Cardiac Xenotransplantation

心脏异种移植

• 批准号: 7321783
• 负责人: Keith A Horvath
• 金额: --
• 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/7321783
• 关键词: Cardiac; Xenotransplantation

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. Methods: 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. Results: 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 naive Tregs. Tregs also suppressed the cytokine production by the CD25- cells in response to pig PBMCs, suggesting a possible mechanism of Treg function. Conclusion: 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.

异种移植为供体器官短缺提供了有效的解决方案，但跨越物种屏障的强烈免疫反应限制了其成功。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并诱导免疫耐受或适应的方法。最近鉴定的 CD4+CD25+ T 调节细胞 (Treg) 为诱导供体特异性免疫调节而没有太大致死性提供了一些希望。它们的治疗用途的局限性之一是它们的数量非常少。在这项研究中，我们尝试分离和扩增狒狒 Tregs，并测量它们在抑制猪对狒狒异种反应方面的有效性。 方法：首先通过磁珠富集CD4+细胞，然后通过FACS分选CD4+CD25+细胞，从健康狒狒的外周血、脾脏和淋巴结中分离Tregs。通过添加到受辐射的猪 PBMC 和 CD4+ CD25- 细胞的共培养物中，测试这些分选的细胞的抑制潜力。分离的 Tregs 还在 IL2 和经照射的猪 PBMC 存在的情况下在培养物中扩增 4 周，并评估其抑制效果。此外，还对细胞内细胞因子和各种激活标记物的表面表达进行了流式细胞术分析，以研究这些 Tregs 在该异种移植模型中的作用机制。 结果：Treg 分离技术非常有效，分离出 97% 的纯 Treg（1-2% 的 CD4+ T 细胞）。这些分离的细胞有效抑制了对受辐射的猪 PBMC 的剧烈 CD4+CD25- 细胞增殖反应（85% 抑制）。这些细胞还表达高水平的 FoxP3，这是 Tregs 的有效标志物。分离的Tregs在培养物中扩增150-200倍，并且还能够以与初始Tregs类似的方式抑制CD4+CD25-细胞。 Treg 还抑制了 CD25- 细胞响应猪 PBMC 产生的细胞因子，这表明 Treg 功能的可能机制。 结论：通过上述实验，可以清楚地看出Tregs是猪对狒狒异种反应的有效抑制因子，并且它们还可以在体外扩增以达到足够的数量而不失去其抑制潜力。因此，Tregs 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命替代方案。