GPI-anchored Molecules Research Articles

Mesenchymal Stem Cells Directly Interact with Breast Cancer Cells and Promote Tumor Cell Growth In Vitro and In Vivo

Cellular interactions were investigated between human mesenchymal stem cells (MSC) and human breast cancer cells. Co-culture of the two cell populations was associated with an MSC-mediated growth stimulation of MDA-MB-231 breast cancer cells. A continuous expansion of tumor cell colonies was progressively surrounded by MSC(GFP) displaying elongated cell bodies. Moreover, some MSC(GFP) and MDA-MB-231(cherry) cells spontaneously generated hybrid/chimeric cell populations, demonstrating a dual (green fluorescent protein+cherry) fluorescence. During a co-culture of 5-6 days, MSC also induced expression of the GPI-anchored CD90 molecule in breast cancer cells, which could not be observed in a transwell assay, suggesting the requirement of direct cellular interactions. Indeed, MSC-mediated CD90 induction in the breast cancer cells could be partially blocked by a gap junction inhibitor and by inhibition of the notch signaling pathway, respectively. Similar findings were observed in vivo by which a subcutaneous injection of a co-culture of primary MSC with MDA-MB-231(GFP) cells into NOD/scid mice exhibited an about 10-fold increased tumor size and enhanced metastatic capacity as compared with the MDA-MB-231(GFP) mono-culture. Flow cytometric evaluation of the co-culture tumors revealed more than 90% of breast cancer cells with about 3% of CD90-positive cells, also suggesting an MSC-mediated in vivo induction of CD90 in MDA-MB-231 cells. Furthermore, immunohistochemical analysis demonstrated an elevated neovascularization and viability in the MSC/MDA-MB-231(GFP)-derived tumors. Together, these data suggested an MSC-mediated growth stimulation of breast cancer cells in vitro and in vivo by which the altered MSC morphology and the appearance of hybrid/chimeric cells and breast cancer-expressing CD90(+) cells indicate mutual cellular alterations.

Neuritin promotes the expansion and persistence of regulatory T cells in vitro and in vivo. (P1051)

Abstract Regulatory T cells (Tregs) represent a major force for immune control and the prevention of excessive or self-directed responses. These cells are marked by expression of the transcription factor Foxp3 and their ability to suppress leukocyte activation. Deficits in Treg function or numbers result in immune mediated pathologies, reflecting their importance as a layer of immune regulation. However the factors and mechanisms responsible for maintaining these cells are incompletely understood. Here, we report that a GPI-anchored molecule known as neuritin is highly and uniquely expressed by Treg and this molecule potently drives expansion and persistence of the ‘Treg-pool’. Transgenic expression of neuritin by Tregs enhanced their division in vitro while treatment with anti-neuritin antibody stunted Treg proliferation. Further suggesting a role in maintaining the Treg population, neuritin transgenic mice displayed expedited recovery in the EAE model of autoimmune disease accompanied by elevated frequencies of Foxp3+ Tregs. Conversely, antibody mediated neuritin blockade prolonged disease and stunted proliferation of Foxp3+ cells during EAE. Further dissection of neuritin activity revealed the involvement of paradoxically enhanced IL-2 production by this Treg specific factor. In all these findings strongly implicate neuritin as a Treg derived factor responsible for the persistence of these cells and their control of immune responses.

The role of surface glycoconjugates inLeishmaniamidgut attachment examined by competitive binding assays and experimental development in sand flies

Binding of promastigotes to the sand fly midgut epithelium is regarded as an essential part of the Leishmania life cycle in the vector. Among Leishmania surface molecules putatively involved in attachment to the sand fly midgut, two GPI-anchored molecules are the most prominent: lipophosphoglycan (LPG) and promastigote surface protease gp63. In this work, we examined midgut attachment of Leishmania lines mutated in GPI-anchored molecules and compared results from 2 different techniques: in vivo development in sand flies and in vitro competitive binding assays using fluorescently labelled parasites. In combination with previous studies, our data provide additional support for (1) an LPG-independent parasite-binding mechanism of Leishmania major within the midgut of the permissive vector Phlebotomus perniciosus, and provide strong support for (2) the crucial role of L. major LPG in specific vector Phlebotomus papatasi, and (3) a role for Leishmania amazonensis gp63 in Lutzomyia longipalpis midgut binding. Moreover, our results suggest a critical role for GPI-anchored proteins and gp63 in Leishmania mexicana attachment to L. longipalpis midguts, as the wild type (WT) line accounted for over 99% of bound parasites.

