BACKGROUND/AIM: Spontaneous regression (SR) of tumours is a rare phenomenon not yet fully understood. The aim of this study was to investigate immune cells infiltrating progressive and SR tumours in a Lewis rat sarcoma model. MATERIALS AND METHODS: Rats were subcutaneously inoculated with rat sarcoma R5-28 (clone C4) cells. Developing tumours were obtained on day 42 and cryosections were immunohistochemically processed for detection of immune cells. RESULTS: A high density of granulocytes was found in the necrotic areas of both progressive and SR tumours. CD4+ cells and CD8+ cells were rare and sparsely dispersed in the tumour tissue without clear difference between the two types of tumours. On the contrary, CD161+ cells were abundant and evenly distributed in SR tumours, but these cells were very rare in progressive tumours. CONCLUSION: Based on the differences in number and distribution of the immune cell subpopulations, we believe that natural killer (CD161+) cells play a major role in the destruction of cancer cells during SR of tumours in this Lewis rat model.

• Keywords
• CD161, CD4, CD8, Tumour, immunohistochemistry, spontaneous regression,
• MeSH
• Killer Cells, Natural pathology   MeSH
• CD4-Positive T-Lymphocytes pathology   MeSH
• CD8-Positive T-Lymphocytes pathology   MeSH
• Immunohistochemistry MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B genetics   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Rats, Inbred Lew MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Sarcoma genetics   pathology   MeSH
• Neoplasm Regression, Spontaneous genetics   pathology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NK Cell Lectin-Like Receptor Subfamily B MeSH

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

• Keywords
• CD8+CD45RA+CD27+CD161+ T cells, Seurat, T cells, cord blood mononuclear cells (CBMCs), high‐dimensional flow cytometry, peripheral blood mononuclear cells (PBMCs),
• MeSH
• Single-Cell Analysis methods   MeSH
• Tumor Necrosis Factor Receptor Superfamily, Member 7 metabolism   immunology   MeSH
• Leukocyte Common Antigens * metabolism   immunology   MeSH
• CD8-Positive T-Lymphocytes * immunology   MeSH
• Adult MeSH
• Fetal Blood * immunology   cytology   MeSH
• Immunophenotyping methods   MeSH
• NK Cell Lectin-Like Receptor Subfamily B immunology   metabolism   MeSH
• Humans MeSH
• Flow Cytometry * methods   MeSH
• Software MeSH
• T-Lymphocyte Subsets immunology   metabolism   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Necrosis Factor Receptor Superfamily, Member 7 MeSH
• Leukocyte Common Antigens * MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH

We investigated the role of natural killer (NK) cells and CD161, their primary C-type-lectin-like receptor in rheumatoid arthritis (RA). Samples were compared with healthy donors (HD), dermatomyositic (DM), polymyositic (PM), and osteoarthritic (OA) patients. RA, PM, and DM NK cell cytotoxicities significantly decreased relative to the HD and OA NK cells (p<0.0001). These results correlated with an increased expression of NK cell inhibitory receptor CD161, in active disease RA patients. We demonstrated that NK cells are able to respond to mutated citrullinated vimentin (MCV), an RA-specific autoantigen, leading to increases in both PAD4 enzyme and CD161 mRNA expression. MGAT5 glycosidase involvement was detected in GlcNAc metabolism within the synoviocytes of RA patients. Our findings reveal a functional relationship between CD161 expression and NK cell cytotoxicity as well as reactivity to glycans and MCV, thus providing new insight into the pathogenesis of RA and confirming the involvement of surface glycosylation.

