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Papers by Jacob Hanna

Research paper thumbnail of Correction: novel insights on human NK cells' immunological modalities revealed by gene expression profiling

Journal of immunology (Baltimore, Md. : 1950), 2015

CD56 bright CD16 − Killer Ig-Like Receptor − NK Cells Display Longer Telomeres and Acquire Featur... more CD56 bright CD16 − Killer Ig-Like Receptor − NK Cells Display Longer Telomeres and Acquire Features of CD56 dim NK Cells upon Activation

Research paper thumbnail of Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4

Proceedings of the National Academy of Sciences of the United States of America, Jan 2, 2009

Ectopic expression of defined transcription factors can reprogram somatic cells to induced plurip... more Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such...

Research paper thumbnail of Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors

Stem Cells, 2011

Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming t... more Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process ...

Research paper thumbnail of Reprogramming of murine and human somatic cells using a single polycistronic vector

Proceedings of the National Academy of Sciences, 2008

Directed reprogramming of somatic cells by defined factors provides a novel method for the genera... more Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus...

Research paper thumbnail of Correction: Harnessing Soluble NK Cell Killer Receptors for the Generation of Novel Cancer Immune Therapy

PLoS ONE, 2008

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly o... more The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.

Research paper thumbnail of Single-gene transgenic mouse strains for reprogramming adult somatic cells

Nature Methods, 2009

We report a novel transgenic mouse model for direct reprogramming of somatic cells by expressing ... more We report a novel transgenic mouse model for direct reprogramming of somatic cells by expressing four reprogramming factors from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types explanted from different tissues can generate induced pluripotent stem cells (iPSC) by culture in doxycycline (Dox). Because the reprogramming factors are carried on a single polycistronic construct the transgenic mice can be easily maintained and transferred into another background.

Research paper thumbnail of Decidual NK cells regulate key developmental processes at the human fetal-maternal interface

Nature Medicine, 2006

Human CD56 bright NK cells accumulate in the maternal decidua during pregnancy and are found in d... more Human CD56 bright NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.

Research paper thumbnail of Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1

Research paper thumbnail of Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus

Nature Immunology, 2005

Human cytomegalovirus, a chief pathogen in immunocompromised people, can persist in a healthy imm... more Human cytomegalovirus, a chief pathogen in immunocompromised people, can persist in a healthy immunocompetent host throughout life without being eliminated by the immune system. Here we show that pp65, the main tegument protein of human cytomegalovirus, inhibited natural killer cell cytotoxicity by an interaction with the activating receptor NKp30. This interaction was direct and specific, leading to dissociation of the linked CD3zeta from NKp30 and, consequently, to reduced killing. Thus, pp65 is a ligand for the NKp30 receptor and demonstrates a unique mechanism by which an intracellular viral protein causes general suppression of natural killer cell cytotoxicity by specific interaction with an activating receptor.

Research paper thumbnail of Transgenic mice with defined combinations of drug-inducible reprogramming factors

Nature Biotechnology, 2009

Drug-inducible lentiviruses encoding Oct4, Sox2, Klf4 and c-Myc were used to derive "primary" iPS... more Drug-inducible lentiviruses encoding Oct4, Sox2, Klf4 and c-Myc were used to derive "primary" iPS cells and segregated through germline transmission generating mice and fibroblasts carrying subsets of the reprogramming factors. Drug treatment of the cells resulted in "secondary" iPS cell derivation only when the missing factor was introduced. This creates a defined platform for studying reprogramming mechanisms and allows screening of genetically homogenous cells for compounds that replace any transcription factor required for iPS cell derivation. The generation of induced pluripotent stem (iPS) cells from mouse and human somatic cells through the forced expression of defined transcription factors1-4 constitutes a major breakthrough in regenerative biology5. However, current reprogramming strategies require viral transduction with potentially oncogenic transcription factors. Understanding the molecular changes underlying iPS cell derivation will be required to devise alternative and safer strategies for reprogramming e.g., by replacing the viral transduced factors with small molecules6-8. Screening approaches using infected cells are hampered by the genetic variability caused by the random integrations of multiple proviral copies9,10. Recently, we generated a "secondary" transgenic system that eliminates such heterogeneity9,10. In this approach, mouse embryonic fibroblasts (MEFs) heterozygous for the ROSA26-M2 reverse tetracycline transactivator (M2-rtTA) were infected with Doxycycline (Dox)-inducible lentiviruses carrying the four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) and induced to generate "primary" iPS cells by addition of Dox. These cells were used to obtain chimeric mice with genetically identical somatic cells that can be isolated and reprogrammed in vitro by addition of Dox. However, such "secondary" somatic cells require isolation from Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Research paper thumbnail of A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

