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1.
Eur J Immunol ; 50(5): 656-665, 2020 05.
Article in English | MEDLINE | ID: mdl-32027754

ABSTRACT

Natural Killer (NK) cell responses are regulated by a variety of different surface receptors. While we can determine the overall positive or negative effect of a given receptor on NK cell functions, investigating NK cell regulation in a quantitative way is challenging. To quantitatively investigate individual receptors for their effect on NK cell activation, we chose to functionalize latex beads that have approximately the same size as lymphocytes with defined amounts of specific antibodies directed against distinct activating receptors. This enabled us to investigate NK cell reactivity in a defined, clean, and controllable system. Only CD16 and NKp30 could activate the degranulation of resting human NK cells. CD16, NKG2D, NKp30, NKp44, and NKp46 were able to activate cultured NK cells. NK cell activation resulted in the induction of polyfunctional cells that degranulated and produced IFN-γ and MIP-1ß. Interestingly, polyfunctional NK cells were only induced by triggering ITAM-coupled receptors. NKp44 showed a very sensitive response pattern, where a small increase in receptor stimulation caused maximal NK cell activity. In contrast, stimulation of 2B4 induced very little NK cell degranulation, while providing sufficient signal for NK cell adhesion. Our data demonstrate that activating receptors differ in their effectiveness to stimulate NK cells.


Subject(s)
Antibodies/pharmacology , Gene Expression Regulation/drug effects , Killer Cells, Natural/drug effects , Lymphocyte Activation/drug effects , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Antibodies/chemistry , Cell Adhesion/drug effects , Cell Degranulation/drug effects , Cells, Cultured , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Microspheres , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/immunology , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 1/immunology , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 3/genetics , Natural Cytotoxicity Triggering Receptor 3/immunology , Protein Binding , Receptors, IgG/genetics , Receptors, IgG/immunology , Signal Transduction
2.
Front Immunol ; 10: 909, 2019.
Article in English | MEDLINE | ID: mdl-31134055

ABSTRACT

The Natural Cytotoxicity Receptors (NCRs), NKp46, NKp44, and NKp30, were some of the first human activating Natural Killer (NK) cell receptors involved in the non-MHC-restricted recognition of tumor cells to be cloned over 20 years ago. Since this time many host- and pathogen-encoded ligands have been proposed to bind the NCRs and regulate the cytotoxic and cytokine-secreting functions of tissue NK cells. This diverse set of NCR ligands can manifest on the surface of tumor or virus-infected cells or can be secreted extracellularly, suggesting a remarkable NCR polyfunctionality that regulates the activity of NK cells in different tissue compartments during steady state or inflammation. Moreover, the NCRs can also be expressed by other innate and adaptive immune cell subsets under certain tissue conditions potentially conferring NK recognition programs to these cells. Here we review NCR biology in health and disease with particular reference to how this important class of receptors regulates the functions of tissue NK cells as well as confer NK cell recognition patterns to other innate and adaptive lymphocyte subsets. Finally, we highlight how NCR biology is being harnessed for novel therapeutic interventions particularly for enhanced tumor surveillance.


Subject(s)
Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 1/immunology , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 3/immunology , Neoplasms/immunology , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Cytotoxicity, Immunologic/genetics , Cytotoxicity, Immunologic/immunology , Humans , Killer Cells, Natural/metabolism , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 1/metabolism , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/metabolism , Natural Cytotoxicity Triggering Receptor 3/genetics , Natural Cytotoxicity Triggering Receptor 3/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism
3.
J Autoimmun ; 93: 139-144, 2018 09.
Article in English | MEDLINE | ID: mdl-30055825

