Oscar A. Aguilar

Poxvirus Infection-Associated Downregulation of C-Type Lectin-Related-b Prevents NK Cell Inhibition by NK Receptor Protein-1B

Innate immune recognition of virus-infected cells includes NK cell detection of changes to endogenous cell-surface proteins through inhibitory receptors. One such receptor system is the NK cell receptor protein-1B (NKR-P1B) and its ligand C-type lectin-related-b (Clr-b). NKR-P1B and Clr-b are encoded within the NK cell gene complex, a locus that has been linked to strain-dependent differences in susceptibility to infection by poxviruses. In this study, we report the impact of vaccinia virus (VV) and ectromelia virus infection on expression of Clr-b and Clr-b–mediated protection from NK cells. We observed a loss of Clr-b cell-surface protein upon VV and ectromelia virus infection of murine cell lines and bone marrow-derived macrophages. The reduction of Clr-b is more rapid than MHC class I, the prototypic ligand of NK cell inhibitory receptors. Reduction of Clr-b requires active viral infection but not expression of late viral genes, and loss of mRNA appears to lag behind loss of Clr-b surface protein. Clr-b–mediated protection from NK cells is lost following VV infection. Together, these results provide the second example of Clr-b modulation during viral infection and suggest reductions of Clr-b may be involved in sensitizing poxvirus-infected cells to NK cells.

Analysis of the mouse 129-strain Nkrp1-Clr gene cluster reveals conservation of genomic organization and functional receptor–ligand interactions despite significant allelic polymorphism

The Nkrp1 (Klrb) family of NK cell receptors and their genetically linked Clr (Clec2) ligands are conserved between rodents and humans. Nonetheless, certain mouse and rat Nkrp1 genes exhibit significant allelic polymorphism between inbred strains. We previously demonstrated that the Nkrp1–Clr recognition system is genetically and functionally conserved between the B6 and BALB/c strains, with focused sequence divergence evident in certain genes (e.g., Nkrp1b,c). Here, we extend this finding by mapping the 129-strain Nkrp1–Clr gene cluster, which is structurally conserved yet displays significant sequence divergence relative to the B6 haplotype. In addition, we show that 129-strain NK cells possess comparable Nkrp1 and Clr transcript expression, and characterize several NKR-P1:Clr interactions that are functionally conserved between the B6 and 129 strains, including documented and novel receptor–ligand pairs. Thus, despite significant allelic polymorphism observed in the Nkrp1–Clr region, the overall genetic organization and functional repertoire appear to be conserved among mouse strains, in contrast to the striking variation observed in the corresponding Ly49 region. These data extend our knowledge of the complex genetically linked Nkrp1–Clr NK recognition system in mice.

An in vitro model of innate lymphoid cell function and differentiation.

Innate lymphoid cells (ILC) are RAG-independent lymphocytes with important roles in innate immunity, and include group-1 (natural killer (NK) cell, ILC1), group-2 (ILC2), and group-3 (lymphoid tissue inducer (LTi), NCR+ ILC3) subsets. Group-3 ILC express Rorγt, produce interleukin (IL)-22, and are critically important in the normal function of mucosal tissues. Here, we describe a novel model cell line for the study of ILC function and differentiation. The parental MNK cell line, derived from NKR-P1B+ fetal thymocytes, shows a capacity to differentiate in γc cytokines. One IL-7-responsive subline, designated MNK-3, expresses Rorγt and produces high levels of IL-22 in response to IL-23 and IL-1β stimulation. MNK-3 cells display surface markers and transcript expression characteristic of group-3 ILC, including IL-7Rα (CD127), c-kit (CD117), CCR6, Thy1 (CD90), RANK, RANKL, and lymphotoxin (LTα1β2). Using an in vitro assay of LTi cell activity, MNK-3 cells induce ICAM-1 and VCAM-1 expression on stromal cells in a manner dependent upon LTα1β2 expression. A second IL-2-responsive subline, MNK-1, expresses several NK cell receptors, perforin and granzymes, and shows some cytotoxic activity. Thus, MNK-1 cells serve as a model of ILC1/NK development and differentiation, whereas MNK-3 cells provide an attractive in vitro system to study the function of ILC3/LTi cells.

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-2Db−/− 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-P1Bhi NK cells, leaving the remaining NKR-P1Blo 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.

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.

Expansion and Protection by a Virus-Specific NK Cell Subset Lacking Expression of the Inhibitory NKR-P1B Receptor during Murine Cytomegalovirus Infection