Nature Biotechnology, 2008

The study of induced pluripotency is complicated by the need for infection with high-titer retrov... more The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25-to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming. It has recently been shown that mouse 1-4 and human 5-8 fibroblasts can be reprogrammed to a pluripotent state through retroviral introduction of four transcription factors such as Oct4, Sox2, Klf4 and c-Myc. Reprogramming can also be achieved in the absence of the tumorigenic factor c-Myc, although with decreased efficiency 9,10. Nevertheless, with these approaches only a very small fraction of cells infected with all four factors will eventually reprogram 11. The random viral infection results in genetic heterogeneity in the infected cell culture that likely contributes to the low observed frequency of induced pluripotent stem (iPS) cell formation. Therefore, reprogrammed cells must be selected for by the reactivation of endogenous pluripotency genes 1-3 or based on morphological criteria 11,12. The reprogramming process has been shown to require ~10 to 12 d of sustained transgene expression after viral transduction and to follow a sequential activation of pluripotency markers, with initial activation of alkaline Correspondence should be addressed to R.J.

Research paper thumbnail of Direct cell reprogramming is a stochastic process amenable to acceleration

Nature, 2009

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved... more Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority

Research paper thumbnail of Dissecting direct reprogramming through integrative genomic analysis

Nature, 2008

In both the Methods Summary and the online-only Methods (cell culture section), the concentration... more In both the Methods Summary and the online-only Methods (cell culture section), the concentration of AZA is incorrectly listed as 0.5 mM. The correct final concentration is 0.5 mM.

Research paper thumbnail of Proteomic analysis of human natural killer cells: insights on new potential NK immune functions

Molecular Immunology, 2005

Applying high-throughput proteomic analysis of mammalian cells can facilitate the identification ... more Applying high-throughput proteomic analysis of mammalian cells can facilitate the identification of a large number of proteins expressed in the examined samples. Moreover, extensive research efforts are being made to perform large-scale characterization of membrane proteins. Here we use mass spectrometry-based proteomic strategy to characterize protein expression in membrane-enriched fractions derived from human NK lymphoma cell line YTS. This query yielded a list of over 1000 identified proteins, and provided us with new insights on NK cell biology. We highlight the expression of CD86 on YTS and its ability to co-stimulate TCR responses of human CD4+ T-cells, providing an unexpected link between innate and adaptive immune systems.

Research paper thumbnail of Complexes of HLA-G Protein on the Cell Surface Are Important for Leukocyte Ig-Like Receptor-1 Function

The Journal of Immunology, 2003

The nonclassical class I MHC molecule HLA-G is selectively expressed on extravillous cytotrophobl... more The nonclassical class I MHC molecule HLA-G is selectively expressed on extravillous cytotrophoblast cells at the maternal-fetal interface during pregnancy. HLA-G can inhibit the killing mediated by NK cells via interaction with the inhibitory NK cell receptor, leukocyte Ig-like receptor-1 (LIR-1). Comparison of the sequence of the HLA-G molecule to other class I MHC proteins revealed two unique cysteine residues located in positions 42 and 147. Mutating these cysteine residues resulted in a dramatic decrease in LIR-1 Ig binding. Accordingly, the mutated HLA-G transfectants were less effective in the inhibition of NK killing and RBL/LIR-1 induced serotonin release. Immunoprecipitation experiments demonstrated the involvement of the cysteine residues in the formation of HLA-G protein oligomers on the cell surface. The cysteine residue located at position 42 is shown to be critical for the expression of such complexes. These oligomers, unique among the class I MHC proteins, probably b...

