Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition

Abstract

Natural killer (NK) cells are innate lymphocytes that aid in self-nonself discrimination by recognizing cells undergoing pathological alterations. The NKR-P1B inhibitory receptor recognizes Clr-b, a self-encoded marker of cell health downregulated during viral infection. Here, we show that Clr-b loss during mouse cytomegalovirus (MCMV) infection is predicated by a loss of Clr-b (Clec2d) promoter activity and nascent transcripts, driven in part by MCMV ie3 (M122) activity. In contrast, uninfected bystander cells near MCMV-infected fibroblasts reciprocally upregulate Clr-b expression due to paracrine type-I interferon (IFN) signaling. Exposure of fibroblasts to type-I IFN augments Clec2d promoter activity and nascent Clr-b transcripts, dependent upon a cluster of IRF3/7/9 motifs located ∼200 bp upstream of the transcriptional start site. Cells deficient in type-I IFN signaling components revealed IRF9 and STAT1 as key transcription factors involved in Clr-b upregulation. In chromatin immunoprecipitation experiments, the Clec2d IRF cluster recruited STAT2 upon IFN-α exposure, confirming the involvement of ISGF3 (IRF9/STAT1/STAT2) in positively regulating the Clec2d promoter. These findings demonstrate that Clr-b is an IFN-stimulated gene on healthy bystander cells, in addition to a missing-self marker on MCMV-infected cells, and thereby enhances the dynamic range of innate self-nonself discrimination by NK cells.

The Inhibitory NKR-P1B:Clr-b Recognition Axis Facilitates Detection of Oncogenic Transformation and Cancer Immunosurveillance

“…Hence, oncogenic Ras signaling downregulates Clr-b transcripts in a manner consistent with that previously observed following viral infection or genotoxic stress (9,(11)(12)(13)(14), perhaps indicative of an oncogenic stress pathway. To assess this effect at the Clec2d promoter level, a dualluciferase reporter assay (DLRA) was performed using the pGL3 vector harboring incremental fragments of the Clec2d promoter (100-500 bp upstream of transcriptional start site, TSS), and luciferase activity was measured relative to control Renilla luciferase activity in the presence of Ras G12V or Ras S17N cotransfection into NIH3T3 fibroblasts (12). Here, the activity of the 200 bp Clec2d fragment was significantly decreased by CA Ras G12V cotransfection relative to DN Ras S17N (Fig.…”

“…It was recently shown that Clr-b (Clec2d) promoter activity, nascent transcripts, and surface protein expression are downregulated in response to viral infection (9,(11)(12)(13), and that Clr-b transcripts are lost in response to genotoxic/cellular stress Figure 3. Ras G12V -mediated Clr-b loss requires MEK1, MEK2, and ERK2.…”

“…More recently, the generation of Clr-b -/and NKR-P1B -/mice have revealed a role for this system in hematopoietic transplants, where NKR-P1B: Clr-b interactions function to inhibit NK-cell-mediated rejection of healthy syngeneic and allogeneic cells (8,15). On the other hand, cytomegaloviruses have been shown to encode novel decoy or Clr-like surrogate ligands used to evade innate immune detection (9,(11)(12)(13). Moreover, enforced maintenance of Clr-b ligand levels during tumor development has been postulated to play a role in immunoediting and immune escape of malignant cells in the Em-cMyc spontaneous lymphoma model, whereby this selective pressure is mitigated in Nkrp1b -/receptor-deficient mice (15).…”

Herpesvirus Evasion of Natural Killer Cells

“…Particularly, the broadly expressed self Clr-b is recognized by the inhibitory NKR-P1B (115). MCMV infection leads to loss of Clr-b gene expression, in part by the activity of the viral m122 protein, which would be expected to yield an NK cell-activating signal (116,117). Recently, however, it was shown that cell surface-expressed m12 directly engages NKR-P1B, thereby suppressing NK cell effector activity in vitro and in vivo (118).…”

Catch Me if You Can or Actors on the Run

“…This process seems to engage an involvement of the interferons IFN-β and IFN-λs, respectively. Within the last years, Journal of Immunity has published many articles involving interferon signaling [9-13], emphasizing the important role of these inflammatory immune mediators in fighting viral infections. Future work will hopefully bring us the answer whether IFN-λs can be used as therapeutic target for the treatment of HIV infection.…”