fluorescence-activated-cell-sorting-for-isolation-scleractinian-cell

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Ben Gurion University of the Negev

Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve NK cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein; it also binds to WNV virus-like particles. These WNV-virus-like particles and WNV-domain III of WNV envelope protein directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK degranulation. Finally, WNV infection of cells results in increased binding of rNKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFN-gamma secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 Abs. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein.

NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells.

NK cells play an important role in the early immune response to cancer. The NKp44 activating receptor is the only natural cytotoxicity receptor that is expressed exclusively by primate NK cells, yet its cellular ligands remain largely unknown. Proliferating cell nuclear Ag (PCNA) is overexpressed in cancer cells. In this study, we show that the NKp44 receptor recognizes PCNA. Their interaction inhibits NK cell function through NKp44/ITIM. The physical interaction of NKp44 and PCNA is enabled by recruitment of target cell PCNA to the NK immunological synapse. We demonstrate that PCNA promotes cancer survival by immune evasion through inhibition of NKp44-mediated NK cell attack.

Proliferating cell nuclear antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44.

Unlike many other viruses that downregulate MHC class I expression on infected cell membranes, flaviviruses were reported to upregulate the MHC class I expression. Dengue virus was shown to induce HLA class I expression; however, the precise transcriptional mechanism that is used by the virus remains unclear. This article assessed the findings of a recently published report describing the mechanism used by dengue virus to induce HLA-A2 expression and characterizing the transcription factors that are involved. The study showed that p50/p65 and p65/65 NF-κB dimers bind to the class I regulatory complex within the HLA-A2 promoter. This finding and its significance for the design of possible antiviral therapeutic agents are discussed in this article.

Upregulation of MHC class I expression following dengue virus infection: the mechanism at the promoter level.

The matter of the pathogen- and cancer-associated ligands recognized by the Natural Cytotoxicity Receptors (NCRs) has been a subject of intense research ever since the identification of the NCRs more than 12 years ago by Alessandro and Lorenzo Moretta: NKp46 in 1997, NKp44 in 1998, and finally NKp30 in 1999. Expression patterns recognized by NCRs include pathogen-derived, pathogen-induced, and cancer-associated cellular 'self' ligands. Pathogen-exposed cells may exhibit both types of pathogen-associated ligands. Transformed cells, in contrast, exhibit only 'self' ligands which are derived from both the intracellular- and membrane-associated milieu of self molecules. These expression patterns allow for NCR-based NK cell discrimination between healthy and affected cells, in the realms of both pathogenic infection and potential tumorigenesis. The focus of this review is on the current knowledge regarding the identities of NCR ligands and the type of target cells expressing these ligands.

Human NK cell recognition of target cells in the prism of natural cytotoxicity receptors and their ligands.

NKp46 is a primary activating receptor of NK cells that is involved in lysis of target cells by NK cells. Previous studies showed that the membrane-proximal domain of NKp46 (NKp46D2) retained the binding of NKp46 to its ligands and is involved in lysis. We studied NKp46D2 by using a peptide-based epitope mapping approach and identified an NKp46D2-derived linear epitope that inhibited NKp46-mediated lysis. The epitope, designated as pep4 (aa 136-155), interacted with NKp46, and lysis by NK cells was inhibited by the presence of pep4. Through modeling and mutagenesis, we showed that pep4 could be involved in NKp46 homodimerization. R145 and D147 contribute to the function of pep4, and R145Q mutation in recombinant NKp46 reduced its binding to target cells. At the cellular level, fluorescent resonance energy transfer analysis revealed that pep4 is indeed involved in dimerization of cell membrane-associated NKp46. We suggest that the NKp46-derived pep4 site is part of the dimerization surface of NKp46 and that NKp46 dimerization contributes to NKp46-mediated lysis by NK cells.

