NK Cell Receptor NKp46 Regulates Graft-versus-Host Disease

SummaryHematopoietic stem cell transplantation (HSCT) is often the only curative treatment for a wide variety of hematologic malignancies. Donor selection in these diseases is crucial, given that transplanted cells can mediate not only the desired graft-versus-leukemia effect but also graft-versus-host disease (GVHD). Here, we demonstrate that in the absence of NKp46, a major killer receptor expressed by human and mouse natural killer (NK) cells, GVHD is greatly exacerbated, resulting in rapid mortality of the transplanted animals because of infection with commensal bacteria. Furthermore, we demonstrate that the exacerbated GVHD is the result of an altered ability of immune cells to respond to stimulation by immature dendritic cells. Because high and low expression of NKp46 on NK cells is observed in different individuals, our data indicate that choosing NKp46-high donors for the treatment of different hematologic malignancies might lead to better tumor eradication while minimizing GVHD

Direct Recognition of Fusobacterium nucleatum by the NK Cell Natural Cytotoxicity Receptor NKp46 Aggravates Periodontal Disease

Periodontitis is a common human chronic inflammatory disease that results in the destruction of the tooth attachment apparatus and tooth loss. Although infections with periopathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are essential for inducing periodontitis, the nature and magnitude of the disease is determined by the host's immune response. Here, we investigate the role played by the NK killer receptor NKp46 (NCR1 in mice), in the pathogenesis of periodontitis. Using an oral infection periodontitis model we demonstrate that following F. nucleatum infection no alveolar bone loss is observed in mice deficient for NCR1 expression, whereas around 20% bone loss is observed in wild type mice and in mice infected with P. gingivalis. By using subcutaneous chambers inoculated with F. nucleatum we demonstrate that immune cells, including NK cells, rapidly accumulate in the chambers and that this leads to a fast and transient, NCR1-dependant TNF-α secretion. We further show that both the mouse NCR1 and the human NKp46 bind directly to F. nucleatum and we demonstrate that this binding is sensitive to heat, to proteinase K and to pronase treatments. Finally, we show in vitro that the interaction of NK cells with F. nucleatum leads to an NCR1-dependent secretion of TNF-α. Thus, the present study provides the first evidence that NCR1 and NKp46 directly recognize a periodontal pathogen and that this interaction influences the outcome of F. nucleatum-mediated periodontitis

HSV-2 specifically down regulates HLA-C expression to render HSV-2-infected DCs susceptible to NK cell killing.

Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity

HSV-2 specifically down regulates HLA-C expression.

<p>(A) 721.221 (221) cells expressing various HLA proteins (indicated in the histograms) were infected with HSV-2 (MOI of 0.5) and the levels of the various HLA proteins expression were determined by FACS (black empty histograms represents the staining of infected cells). (B) Two clinical isolates of HSV-2 (HSV2 Iso 2 and HSV2 Iso 3) and one clinical isolate of HSV-1 were used to infect the various 721.221 transfectants indicated in the figures (MOI of 0.5 for all infections) and the levels of the HLA expression were measured FACS (black empty histograms represent the infected cell staining). (C) 221 expressing HLA-B73 or HLA-Cw6 cells were infected with HSV-2 (MOI of 1) and HLA expression was assessed right at the end of the infection, and at the indicated time points post infection (pi). In A–C the dark gray shaded histograms represent staining of the corresponding uninfected cells and the background levels (light grey shaded histogram) are the APC-conjugated Abs. For all panels, one representative experiment is shown out of at least three performed. (D) Quantification of all experiments performed in (A). The expression of each HLA allele without infection was set up to be 100%. Shown are relative average MFI ± S.D. from four to eight independent experiments.</p

The KIR2DL1-mediated inhibition is abolished in HSV-2 infected cells.

<p>(A) Top histograms: 221 cells expressing either HLA-B8 or HLA-Cw6 were infected with HSV-2 (MOI of 0.5). The levels of HLAs expression were determined by FACS (black empty histograms represent infected cells). The dark gray shaded histograms represent staining of the corresponding uninfected cells. Background levels (light grey shaded histogram) are the APC-conjugated Abs. Lower histogram: Expression of KIR2DL1 (black empty histograms) was detected with anti-KIR2DL1 antibody. Background levels (grey shaded histogram) are the APC-conjugated Abs. (B) Killing assay. 221 and 221 expressing either HLA-B8 or HLA-Cw6 were infected with HSV-2 (MOI of 0.5). Uninfected and infected (indicated in the figure as HSV2) 221 cells were radioactively labeled and incubated with primary KIR2DL1-positive NK clones for 5 hours at an E:T ratio of 10∶1. The killing assays were performed 48 hours post infection and the NK clone was incubated either with a control mAb (Ctrl, grey columns) or with an anti-KIR2DL1 blocking antibody (white columns). Shown are mean values ± SD. Statistically significant differences are indicated (_* p≤0.0005, by one-tailed t test). Error bars (SD) are derived from triplicates. For all panels, one representative experiment is shown out of at least three performed.</p

The ICP47 protein of HSV-2 reduces HLA-C expression.

<p>Various 221 cells expressing wild type (A) and mutants (B) HLA proteins (indicated in the figure) were transduced with lenti virus vectors expressing ICP47 and GFP (indicative for the transfection efficiency). The levels of the various HLAs were determined by FACS (black empty histograms represent the ICP47 transduced cells). The dark gray shaded histograms represent staining of the corresponding cells transduced with a control lenti virus vector. Background levels (light grey shaded histogram) are the APC-conjugated Abs. Figure show one representative experiment out of 3 performed.</p

HSV-2 specifically downregulates MHC-I expression.

<p>Time course for the expression of various NK cell ligands following HSV-2 infection of HeLa cells (MOI of 0.1). The expression levels of NK cells ligands (indicated on top of the histograms) were determined by staining with specific antibodies. Expression following infection is indicated by the black empty histograms. The dark gray shaded histograms represent staining of the corresponding uninfected cells. Background levels (light grey shaded histogram) are the secondary FITC-conjugated Abs staining. One representative experiment is shown out of three performed.</p