# Methodological approach for allele-specific antibody responses to HEK-293T-based cell lines expressing single MHC class I chain-related gene B antigens

## TL;DR

Researchers developed a cell-based platform to study antibodies against specific MICB antigens, which could help understand their role in transplant rejection.

## Contribution

A novel cell-based platform using HEK-293T cells expressing single MICB alleles was developed to study allele-specific anti-MICB antibody responses.

## Key findings

- Five HEK-293T cell lines expressing individual MICB antigens were successfully established and validated.
- No anti-MICB antibodies were detected in 64 pre-transplant sera, suggesting low sensitization in this population.
- The platform offers a promising tool for future studies on MICB immunogenicity in transplantation.

## Abstract

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.

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.

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.

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.

The online version contains supplementary material available at 10.1186/s12860-025-00549-5.