Regulation of humoral immunity against glycan antigens in glycan-incompatible transplantation

Abstract

Although clinical transplantation is the most effective therapy for patients with end-stage organ failure, this treatment has a limitation caused by the severe shortage of organ resources. Glycan-incompatible transplantation such as ABO-incompatible (ABOi) allotransplantation and xenotransplantation using pig organs has been considered as one of solutions to alleviate the limitation. However, there are a number of problems that need to be overcome for glycan-incompatible transplantation to be successful, in particular antibody-mediated responses toward glycan antigens on donor endothelium. Firstly, owing to the molecular incompatibility between human and pig, a slight binding of human antibody to pig endothelium can activate complement and coagulation system. To overcome a series of transplant rejections caused by molecular incompatibility, transgenic pigs expressing multiple human genes are required. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) was adopted. To evaluate the effect of T2A expression system, I constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and analyzed the expression pattern of target genes using porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein and the adequate expression of downstream genes can be achieved if the expression of the upstream gene is efficient. Therefore, T2A expression system is a promising tool for generating transgenic pigs that express multiple target genes for xenotransplantation. Secondly, humoral immune responses caused by preformed antibodies against pig glycan antigens are critical hurdles that need to be overcome. Despite the development of α1,3-galactosyl transferase-knockout (GT-KO) pigs, acute humoral xenograft rejection caused by antibodies against non-Gal antigens have been observed. Among non-Gal antigens, N-glycolylneuraminic acid (Neu5Gc) is considered to play an important role in xenograft rejection in human. To study human preformed antibody responses to Neu5Gc, I generated human embryonic kidney 293 (HEK293) cells that expressed xenogeneic Neu5Gc (HEK293-pCMAH) or α1,3Gal (HEK293-pGT) antigen and investigated the degree of human antibody binding and complement-dependent cytotoxicity (CDC) against these antigens using 100 individual human sera. Both IgM and IgG bound to α1,3Gal, while only IgG bound to Neu5Gc. Although the antibody reactivity to Neu5Gc was highly variable among individuals, severe CDC was significantly observed in HEK293-pCMAH responded with some human sera. In addition, the severity of CDC against HEK293-pCMAH cells positively correlated with that against GT-KO pig aortic endothelial cells (PAECs), suggesting that Neu5Gc is the main antigen in GT-KO PAECs. These results suggest that additional modifications to the CMAH gene will be required for widespread use of pig organs for human transplants. Thirdly, even though hyperacute rejection (HAR) can be prevented by using antibody desensitization protocols and GT-KO pigs, acute and chronic antibody mediated rejection induced by de novo synthesized antibodies after transplantation can be an another hurdle. For glycan-incompatible transplantation situation, it is unknown that which B cell subsets can recognize certain carbohydrate antigens, whether the responses are T cell dependent or independent, and whether T cells can recognize the certain carbohydrate antigens in antigen presenting molecules. To solve the questions, I established mice model for investigating induced anti-carbohydrate antibody production using target carbohydrate antigen-deficient mice and representative cell lines expressing target carbohydrate antigens, α1,3Gal, Neu5Gc, or blood group A, respectively. The induced antibodies against each carbohydrate antigens (α1,3Gal, Neu5Gc, or blood group A) are specific to target antigen. The mechanism of induced antibodies production against each carbohydrate antigen was quite different from each other. Interestingly, CD4+ T cell depletion effectively inhibited the production of induced antibody against three carbohydrate antigens altogether in mice. However, further study will be required to confirm whether the regulation of CD4+ T cell can ameliorate induced antibody responses in non-human primate and human.