Research paper thumbnail of Pivotal role of CEACAM1 protein in the inhibition of activated decidual lymphocyte functions

Journal of Clinical Investigation, 2002

Lymphocytes in direct contact with embryonic extravillous trophoblasts constitute more than 40% o... more Lymphocytes in direct contact with embryonic extravillous trophoblasts constitute more than 40% of decidual cells and appear to play major roles in implantation and early gestation. A unique subset of NK cells, making up 70-80% of decidual lymphocytes, express high levels of CD56 but lack CD16. We have recently demonstrated a novel class I MHC-independent inhibitory mechanism of NK cell cytotoxicity that is mediated by CEACAM1 homotypic interactions. This mechanism is used by some melanoma cells to avoid attack, mainly by CD16-NK cells. We now demonstrate that CEACAM1 is expressed on primary extravillous trophoblasts and is upregulated on the vast majority of IL-2-activated decidual lymphocytes, including NK, T, and NKT cells. Importantly, we present evidence that CEACAM1 interactions inhibit the lysis, proliferation, and cytokine secretion of activated decidual NK, T, and NKT cells, respectively. In vivo analysis of decidual lymphocytes isolated from cytomegalovirus-infected (CMV-infected) pregnant women revealed a dramatic increase in the expression of CEACAM1. Finally, we suggest that a novel ligand for this adhesion molecule is present on the surface of CMV-infected fibroblasts. These combined results demonstrate a major role for the CEACAM1 protein in controlling local decidual immune responses.

Research paper thumbnail of The involvement of NK cells in ankylosing spondylitis

International Immunology, 2005

A role for NK cells in the regulation of autoimmunity has been demonstrated. Since there is a str... more A role for NK cells in the regulation of autoimmunity has been demonstrated. Since there is a strong association between Ankylosing Spondylitis (AS) and HLA-B27, which is specifically recognized by the NK-inhibitory receptor KIR3DL1, this study evaluated the potential involvement of NK cells in AS. We studied 19 AS patients and 22 healthy volunteer donors and assessed the percentage, activity and receptor expression of peripheral blood NK cells. We also evaluated candidate-inflammatory mediators in sera. We found that AS patients have significantly higher percentages of NK cells. However, we found no differences between the ability of NK cells derived from AS and healthy controls to recognize target cells expressing HLA-B27. Remarkably, we observed that the NK-inhibitory receptor CEACAM1 (carcino-embryonic antigen-cell adhesion molecule) is highly expressed among AS-derived NK cells. Furthermore, engagement of CEACAM1 inhibited NK activity in these patients. Finally, we demonstrated that CEACAM1 expression is induced by IL-8 and SDF-1 (stromal cell derived factor), both of which are present in high levels in the sera of AS patients. These results may indicate that NK cells and CEACAM1 play a role in AS pathogenesis and implicate chemokines in the mechanism of CEACAM1 expression.

Research paper thumbnail of Special organization of the HLA-G protein on the cell surface

Human Immunology, 2003

The human leukocyte antigen G (HLA-G) molecule possesses unique properties such as low polymorphi... more The human leukocyte antigen G (HLA-G) molecule possesses unique properties such as low polymorphism and restricted distribution mainly to the extravillous cytotrophoblast (EVT) cells. The EVT cells vigorously penetrate into the maternal decidual tissues and are found in contact with maternal lymphocytes, mainly with natural killer (NK) cells. The HLA-G molecule inhibits the effector function of maternal NK cells via interaction with the KIR2DL4 and the ILT-2 inhibitory NK receptors. Previously, we have demonstrated that complexes of the HLA-G protein are expressed on the cell surface. We reported that these complexes are formed due to the presence of two unique cysteine residues located at positions 42 and 147. Finally, we demonstrated that efficient binding and function of ILT-2 is dependent on the presence of HLA-G complexes on the cell surface. Here we expand the significance of these observations by revealing that complexes of HLA-G are present on the cell surface using different assays and cell lines and further demonstrate that complexes of HLA-G might be present in a soluble form after interaction with ILT-2. Therefore, the HLA-G molecule has developed a special mechanism to increase the avidity of NK receptors to the HLA-G molecule, which provides better protection for the fetus from maternal NK rejection.

Research paper thumbnail of Biological function of the soluble CEACAM1 protein and implications in TAP2-deficient patients

European Journal of Immunology, 2004

Interactions of natural killer (NK) cells with MHC class I proteins provide the main inhibitory s... more Interactions of natural killer (NK) cells with MHC class I proteins provide the main inhibitory signals controlling NK killing activity. It is therefore surprising to learn that TAP2-deficient patients suffer from autoimmune manifestations only occasionally in later stages of life. We have previously described that the CEACAM1-mediated inhibitory mechanism of NK cytotoxicity plays a major role in controlling NK autoreactivity in three newly identified TAP2-deficient siblings. This novel mechanism probably compensates for the lack of MHC class I-mediated inhibition. The CEACAM1 protein can also be present in a soluble form and the biological function of the soluble form of CEACAM1 with regard to NK cells has not been investigated. Here we show that the homophilic CEACAM1 interactions are abrogated in the presence of soluble CEACAM1 protein in a dose-dependent manner. Importantly, the amounts of soluble CEACAM1 protein detected in sera derived from the TAP2-deficient patients were dramatically reduced as compared to healthy controls. This dramatic reduction does not depend on the membrane-bound metalloproteinase activity. Thus, the expression of CEACAM1 and the absence of soluble CEACAM1 observed in the TAP2-deficient patients practically maximize the inhibitory effect and probably help to minimize autoimmunity in these patients.