Stable incorporation of functionally active purified GPI-anchored immunostimulatory molecules on H5 influenza VLP surfaces by protein transfer. (113.33)

Abstract H5N1 influenza, a bilayered lipid enveloped virus, is highly pathogenic and can lead to a pandemic threat; therefore, a potent vaccine is required. The use of virus-like particles (VLPs) as a vaccine has recently gained importance as they are safe and highly immunogenic. VLPs are similar in size and structure to their viral counterparts but do not contain the viral genome required for replication. Although VLPs are immunogenic, full protection and cross-reactivity to other influenza strains does not always occur; therefore, we aim to modify H5 influenza VLPs to incorporate immunostimulatory molecules (ISMs) by using a protein transfer method. Previously, we have shown that GPI-anchored ISMs (ICAM-1 and IL-12) are able to incorporate onto H5 influenza VLPs by a simple and easy protein transfer method. In this study, we show that purification of GPI-IL-12 does not alter the functionality of the protein in stimulating proliferation of ConA-activated splenocytes suggesting that purified GPI-ISMs remain functionally active. Furthermore, the detection of incorporated GPI-ISMs onto the surface of H5 influenza VLPs by protein transfer was conducted by using immunogold staining electron microscopy and incorporation occurred uniformly as detected by flow cytometry analysis. Moreover, these GPI-ISMs remain stably expressed on the VLP surface for at least a week at 4oC. This study suggests that H5 influenza VLPs can be modified to stably express ISMs on its surface by protein transfer.

Abstract 3537: Manipulation of local and systemic immune suppression by GPI-anchored immune stimulatory proteins

Abstract Each day in the United States alone, approximately 3400 people are diagnosed while another 1500 people die from cancer. Breast cancer is the most common form of cancer among females and accounts for the second leading cause of death among female cancer patients. In order to be effective in a therapeutic setting, current immunotherapies must be able to overcome the multiple layers of tumor-induced immune suppression such as modulation of MHC expression and costimulatory molecules (CD80/CD86), expression of inhibitory molecules PDL-1 and GITRL and the secretion of immunosuppressive factors such as VEGF and TGF-β into the tumor microenvironment. These immunosuppressive factors promote the recruitment, differentiation and expansion of regulatory immune cells such as myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) into the tumor microenvironment. Using the 4T07 murine breast cancer model we investigated the effects of expressing GPI-anchored immune stimulatory molecules (GPI-ISMs), namely cytokines (IL-2, IL-12) and the costimulatory protein B7-1, on the surface of the tumor cells. BALB/c mice were challenged subcutaneously (s.c.) with either wild-type 4T07 cells (4T07-WT) or 4T07 cells expressing GPI-ISMs. We observed significant splenomegaly in the mice challenged with 4T07-WT cells relative to the mice challenged with 4T07 cells expressing GPI-ISMs. This observed splenomegaly correlated with tumor size and a 4-5 fold increase in the percentage of splenic CD11b+Gr1+ MDSCs indicating the role of active immune suppression in the tumorigenicity of 4T07 breast cancer cells. We then conducted studies to analyze the effect of GPI-ISMs on infiltrating cells into the tumor microenvironment as well as in the spleen and draining lymph nodes (dLNs). Three groups of mice were challenged (s.c.) with the following cells mixed in a 1:1 ratio with BD Matrigel™ (a solubilized basement membrane preparation derived from a mouse sarcoma): 4T07-WT, 4T07-B7/IL-12 or PBS (control). Seven days post challenge, the Matrigel/tumor, spleen and dLNs were harvested from the mice, digested and analyzed for cellular infiltrates by flow cytometry. We observed that the expression of GPI-ISMs on the surface of tumor cells led to reduced angiogenesis as evidenced by a reduced level of blood vessels and decreased presence of CD4+CD25+FOXP3+ regulatory T cells and CD11b+Gr1+ MDSCs locally at the tumor site and dLNs as well as systemically in the spleen. Additionally, there was a decrease in CD8+PD1+ exhausted T cells at the tumor site. Along with the inhibition of immune suppressive cell populations, the GPI-ISMs increased the presence of CD4+ and CD8+ T cells as well as dendritic cells and B cells. These observations suggest that components of the active immune suppression evident in this model can be inhibited by expressing GPI-ISMs on the surface of the 4T07 tumor cells and could be effective in a therapeutic setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3537. doi:1538-7445.AM2012-3537

3PT172 ラフト親和性GPIアンカー型分子プリオンタンパク質の神経細胞膜でのダイナミクス(日本生物物理学会第50回年会(2012年度))

3PT172 ラフト親和性GPIアンカー型分子プリオンタンパク質の神経細胞膜でのダイナミクス(日本生物物理学会第50回年会(2012年度))

The Mouse IAPE Endogenous Retrovirus Can Infect Cells through Any of the Five GPI-Anchored EphrinA Proteins

The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family –but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.