ABSTRACT

Innate lymphoid cells (ILCs) have been characterized as innate immune cells capable to modulate the immune response in the mucosae. Human ILCs have been rarely described in secondary lymphoid organs except in tonsils. Moreover, their function and phenotype in human secondary lymphoid organs during autoimmune diseases have never been studied. We took advantage of splenectomy as a treatment of immune thrombocytopenia (ITP) to describe and compare splenic ILC from 18 ITP patients to 11 controls. We first confirmed that ILC3 represented the most abundant ILC subset in human non-inflamed spleens, accounting for 90% of total ILC, and that they were mostly constituted of NKp44- cells. On the contrary, proportions of ILC1 and ILC2 in spleens were lower than in blood. Splenic IL-2- and IFN-γ-producing ILC1 were increased in ITP. While the frequencies of total splenic ILC3 were similar in the two groups, splenic GM-CSF-producing ILC3 were increased in ITP. This is the first description of human ILC in a major secondary lymphoid organ during an autoimmune disease, ITP. We observed an expansion of splenic ILC1 that could participate to the Th1 skewing, while the increased production of GM-CSF by splenic ILC3 could stimulate splenic macrophages which play a key role in ITP pathophysiology.


Subject(s)
Lymphocytes/immunology , Purpura, Thrombocytopenic, Idiopathic/immunology , Spleen/immunology , Splenectomy , Adult , Case-Control Studies , Cell Differentiation , Female , Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Humans , Immunity, Innate , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/immunology , Lymphocyte Count , Lymphocytes/pathology , Macrophages/immunology , Macrophages/pathology , Male , Middle Aged , Natural Cytotoxicity Triggering Receptor 2/deficiency , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Purpura, Thrombocytopenic, Idiopathic/pathology , Purpura, Thrombocytopenic, Idiopathic/surgery , Spleen/pathology , Spleen/surgery
4.
J Gen Virol ; 99(6): 763-767, 2018 06.
Article in English | MEDLINE | ID: mdl-29683419

ABSTRACT

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.


Subject(s)
HN Protein/chemistry , Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 1/metabolism , Natural Cytotoxicity Triggering Receptor 2/metabolism , Parainfluenza Virus 3, Human/chemistry , T-Lymphocytes/cytology , Cell Proliferation , Cells, Cultured , Gene Expression Regulation , HN Protein/genetics , Humans , Lipopolysaccharide Receptors/metabolism , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 2/genetics , Parainfluenza Virus 3, Human/genetics , Receptors, Natural Killer Cell/genetics , Receptors, Natural Killer Cell/metabolism , T-Lymphocytes/immunology
5.
Eur J Immunol ; 47(8): 1280-1294, 2017 08.
Article in English | MEDLINE | ID: mdl-28613415

ABSTRACT

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44- ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.


Subject(s)
Liver Cirrhosis/immunology , Liver/cytology , Liver/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Cytokines/immunology , Cytokines/metabolism , Fetus/immunology , Hepatic Stellate Cells/immunology , Hepatocytes/immunology , Humans , Immunity, Innate , Interleukin-13/immunology , Interleukin-13/metabolism , Interleukin-33/genetics , Interleukin-33/immunology , Interleukin-33/metabolism , Kupffer Cells/immunology , Liver/embryology , Liver/pathology , Lymphocytes/classification , Natural Cytotoxicity Triggering Receptor 2/deficiency , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Toll-Like Receptor 3/immunology , Toll-Like Receptor 3/metabolism , Thymic Stromal Lymphopoietin
6.
PLoS One ; 12(2): e0172625, 2017.
Article in English | MEDLINE | ID: mdl-28235099

ABSTRACT

West Nile virus (WNV) typically leads to asymptomatic infection but can cause severe neuroinvasive disease or death, particularly in the elderly. Innate NK cells play a critical role in antiviral defenses, yet their role in human WNV infection is poorly defined. Here we demonstrate that NK cells mount a robust, polyfunctional response to WNV characterized by cytolytic activity, cytokine and chemokine secretion. This is associated with downregulation of activating NK cell receptors and upregulation of NK cell activating ligands for NKG2D. The NK cell response did not differ between young and old WNV-naïve subjects, but a history of symptomatic infection is associated with more IFN-γ producing NK cell subsets and a significant decline in a specific NK cell subset. This NK repertoire skewing could either contribute to or follow heightened immune pathogenesis from WNV infection, and suggests that NK cells could play an important role in WNV infection in humans.


