Identification of human preformed antibody targets in GTKO pigs

Christopher Burlak, Zheng Yu Wang, Ray K. Chihara, Andrew J. Lutz, Yueren Wang, Jose L. Estrada, A. Joseph Tector

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Background: Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α-galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver. Methods: Human serum was analyzed for the concentration of IgG, IgM, anti-αgal antibody, anti-non-αgal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation. Results: Human serum used in this study contained 2.06 μg/ml IgG and 0.013 μg/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose-dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were α-enolase, CFTR, and E-cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured α-enolase, CFTR, and E-cadherin by immunoprecipitation validating the proteomic identification. Conclusion: These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody-mediated xenograft rejection are necessary.

Original languageEnglish
Pages (from-to)92-101
Number of pages10
JournalXenotransplantation
Volume19
Issue number2
DOIs
StatePublished - Mar 2012

Fingerprint

Galactosyltransferases
Forensic Anthropology
Swine
Antibodies
Immunoglobulin M
Immunoglobulin G
Liver
Endothelial Cells
Phosphopyruvate Hydratase
Cadherins
Immunoprecipitation
Antigens
Heterografts
Proteomics
Mass Spectrometry
Fibroblasts
Serum
Proteins
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Confocal Microscopy

Keywords

  • α- galactosyltransferase knockout pigs
  • Xenoantigens
  • Xenograft rejection
  • Xenotransplantation

ASJC Scopus subject areas

  • Transplantation
  • Immunology

Cite this

Burlak, C., Wang, Z. Y., Chihara, R. K., Lutz, A. J., Wang, Y., Estrada, J. L., & Tector, A. J. (2012). Identification of human preformed antibody targets in GTKO pigs. Xenotransplantation, 19(2), 92-101. https://doi.org/10.1111/j.1399-3089.2012.00695.x

Identification of human preformed antibody targets in GTKO pigs. / Burlak, Christopher; Wang, Zheng Yu; Chihara, Ray K.; Lutz, Andrew J.; Wang, Yueren; Estrada, Jose L.; Tector, A. Joseph.

In: Xenotransplantation, Vol. 19, No. 2, 03.2012, p. 92-101.

Research output: Contribution to journalArticle

Burlak, C, Wang, ZY, Chihara, RK, Lutz, AJ, Wang, Y, Estrada, JL & Tector, AJ 2012, 'Identification of human preformed antibody targets in GTKO pigs', Xenotransplantation, vol. 19, no. 2, pp. 92-101. https://doi.org/10.1111/j.1399-3089.2012.00695.x
Burlak C, Wang ZY, Chihara RK, Lutz AJ, Wang Y, Estrada JL et al. Identification of human preformed antibody targets in GTKO pigs. Xenotransplantation. 2012 Mar;19(2):92-101. https://doi.org/10.1111/j.1399-3089.2012.00695.x
Burlak, Christopher ; Wang, Zheng Yu ; Chihara, Ray K. ; Lutz, Andrew J. ; Wang, Yueren ; Estrada, Jose L. ; Tector, A. Joseph. / Identification of human preformed antibody targets in GTKO pigs. In: Xenotransplantation. 2012 ; Vol. 19, No. 2. pp. 92-101.
@article{a7f4ffeba0f64f86882e6a8fa8eaa1da,
title = "Identification of human preformed antibody targets in GTKO pigs",
abstract = "Background: Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α-galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver. Methods: Human serum was analyzed for the concentration of IgG, IgM, anti-αgal antibody, anti-non-αgal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation. Results: Human serum used in this study contained 2.06 μg/ml IgG and 0.013 μg/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose-dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were α-enolase, CFTR, and E-cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured α-enolase, CFTR, and E-cadherin by immunoprecipitation validating the proteomic identification. Conclusion: These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody-mediated xenograft rejection are necessary.",
keywords = "α- galactosyltransferase knockout pigs, Xenoantigens, Xenograft rejection, Xenotransplantation",
author = "Christopher Burlak and Wang, {Zheng Yu} and Chihara, {Ray K.} and Lutz, {Andrew J.} and Yueren Wang and Estrada, {Jose L.} and Tector, {A. Joseph}",
year = "2012",
month = "3",
doi = "10.1111/j.1399-3089.2012.00695.x",
language = "English",
volume = "19",
pages = "92--101",
journal = "Xenotransplantation",
issn = "0908-665X",
publisher = "Wiley-Blackwell",
number = "2",

}

TY - JOUR

T1 - Identification of human preformed antibody targets in GTKO pigs

AU - Burlak, Christopher

AU - Wang, Zheng Yu

AU - Chihara, Ray K.

AU - Lutz, Andrew J.

AU - Wang, Yueren

AU - Estrada, Jose L.

AU - Tector, A. Joseph

PY - 2012/3

Y1 - 2012/3

N2 - Background: Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α-galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver. Methods: Human serum was analyzed for the concentration of IgG, IgM, anti-αgal antibody, anti-non-αgal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation. Results: Human serum used in this study contained 2.06 μg/ml IgG and 0.013 μg/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose-dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were α-enolase, CFTR, and E-cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured α-enolase, CFTR, and E-cadherin by immunoprecipitation validating the proteomic identification. Conclusion: These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody-mediated xenograft rejection are necessary.

AB - Background: Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α-galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver. Methods: Human serum was analyzed for the concentration of IgG, IgM, anti-αgal antibody, anti-non-αgal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation. Results: Human serum used in this study contained 2.06 μg/ml IgG and 0.013 μg/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose-dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were α-enolase, CFTR, and E-cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured α-enolase, CFTR, and E-cadherin by immunoprecipitation validating the proteomic identification. Conclusion: These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody-mediated xenograft rejection are necessary.

KW - α- galactosyltransferase knockout pigs

KW - Xenoantigens

KW - Xenograft rejection

KW - Xenotransplantation

UR - http://www.scopus.com/inward/record.url?scp=84859854200&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84859854200&partnerID=8YFLogxK

U2 - 10.1111/j.1399-3089.2012.00695.x

DO - 10.1111/j.1399-3089.2012.00695.x

M3 - Article

C2 - 22497511

AN - SCOPUS:84859854200

VL - 19

SP - 92

EP - 101

JO - Xenotransplantation

JF - Xenotransplantation

SN - 0908-665X

IS - 2

ER -