Dimerization of NKp46 receptor is essential for NKp46-mediated lysis: characterization of the dimerization site by epitope mapping.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by degeneration of upper and lower motor neurons. To date, glycosylation patterns of glycoproteins in fluids of ALS patients have not been described. Moreover, the aberrant glycosylation related to the pathogenesis of other neurodegenerative diseases encouraged us to explore the glycome of ALS patient sera. We found high levels of sialylated glycans and low levels of core fucosylated glycans in serum-derived N-glycans of patients with ALS, compared to healthy volunteer sera. Based on these results, we analyzed the IgG Fc N(297)-glycans, as IgG are major serum glycoproteins affected by sialylation or core fucosylation and are found in the motor cortex of ALS patients. The analyses revealed a distinct glycan, A2BG2, in IgG derived from ALS patient sera (ALS-IgG). This glycan increases the affinity of IgG to CD16 on effector cells, consequently enhancing Antibody-Dependent Cellular Cytotoxicity (ADCC). Therefore, we explore whether the Fc-N(297)-glycans of IgG may be involved in ALS disease. Immunostaining of brain and spinal cord tissues revealed over-expression of CD16 and co-localization of intact ALS-IgG with CD16 and in brain with activated microglia of G93A-SOD1 mice. Intact ALS-IgG enhanced effector cell activation and ADCC reaction in comparison to sugar-depleted or control IgG. ALS-IgG were localized in the synapse between brain microglia and neurons of G93A-SOD1 mice, manifesting a promising in vivo ADCC reaction. Therefore, glycans of ALS-IgG may serve as a biomarker for the disease and may be involved in neuronal damage.

Glycans in sera of amyotrophic lateral sclerosis patients and their role in killing neuronal cells.

We recently reported proliferating cell nuclear antigen (PCNA) as a ligand for the NK cell activating receptor, NKp44, which unexpectedly triggers inhibition. The recognition of nuclear proteins such as PCNA, by related NK cell receptors has been reported. Widespread upregulation of PCNA in tumor cells may therefore promote immune evasion.

A novel mechanism for cancer cells to evade immune attack by NK cells: The interaction between NKp44 and proliferating cell nuclear antigen.

The study of the Plasmodium falciparum heavy metal transporter gene pfmdr2 employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank considerably during the last few years, resulting in the cessation of the production of some isotopes, amongst them Cadmium109 which was used for that purpose, a different approach had to be developed. Herein, a dual fluorescent labelling of heavy metals accumulation in the P. falciparum parasite is proposed as an alternative to the use of radioactive labelled heavy metals.

Dual fluorescent labelling of the human malaria parasite Plasmodium falciparum for the analysis of the ABC type transporter pfmdr2.

KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56(high) subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cell-surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0 domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4), and induces differential localization of KIR2DL4 to rab5(+) and rab7(+) endosomes, thus leading to downregulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS proteoglycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking.

Genome-Wide siRNA Screen Reveals a New Cellular Partner of NK Cell Receptor KIR2DL4: Heparan Sulfate Directly Modulates KIR2DL4-Mediated Responses.

The Ral (Ras-like) GTP-binding proteins (RalA and RalB), as effectors of the proto-oncogene Natural killer (NK) cells are an important component of the anti-tumor response. Tumor recognition by NK cells was found to be partly triggered by molecules termed natural cytotoxic receptors (NCRs). Adoptive transfer of genetically-engineered tumor-reactive T-lymphocytes can mediate remarkable tumor regressions mostly in melanoma and leukemia patients. Yet, the application of such treatments to other cancers is needed and dependent on the isolation of receptors that could facilitate efficient recognition of these malignancies. Herein, we aimed at combining NK tumor recognition capability with the genetic modification of T-cells to provide the latter with a means to recognize several tumors in a non-MHC restricted way. Consequently, we generated and evaluated several chimeric receptors based on the extracellular domain of NCR1 (NKp46) fused to multiple signaling moieties and assess their antitumor activity when retrovirally expressed in T-cells. Following co-culture with different tumors, primary human T-lymphocytes expressing a chimeric NCR1 molecule recognized target cells derived from lung, cervical carcinoma, leukemia and pancreatic cancer. In addition, this receptor mediated an upregulation of surface activation markers and significant antitumor cytotoxicity both in vitro and in vivo. These results have meaningful implications for the immunotherapeutic treatment of cancer using gene-modified T-cells. 

An NCR1-based chimeric receptor endows T-cells with multiple anti-tumor specificities.

Capsule: We observed that first trimester pregnancy loss is associated with an altered expression profile of the three isoforms of the NK receptor NKp30 expressed by NKs in PBMC and placental tissue. In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms) in maternal peripheral blood or placental tissue. We conducted a prospective case-control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group comprises women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expressions were mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms-a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. By contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10, and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss. 

First Trimester Pregnancy Loss and the Expression of Alternatively Spliced NKp30 Isoforms in Maternal Blood and Placental Tissue.

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. 

Survival in acute myeloid leukemia is associated with NKp44 splice variants.

NKp44 and NKp30 splice variant profiles have been shown to promote diverse cellular functions. Moreover, microenvironment factors such as TGF-β, 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.