Subject(s)
Immunity, Innate , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily K/immunology , West Nile Fever/immunology , West Nile virus/immunology , Adult , Age Factors , Aged , Aged, 80 and over , Antigens, CD/genetics , Antigens, CD/immunology , Asymptomatic Diseases , Female , Gene Expression Regulation , Humans , Immunophenotyping , Interferon-gamma/genetics , Interferon-gamma/immunology , Killer Cells, Natural/virology , Lymphocyte Activation , Lymphocyte Count , Middle Aged , NK Cell Lectin-Like Receptor Subfamily C/genetics , NK Cell Lectin-Like Receptor Subfamily C/immunology , NK Cell Lectin-Like Receptor Subfamily K/genetics , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 1/immunology , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 3/genetics , Natural Cytotoxicity Triggering Receptor 3/immunology , Primary Cell Culture , Severity of Illness Index , West Nile Fever/diagnosis , West Nile Fever/genetics , West Nile Fever/virology , West Nile virus/growth & development
7.
Oncotarget ; 7(43): 70912-70923, 2016 Oct 25.
Article in English | MEDLINE | ID: mdl-27765926

ABSTRACT

NKp44 and NKp30 splice variant profiles have been shown to promote diverse cellular functions. Moreover, microenvironment factors such as TGF-ß, IL-15 and IL-18 are able to influence both NKp44 and NKp30 splice variant profiles, leading to cytokine-associated profiles. Placenta and cancerous tissues have many similarities; both are immunologically privileged sites and both share immune tolerance mechanisms to support tissue development. Therefore, we studied the profiles of NKp44 and NKp30 splice variants in these states by comparing (i) decidua from pregnancy disorder and healthy gestation and (ii) matched normal and cancer tissue. Decidua samples had high incidence of both NKp44 and NKp30. In cancerous state it was different; while NKp30 expression was evident in most cancerous and matched normal tissues, NKp44 incidence was lower and was mostly associated with the cancerous tissues. A NKp44-1dominant inhibitory profile predominated in healthy pregnancy gestation. Interestingly, the NKp44-2/3 activation profile becomes the leading profile in spontaneous abortions, whereas balanced NKp44 profiles were observed in preeclampsia. In contrast, a clear preference for the NKp30a/b profile was evident in the 1st trimester decidua, yet no significant differences were observed for NKp30 profiles between healthy gestation and spontaneous abortions/preeclampsia. Both cancerous and matched normal tissues manifested balanced NKp30c inhibitory and NKp30a/b activation profiles with a NKp44-1dominant profile. However, a shift in NKp30 profiles between matched normal and cancer tissue was observed in half of the cases. To summarize, NKp44 and NKp30 splice variants profiles are tissue/condition specific and demonstrate similarity between placenta and cancerous tissues.


Subject(s)
Decidua/metabolism , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 3/genetics , Neoplasms/metabolism , RNA Splicing , Abortion, Spontaneous/immunology , Abortion, Spontaneous/pathology , Decidua/immunology , Decidua/pathology , Female , Flow Cytometry , Humans , Immune Privilege , Interleukin-15/metabolism , Interleukin-18/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Natural Cytotoxicity Triggering Receptor 2/immunology , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Pre-Eclampsia/immunology , Pre-Eclampsia/pathology , Pregnancy , Transforming Growth Factor beta/metabolism , Tumor Microenvironment/immunology
8.
Oncotarget ; 7(22): 32933-45, 2016 May 31.
Article in English | MEDLINE | ID: mdl-27102296

ABSTRACT

NKp44 is a receptor encoded by the NCR2 gene, which is expressed by cytokine-activated natural killer (NK) cells that are involved in anti-AML immunity. NKp44 has three splice variants corresponding to NKp44ITIM+ (NKp44-1) and NKp44ITIM- (NKp44-2, and NKp44-3) isoforms. RNAseq data of AML patients revealed similar survival of NKp46+NKp44+ and NKp46+NKp44- patients. However, if grouped according to the NKp44 splice variant profile, NKp44-1 expression was significantly associated with poor survival of AML patients. Moreover, activation of PBMC from healthy controls showed co-dominant expression of NKp44-1 and NKp44-3, while primary NK clones show more diverse NKp44 splice variant profiles. Cultured primary NK cells resulted in NKp44-1 dominance and impaired function associated with PCNA over-expression by target cells. This impaired functional phenotype could be rescued by blocking of NKp44 receptor. Human NK cell lines revealed co-dominant expression of NKp44-1 and NKp44-3 and showed a functional phenotype that was not inhibited by PCNA over-expression. Furthermore, transfection-based overexpression of NKp44-1, but not NKp44-2/NKp44-3, reversed the endogenous resistance of NK-92 cells to PCNA-mediated inhibition, and resulted in poor formation of stable lytic immune synapses. This research contributes to the understanding of AML prognosis by shedding new light on the functional implications of differential splicing of NKp44.


