模型中的作用机制。 Treg分离技术是非常有效的，并且分离了97%纯的Treg（1-2%的CD 4 + T细胞）。这些分离的细胞有效地抑制了对辐照的猪PBMC的强烈的CD 4 + CD 25-细胞增殖反应（85%抑制）。 这些细胞还表达高水平的FoxP 3，这是一种有效的TGFAP标志物。分离的TcB在培养物中扩增150-200倍，并且还能够以与未分离的TcB类似的方式抑制CD 4 + CD 25-细胞。TdR还抑制CD 25-细胞响应于猪PBMC的细胞因子产生，表明Treg功能的可能机制。 通过上述实验，清楚的是，Tactobacillus是猪对狒狒异种反应的有效抑制剂，并且它们也可以在体外扩增以达到足够的量而不失去其抑制潜力。因此，TdR为目前用于克服异种移植排斥的非特异性免疫抑制提供了潜在的非致死性替代方案。 除了这些细胞研究，我们已经开始进行猪到狒狒原位异种心脏移植。 因此，我们正在验证该动物模型的稳定性，并建立一组基线对照结果，以比较使用药理学和基于细胞的免疫抑制治疗的其他移植物。