Cardiac Xenotransplantation

异种移植是解决供体器官短缺的有效方法，但跨越物种屏障的强烈免疫反应限制了它的成功。目前用于延长异种移植物存活的免疫抑制水平是致命的，也不适用于临床。因此，迫切需要开发方法来限制免疫抑制和诱导免疫耐受或调节。最近鉴定的CD4+CD25+T调节细胞(Tregs)为诱导供者特异性免疫调节提供了一些希望，而不会产生太大的致命性。它们治疗使用的局限性之一是它们的存在数量非常少。在这项研究中，我们试图分离和扩大狒狒树突起，并测试它们对猪对狒狒异种反应的抑制效果。 首先用磁珠富集外周血、脾和淋巴结中的CD_4~+细胞，然后用流式细胞仪分选CD_4~+CD25~+细胞。这些分选的细胞通过与照射后的猪PBMC和CD4+CD25-细胞共培养来测试它们的抑制潜力。将分离的Tregs与IL2和照射后的猪PBMCs共同培养4周，观察其抑制作用。此外，还采用流式细胞术检测细胞内细胞因子和各种激活标志物的表面表达，以研究这些Tregs在异种移植模型中的作用机制。 Treg分离技术非常有效，可分离出97%的纯Tregs(1-2%的CD4+T细胞)。这些分离的细胞有效地抑制了强烈的CD4+CD25-细胞对辐照猪PBMC的增殖反应(抑制率为85%)。这些细胞还表达高水平的FoxP3，这是Tregs的一个有效标记。分离的Tregs在培养中扩增150-200倍，也能够以与NAve Tregs相似的方式抑制CD4+CD25-细胞。Treg还抑制CD25-细胞对猪PBMCs的细胞因子的产生，提示Treg功能的可能机制。 以上实验表明，Tregs是猪对狒狒异种反应的有效抑制因子，它们也可以在体外扩增以获得足够的数量而不失去它们的抑制潜力。因此，Treg为目前用于克服异种移植排斥反应的非特异性免疫抑制提供了一种潜在的非致死性选择。 除了这些细胞研究外，我们还开始了猪到狒狒的同种异种心脏移植。因此，我们正在验证这一动物模型的稳定性，并建立一套基线对照结果，以比较药物和基于细胞的免疫抑制治疗的其他移植。

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.