Subject(s)
Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Natural Cytotoxicity Triggering Receptor 2/genetics , Adult , Case-Control Studies , HeLa Cells , Humans , K562 Cells , Leukemia, Myeloid, Acute/metabolism , Natural Cytotoxicity Triggering Receptor 2/metabolism , Protein Isoforms , Survival Rate
9.
Clin Exp Immunol ; 185(1): 42-9, 2016 07.
Article in English | MEDLINE | ID: mdl-26850369

ABSTRACT

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees.


Subject(s)
Cell Lineage/immunology , Cytotoxicity, Immunologic , Hepacivirus/immunology , Hepatitis C/immunology , Killer Cells, Natural/immunology , Animals , Ape Diseases , Cell Degranulation/drug effects , Flow Cytometry , Gene Expression Regulation , Hepatitis C/pathology , Hepatitis C/virology , Immunophenotyping , Interleukin-2/pharmacology , Interleukins/pharmacology , Killer Cells, Natural/classification , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Lymphocyte Activation/drug effects , Lysosomal-Associated Membrane Protein 1/genetics , Lysosomal-Associated Membrane Protein 1/immunology , NK Cell Lectin-Like Receptor Subfamily D/genetics , NK Cell Lectin-Like Receptor Subfamily D/immunology , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 3/genetics , Natural Cytotoxicity Triggering Receptor 3/immunology , Pan troglodytes , Receptors, IgG/genetics , Receptors, IgG/immunology
10.
J Leukoc Biol ; 96(6): 1119-29, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25139289

ABSTRACT

Although the mechanisms underlying the cytotoxic effect of NK cells on tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell-surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/physiology , Bacteriolysis , Killer Cells, Natural/immunology , Mycobacterium kansasii , Mycobacterium tuberculosis , Perforin/metabolism , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Differentiation, T-Lymphocyte/pharmacology , Bacteriolysis/drug effects , Bacteriolysis/physiology , Cell Line, Tumor , Cell Wall/drug effects , Humans , Killer Cells, Natural/metabolism , Killer Cells, Natural/ultrastructure , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , NK Cell Lectin-Like Receptor Subfamily K/antagonists & inhibitors , NK Cell Lectin-Like Receptor Subfamily K/biosynthesis , NK Cell Lectin-Like Receptor Subfamily K/genetics , Nanotubes , Natural Cytotoxicity Triggering Receptor 2/antagonists & inhibitors , Natural Cytotoxicity Triggering Receptor 2/biosynthesis , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 3/antagonists & inhibitors , Natural Cytotoxicity Triggering Receptor 3/biosynthesis , Natural Cytotoxicity Triggering Receptor 3/genetics , Perforin/biosynthesis , Perforin/genetics , Perforin/pharmacology , RNA Interference , RNA, Small Interfering/pharmacology , Transcription, Genetic/drug effects
11.
Toxins (Basel) ; 5(11): 1932-47, 2013 Oct 28.
Article in English | MEDLINE | ID: mdl-24169587

ABSTRACT

We describe here the effects of three drugs that are either approved or have the potential for treating multiple sclerosis (MS) patients through the in vitro activities of human natural killer (NK) cells and dendritic cells (DCs). Our results indicate that 1,25(OH)2D3, the biologically active metabolite of vitamin D3, calcipotriol and FTY720 augment IL-2-activated NK cell lysis of K562 and RAJI tumor cell lines as well as immature (i) and mature (m) DCs, with variable efficacies. These results are corroborated with the ability of the drugs to up-regulate the expression of NK cytotoxicity receptors NKp30 and NKp44, as well as NKG2D on the surfaces of NK cells. Also, they down-regulate the expression of the killer inhibitory receptor CD158. The three drugs down-regulate the expression of CCR6 on the surface of iDCs, whereas vitamin D3 and calcipotriol tend to up-regulate the expression of CCR7 on mDCs, suggesting that they may influence the migration of DCs into the lymph nodes. Finally, vitamin D3, calcipotriol and FTY720 enhance NK17/NK1 cell lysis of K562 cells, suggesting that a possible mechanism of action for these drugs is via activating these newly described cells. In conclusion, our results show novel mechanisms of action for vitamin D3, calcipotriol and FTY720 on cells of the innate immune system.


