Human antibody reactivity against xenogeneic <i>N</i>‐glycolylneuraminic acid and galactose‐α‐1,3‐galactose antigen

Hurh, Sunghoon; Kang, Byung Hee; Choi, Inho; Cho, Bumrae; Lee, Eun Mi; Kim, Hwajung; Kim, Yong Baek; Chung, Yun Shin; Jeong, Jong Cheol; Hwang, Jong Ik; Kim, Jae Young; Lee, Byeong Chun; Surh, Charles D.; Yang, Jaeseok; Ahn, Curie

doi:10.1111/xen.12239

Cited by 26 publications

(18 citation statements)

References 55 publications

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“…In addition, a study of a Korean population demonstrated that IgM andIgG antibodies against Neu5Gc were not significantly different among subjects of various blood groups and age [32]. …”

Section: Discussionmentioning

confidence: 99%

“…The Neu5Gc-PAA conjugate which was used for ELISA in the present study does not present a sufficiently diverse range of antigens to accurately measure the amount of anti-Neu5Gc in the serum, and therefore does not give a complete comparison of the antibody levels in humans. In future experiments, it will be necessary to use more pertinent methodologies for measuring anti-Neu5Gc antibodies [30, 32, 35, 36]. …”

Section: Discussionmentioning

confidence: 99%

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Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

et al. 2017

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Our group previously investigated the levels of anti-Gal and anti-nonGal IgM and IgG in a cohort of 75 healthy humans of various backgrounds, and found some significant differences related to factors such as age, gender, ABO blood group, diet, vaccination history, and geographic location during childhood. We have now expanded our cohort (n = 84) to investigate the levels of anti-Neu5Gc and anti-nonGal/nonNeu5Gc antibodies in healthy humans. Anti-nonGal and anti-nonGal/nonNeu5Gc human IgM and IgG binding to pRBCs and pAECs from GTKO/CD46 and GTKO/CD46/Neu5GcKO pigs were measured by flow cytometry. Anti-Gal and anti-Neu5Gc IgM and IgG levels were measured by ELISA. In summary, (i) the great majority (almost 100%) of humans had anti-Neu5Gc IgM and IgG antibodies that bound to pAECs and approximately 50% had anti-Neu5Gc antibodies that bound to pRBCs, (ii) there was significantly less human antibody binding to pig cells that did not express either Gal or Neu5Gc compared with those that did not express Gal alone, (iii) the levels of both IgM and IgG binding to GTKO/CD46/Neu5GcKO pRBCs and pAECs were low, (iv) the level of anti-Neu5Gc IgG was higher in men than women, (v) the level did not change with age or diet, and there was some variability associated with (vi) previous vaccination history and (vii) the geographic region in which the individual spent his or her childhood. Our study confirms that human antibody binding to RBCs and AECs from GTKO/CD46/Neu5GcKO pigs is greatly reduced compared to binding to GTKO/CD46 cells. However, all humans appear to have a low level of antibody that binds to pAECs that is not directed to either Gal or Neu5Gc. Our findings require consideration in planning clinical trials of xenotransplantation.

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“…In addition, a study of a Korean population demonstrated that IgM andIgG antibodies against Neu5Gc were not significantly different among subjects of various blood groups and age [32]. …”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

et al. 2017

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“…The complement-mediated early rejection is mainly caused by preformed antibodies directed against galactose-α1,3-galactose-β1,4-N-acetylglucosamine-R epitope (αGal) 1,2 and other non-Gal carbohydrate epitopes such as Neu5Gc [3][4][5] or the glycan produced by porcine β1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) 6 on porcine cells, which in turn rapidly destroys the porcine graft. The development of pigs deficient in αGal (GGTA1-Ko) and Neu5Gc (CMAH-Ko) 7,8 and the transgenic expression of human complement regulatory proteins [9][10][11][12][13][14] helped to overcome these barriers and shifted the research focus to the following cellular and late rejection processes against porcine tissue.…”

Section: Introductionmentioning

confidence: 99%

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class Ilow phenotype—Effects on immune status and susceptibility to human immune responses

Hein

Sake

Pokoyski

et al. 2020

American Journal of Transplantation

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Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9‐mediated deletion of β2‐microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8+ T cell subset was observed in B2Mlow pigs. Cells from B2Mlow pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8+ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4+ T cells, natural killer cells) lysed targets from both SLA class I+ wildtype and SLA class Ilow pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8+ T cells during the induction phase of an anti‐xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.

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“…Acute vascular rejection occurs within a few days or weeks and is characterized by proinflammatory and procoagulatory activation of the vascular endothelium, complement activation, thrombotic microangiopathy and infiltration of innate immune cells into the graft . This response is also initiated by preformed antibodies, with evidence indicating a role for non‐Gal porcine antigens including N‐glycolylneuraminic acid (Neu5Gc), synthesized by cytidine monophospho‐N‐acetylneuraminic acid hydroxylase ( CMAH ), and surface glycans including the Sd(a) blood group antigen produced by β‐1,4‐N‐acetyl‐galactosaminyl transferase 2 ( B4GALNT2 ) …”

Section: Introductionmentioning

confidence: 99%

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2

Fischer

Rieblinger

Hein

et al. 2019

Xenotransplantation

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Background Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α‐1,3 Gal (Galactose‐alpha‐1,3‐galactose) causing hyperacute rejection, also Neu5Gc (N‐glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross‐reacted with swine leucocyte antigen class I (SLA‐I). We previously demonstrated efficient generation of pigs with multiple xeno‐transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi‐transgenic background further reduces antibody deposition and complement activation. Methods Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one‐ to four‐fold knockout animals, and from multi‐transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four‐fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. Results Pigs were generated carrying four‐fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi‐transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. Conclusion We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

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Human antibody reactivity against xenogeneic N‐glycolylneuraminic acid and galactose‐α‐1,3‐galactose antigen

Cited by 26 publications

References 55 publications

Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class Ilow phenotype—Effects on immune status and susceptibility to human immune responses

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2

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