• MeSH
• Autoantigens pharmacology   MeSH
• Autoimmune Diseases immunology   MeSH
• Autoantibodies blood   immunology   MeSH
• Killer Cells, Natural cytology   drug effects   immunology   metabolism   MeSH
• Cytotoxicity, Immunologic drug effects   immunology   MeSH
• Dermatomyositis immunology   MeSH
• Adult MeSH
• Gene Expression drug effects   genetics   MeSH
• Glucocorticoids therapeutic use   MeSH
• Glycoconjugates pharmacology   MeSH
• Hydrolases genetics   MeSH
• Immunosuppressive Agents therapeutic use   MeSH
• NK Cell Lectin-Like Receptor Subfamily B agonists   genetics   metabolism   MeSH
• Leukocytes, Mononuclear cytology   drug effects   immunology   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• N-Acetylglucosaminyltransferases genetics   MeSH
• Osteoarthritis immunology   MeSH
• Protein-Arginine Deiminase Type 4 MeSH
• Protein-Arginine Deiminases MeSH
• Cell Count MeSH
• Polymyositis immunology   MeSH
• Polysaccharides pharmacology   MeSH
• Arthritis, Rheumatoid diagnosis   drug therapy   immunology   MeSH
• Aged MeSH
• Synovial Fluid cytology   metabolism   MeSH
• Vimentin pharmacology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase MeSH Browser
• Autoantigens MeSH
• Autoantibodies MeSH
• Glucocorticoids MeSH
• Glycoconjugates MeSH
• Hydrolases MeSH
• Immunosuppressive Agents MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• N-Acetylglucosaminyltransferases MeSH
• PADI4 protein, human MeSH Browser
• Protein-Arginine Deiminase Type 4 MeSH
• Protein-Arginine Deiminases MeSH
• Polysaccharides MeSH
• Vimentin MeSH

Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor; and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFNγ. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches. We tested effect of different protein tags (SUMO, TRX, GST, MsyB) on expression of soluble NKR-P1 in E. coli. Then we optimized the expression construct of soluble NKR-P1 by preparing a library of expression constructs in pOPING vector containing the extracellular lectin-like domain with different length of the putative N-terminal stalk region and tested its expression in Sf9 and HEK293 cells. Finally, a high-level expression of soluble NKR-P1 was achieved by stable expression in suspension-adapted HEK293S GnTI- cells utilizing pOPINGTTneo expression vector. Purified soluble NKR-P1 is homogeneous, deglycosylatable, crystallizable and monomeric in solution, as shown by size-exclusion chromatography, multi-angle light scattering and analytical ultracentrifugation.

• Keywords
• CD161, HEK293, LLT1, NK cells, NKR-P1, klrb1,
• MeSH
• Bioreactors MeSH
• Killer Cells, Natural metabolism   MeSH
• Th17 Cells metabolism   MeSH
• Escherichia coli genetics   MeSH
• HEK293 Cells MeSH
• NK Cell Lectin-Like Receptor Subfamily B biosynthesis   genetics   isolation & purification   MeSH
• Lectins, C-Type metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Receptors, Cell Surface metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CLEC2D protein, human MeSH Browser
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Ligands MeSH
• Receptors, Cell Surface MeSH

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5 glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

• Keywords
• C-type lectin-like ligand, LLT1,
• MeSH
• Glycosylation MeSH
• Protein Structure, Quaternary MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   genetics   metabolism   MeSH
• Lectins, C-Type chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Protein Multimerization * MeSH
• Receptors, Cell Surface chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CLEC2D protein, human MeSH Browser
• KLRB1 protein, human MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Receptors, Cell Surface MeSH

Natural killer (NK) and T cells express a superfamily of proteins with structural features of C-type lectins. Recombinantly prepared soluble form of rat NKR-P1 (CD161) recognized carbohydrate GalNac and GlcNac moieties. Ganglioside GM2 and heparin related-IS oligosaccharides representing the high affinity ligands for this receptor, increased the sensitivity of targets for killing by the rat effectors isolated from blood and spleen in vitro. Based on these results, we investigated in vivo the possible therapeutic effect of GM2 and IS carried by liposomes during induced rat colorectal carcinogenesis. The reduction of cancer incidence versus the controls (50% vs 88.88%), approached the 5-fluorouracil treatment (41.66%).