Research paper thumbnail of Involvement of the CXCL12/CXCR4 pathway in the advanced liver disease that is associated with hepatitis C virus or hepatitis B virus

European Journal of Immunology, 2004

Chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is accompanied by inflammat... more Chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is accompanied by inflammation and fibrosis eventually leading to cirrhosis. The chemokine CXCL12 is involved in chronic inflammatory conditions. The role of the CXCL12/CXCR4 pathway in HCV-and HBV-associated liver inflammation and fibrosis was therefore studied. The levels and tissue localization of CXCL12 in liver and plasma of HCV and HBV patients were tested using immunohistochemistry and ELISA. The expression and function of CXCR4 on liver-infiltrating lymphocytes (LIL) were tested by FACS and transwell migration assays. We found that CXCL12 is expressed by bile duct epithelial cells in normal liver tissue. Bile duct proliferation and liver fibrosis in chronic HCV and HBV infection result in the anatomical redistribution of CXCL12 in the liver. Moreover, CXCL12 is up-regulated in the endothelium of neo-bloodvessels formed in active inflammatory foci and is significantly elevated, compared with controls, in the plasma of patients with advanced liver fibrosis. Complementing these observations were others indicating that over 50% of LIL express CXCR4 and, in response to CXCL12, migrated and adhered to fibronectin. These observations suggest an important role for the CXCL12/CXCR4 pathway in recruitment and retention of immune cells in the liver during chronic HCV and HBV infection.

Research paper thumbnail of Correction: novel insights on human NK cells' immunological modalities revealed by gene expression profiling

Journal of immunology (Baltimore, Md. : 1950), 2015

CD56 bright CD16 − Killer Ig-Like Receptor − NK Cells Display Longer Telomeres and Acquire Featur... more CD56 bright CD16 − Killer Ig-Like Receptor − NK Cells Display Longer Telomeres and Acquire Features of CD56 dim NK Cells upon Activation

Research paper thumbnail of Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4

Proceedings of the National Academy of Sciences of the United States of America, Jan 2, 2009

Ectopic expression of defined transcription factors can reprogram somatic cells to induced plurip... more Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such...

Research paper thumbnail of Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors

Stem Cells, 2011

Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming t... more Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process ...

Research paper thumbnail of Reprogramming of murine and human somatic cells using a single polycistronic vector

Proceedings of the National Academy of Sciences, 2008

Directed reprogramming of somatic cells by defined factors provides a novel method for the genera... more Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. Here we report a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, which support efficient polycistronic expression from a single promoter. We find that up to four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus...

Research paper thumbnail of Correction: Harnessing Soluble NK Cell Killer Receptors for the Generation of Novel Cancer Immune Therapy

PLoS ONE, 2008

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly o... more The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.

Research paper thumbnail of Single-gene transgenic mouse strains for reprogramming adult somatic cells

Nature Methods, 2009

We report a novel transgenic mouse model for direct reprogramming of somatic cells by expressing ... more We report a novel transgenic mouse model for direct reprogramming of somatic cells by expressing four reprogramming factors from a single genomic locus using a drug-inducible transgene. Multiple somatic cell types explanted from different tissues can generate induced pluripotent stem cells (iPSC) by culture in doxycycline (Dox). Because the reprogramming factors are carried on a single polycistronic construct the transgenic mice can be easily maintained and transferred into another background.

Research paper thumbnail of Decidual NK cells regulate key developmental processes at the human fetal-maternal interface

Nature Medicine, 2006

Human CD56 bright NK cells accumulate in the maternal decidua during pregnancy and are found in d... more Human CD56 bright NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.

