Establishment of HLA class I and MICA/B null HEK-293T panel expressing single MICA alleles to detect anti-MICA antibodies

Abstract

Pre- and post-transplantation anti-MICA antibody detection development are associated with an increased rejection risk and low graft survival. We previously generated HLA class I null HEK-293T using CRISPR/Cas9, while MICA and MICB genes were removed in this study. A panel of 11 cell lines expressing single MICA alleles was established. Anti-MICA antibody in the sera of kidney transplant patients was determined using flow cytometric method (FCM) and the Luminex method. In the 44 positive sera, the maximum FCM value was 2879 MFI compared to 28,135 MFI of Luminex method. Eleven sera (25%) were determined as positive by FCM and 32 sera (72%) were positive by the Luminex method. The sum of total MICA antigens, MICA*002, *004, *009, *019, and *027 correlation showed a statistically significant between the two methods (P = 0.0412, P = 0.0476, P = 0.0019, P = 0.0098, P = 0.0467, and P = 0.0049). These results demonstrated that HEK-293T-based engineered cell lines expressing single MICA alleles were suitable for measuring specific antibodies against MICA antigens in the sera of transplant patients. Studies of antibodies to MICA antigens may help to understand responses in vivo and increase clinical relevance at the cellular level such as complement-dependent cytotoxicity.

Serological Antibodies against Kidney, Liver, and Spleen Membrane Antigens as Potential Biomarkers in Patients with Immune Disorders

“…The inclusion of these cells was based on studies reported in the literature validating their use in various areas of experimentation [65]. These areas encompass studies on protein expression and interaction, viral packaging, and even antibody detection [65,66]. Moreover, since these cell lines are more readily available from commercial suppliers compared to tissues from both humans and animals, their incorporation into the study would enhance result reproducibility.…”

“…The results demonstrated a significant binding of IgG from the sera of KT patients to HEK 293 cells and certain human kidney tissues. Although HEK 293 cell lines are used as kidney epithelial cell lines [66], they also express some proteins specific to immature neurons, e.g., neurofilament proteins or alphainternexin [67]. Hence, it is still unclear whether they should be considered a model of kidney cells or whether they should be considered instead as immature neurons [65,[67][68][69].…”

Immune Editing: Overcoming Immune Barriers in Stem Cell Transplantation

“…While the expression of the classical costimulatory ligands B7-1 and B7-2 is fairly limited to professional APC, other activating ligands are rather expressed under inflammatory or other stress conditions that mark virus-infected or, otherwise, aberrant cells for destruction by NK cells [42]. Deletion of the stress signals MICA/B has recently been reported, [91] which could potentially help to evade NK cell responses. CD155 or poliovirus receptor (PVR), another costimulatory ligand for NK, has recently been deleted in HLA-deficient iPSC that were successfully differentiated into T cells [40•].…”