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.

Increased programmed death-1 molecule expression in cytomegalovirus disease and acute graft-versus-host disease after allogeneic hematopoietic cell transplantation.

To study the role of the programmed death-1 molecule (PD-1) in cytomegalovirus (CMV) infection and disease after allogeneic hematopoietic cell transplantation (HCT), 206 subjects were followed prospectively for immune response to CMV and assigned to 3 groups based on CMV outcome. The subjects were analyzed retrospectively for PD-1 expression in cryopreserved CD4+ and CD8+T cells collected at days 40, 90, 120, 150, 180, and 360 posttransplantation. HCT recipients with CMV disease (n=14) were compared with recipients with prolonged CMV infection, but no CMV disease (median duration of infection, 3 months; n=14) and with controls with no CMV infection who received similar transplants (n=22). The CMV disease group had a significantly higher mean fluorescein intensity of PD-1 in CD4+ (P < .05) and CD8+ (P < .05) lymphocytes at all time points studied. PD-1 expression also was significantly elevated in those with severe acute graft-versus-host disease (aGVHD), including the no-viremia group. The data suggest that PD-1 is induced by aGVHD even in the absence of CMV infection. This enhanced PD-1 expression during severe aGVHD and with CMV reactivation could explain the known role of aGVHD as a risk factor for CMV disease.

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.

Biologic and immunologic effects of knockout of human cytomegalovirus pp65 nuclear localization signal.

The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.

Cytomegalovirus immune reconstitution occurs in recipients of allogeneic hematopoietic cell transplants irrespective of detectable cytomegalovirus infection.

The question of when immune reconstitution of cytomegalovirus (CMV)-specific CD8 T cells occurs after hematopoietic cell transplantation and, more specifically, to which CMV targets this immunity is likely to be directed remains poorly understood. The dependence of immune reconstitution on CMV reactivation is even less clear. To better understand these events, 44 CMV-seropositive HLA-A*0201 subjects were followed up at approximately days 40, 90, 120, 150, 180, and 360 after hematopoietic cell transplantation for CMV immunity as measured by 2 types of assays: (1) an HLA-A*0201 tetramer-binding assay for both CMV pp65 (pp65) and immediate-early 1 (IE-1) or (2) intracellular cytokine interferon gamma responses induced by pp65 or IE-1-derived peptides. To verify the reliability of IE-1-specific assays relative to the pp65-based assays, a pilot study first compared the development of IE-1-specific immunity in a subgroup by using multiple HLA-A*0201-restricted peptides, and then these recipients were followed up for 1 year for immunologic function and for CMV infection. The IE-1-specific response occurred to each of the 3 HLA-A*0201-restricted peptides studied (IE-1-256, -297, and -316), and there was no predominant IE peptide response. However, the immunodominant HLA-A*0201-restricted pp65 peptide was recognized significantly more frequently than these IE-1 peptides. When this was compared with the occurrence of CMV infection, the overall immune reactivity, as measured by the mean or median number of CD8+ T cells reactive to either pp65 or IE-1 peptides by intracellular cytokine or tetramer binding assay, was not significantly different in those with and without CMV infection. For patients who demonstrated reconstituted immunity to CMV at 1 year, all were reconstituted by 6 months, and the timing of the first observed immune reactivity to either of the pp65 or the IE peptides was not different in those with and without detectable CMV infection.

DNA and low titer, helper-free, recombinant AAV prime-boost vaccination for cytomegalovirus induces an immune response to CMV-pp65 and CMV-IE1 in transgenic HLA A*0201 mice.

A prime-boost immunization regimen allowed the use of low titer, helper-free rAAV-pp65mII and rAAV-IE1 virus to elicit specific humoral and cellular responses to two important cytomegalovirus (CMV) antigens: the immediate-early 1 (IE-1) and pp65 proteins. Simultaneous immunization of both CMV proteins, using DNA vaccine priming followed by rAAV boost, induced antibody (Ab) response, CD8 lymphocytes with cytotoxic function, and detectible binding of the cognate peptide epitopes for human HLA A*0201 restriction using tetramer technology.