Research paper thumbnail of Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1

Research paper thumbnail of Inhibition of the NKp30 activating receptor by pp65 of human cytomegalovirus

Nature Immunology, 2005

Human cytomegalovirus, a chief pathogen in immunocompromised people, can persist in a healthy imm... more Human cytomegalovirus, a chief pathogen in immunocompromised people, can persist in a healthy immunocompetent host throughout life without being eliminated by the immune system. Here we show that pp65, the main tegument protein of human cytomegalovirus, inhibited natural killer cell cytotoxicity by an interaction with the activating receptor NKp30. This interaction was direct and specific, leading to dissociation of the linked CD3zeta from NKp30 and, consequently, to reduced killing. Thus, pp65 is a ligand for the NKp30 receptor and demonstrates a unique mechanism by which an intracellular viral protein causes general suppression of natural killer cell cytotoxicity by specific interaction with an activating receptor.

Research paper thumbnail of Transgenic mice with defined combinations of drug-inducible reprogramming factors

Nature Biotechnology, 2009

Drug-inducible lentiviruses encoding Oct4, Sox2, Klf4 and c-Myc were used to derive "primary" iPS... more Drug-inducible lentiviruses encoding Oct4, Sox2, Klf4 and c-Myc were used to derive "primary" iPS cells and segregated through germline transmission generating mice and fibroblasts carrying subsets of the reprogramming factors. Drug treatment of the cells resulted in "secondary" iPS cell derivation only when the missing factor was introduced. This creates a defined platform for studying reprogramming mechanisms and allows screening of genetically homogenous cells for compounds that replace any transcription factor required for iPS cell derivation. The generation of induced pluripotent stem (iPS) cells from mouse and human somatic cells through the forced expression of defined transcription factors1-4 constitutes a major breakthrough in regenerative biology5. However, current reprogramming strategies require viral transduction with potentially oncogenic transcription factors. Understanding the molecular changes underlying iPS cell derivation will be required to devise alternative and safer strategies for reprogramming e.g., by replacing the viral transduced factors with small molecules6-8. Screening approaches using infected cells are hampered by the genetic variability caused by the random integrations of multiple proviral copies9,10. Recently, we generated a "secondary" transgenic system that eliminates such heterogeneity9,10. In this approach, mouse embryonic fibroblasts (MEFs) heterozygous for the ROSA26-M2 reverse tetracycline transactivator (M2-rtTA) were infected with Doxycycline (Dox)-inducible lentiviruses carrying the four reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) and induced to generate "primary" iPS cells by addition of Dox. These cells were used to obtain chimeric mice with genetically identical somatic cells that can be isolated and reprogrammed in vitro by addition of Dox. However, such "secondary" somatic cells require isolation from Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Research paper thumbnail of A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

Nature Biotechnology, 2008

The study of induced pluripotency is complicated by the need for infection with high-titer retrov... more The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25-to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming. It has recently been shown that mouse 1-4 and human 5-8 fibroblasts can be reprogrammed to a pluripotent state through retroviral introduction of four transcription factors such as Oct4, Sox2, Klf4 and c-Myc. Reprogramming can also be achieved in the absence of the tumorigenic factor c-Myc, although with decreased efficiency 9,10. Nevertheless, with these approaches only a very small fraction of cells infected with all four factors will eventually reprogram 11. The random viral infection results in genetic heterogeneity in the infected cell culture that likely contributes to the low observed frequency of induced pluripotent stem (iPS) cell formation. Therefore, reprogrammed cells must be selected for by the reactivation of endogenous pluripotency genes 1-3 or based on morphological criteria 11,12. The reprogramming process has been shown to require ~10 to 12 d of sustained transgene expression after viral transduction and to follow a sequential activation of pluripotency markers, with initial activation of alkaline Correspondence should be addressed to R.J.

Research paper thumbnail of Direct cell reprogramming is a stochastic process amenable to acceleration

Nature, 2009

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved... more Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority

Research paper thumbnail of Dissecting direct reprogramming through integrative genomic analysis

Nature, 2008

In both the Methods Summary and the online-only Methods (cell culture section), the concentration... more In both the Methods Summary and the online-only Methods (cell culture section), the concentration of AZA is incorrectly listed as 0.5 mM. The correct final concentration is 0.5 mM.

