KIR2DS2 and KIR2DS4 promoter hypomethylation patterns in patients undergoing hematopoietic cell transplantation (HCT)

Abstract

The killer cell Ig-like receptor (KIR)-MHC class I pathway is an integral part of natural killer cell immunity, and its role in host protection from both cancer and infection is important. In addition, we have shown elevated KIR2DS2 and 2DS4 expression in PBMCs of patients undergoing hematopoietic cell transplantation (HCT) [1]. Since all inhibitory KIR promoters are known to be heavily methylated, the question asked here is how and when KIR2DS2 and 2DS4 promoters had changed their methylation profile in association with HCT. Genomic DNA, extracted from 20 KIR2DS2/4+ donor and recipient cells, was treated with sodium bisulfate that will modify the unmethylated cytosine into uracil. Sequencing chromatographs were examined for C/T double peak indicative of base conversion. A CpG island in KIR2DS2 promoter spans from -160 to +26 with six cytosine sites. In contrast, the KIR2DS4 promoter CpG island contains three cytosine sites. The noted increase of unmethylated sites was associated with increased KIR expression as measured by mRNA-cDNA Q-PCR. In addition, the frequency of unmethylated sites in the CpG island was increased after HCT. The mechanism through which hypomethylation occurs after HCT is not known but it suggests a linkage to NK clonal expansion during the process of NK education in response to transplant therapy or viral infection.

Figure 1. Location of minimal promoter activity for KIR2DS2 and KIR2DS4

A) and C) show the schema of the various domains of the promoters that were amplified by PCR and inserted in pGL3. NK92 cells were transfected with the various constructs and tested for luciferase activity after 48hrs. A) and C) show the schematic intergenic region and PCR primers (arrows) for the KIR2DS2 promoter and the KIR2DS4 promoter, respectively. B) and D) show the luciferase activity in fold-increase above the reference control, generated by the cloned regions of KIR2DS2 promoter and KIR2DS4 promoter, respectively. The luciferase activity has been normalized to the value of the 2K putative promoter fragment as reference.

Figure 2. Promoter demethylation and KIR expression

NK92 cells (panel A) and PBMCs (panel B) from 3 donors were treated with 2.5μM 5-Aza-dC for 48 hours or as indicated and the expression of KIR2DS2 and KIR2DS4, as measured by mRNA cDNA Q-PCR is reported as copy number of mRNA-cDNA adjusted to 1e6 copies of actin mRNA. Untreated cells are labeled as controls.

Figure 3. CpG islands in the KIR2DS2 and KIR2DS4 promoter region

The CpG islands were found using Methyl Primer Express v1.0 (Applied Biosystems) and the numbers represent the location of CpGs in the sequence relative to the start site. A) The numbers surrounded by a circle were the sites found experimentally to be frequently unmethylated for KIR2DS2 and KIR2DS4. B) The frequency of KIR2DS2 unmethylated CpG sites are shown in 6 locations around the start site for 10 donors genotyped for KIR2DS2; C) The frequency of unmethylated CpG sites for 19 donors genotyped for KIR2DS4; D) and E) Frequency of unmethylated CpG for HCT recipients at various time post-HCT for KIR2DS2 and KIR2DS4 respectively.

Figure 4. Relationship between unmethylated profile of CpG sites in KIR2DS2/4 promoter and observed KIR expression

Blood samples from HCT recipients were analyzed for CpG KIR profile and for KIR expression using the mRNA-cDNA Q-PCR described in Materials and Methods. 32 samples were tested for KIR2DS2 (A and C) and 61 for KIR2DS4 (B and D). The relationship was examined using the contingency graphs (E) and the Fisher’s Exact Test where an (*) indicates the p value <0.05, and Odds Ratio are reported.