Modulation of Clr Ligand Expression and NKR-P1 Receptor Function during Murine Cytomegalovirus Infection.

Viruses are known to induce pathological cellular states that render infected cells susceptible or resistant to immune recognition. Here, we characterize an MHC-I-independent natural killer (NK) cell recognition mechanism that involves modulation of inhibitory NKR-P1B:Clr-b receptor-ligand interactions in response to mouse cytomegalovirus (MCMV) infection. We demonstrate that mouse Clr-b expression on healthy cells is rapidly lost at the cell surface and transcript levels in a time- and dose-dependent manner upon MCMV infection. In addition, cross-species infections using rat cytomegalovirus (RCMV) infection of mouse fibroblasts and MCMV infection of rat fibroblasts suggest that this response is conserved during host-pathogen interactions. Active viral infection appears to be necessary for Clr-b loss, as cellular stimulation using UV-inactivated whole virus or agonists of many innate pattern recognition receptors failed to elicit efficient Clr-b downregulation. Notably, Clr-b loss could be partially blocked by titrated cycloheximide treatment, suggesting that early viral or nascent host proteins are required for Clr-b downregulation. Interestingly, reporter cell assays suggest that MCMV may encode a novel Clr-b-independent immunoevasin that functionally engages the NKR-P1B receptor. Together, these data suggest that Clr-b modulation is a conserved innate host cell response to virus infection that is subverted by multiple CMV immune evasion strategies.

Genetic investigation of MHC-independent missing-self recognition by mouse NK cells using an in vivo bone marrow transplantation model.

MHC-I-specific receptors play a vital role in NK cell-mediated "missing-self" recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b(-/-) bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2D(b-/-) MHC-I-deficient BM cells. Selective rejection of Clr-b(-/-) BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b(-/-) BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1B(hi) NK cells, leaving the remaining NKR-P1B(lo) NK subset and MHC-I-dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b(-/-) hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I-deficient NK cells, Clr-b(-/-) NK cells were hyporesponsive to both NK1.1 (NKR-P1C)-stimulated and IL-12/18 cytokine-primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.

Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.