Research paper thumbnail of Proteomic analysis of human natural killer cells: insights on new potential NK immune functions

Molecular Immunology, 2005

Applying high-throughput proteomic analysis of mammalian cells can facilitate the identification ... more Applying high-throughput proteomic analysis of mammalian cells can facilitate the identification of a large number of proteins expressed in the examined samples. Moreover, extensive research efforts are being made to perform large-scale characterization of membrane proteins. Here we use mass spectrometry-based proteomic strategy to characterize protein expression in membrane-enriched fractions derived from human NK lymphoma cell line YTS. This query yielded a list of over 1000 identified proteins, and provided us with new insights on NK cell biology. We highlight the expression of CD86 on YTS and its ability to co-stimulate TCR responses of human CD4+ T-cells, providing an unexpected link between innate and adaptive immune systems.

Research paper thumbnail of Complexes of HLA-G Protein on the Cell Surface Are Important for Leukocyte Ig-Like Receptor-1 Function

The Journal of Immunology, 2003

The nonclassical class I MHC molecule HLA-G is selectively expressed on extravillous cytotrophobl... more The nonclassical class I MHC molecule HLA-G is selectively expressed on extravillous cytotrophoblast cells at the maternal-fetal interface during pregnancy. HLA-G can inhibit the killing mediated by NK cells via interaction with the inhibitory NK cell receptor, leukocyte Ig-like receptor-1 (LIR-1). Comparison of the sequence of the HLA-G molecule to other class I MHC proteins revealed two unique cysteine residues located in positions 42 and 147. Mutating these cysteine residues resulted in a dramatic decrease in LIR-1 Ig binding. Accordingly, the mutated HLA-G transfectants were less effective in the inhibition of NK killing and RBL/LIR-1 induced serotonin release. Immunoprecipitation experiments demonstrated the involvement of the cysteine residues in the formation of HLA-G protein oligomers on the cell surface. The cysteine residue located at position 42 is shown to be critical for the expression of such complexes. These oligomers, unique among the class I MHC proteins, probably b...

Research paper thumbnail of Pivotal role of CEACAM1 protein in the inhibition of activated decidual lymphocyte functions

Journal of Clinical Investigation, 2002

Lymphocytes in direct contact with embryonic extravillous trophoblasts constitute more than 40% o... more Lymphocytes in direct contact with embryonic extravillous trophoblasts constitute more than 40% of decidual cells and appear to play major roles in implantation and early gestation. A unique subset of NK cells, making up 70-80% of decidual lymphocytes, express high levels of CD56 but lack CD16. We have recently demonstrated a novel class I MHC-independent inhibitory mechanism of NK cell cytotoxicity that is mediated by CEACAM1 homotypic interactions. This mechanism is used by some melanoma cells to avoid attack, mainly by CD16-NK cells. We now demonstrate that CEACAM1 is expressed on primary extravillous trophoblasts and is upregulated on the vast majority of IL-2-activated decidual lymphocytes, including NK, T, and NKT cells. Importantly, we present evidence that CEACAM1 interactions inhibit the lysis, proliferation, and cytokine secretion of activated decidual NK, T, and NKT cells, respectively. In vivo analysis of decidual lymphocytes isolated from cytomegalovirus-infected (CMV-infected) pregnant women revealed a dramatic increase in the expression of CEACAM1. Finally, we suggest that a novel ligand for this adhesion molecule is present on the surface of CMV-infected fibroblasts. These combined results demonstrate a major role for the CEACAM1 protein in controlling local decidual immune responses.

Research paper thumbnail of The involvement of NK cells in ankylosing spondylitis

International Immunology, 2005

A role for NK cells in the regulation of autoimmunity has been demonstrated. Since there is a str... more A role for NK cells in the regulation of autoimmunity has been demonstrated. Since there is a strong association between Ankylosing Spondylitis (AS) and HLA-B27, which is specifically recognized by the NK-inhibitory receptor KIR3DL1, this study evaluated the potential involvement of NK cells in AS. We studied 19 AS patients and 22 healthy volunteer donors and assessed the percentage, activity and receptor expression of peripheral blood NK cells. We also evaluated candidate-inflammatory mediators in sera. We found that AS patients have significantly higher percentages of NK cells. However, we found no differences between the ability of NK cells derived from AS and healthy controls to recognize target cells expressing HLA-B27. Remarkably, we observed that the NK-inhibitory receptor CEACAM1 (carcino-embryonic antigen-cell adhesion molecule) is highly expressed among AS-derived NK cells. Furthermore, engagement of CEACAM1 inhibited NK activity in these patients. Finally, we demonstrated that CEACAM1 expression is induced by IL-8 and SDF-1 (stromal cell derived factor), both of which are present in high levels in the sera of AS patients. These results may indicate that NK cells and CEACAM1 play a role in AS pathogenesis and implicate chemokines in the mechanism of CEACAM1 expression.