Subject(s)
Calcitriol/analogs & derivatives , Cholecalciferol/pharmacology , Dendritic Cells/drug effects , Killer Cells, Natural/drug effects , Membrane Proteins/metabolism , Propylene Glycols/pharmacology , Sphingosine/analogs & derivatives , Calcitriol/pharmacology , Cell Line, Tumor , Cytotoxicity, Immunologic/drug effects , Dendritic Cells/cytology , Down-Regulation , Fingolimod Hydrochloride , Gene Expression Regulation, Neoplastic , Humans , Interleukin-2/pharmacology , K562 Cells , Killer Cells, Natural/cytology , Membrane Proteins/genetics , Multiple Sclerosis/drug therapy , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/metabolism , Natural Cytotoxicity Triggering Receptor 3/genetics , Natural Cytotoxicity Triggering Receptor 3/metabolism , Receptors, CCR6/genetics , Receptors, CCR6/metabolism , Receptors, CCR7/genetics , Receptors, CCR7/metabolism , Sphingosine/pharmacology , Up-Regulation
12.
Blood ; 122(17): 2935-42, 2013 Oct 24.
Article in English | MEDLINE | ID: mdl-23958951

ABSTRACT

With an array of activating and inhibitory receptors, natural killer (NK) cells are involved in the eradication of infected, transformed, and tumor cells. NKp44 is a member of the natural cytotoxicity receptor family, which is exclusively expressed on activated NK cells. Here, we identify natural cytotoxicity receptor NKp44 (NKp44L), a novel isoform of the mixed-lineage leukemia-5 protein, as a cellular ligand for NKp44. Unlike the other MLL family members, NKp44L is excluded from the nucleus, but expressed at the cell-surface level; its subcellular localization is being associated with the presence of a specific C-terminal motif. Strikingly, NKp44L has not been detected on circulating cells isolated from healthy individuals, but it is expressed on a large panel of the tumor and transformed cells. The sharply decreased NK lysis activity induced by anti-NKp44L antibodies directly demonstrates the role of NKp44L in cytotoxicity. Taken together, these results show that NKp44L could be critical for NK cell-mediated innate immunity. The identification and cellular distribution of NKp44L highlight the role of this self-molecule as a danger signal to alert the NK cell network.


Subject(s)
Cytotoxicity, Immunologic/genetics , DNA-Binding Proteins/immunology , Immunity, Innate , Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 2/immunology , Amino Acid Motifs , Antibodies, Neutralizing/pharmacology , Cell Line, Tumor , Cells, Cultured , Cytotoxicity, Immunologic/drug effects , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Gene Library , Humans , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Ligands , Lymphocyte Activation , Natural Cytotoxicity Triggering Receptor 2/genetics , Organ Specificity , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Structure, Tertiary , Two-Hybrid System Techniques
13.
J Immunol ; 190(5): 2381-90, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23345327

ABSTRACT

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.


Subject(s)
Adenocarcinoma/immunology , Colonic Neoplasms/immunology , Histocompatibility Antigens Class I/immunology , Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 3/immunology , Neoplastic Stem Cells/immunology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Cell Lineage/immunology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Cytotoxicity, Immunologic , Gene Expression , Histocompatibility Antigens Class I/genetics , Humans , Killer Cells, Natural/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Microscopy, Confocal , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 3/genetics , Neoplastic Stem Cells/pathology , Neoplastic Stem Cells/transplantation , Organ Specificity , Tumor Cells, Cultured
14.
Mol Immunol ; 54(3-4): 296-301, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23328088