The mouse IAPE endogenous retrovirus can infect cells through any of the five GPI-anchored EphrinA proteins

The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family –but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.

Receptor-like Protein-tyrosine Phosphatase α Enhances Cell Surface Expression of Neural Adhesion Molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

Adjuvanting influenza virus-like particles with glycophosphatidylinosital-anchored immunostimulatory molecules by protein transfer to enhance antiviral immune responses (106.14)

Abstract The effortless nature through which influenza, a bilayered lipid enveloped virus, can spread via an aerosol route and cause mortality is distressing. The use of virus-like particles (VLPs), that consist of a virus’ capsid proteins but not the viral genome required for replication, as vaccine candidates proves beneficial due to their safe nature and structural similarity with the live virus. In this study, we show that the incorporation of GPI-anchored immunostimulatory molecules onto H5 influenza VLPs can be accomplished through a quick, easy and stable protein transfer method. Affinity chromatography was used to purify the GPI-proteins (ICAM-1 and IL-12) from CHO-K1 cell lysates and incorporation onto the VLPs was determined by western blot analysis. To verify that incorporation occurred specifically via the GPI-anchor, PI-PLC treatment before and after incorporation and inhibition by fatty-acid binding proteins was carried out. Finally, electron microscopy was used to determine whether the structural integrity of the VLPs was altered following protein transfer. Our results indicate that incorporation of both GPI-proteins onto influenza H5 VLPs occurred following a 2 hour incubation at 37°C without disturbing the structural integrity of the VLPs. Therefore, this protein transfer method can be used to enhance the immunogenicity of VLPs easily and quickly to then be used as a potential influenza vaccine.

IgLONs form heterodimeric complexes on forebrain neurons

IgLONs are a family of four GPI-anchored cell adhesion molecules that regulate neurite outgrowth and synaptogenesis and may act as tumour suppressor genes. Recently we have proposed that two members of the IgLON family act as a heterodimeric complex termed DIgLON. Neurons isolated from chick forebrain co-express all six combinations of IgLONs and the intensity of fluorescence for each pair of IgLONs was highly correlated. Antibody-patching experiments on forebrain neurons show complex formation for IgLON pairs but not between unrelated GPI-anchored glycoproteins. Thus IgLONs are the first GPI-anchored family of glycoproteins shown to form heterodimeric complexes in the plane of the membrane.

Influence of Quantum Dot Labels on Single Molecule Movement in the Plasma Membrane

Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues in labeling single molecules with QDs (and other particles e.g. gold particles) are induction of cross-linking of the target molecules, which can cause activation of signaling pathways or reduced mobility, and steric hindrance as a result of the probe size. Cross-linking can be a result of the multivalent functionalization tag (e.g. streptavidin (sAv)) or the presence of multiple mono- or multivalent functionalization tags per QD. In this work, we have compared commercially available sAv-QDs of different sizes with custom prepared Co enzyme A (CoA)-QDs both targeting a GPI-anchored protein modified with either a biotin ligase acceptor peptide (BLAP) or an acyl carrier protein (ACP) tag, respectively. Trajectories of the differently labeled GPI-anchored molecules were recorded simultaneously in dual-color experiments at rates of ∼25 -∼1500 Hz. Knowing the effect of different labels is of utmost importance for simultaneous investigations of different plasma membrane species in order to discriminate the effect of the label from differences in movement of the target molecules.

