performed KIR genetic analysis. CD45RA+ (CCR7CCD45RA+) cells in the CD8+ T?cell compartment (24.1 14.4 versus 32.3 16.9, p?=?0.014), whereas no significant changes were observed for any of the other CD8 T?cell subsets studied (Figures S1A and S1B). Interestingly, the accumulation of mature CD8 T?cells was particularly visible in young and middle-age individuals (17.8 9.6 versus 32.07 17.2, p?= 0.001; Figures 1AC1C). However, CD8 T?cell responses following stimulation with overlapping peptide pools derived from the HCMV proteins IE-1, IE-2, and pp65 were identical in deletion was not associated with any significant phenotypic or functional differences in CD4+ T?cells (Figure?S2) and did not imprint B cell differentiation (Figure?S3). Thus, despite an accumulation of terminally differentiated CD8 T?cells in young NKG2C?/? individuals, our results show that no major reshaping of T and B cell immunity to HCMV takes place in NKG2C-deficient individuals. Open in a separate window Figure?1 Homozygous Deletion Is Associated with Accumulation of Terminally Differentiated Effector Memory CD45RA+ T?Cells (A and B) Frequency of CD63 EMRA CD8 T?cells in?HCMV+deletion. (D) Frequency of IFN-+ CD8 T?cells after overnight?stimulation with pp65 overlapping peptide pools. (E) Frequency of HCMV-specific CD8 T?cells as defined by HLA-A?02 or HLA-B?07 tetramers refolded with pp65-derived peptides. Gray lines represent the median value within each group. Adaptive NK Cell Response to HCMV in locus (Figure?3H), which was shown to be exclusively demethylated in?NKG2C-expressing expansions from HCMV+ individuals (Luetke-Eversloh et?al., 2014). Open in a separate window Figure?3 Adaptive NK Cells in raised the question of which potential activating receptors might contribute to the expansion of this subset. Among other genes, the NK gene complex on chromosome 12 encodes NKG2E, an activating receptor that also forms functional heterodimers with CD94 and recognizes HLA-E (Lanier et?al., 1998, Lazetic et?al., 1996). Since CD94 was at least weakly expressed on all NK cells in both deletion (Bziat et?al., 2013, Della Chiesa et?al., 2014). Accordingly, we examined the relative contribution of NKG2C and activating KIRs to the adaptive NK cell pool in each donor (Figure?4E). Seletalisib (UCB-5857) In deletion and seemed to be independent of the activating receptor composition (Figure?4F). Although our phenotypic analysis did not include KIR2DS3 and KIR2DS5, the detection of three haplotype A/A donors among the 11 gene allowed us to address these possibilities in the human. Here, adaptive NK cell responses in donors displayed similar frequencies of CMV-specific T?cells as the gene. These results suggest that, despite a high level of redundancy within the NK cell compartment itself, the lack of might also be Seletalisib (UCB-5857) partly compensated for by enhanced T and B cell responses, particularly during the early phases of HCMV infection. Possibly, an effective adaptive NK cell immunity helps to control the burden of HCMV infection before the emergence of efficient T and B cell immunity. Although adaptive NK?cells displayed reduced degranulation Seletalisib (UCB-5857) responses, their enhanced ability to release cytokines in response to antibody-coated targets might help to fulfill this role and contribute to maintaining the virus silent during latency. The plasticity of adaptive NK cell responses in the absence of activating KIRs and NKG2C points to the importance of such responses within the innate immune system. Experimental Procedures Human Participants and Cells This study was conducted in accordance with the Declaration of Helsinki and?was approved by the ethics committee in Stockholm, Sweden. 2,208 random healthy blood donors were screened for NKG2C expression by flow cytometry. Donors lacking NKG2C expression were confirmed by PCR using the protocol described by Moraru et?al. verifying homozygous deletion of gene (Moraru et?al., 2012a). 60 controls expressing NKG2C and 60 donors lacking the gene were identified and enrolled in the study. For all donors, peripheral blood mononuclear cells (PBMCs) were cryopreserved for later use. Genomic DNA was isolated using the DNeasy Blood and Tissue Kit (QIAGEN). KIR and KIR-Ligand Typing and HCMV Serology KIR ligands were determined using the KIR HLA ligand kit (Olerup SSP; QIAGEN) for detection of the HLA-Bw4, HLA-C1, and HLA-C2 motifs. KIR genotyping was performed by using quantitative KIR automated typing (qKAT) (Jiang et?al., 2012). HCMV serology was determined using an ELISA-based assay on plasma obtained during sample preparation. Purified nuclear CMV antigen (AD 169) was used, and the cut-off level for seropositivity was an absorbance of 0.2 at a dilution of 1/100. Flow Cytometry A list of fluorochrome-conjugated reagents used for stainings can be found in the Supplemental Experimental Procedures. Detailed protocols of flow cytometry staining, Stochastic neighbor embedding (SNE) analysis, functional flow cytometry.
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