Research paper thumbnail of Special organization of the HLA-G protein on the cell surface

Human Immunology, 2003

The human leukocyte antigen G (HLA-G) molecule possesses unique properties such as low polymorphi... more The human leukocyte antigen G (HLA-G) molecule possesses unique properties such as low polymorphism and restricted distribution mainly to the extravillous cytotrophoblast (EVT) cells. The EVT cells vigorously penetrate into the maternal decidual tissues and are found in contact with maternal lymphocytes, mainly with natural killer (NK) cells. The HLA-G molecule inhibits the effector function of maternal NK cells via interaction with the KIR2DL4 and the ILT-2 inhibitory NK receptors. Previously, we have demonstrated that complexes of the HLA-G protein are expressed on the cell surface. We reported that these complexes are formed due to the presence of two unique cysteine residues located at positions 42 and 147. Finally, we demonstrated that efficient binding and function of ILT-2 is dependent on the presence of HLA-G complexes on the cell surface. Here we expand the significance of these observations by revealing that complexes of HLA-G are present on the cell surface using different assays and cell lines and further demonstrate that complexes of HLA-G might be present in a soluble form after interaction with ILT-2. Therefore, the HLA-G molecule has developed a special mechanism to increase the avidity of NK receptors to the HLA-G molecule, which provides better protection for the fetus from maternal NK rejection.

Research paper thumbnail of Biological function of the soluble CEACAM1 protein and implications in TAP2-deficient patients

European Journal of Immunology, 2004

Interactions of natural killer (NK) cells with MHC class I proteins provide the main inhibitory s... more Interactions of natural killer (NK) cells with MHC class I proteins provide the main inhibitory signals controlling NK killing activity. It is therefore surprising to learn that TAP2-deficient patients suffer from autoimmune manifestations only occasionally in later stages of life. We have previously described that the CEACAM1-mediated inhibitory mechanism of NK cytotoxicity plays a major role in controlling NK autoreactivity in three newly identified TAP2-deficient siblings. This novel mechanism probably compensates for the lack of MHC class I-mediated inhibition. The CEACAM1 protein can also be present in a soluble form and the biological function of the soluble form of CEACAM1 with regard to NK cells has not been investigated. Here we show that the homophilic CEACAM1 interactions are abrogated in the presence of soluble CEACAM1 protein in a dose-dependent manner. Importantly, the amounts of soluble CEACAM1 protein detected in sera derived from the TAP2-deficient patients were dramatically reduced as compared to healthy controls. This dramatic reduction does not depend on the membrane-bound metalloproteinase activity. Thus, the expression of CEACAM1 and the absence of soluble CEACAM1 observed in the TAP2-deficient patients practically maximize the inhibitory effect and probably help to minimize autoimmunity in these patients.

Research paper thumbnail of Involvement of the CXCL12/CXCR4 pathway in the advanced liver disease that is associated with hepatitis C virus or hepatitis B virus

European Journal of Immunology, 2004

Chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is accompanied by inflammat... more Chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is accompanied by inflammation and fibrosis eventually leading to cirrhosis. The chemokine CXCL12 is involved in chronic inflammatory conditions. The role of the CXCL12/CXCR4 pathway in HCV-and HBV-associated liver inflammation and fibrosis was therefore studied. The levels and tissue localization of CXCL12 in liver and plasma of HCV and HBV patients were tested using immunohistochemistry and ELISA. The expression and function of CXCR4 on liver-infiltrating lymphocytes (LIL) were tested by FACS and transwell migration assays. We found that CXCL12 is expressed by bile duct epithelial cells in normal liver tissue. Bile duct proliferation and liver fibrosis in chronic HCV and HBV infection result in the anatomical redistribution of CXCL12 in the liver. Moreover, CXCL12 is up-regulated in the endothelium of neo-bloodvessels formed in active inflammatory foci and is significantly elevated, compared with controls, in the plasma of patients with advanced liver fibrosis. Complementing these observations were others indicating that over 50% of LIL express CXCR4 and, in response to CXCL12, migrated and adhered to fibronectin. These observations suggest an important role for the CXCL12/CXCR4 pathway in recruitment and retention of immune cells in the liver during chronic HCV and HBV infection.