Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens
Abstract Background Antibodies against non-HLA antigens, such as MICA and MICB, have emerged as potential contributors to antibody-mediated rejection and graft failure. While MICA antibodies are well characterized, MICB-specific antibodies remain poorly understood due to the lack of standardized det...
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BMC
2025-07-01
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| Series: | BMC Molecular and Cell Biology |
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| Online Access: | https://doi.org/10.1186/s12860-025-00549-5 |
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| author | Ji-Ho Jeon Cheol-Hwa Hong You-Seok Hyun Hyeyoung Lee Eun-Jee Oh Tai-Gyu Kim In-Cheol Baek |
| author_facet | Ji-Ho Jeon Cheol-Hwa Hong You-Seok Hyun Hyeyoung Lee Eun-Jee Oh Tai-Gyu Kim In-Cheol Baek |
| author_sort | Ji-Ho Jeon |
| collection | DOAJ |
| description | Abstract Background Antibodies against non-HLA antigens, such as MICA and MICB, have emerged as potential contributors to antibody-mediated rejection and graft failure. While MICA antibodies are well characterized, MICB-specific antibodies remain poorly understood due to the lack of standardized detection tools. To address this gap, we aimed to develop a cell-based platform expressing individual MICB antigens to evaluate the feasibility of detecting allele-specific anti-MICB antibodies in pre-kidney transplant sera. Methods HLA class I, MICA, and MICB-null human embryonic kidney (HEK)-293T cells were previously generated by CRISPR/Cas9. We established five cell lines expressing single MICB antigens (each MICB*002, *003, *004, *005:02, and *008 allele). A total of 64 pre-kidney transplant sera were tested to assess anti-MICB antibody responses to the five cell lines using flow cytometry. Results We successfully established and validated five HEK-293T cell lines each expressing a single MICB antigen using anti-MICB monoclonal antibody staining. No anti-MICB antibodies were detected in any of the 64 pre-transplant sera tested. This finding may reflect a low incidence of sensitization to MICB in this patient population and suggests the need for larger, more diverse cohorts in future studies to fully assess the prevalence of anti-MICB responses. The established cell lines provide a promising tool for future investigation of allele-specific anti-MICB antibody responses. Conclusions While the present study did not detect allele-specific anti-MICB antibody responses, establishing HEK-293T cell lines expressing single MICB antigens represents a significant methodological advance. This platform enables the potential assessment of immune responses targeted to individual MICB allotypes, thus offering new avenues for the future study of MICB immunogenicity in transplantation settings. |
| format | Article |
| id | doaj-art-fd48847568984d77bc42ac073bac1631 |
| institution | Kabale University |
| issn | 2661-8850 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | BMC |
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| series | BMC Molecular and Cell Biology |
| spelling | doaj-art-fd48847568984d77bc42ac073bac16312025-08-20T03:46:24ZengBMCBMC Molecular and Cell Biology2661-88502025-07-012611810.1186/s12860-025-00549-5Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigensJi-Ho Jeon0Cheol-Hwa Hong1You-Seok Hyun2Hyeyoung Lee3Eun-Jee Oh4Tai-Gyu Kim5In-Cheol Baek6Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of KoreaDepartment of Microbiology, College of Medicine, The Catholic University of KoreaDepartment of Microbiology, College of Medicine, The Catholic University of KoreaDepartment of Laboratory Medicine, College of Medicine, International St. Mary’s Hospital, Catholic Kwandong UniversityDepartments of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of KoreaDepartment of Microbiology, College of Medicine, The Catholic University of KoreaCatholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of KoreaAbstract Background Antibodies against non-HLA antigens, such as MICA and MICB, have emerged as potential contributors to antibody-mediated rejection and graft failure. While MICA antibodies are well characterized, MICB-specific antibodies remain poorly understood due to the lack of standardized detection tools. To address this gap, we aimed to develop a cell-based platform expressing individual MICB antigens to evaluate the feasibility of detecting allele-specific anti-MICB antibodies in pre-kidney transplant sera. Methods HLA class I, MICA, and MICB-null human embryonic kidney (HEK)-293T cells were previously generated by CRISPR/Cas9. We established five cell lines expressing single MICB antigens (each MICB*002, *003, *004, *005:02, and *008 allele). A total of 64 pre-kidney transplant sera were tested to assess anti-MICB antibody responses to the five cell lines using flow cytometry. Results We successfully established and validated five HEK-293T cell lines each expressing a single MICB antigen using anti-MICB monoclonal antibody staining. No anti-MICB antibodies were detected in any of the 64 pre-transplant sera tested. This finding may reflect a low incidence of sensitization to MICB in this patient population and suggests the need for larger, more diverse cohorts in future studies to fully assess the prevalence of anti-MICB responses. The established cell lines provide a promising tool for future investigation of allele-specific anti-MICB antibody responses. Conclusions While the present study did not detect allele-specific anti-MICB antibody responses, establishing HEK-293T cell lines expressing single MICB antigens represents a significant methodological advance. This platform enables the potential assessment of immune responses targeted to individual MICB allotypes, thus offering new avenues for the future study of MICB immunogenicity in transplantation settings.https://doi.org/10.1186/s12860-025-00549-5MHC class I chain-related gene BMICB antibodyAllotype responsePanel reactive antibody testKidney transplant |
| spellingShingle | Ji-Ho Jeon Cheol-Hwa Hong You-Seok Hyun Hyeyoung Lee Eun-Jee Oh Tai-Gyu Kim In-Cheol Baek Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens BMC Molecular and Cell Biology MHC class I chain-related gene B MICB antibody Allotype response Panel reactive antibody test Kidney transplant |
| title | Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens |
| title_full | Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens |
| title_fullStr | Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens |
| title_full_unstemmed | Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens |
| title_short | Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens |
| title_sort | methodological approach for allele specific antibody responses to hek 293t based cell lines expressing single mhc class i chain related gene b antigens |
| topic | MHC class I chain-related gene B MICB antibody Allotype response Panel reactive antibody test Kidney transplant |
| url | https://doi.org/10.1186/s12860-025-00549-5 |
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