ABSTRACT

DNA hypermethylation resulting in aberrant epigenetic silencing plays an important role in the oncogenesis of many cancer types, including acute myelogenous leukemia (AML).(4) The modulation of NK cell receptors and their cognate ligands is a known mechanism of immune escape in AML, and some membrane proteins, such as killer immunoglobulin-like receptors (KIR), are known to be transcriptionally regulated by DNA methylation of their promoter regions. Thus, restoring proper expression of immunoreceptors or their ligands with immunosensitizing drugs is an attractive approach to improving cancer immunotherapy. The cytidine analog 5-aza-2'-deoxycytidine (decitabine, DAC) has both a hypomethylating effect at low doses when incorporated into DNA and a cytotoxic effect at higher doses as a result of interfering with translation when incorporated into RNA. Thus, decitabine has been used at higher doses for its direct anti-leukemic effect, and is being tested at low doses for its ability to correct the malignant gene expression phenotype. A known benefit of hypomethylating agents is their ability to sensitize AML blasts to lysis by NK cells. However, there is little information on the direct effect of hypomethylating agents on NK cell phenotype, proliferation, survival, or function. We recently described a method for inducing robust proliferation of NK cells, enabling us to study the hypomethylating effects of decitabine. To distinguish direct toxicity of the decitabine from its hypomethylating effect, and promote hypomethylation during proliferation, decitabine was added to human peripheral blood NK cells at concentrations from 0.02 to 5µM under either static or proliferation-inducing culture conditions. After 5 days, NK cells were assessed for viability, proliferation, cytotoxicity, expression of major activating and inhibitory receptors, and global DNA methylation. Increasing concentrations of decitabine not only causes increased expression of KIR and the activating receptor NKp44, but also causes decreased viability, proliferation, and expression of the activating receptor NKG2D. Decitabine treatment results in a biphasic effect in overall NK cell lytic function, which correlates with a biphasic pattern of global hypomethylation. Decitabine affects the expression of activating and inhibitory receptors in NK cells at low concentrations when exposed during cell proliferation. High doses of decitabine decrease NK cell proliferation and viability, likely through direct inhibition of mRNA transcription. The results of these combined effects leads to a biphasic response in hypomethylation and cytotoxicity. This suggests that optimal immunomodulation with decitabine occurs at low dose ranges and that high doses abrogate this effect through inhibition of proliferation and direct toxicity to NK cells.


Subject(s)
Azacitidine/analogs & derivatives , Killer Cells, Natural/drug effects , Antimetabolites, Antineoplastic/pharmacology , Azacitidine/pharmacology , Cell Growth Processes/drug effects , Cell Growth Processes/genetics , Cell Growth Processes/immunology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Cell Survival/immunology , Cytotoxicity, Immunologic/drug effects , DNA Methylation/drug effects , Decitabine , Humans , K562 Cells , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/metabolism , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 2/metabolism , Phenotype , RNA, Messenger/genetics , RNA, Messenger/immunology , Receptors, KIR/genetics , Receptors, KIR/immunology , Receptors, KIR/metabolism , Receptors, Natural Killer Cell/genetics , Receptors, Natural Killer Cell/immunology , Receptors, Natural Killer Cell/metabolism , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Transcription, Genetic/immunology
15.
Transfusion ; 51(2): 293-305, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20723170

ABSTRACT

BACKGROUND: The human cytokine granulocyte-colony stimulatory factor (G-CSF) has found widespread application in the medical treatment of neutropenia and to mobilize hematopoietic stem cells used for transplantation. So far, the effect of G-CSF on natural killer (NK) cells has not been fully investigated. STUDY DESIGN AND METHODS: The effect of G-CSF on the phenotype, cytokine secretion profile, and cytotoxicity of NK cells was assessed. NK cells incubated in vitro in presence of G-CSF for 48 hours as well as NK cells isolated from peripheral blood of G-CSF-mobilized stem cell donors (in vivo) were used. RESULTS: In vitro, G-CSF caused a strongly altered phenotype in NK cells with 49% down regulation of NKp44 frequency. Furthermore, the expression of the activating receptors NKp46 and NKG2D decreased 40 and 64%, respectively. The expression of KIR2DL1 and KIR2DL2 decreased by 46% each. In cytotoxicity assays, the lytic capacity of G-CSF-exposed NK cells is reduced by up to 68% in vitro and up to 83% in vivo. Accordingly, granzyme B levels of in vivo G-CSF-exposed NK cells were reduced by up to 87% in comparison to nonstimulated NK cells. Cytokine production of in vitro and in vivo incubated NK cells was strongly decreased for interferon-γ, tumor necrosis factor-α, and granulocyte macrophage colony-stimulating factor as well as interleukin (IL)-6 and IL-8. Furthermore, we observed a reduction in proliferation and a positive feedback loop that increased the expression of the G-CSF receptor. CONCLUSION: G-CSF was demonstrated to be a strong inhibitor of NK cells activity and may prevent their graft-versus-leukemia effect after transplantation.


