Ketevan Gendzekhadze

Expression of Activating KIR2DS2 and KIR2DS4 Genes after Hematopoietic Cell Transplantation: Relevance to Cytomegalovirus Infection

The important role of activating killer immunoglobulin-like receptors (KIRs) in protecting against cytomegalovirus (CMV) reactivation has been described previously in patients undergoing hematopoietic cell transplantation (HCT). More specifically, the presence of multiple activating KIRs and the presence of at least KIR2DS2 and KIR2DS4 in the donor genotype identified a group of HCT patients at low risk for CMV reactivation. However, CMV infection still occurs in patients with the KIR protective genotype, and the question has been raised as to whether this is related to the lack of KIR expression. In this report, expression of the KIR2DS2 and KIR2DS4 genes, as measured by mRNA-based quantitative polymerase chain reaction in both the donor cells and the HCT recipient cells, was studied relative to CMV reactivation. In the control samples from healthy donors, the median range for KIR2DS2 and KIR2DS4 expression was low, with 35% of donors considered null-expressers. Interestingly, KIR2DS2 and KIR2DS4 expression was elevated after HCT compared with donor expression before HCT, and was significantly elevated in CMV viremic compared with CMV nonviremic HCT recipients. The CMV seropositivity of donors was not associated with activating KIR expression, and donor null expression in those with the KIR2DS2 or KIR2DS4 genotype was not predictive for CMV reactivation in the recipient. After controlling for other transplant factors, including donor type (sibling or unrelated), transplant source (bone marrow or peripheral blood stem cells), and acute GVHD grade, regression analysis of elevated KIR gene expression found an association for both KIR2DS2 and KIR2DS4, with a 7-fold increase in risk for CMV reactivation. We speculate that the elevated activating KIR expression in CMV-viremic HCT recipients is either coincidental with factors that activate CMV or is initiated by CMV or cellular processes responsive to such CMV infection reactivation.

Protective effect of the KIR2DS1 gene in atopic dermatitis.

Atopic dermatitis (AD) is a common skin disease of complex etiology including affected humoral and cellular immune responses. The role of NK cells in development of this disease has been recently postulated, but is still poorly documented. The current study was undertaken to determine the impact of genes for the most polymorphic NK cell receptors, known as killer cell immunoglobulin-like receptors (KIRs), on the development of AD. We compared 240 patients suffering from AD with 570 healthy controls. Frequencies of the great majority of KIR genes did not differ between patients and controls, except for KIR2DS1, whose frequency was significantly (OR=0.629, CI95% (0.45; 0.87), pcorr=0.0454) lower in patients than in controls. These results were confirmed in a second cohort of 201 patients. When both patient groups were combined and compared to the control group, the result for KIR2DS1 achieved even higher significance (OR=0.658, CI95% (0.5; 0.86), pcorr=0.0158). To the best of our knowledge, this is the first report on KIR gene contribution to AD, and to allergy in general.

Chimerism Testing by Quantitative PCR Using Indel Markers

Engraftment monitoring is critical for patients after Hematopoietic Stem Cell Transplantation (HSCT). Complete donor chimerism is the goal; therefore, early detection of rejection and relapse is crucial for guiding the patient post HSCT treatment. Quantitative PCR for chimerism testing has been reported to be highly sensitive. In this chapter we discuss the quantitative PCR (qPCR) method using 34 Indel (Insertion and Deletion) genetic markers spread over 20 different chromosomes.

Favorable impact of allogeneic stem cell transplantation in patients with therapy-related myelodysplasia regardless of TP53 mutational status

Therapy-related myelodysplastic syndrome is a long-term complication of cancer treatment in patients receiving cytotoxic therapy, characterized by high-risk genetics and poor outcomes. Allogeneic hematopoietic cell transplantation is the only potential cure for this disease, but the prognostic impact of pre-transplant genetics and clinical features has not yet been fully characterized. We report here the genetic and clinical characteristics and outcomes of a relatively large cohort of patients with therapy-related myelodysplastic syndrome (n=67) who underwent allogeneic transplantation, comparing these patients to similarly treated patients with de novo disease (n=199). The 5-year overall survival was not different between patients with therapy-related and de novo disease (49.9% versus 53.9%; P=0.61) despite a higher proportion of individuals with an Intermediate-2/High International Prognostic Scoring System classification (59.7% versus 43.7%; P=0.003) and high-risk karyotypes (61.2% versus 30.7%; P<0.01) among the patients with therapy-related disease. In mutational analysis, TP53 alteration was the most common abnormality in patients with therapy-related disease (n=18: 30%). Interestingly, the presence of mutations in TP53 or in any other of the high-risk genes (EZH2, ETV6, RUNX1, ASXL1: n=29: 48%) did not significantly affect either overall survival or relapse-free survival. Allogeneic stem-cell transplantation is, therefore, a curative treatment for patients with therapy-related myelodysplastic syndrome, conferring a similar long-term survival to that of patients with de novo disease despite higher-risk features. While TP53 alteration was the most common mutation in therapy-related myelodysplastic syndrome, the finding was not detrimental in our case-series.

The novel HLA‐B*15:180 allele appears to be a recombinant B*08/B*15 allele

Human leukocyte antigen B-*15:180 is a B*08/B*15 recombinant allele similar to B*15:29 with substitutions positions at 97, 292, 538, 539.

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

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.