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Abundant CpG-sequences in human genomes inhibit KIR3DL2-expressing NK cells

ABSTRACT: Killer Immunoglobulin-like Receptors (KIR) comprise a diverse, highly polymorphic family of cell-surface glycoproteins that are principally expressed by Natural Killer (NK) cells. These innate immune lymphocytes fulfill vital functions in human reproduction and immune responses to viral infection. KIR3DL2 is an inhibitory NK cell receptor that recognizes a common epitope of the HLA-A3 and HLA-A11 class I glycoproteins of the major histocompatibility complex. KIR3DL2 also binds exogenous DNA containing the CpG motif. This interaction causes internalization of the KIR-DNA. Exogenous CpG-DNA typically activates NK cells, but the specificity of KIR3DL2-DNA binding and internalization is unclear. We hypothesized that KIR3DL2 binds exogenous DNA in a sequence-specific manner that differentiates pathogen DNA from self-DNA. In testing this hypothesis, we surveyed octameric CpG-DNA sequences in the human genome, and in reference genomes of all bacteria, fungi, viruses, and parasites, with focus on medically relevant species. Among all pathogens, the nucleotides flanking CpG motifs in the genomes of parasitic worms that infect humans are most divergent from those in the human genome. We cultured KIR3DL2+NKL cells with the commonest CpG-DNA sequences in either human or pathogen genomes. DNA uptake was negatively correlated with the most common CpG-DNA sequences in the human genome. These CpG-DNA sequences induced inhibitory signaling in KIR3DL2+NKL cells. In contrast, KIR3DL2+NKL cells lysed more malignant targets and produced more IFNγ after culture with CpG-DNA sequences prevalent in parasitic worms. By applying functional immunology to evolutionary genomics, we conclude that KIR3DL2 allows NK cells to differentiate self-DNA from pathogen DNA.

SUBMITTER: Pugh J

PROVIDER: S-EPMC8574216 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells ex vivo using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction. Methods: K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4-5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells. Results: The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation. Conclusion: Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.

Project description:BACKGROUND: The Human Papillomavirus (HPV) genome is divided into early and late coding sequences, including 8 open reading frames (ORFs) and a regulatory region (LCR). Viral gene expression may be regulated through epigenetic mechanisms, including cytosine methylation at CpG dinucleotides. We have analyzed the distribution of CpG sites and CpG islands/clusters (CGI) among 92 different HPV genomes grouped in function of their preferential tropism: cutaneous or mucosal. We calculated the proportion of CpG sites (PCS) for each ORF and calculated the expected CpG values for each viral type. RESULTS: CpGs are underrepresented in viral genomes. We found a positive correlation between CpG observed and expected values, with mucosal high-risk (HR) virus types showing the smallest O/E ratios. The ranges of the PCS were similar for most genomic regions except E4, where the majority of CpGs are found within islands/clusters. At least one CGI belongs to each E2/E4 region. We found positive correlations between PCS for each viral ORF when compared with the others, except for the LCR against four ORFs and E6 against three other ORFs. The distribution of CpG islands/clusters among HPV groups is heterogeneous and mucosal HR-HPV types exhibit both lower number and shorter island sizes compared to cutaneous and mucosal Low-risk (LR) HPVs (all of them significantly different). CONCLUSIONS: There is a difference between viral and cellular CpG underrepresentation. There are significant correlations between complete genome PCS and a lack of correlations between several genomic region pairs, especially those involving LCR and E6. L2 and L1 ORF behavior is opposite to that of oncogenes E6 and E7. The first pair possesses relatively low numbers of CpG sites clustered in CGIs while the oncogenes possess a relatively high number of CpG sites not associated to CGIs. In all HPVs, E2/E4 is the only region with at least one CGI and shows a higher content of CpG sites in every HPV type with an identified E4. The mucosal HR-HPVs show either the shortest CGI size, followed by the mucosal LR-HPVs and lastly by the cutaneous viral subgroup, and a trend to the lowest CGI number, followed by the cutaneous viral subgroup and lastly by the mucosal LR-HPVs.

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Abundant CpG-sequences in human genomes inhibit KIR3DL2-expressing NK cells

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