Hultman C S, Hunt J P, Yamamoto H, Giannopoulos A, deSerres S, Frelinger J A, Meyer A A
Department of Surgery, University of North Carolina School of Medicine, Chapel Hill 27599-7210, USA.
J Trauma. 1998 Jul;45(1):25-33; discussion 33-4. doi: 10.1097/00005373-199807000-00005.
Full-thickness (FT) and cultured keratinocyte (CK) allografts have been used as temporary skin replacements in patients with massive burns, but these grafts are ultimately rejected after restoration of host immunocompetence. Genetic engineering has permitted the creation of knockout (KO) mice deficient in class I or class II major histocompatibility antigens. This study examines the immunogenicity of such grafts to determine if these genetically modified keratinocytes could be used for permanent wound coverage.
Host sensitization to alloantigen was assessed by second-set rejection. CBA mice (n = 111) were primed with flank grafts consisting of FT and CK allografts from normal C57BL/6 donors, FT and CK class I KO allografts, FT and CK class II KO allografts, and CK autografts. Three weeks later, hosts were challenged with normal tail allografts and observed for second-set rejection. Median graft survival was analyzed by chi2 and Wilcoxon rank tests. In the second experiment, cytotoxic T lymphocytes (CTLs) were harvested from CBA mice (n = 28) 3 weeks after flank grafting. CTL effectors were tested on radiolabeled targets at various ratios in a 51Cr release assay. Dilution curves of CTL activity were compared by analysis of variance.
Hosts primed with CK or FT allografts demonstrated accelerated rejection of second-set tail grafts compared with hosts covered with CBA autografts. CK knockout grafts were less immunogenic than FT knockout skin; class II KO allografts were considerably less immunogenic than class I KO allografts. CTL activity against the knockout CK allografts was negligible compared with that of hosts primed with normal allografts or FT knockout allografts.
Although full-thickness knockout skin retains substantial immunogenicity, cultured keratinocytes deficient in class II antigens fail to prime for accelerated second-set rejection and do not elicit a CTL response in the graft recipient. This lack of immunogenicity may permit the indefinite survival of allogeneic knockout keratinocytes in patients requiring massive wound excision and coverage.
全层（FT）和培养的角质形成细胞（CK）同种异体移植物已被用作大面积烧伤患者的临时皮肤替代物，但这些移植物在宿主免疫功能恢复后最终会被排斥。基因工程已使得能够创建缺乏I类或II类主要组织相容性抗原的基因敲除（KO）小鼠。本研究检查此类移植物的免疫原性，以确定这些基因修饰的角质形成细胞是否可用于永久性伤口覆盖。
通过二次排斥评估宿主对同种异体抗原的致敏情况。将111只CBA小鼠用来自正常C57BL/6供体的FT和CK同种异体移植物、FT和I类KO同种异体移植物、FT和II类KO同种异体移植物以及CK自体移植物组成的侧腹移植物进行致敏。三周后，用正常尾同种异体移植物对宿主进行攻击，并观察二次排斥情况。通过卡方检验和威尔科克森秩和检验分析移植物存活中位数。在第二个实验中，在侧腹移植三周后从28只CBA小鼠中收获细胞毒性T淋巴细胞（CTL）。在51Cr释放试验中以不同比例在放射性标记的靶标上测试CTL效应细胞。通过方差分析比较CTL活性的稀释曲线。
与覆盖CBA自体移植物的宿主相比，用CK或FT同种异体移植物致敏的宿主表现出二次尾移植物的加速排斥。CK基因敲除移植物的免疫原性低于FT基因敲除皮肤；II类KO同种异体移植物的免疫原性明显低于I类KO同种异体移植物。与用正常同种异体移植物或FT基因敲除同种异体移植物致敏的宿主相比，针对基因敲除CK同种异体移植物的CTL活性可忽略不计。
尽管全层基因敲除皮肤保留了相当大的免疫原性，但缺乏II类抗原的培养角质形成细胞不会引发加速的二次排斥致敏，也不会在移植物接受者中引发CTL反应。这种免疫原性的缺乏可能允许同种异体基因敲除角质形成细胞在需要大面积伤口切除和覆盖的患者中无限期存活。