• MeSH
• Antigens, Surface * drug effects   MeSH
• Antimetabolites, Antineoplastic therapeutic use   MeSH
• Azoxymethane MeSH
• Escherichia coli MeSH
• Fluorouracil therapeutic use   MeSH
• G(M2) Ganglioside therapeutic use   MeSH
• Carcinogens MeSH
• Colorectal Neoplasms chemically induced   drug therapy   MeSH
• Rats MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type * MeSH
• Drug Screening Assays, Antitumor MeSH
• Liposomes MeSH
• Drug Carriers MeSH
• Oligosaccharides therapeutic use   MeSH
• Rats, Sprague-Dawley MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface * MeSH
• Antimetabolites, Antineoplastic MeSH
• Azoxymethane MeSH
• Fluorouracil MeSH
• G(M2) Ganglioside MeSH
• Carcinogens MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type * MeSH
• Liposomes MeSH
• Drug Carriers MeSH
• Oligosaccharides MeSH

The structure of the H107R variant of the extracellular domain of the mouse natural killer cell receptor NKR-P1A has been determined by X-ray diffraction at 2.3 Å resolution from a merohedrally twinned crystal. Unlike the structure of the wild-type receptor in space group I4(1)22 with a single chain per asymmetric unit, the crystals of the variant belonged to space group I4(1) with a dimer in the asymmetric unit. Different degrees of merohedral twinning were detected in five data sets collected from different crystals. The mutation does not have a significant impact on the overall structure, but led to the binding of an additional phosphate ion at the interface of the molecules.

• MeSH
• Extracellular Space chemistry   MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   genetics   MeSH
• Models, Molecular MeSH
• Mutation * MeSH
• Mice MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NK Cell Lectin-Like Receptor Subfamily B MeSH

The cytotoxicity of mouse natural killer (NK) cells in response to pathological changes in target cells is regulated via the Nkrp1b receptor. Here, we characterized the Nkrp1b structure and structural features (stalk, loop, and oligomerization state) that affect its interactions. To study the Nkrp1b protein structure and the functional importance of its stalk, two Nkrp1b protein variants differing by the presence of the stalk were prepared. These variants were studied using a combination of structural mass spectrometry approaches with computational modeling to derive structural models. In addition, information about biological activity and localization in mammalian cells was acquired using scanning microscopy techniques and western blotting. Based on these methods, we obtained the structure of Nkrp1b ectodomain in its monomeric and dimeric conformations, identified the dimerization interface, and determined disulfide connections within the molecule. We found that Nkrp1b occurs as a mixture of monomers and homodimers, both in vitro and in vivo. SIGNIFICANCE: Despite the long-standing assumption that Nkrp1 proteins are homodimers connected by disulfide bonds in the stalk region, our data showed that both Nkrp1b protein variants form monomers and homodimers irrespective of the presence of the stalk. We demonstrated that the stalk is not crucial for protein dimerization or ligand binding and that Nkrp1b interacts with its natural ligands only in its monomeric conformation; therefore, dimers may have another regulatory function. Using a unique combination of computational, biochemical, and biological methods, we revealed the structural conformation and behavior of Nkrp1b in its native state. In addition, it is a first report utilizing the intermolecular chemical cross-linking of light- and heavy-labeled protein chains together with ion mobility-mass spectrometry to design the structural models of protein homodimers.

• Keywords
• Chemical cross-linking, Homodimers, Ion mobility, Natural killer cells, Nkrp1b, Structural mass spectrometry,
• MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   metabolism   MeSH
• Models, Molecular * MeSH
• Protein Multimerization * MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Proteomics * MeSH
• Protein Structure, Secondary MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NK Cell Lectin-Like Receptor Subfamily B MeSH

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.

• MeSH
• Killer Cells, Natural metabolism   MeSH
• X-Ray Diffraction MeSH
• NK Cell Lectin-Like Receptor Subfamily B chemistry   metabolism   MeSH
• Molecular Sequence Data MeSH
• Mice MeSH
• Spectrum Analysis, Raman MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NK Cell Lectin-Like Receptor Subfamily B MeSH

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

• MeSH
• Antigens, Surface MeSH
• Killer Cells, Natural * MeSH
• X-Ray Diffraction MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Humans MeSH
• Ligands MeSH
• Scattering, Small Angle MeSH
• Receptors, Cell Surface * MeSH
• Cluster Analysis MeSH
• Synapses MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Lectins, C-Type MeSH
• Ligands MeSH
• Receptors, Cell Surface * MeSH