Abstract
Purpose :
Human Corneal-Limbal Epithelial (HCLE) cells express high levels of Human Leukocyte Antigens Class I (HLA-I), which make them a target for alloimmune rejection. The chaperon Tapasin (coded from the TAPBP gene) is not only important for the proper folding of the HLA-I molecules, but it also plays a vital role in loading the HLA-I with the antigenic peptides before its exposure on the cell surface. The goal of the project is to analyze how the deletion of the TAPBP gene in HCLE cells affects their expression of HLA-I.
Methods :
We used the CRISPR/Cas9 gene-editing method to generate the TAPBP gene knockout (KO) in HCLE cells. Specifically, we electroporated the HCLE cells with the Cas9 enzyme and 2 guide RNAs targeting the TABPB gene. After the recovery of the cells, we performed limiting dilution to generate monoclonal cell lines. We screened such lines by PCR-based genotyping and Sanger-sequencing to uncover the TAPBP-KO clones. We next performed qRT-PCR to verify the abolished expression of TAPBP. To test the ability of the TAPBP-KO cells to express the HLA-I molecules, we performed FACS analysis and compared the HLA-I expression levels with the HCLE wild type (WT) cells.
Results :
The comparison of the HLA-I expression in HCLE and in human immune cells (monocytes) demonstrated that both cell types express comparable levels of HLA-I even after the IFN-γ stimulation. Next, we embarked upon the CRISPR/Cas9-mediated KO of the TAPBP gene in the HCLE and tested how TAPBP KO affects the HLA-I expression. We found that 2 electroporation pulses at 1400V gave us 55.7% efficiency and 89% of cell viability. After limiting dilution, we selected 3 monoclonal lines for further analysis. FACS analysis showed a substantial decrease in the HLA-I ABC expression in TAPBP-KO cells (66% vs 99% of WT cells), suggesting that TAPBP-KO cells might escape CD8+ T cell-mediated cell death. In contrast, the levels of universally expressed HLA-I E in TAPBP-KO and WT cells remained comparable (93% vs 99%), suggesting TAPBP-KO cells retained their ability to escape from NK cell cytotoxicity.
Conclusions :
The deletion of chaperon Tapasin selectively downregulates HLA-I expression in the corneal-limbal epithelial cells, which could prevent their rejection by the immune system.
This is a 2021 ARVO Annual Meeting abstract.