NK cells play a major role in immune defense against human and murine CMV (MCMV) infection. Although the MCMV genome encodes for MHC class I–homologous decoy ligands for inhibitory NK cell receptors to evade detection, some mouse strains have evolved activating receptors, such as Ly49H, to recognize these ligands and initiate an immune response. In this study, we demonstrate that approximately half of the Ly49H-expressing (Ly49H+) NK cells in the spleen and liver of C57BL/6 mice also express the inhibitory NKR-P1B receptor. During MCMV infection, the NKR-P1B−Ly49H+ NK cell subset proliferates to constitute the bulk of the NK cell population. This NK cell subset also confers better protection against MCMV infection compared with the NKR-P1B+Ly49H+ subset. The two populations are composed of cells that differ in their surface expression of receptors such as Ly49C/I and NKG2A/C/E, as well as developmental markers, CD27 and CD11b, and the high-affinity IL-2R (CD25) following infection. Although the NKR-P1B+ NK cells can produce effector molecules such as IFNs and granzymes, their proliferation is inhibited during infection. A similar phenotype in MCMV-infected Clr-b–deficient mice, which lack the ligand for NKR-P1B, suggests the involvement of ligands other than the host Clr-b. Most interestingly, genetic deficiency of the NKR-P1B, but not Clr-b, results in accelerated virus clearance and recovery from MCMV infection. This study is particularly significant because the mouse NKR-P1B:Clr-b receptor:ligand system represents the closest homolog of the human NKR-P1A:LLT1 system and may have a direct relevance to human CMV infection.

A Truncated Human NKG2D Splice Isoform Negatively Regulates NKG2D-Mediated Function

Natural killer group 2, member D (NKG2D) is a stimulatory receptor expressed by NK cells and a subset of T cells. NKG2D is crucial in diverse aspects of innate and adaptive immune functions. In this study, we characterize a novel splice variant of human NKG2D that encodes a truncated receptor lacking the ligand-binding ectodomain. This truncated NKG2D (NKG2DTR) isoform was detected in primary human NK and CD8+ T cells. Overexpression of NKG2DTR severely attenuated cell killing and IFN-γ release mediated by full-length NKG2D (NKG2DFL). In contrast, specific knockdown of endogenously expressed NKG2DTR enhanced NKG2D-mediated cytotoxicity, suggesting that NKG2DTR is a negative regulator of NKG2DFL. Biochemical studies demonstrated that NKG2DTR was bound to DNAX-activated protein of 10 kDa (DAP10) and interfered with the interaction of DAP10 with NKG2DFL. In addition, NKG2DTR associated with NKG2DFL, which led to forced intracellular retention, resulting in decreased surface NKG2D expression. Taken together, these data suggest that competitive interference of NKG2D/DAP10 complexes by NKG2DTR constitutes a novel mechanism for regulation of NKG2D-mediated function in human CD8+ T cells and NK cells.

Transcriptome analysis reveals similarities between human blood CD3− CD56bright cells and mouse CD127+ innate lymphoid cells

For many years, human peripheral blood natural killer (NK) cells have been divided into functionally distinct CD3− CD56bright CD16− and CD3− CD56dim CD16+ subsets. Recently, several groups of innate lymphoid cells (ILC), distinct from NK cells in development and function, have been defined in mouse. A signature of genes present in mouse ILC except NK cells, defined by Immunological Genome Project studies, is significantly over-represented in human CD56bright cells, by gene set enrichment analysis. Conversely, the signature genes of mouse NK cells are enriched in human CD56dim cells. Correlations are based upon large differences in expression of a few key genes. CD56bright cells show preferential expression of ILC-associated IL7R (CD127), TNFSF10 (TRAIL), KIT (CD117), IL2RA (CD25), CD27, CXCR3, DPP4 (CD26), GPR183, and MHC class II transcripts and proteins. This could indicate an ontological relationship between human CD56bright cells and mouse CD127+ ILC, or conserved networks of transcriptional regulation. In line with the latter hypothesis, among transcription factors known to impact ILC or NK cell development, GATA3, TCF7 (TCF-1), AHR, SOX4, RUNX2, and ZEB1 transcript levels are higher in CD56bright cells, while IKZF3 (AIOLOS), TBX21 (T-bet), NFIL3 (E4BP4), ZEB2, PRDM1 (BLIMP1), and RORA mRNA levels are higher in CD56dim cells.

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

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.

NKR-P1B expression in gut-associated innate lymphoid cells is required for the control of gastrointestinal tract infections

Helper-type innate lymphoid cells (ILC) play an important role in intestinal homeostasis. Members of the NKR-P1 gene family are expressed in various innate immune cells, including natural killer (NK) cells, and their cognate Clr ligand family members are expressed in various specialized tissues, including the intestinal epithelium, where they may play an important role in mucosal-associated innate immune responses. In this study, we show that the inhibitory NKR-P1B receptor, but not the Ly49 receptor, is expressed in gut-resident NK cells, ILC, and a subset of γδT cells in a tissue-specific manner. ILC3 cells constitute the predominant cell subset expressing NKR-P1B in the gut lamina propria. The known NKR-P1B ligand Clr-b is broadly expressed in gut-associated cells of hematopoietic origin. The genetic deletion of NKR-P1B results in a higher frequency and number of ILC3 and γδT cells in the gut lamina propria. However, the function of gut-resident ILC3, NK, and γδT cells in NKR-P1B-deficient mice is impaired during gastrointestinal tract infection by Citrobacter rodentium or Salmonella typhimurium, resulting in increased systemic bacterial dissemination in NKR-P1B-deficient mice. Our findings highlight the role of the NKR-P1B:Clr-b recognition system in the modulation of intestinal innate immune cell functions.