The inhibitory NK cell receptor KIR3DL1, which interacts with Bw4-motif-carrying HLA class I ligands, has been extensively studied in the context of disease associations. In HIV infection, combinations of KIR3DL1 high- and intermediate-expressing genotypes with Bw4-expressing HLA-B*57 alleles have been associated with favorable clinical outcomes. Due to the significant allelic diversity of the KIR3DL1 gene, its genotyping holds considerable interest for genetic association studies. However, existing KIR3DL1 genotyping methods are labor-intensive, requiring multiple distinct PCR reactions. Furthermore, these methods fail to capture certain intronic regions that may impact alternative exon recognition and, consequently, gene expression.
To streamline KIR3DL1 genotyping and sequence analysis, we set out to develop a simplified method by establishing a KIR3DL1 "full-length" PCR protocol followed by Oxford Nanopore Sequencing. As a proof of concept, we genotyped approximately 200 healthy blood donors, comparing the performance of our method to the established protocol and analyzing NK cell expression profiles through flow cytometry. Furthermore, we investigated splicing regulatory motifs, which we validated using whole RNA sequencing of sorted NK cells.
Our method demonstrated robustness and simplicity for both KIR3DL1 genotyping and full-length sequencing. Notably, the alternative exon harbors a premature polyadenylation signal upstream of the KIR3DL1 transmembrane region, resulting in a shorter, potentially soluble KIR3DL1 isoform. In KIR3DL1-positive sorted NK cells, we detected mRNA species lacking the transmembrane region of KIR3DL1 alongside full-length transcripts, further supporting the existence of a soluble KIR3DL1 variant. Finally, using a FACS-based approach, we successfully identified this shorter isoform in the cell culture supernatant of KIR3DL1-positive NK cells.
Since we discovered a soluble KIR3DL1 isoform at both mRNA and protein levels, future investigations will focus on identifying the factors that regulate soluble KIR3DL1 expression and evaluating its functional impact on NK cell activity, particularly in the context of viral infections.