Subject(s)
Cytotoxicity, Immunologic/drug effects , Granulocyte Colony-Stimulating Factor/pharmacology , Killer Cells, Natural/drug effects , Cell Division/drug effects , Cells, Cultured/drug effects , Cells, Cultured/immunology , Cytokines/biosynthesis , Cytokines/genetics , Depression, Chemical , Gene Expression Regulation/drug effects , Graft vs Leukemia Effect/drug effects , Granulocyte Colony-Stimulating Factor/adverse effects , Granzymes/biosynthesis , Granzymes/genetics , Humans , Killer Cells, Natural/metabolism , Lenograstim , NK Cell Lectin-Like Receptor Subfamily K/biosynthesis , NK Cell Lectin-Like Receptor Subfamily K/genetics , Natural Cytotoxicity Triggering Receptor 1/biosynthesis , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 2/biosynthesis , Natural Cytotoxicity Triggering Receptor 2/genetics , Receptors, Granulocyte Colony-Stimulating Factor/biosynthesis , Receptors, Granulocyte Colony-Stimulating Factor/genetics , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacology
16.
PLoS One ; 5(11): e15080, 2010 Nov 30.
Article in English | MEDLINE | ID: mdl-21151495

ABSTRACT

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells endowed with the capacity of producing large amounts of IFNα. Here we show that the Leukocyte-Associated Ig-like Receptor-1 (LAIR-1) is abundantly expressed on pDCs (the highest expression among all leukocytes) and its cross-linking inhibits IFNα production in response to Toll-like receptor ligands. Remarkably, LAIR-1 expression in pDCs is down-regulated in the presence of interleukin (IL)-3, thus indicating coordinated functions with NKp44, another pDC inhibitory receptor, which is conversely induced by IL-3. Nevertheless, the expression of NKp44 in pDCs isolated from secondary lymphoid organs, which is thought to be influenced by IL-3, is not coupled to a decreased expression of LAIR-1. Interestingly, pDCs isolated from peripheral blood of systemic lupus erithematosus (SLE) patients express lower levels of LAIR-1 while displaying slight but consistent expression of NKp44, usually undetectable on pDCs derived from healthy donors. Using sera derived from SLE patients, we show that LAIR-1 and NKp44 display synergistic inhibitory effects on IFNα production by interleukin IL-3 cultured pDCs stimulated with DNA immunocomplexes. In conclusion, our results indicate that the inhibitory function of LAIR-1 may play a relevant role in the mechanisms controlling IFNα production by pDCs both in normal and pathological innate immune responses.


Subject(s)
Dendritic Cells/metabolism , Interferon-alpha/metabolism , Natural Cytotoxicity Triggering Receptor 2/metabolism , Receptors, Immunologic/metabolism , Cells, Cultured , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression , Humans , Killer Cells, Natural/metabolism , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/metabolism , Lymphoid Tissue/metabolism , Natural Cytotoxicity Triggering Receptor 2/genetics , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction
17.
Cytotherapy ; 12(8): 1044-55, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20795758

ABSTRACT

BACKGROUND AIMS: Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality. RESULTS: Significant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 x 109 NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks. CONCLUSIONS: These results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.