NKp44 and NKp30 splice variant profiles in decidua and tumor tissues: a comparative viewpoint.

Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

Complex mammalian-like haematopoietic system found in a colonial chordate.

The adaptive immune response in jawed vertebrates is marked by the ability to diversify somatically specific immune receptor genes. Somatic recombination and hypermutation of gene segments are used to generate extensive repertoires of T and B cell receptors. In contrast, jawless vertebrates utilize a distinct diversification system based on copy choice to assemble their variable lymphocyte receptors. To date, very little evidence for somatic immune gene diversification has been reported in invertebrate species. Here we show that the  ( ; formerly ) immune effector gene family members from individual coelomocytes from purple sea urchins undergo somatic diversification by means of gene deletions, duplications, and acquisitions of single nucleotide polymorphisms. While sperm cells from an individual sea urchin have identical  gene repertoires, single cells from two distinct coelomocyte subpopulations from the same sea urchin exhibit significant variation in the  gene repertoires. Moreover, the highly diverse gene sequences derived from single coelomocytes are all in-frame, suggesting that an unknown mechanism(s) driving these somatic changes involve stringent selection or correction processes for expression of productive  transcripts. Together, our findings infer somatic immune gene diversification strategy in an invertebrate.

Individual Sea Urchin Coelomocytes Undergo Somatic Immune Gene Diversification.

Evolution and selection have shaped diverse immune systems throughout phylogeny, the vast majority of which remain unexplored. Botryllus schlosseri is a colonial tunicate, a sister group to vertebrates, that develops as a chordate, then metamorphoses to an asexually reproductive invertebrate that every week makes the same body plan from budded stem cells. Genetically distinct B. schlosseri colonies can fuse to form a chimera, or reject each other based on allogeneic recognition. In chimeras, circulating germline and somatic stem cells participate in development; stem cells compete in all individuals in the fused colonies, with rejection preventing germline parasitism. Here we review the isolation and characterization of B. schlosseri hematopoietic stem cells (HSC) and their niches, and the role of the immune effector cells in allorecognition.

Article	Year
NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19635919	2009
Proliferating cell nuclear antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 22021614	2011
Upregulation of MHC class I expression following dengue virus infection: the mechanism at the promoter level. Expert review of anti-infective therapy\| PubMed ID: 22397562	2012
Human NK cell recognition of target cells in the prism of natural cytotoxicity receptors and their ligands. Journal of immunotoxicology\| PubMed ID: 22524686	2012
Dimerization of NKp46 receptor is essential for NKp46-mediated lysis: characterization of the dimerization site by epitope mapping. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 22615207	2012
Glycans in sera of amyotrophic lateral sclerosis patients and their role in killing neuronal cells. PloS one\| PubMed ID: 22666317	2012
A novel mechanism for cancer cells to evade immune attack by NK cells: The interaction between NKp44 and proliferating cell nuclear antigen. Oncoimmunology\| PubMed ID: 22754791	2012
Dual fluorescent labelling of the human malaria parasite Plasmodium falciparum for the analysis of the ABC type transporter pfmdr2. Malaria journal\| PubMed ID: 23137170	2012
Genome-Wide siRNA Screen Reveals a New Cellular Partner of NK Cell Receptor KIR2DL4: Heparan Sulfate Directly Modulates KIR2DL4-Mediated Responses. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 24127555	2013
An NCR1-based chimeric receptor endows T-cells with multiple anti-tumor specificities. Oncotarget\| PubMed ID: 25431955	2014
First Trimester Pregnancy Loss and the Expression of Alternatively Spliced NKp30 Isoforms in Maternal Blood and Placental Tissue. Frontiers in immunology\| PubMed ID: 26082773	2015
Survival in acute myeloid leukemia is associated with NKp44 splice variants. Oncotarget\| PubMed ID: 27102296	2016
NKp44 and NKp30 splice variant profiles in decidua and tumor tissues: a comparative viewpoint. Oncotarget\| PubMed ID: 27765926	2016
Complex mammalian-like haematopoietic system found in a colonial chordate. Nature\| PubMed ID: 30518860	2018
Individual Sea Urchin Coelomocytes Undergo Somatic Immune Gene Diversification. Frontiers in immunology\| PubMed ID: 31244844	2019
Evolutionary perspective on the hematopoietic system through a colonial chordate: allogeneic immunity and hematopoiesis. Current opinion in immunology\| PubMed ID: 31954962	2020

Benyamin Rosental

Biography

JoVE Journal Publications

Other Publications