Yolcu E S, Singh N P
Institute for Cellular Therapeutics and Department of Microbiology and Immunology, University of Louisville, Kentucky.
Transplant Proc. 2013 Jun;45(5):1850-2. doi: 10.1016/j.transproceed.2013.01.029.
Allogeneic bone marrow transplantation as a therapeutic approach in the clinic suffers from graft-versus- host disease (GVHD) initiated and perpetuated by donor T cells responding to alloantigens in immunocompromised hosts. Although the depletion of mature T cells from bone marrow inoculum overcomes GVHD, this manipulation is associated with engraftment failure and early post-transplant infection complications. Therefore, approaches that specifically purge out alloreactive T cells in the bone marrow inoculum without major effect on alloantigen-nonreactive T cells may be effective in facilitating engraftment without complications of GVHD and infections.
Inasmuch as Fas/FasL-induced apoptosis plays a critical role in self-tolerance, we tested whether the direct display of a novel form of FasL (SA-FasL) protein chimeric with streptavidin (SA) on the surface of T cells induces apoptosis in such cells in response to alloantigens. BALB/c and C57BL/6 total lymphocytes or purified T cells were biotinylated under physiologic conditions and engineered with SA-FasL protein taking advantage of the high-affinity interaction between biotin and SA.
All engineered cells displayed SA-FasL protein on their surface as determined by flow cytometry. When used as responders against irradiated, unmodified allogeneic stimulators, the SA-FasL-engineered T cells underwent apoptosis, which resulted in minimal proliferation. This effect was specific to SA-FasL; control SA protein-engineered T cells generated a potent proliferative alloresponse without significant apoptosis.
Taken together, these results demonstrate the feasibility of purging out alloreactive T cells by the display of SA-FasL protein on their surface with important implications for the prevention of GVHD associated with allogeneic bone marrow transplantation.
在临床中，同种异体骨髓移植作为一种治疗方法，会受到移植物抗宿主病（GVHD）的影响，这种疾病由供体T细胞对免疫功能低下宿主中的同种异体抗原作出反应而引发并持续存在。尽管从骨髓接种物中去除成熟T细胞可克服GVHD，但这种操作与植入失败和移植后早期感染并发症相关。因此，在不对同种异体抗原无反应性T细胞产生重大影响的情况下，特异性清除骨髓接种物中同种异体反应性T细胞的方法可能有效地促进植入，而不会出现GVHD和感染并发症。
由于Fas/FasL诱导的细胞凋亡在自身耐受中起关键作用，我们测试了在T细胞表面直接展示与链霉亲和素（SA）嵌合的新型FasL（SA-FasL）蛋白是否会诱导此类细胞因同种异体抗原而发生凋亡。在生理条件下对BALB/c和C57BL/6全淋巴细胞或纯化的T细胞进行生物素化，并利用生物素与SA之间的高亲和力相互作用，用SA-FasL蛋白对其进行改造。
通过流式细胞术测定，所有改造后的细胞在其表面均展示了SA-FasL蛋白。当用作针对经辐照的、未修饰的同种异体刺激物的反应细胞时，经SA-FasL改造的T细胞发生凋亡，导致增殖极少。这种作用对SA-FasL具有特异性；对照SA蛋白改造的T细胞产生了强烈的增殖性同种异体反应，而没有明显的凋亡。
综上所述，这些结果证明了通过在T细胞表面展示SA-FasL蛋白来清除同种异体反应性T细胞的可行性，这对预防与同种异体骨髓移植相关的GVHD具有重要意义。