Subject(s)
Immunotherapy, Adoptive , Killer Cells, Natural/metabolism , Multiple Myeloma/therapy , Automation, Laboratory , Bioreactors , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell Proliferation , Cell Separation , Cytotoxicity, Immunologic , Feasibility Studies , Flow Cytometry , Humans , Immunophenotyping , K562 Cells , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 2/metabolism
18.
J Virol ; 83(16): 8108-21, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19515783

ABSTRACT

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3zeta lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.


Subject(s)
Avulavirus Infections/immunology , HN Protein/immunology , Killer Cells, Natural/immunology , Newcastle disease virus/enzymology , Avulavirus Infections/virology , Cell Line, Tumor , HN Protein/genetics , Humans , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 1/immunology , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/immunology , Newcastle disease virus/genetics , Newcastle disease virus/immunology
19.
Cancer Immunol Immunother ; 58(10): 1691-700, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19259669

ABSTRACT

Natural cytotoxicity receptors (NCRs) are major activating receptors involved in NK cytotoxicity. NCR expression varies with the activation state of NK cells, and the expression level correlates with NK cells' natural cytotoxicity. In this study, we found that Gö6983, a PKC inhibitor, induced a remarkable increase of NCR expression on primary NK cells, but other PKC inhibitors and NK cell stimulators such as IL-2 and PMA, did not. Gö6983 increased the expression of NCR in a time- and concentration-dependent manner. Furthermore, Gö6983 strongly upregulated the surface expression of death ligands FasL and TRAIL, but not cytotoxic molecules perforin and granzyme B. Unlike two other NK stimulating molecules, IL-2, and PMA, Gö6983 did not induce NK cell proliferation. Up-regulation of NCRs and death ligands on NK cells by Gö6983 resulted in a significant enhancement of NK cytotoxicity against various cancer cell lines. Most importantly, administration of Gö6983 effectively inhibited pulmonary tumor metastasis in mice in a dose-dependent manner. These results suggest that Gö6983 functions as an NK cell activating molecule (NKAM); this NKAM is a novel anti-cancer and anti-metastasis drug candidate because it enhances NK cytotoxicity against cancer cells in vivo as well as in vitro.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/prevention & control , Indoles/pharmacology , Killer Cells, Natural/immunology , Liver Neoplasms/prevention & control , Lung Neoplasms/prevention & control , Maleimides/pharmacology , Animals , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/secondary , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Fas Ligand Protein/metabolism , Female , Flow Cytometry , Humans , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Lung Neoplasms/immunology , Lung Neoplasms/secondary , Mice , Mice, Inbred C57BL , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 1/metabolism , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 2/metabolism , Natural Cytotoxicity Triggering Receptor 3/genetics , Natural Cytotoxicity Triggering Receptor 3/metabolism , Protein Kinase C/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Cells, Cultured
20.
J Proteome Res ; 8(2): 712-20, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19196184

ABSTRACT

Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One important family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. This study aims to elucidate heparan sulfate structural motifs that are important for NCR binding. Microarray and surface plasmon resonance experiments with a small library of heparan sulfate/heparin oligosaccharides helped to clarify the binding preferences of the three NCRs. We demonstrate that the NCRs interact with highly charged HS/heparin structures, but differ in preferred modification patterns and chain lengths. The affinity of NKp30 and NKp44 for synthetic HS/heparin is approximately one order of magnitude higher than the affinity of NKp46. We further show the relevance of synthetic HS/heparin for the binding of NCRs to tumor cells and for NCR-mediated activation of natural killer cells. In conclusion, NCRs recognize different microdomains on heparan sulfate with different affinities.


Subject(s)
Heparin/chemistry , Heparitin Sulfate/chemistry , Natural Cytotoxicity Triggering Receptor 1/immunology , Natural Cytotoxicity Triggering Receptor 2/immunology , Natural Cytotoxicity Triggering Receptor 3/immunology , Carbohydrate Conformation , Carbohydrate Sequence , Heparin/immunology , Heparitin Sulfate/immunology , Humans , Killer Cells, Natural/immunology , Microarray Analysis/methods , Molecular Sequence Data , Natural Cytotoxicity Triggering Receptor 1/genetics , Natural Cytotoxicity Triggering Receptor 2/genetics , Natural Cytotoxicity Triggering Receptor 3/genetics , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology
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