Activation and Control of CNS Innate Immune Responses in Health and Diseases: A Balancing Act Finely Tuned by Neuroimmune Regulators (NIReg)

Innate immunity is an arsenal of molecules and receptors expressed by professional phagocytes, glial cells and neurons and involved in host defence and clearance of toxic and dangerous cell debris. However, any uncontrolled innate immune responses within the central nervous system (CNS) are widely recognized as playing a major role in the development of autoimmune disorders and neurodegeneration, with multiple sclerosis (MS) and Alzheimer's diseases (AD) being primary examples. Critically, neuroimmune regulatory proteins (NIReg) may control the adverse immune responses in health and diseases. NIRegs are found mainly on neurons, glia, endothelia and ependymal cells and include GPI-anchored molecules (CD24, CD90, complement regulators CD55 and CD59), molecules of the immunoglobulin superfamily (siglec CD22, Siglec 10, CD200, ICAM-5) and others (CD47, fractalkine, TAM receptor tyrosine kinase and complement C3a and factor H). These regulators modulate the innate immune response in the CNS and for instance critically control the level of phagocytosis and inflammation engaged by resident microglia and infiltrating immune cells. Others will sequester and neutralize proinflammatory molecules such as HMGB1 and DNA. Moreover, some NIRegs can instigate the recruitment of stem cells to mediate tissue repair. In the absence of these regulators, when neurons die by apoptosis, become infected or damaged, microglia and infiltrating immune cells are free to cause injury and an adverse inflammatory response in acute and chronic settings. The therapeutic applications of NIRegs should be exploited given their natural and selective healing properties.

DIgLONs inhibit initiation of neurite outgrowth from forebrain neurons via an IgLON-containing receptor complex

IgLONs are a family of four GPI-anchored cell adhesion molecules that regulate neurite outgrowth, synaptogenesis and may act as tumour suppressor genes. IgLONs are thought to function as monomers or homodimers and we have proposed that IgLONs also act as heterodimeric complexes termed Dimeric IgLONs or DIgLONs. Here we show that the initiation of neurite outgrowth is inhibited from a subset of chick embryonic day (E) 7 or 8 forebrain neurons when they are cultured on CHO cell lines expressing DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP but not on CHO cells that express single IgLONs CEPU-1 or OBCAM. Surprisingly at the younger age of E6 forebrain neurons do not respond to DIgLONs. Since there is little difference in expression of IgLONs on the surface of chick forebrain neurons at these two ages we suggest IgLONs alone are not the receptor on the responding forebrain neurons. A DIgLON heterodimeric recombinant protein DIgLON:CEPU-1-OBCAM-Fc also blocked neurite outgrowth from E8 chick forebrain neurons. However, when IgLONs were removed from the surface of these E8 neurons they no longer responded to DIgLON:CEPU-1-OBCAM-Fc substrate, indicating that IgLONs form at least a component of the neuronal cell receptor complex involved in this inhibition of neurite outgrowth. Inhibitors pertussis toxin and Y27632 reversed the inhibition of neurite outgrowth on a DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP substrate. This suggests the involvement of a G-protein coupled receptor and activation of Rho A. In summary we provide evidence that DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP complexes regulate initiation of neurite outgrowth on forebrain neurons via an IgLON-containing receptor complex.

Altered GPI modification of insect AChE improves tolerance to organophosphate insecticides

The olive fruit fly Bactrocera oleae is the most destructive and intractable pest of olives. The management of B. oleae has been based on the use of organophosphate (OP) insecticides, a practice that induced resistance. OP-resistance in the olive fly was previously shown to be associated with two mutations in the acetylcholinesterase (AChE) enzyme that, apparently, hinder the entrance of the OP into the active site. The search for additional mutations in the ace gene that encodes AChE revealed a short deletion of three glutamines (Δ3Q) from a stretch of five glutamines, in the C-terminal peptide that is normally cleaved and substituted by a GPI anchor. We verified that AChEs from B. oleae and other Dipterans are actually GPI-anchored, although this is not predicted by the “big-PI” algorithm. The Δ3Q mutation shortens the unusually long hydrophilic spacer that follows the predicted GPI attachment site and may thus improve the efficiency of GPI anchor addition. We expressed the wild type B. oleae AChE, the natural mutant Δ3Q and a constructed mutant lacking all 5 consecutive glutamines (Δ5Q) in COS cells and compared their kinetic properties. All constructs presented identical K m and k cat values, in agreement with the fact that the mutations did not affect the catalytic domain of the enzyme. In contrast, the mutants produced higher AChE activity, suggesting that a higher proportion of the precursor protein becomes GPI-anchored. An increase in the number of GPI-anchored molecules in the synaptic cleft may reduce the sensitivity to insecticides.

IgSF molecule MDGA1 is involved in radial migration and positioning of a subset of cortical upper‐layer neurons

Mdga1, encoding a GPI-anchored immunoglobulin superfamily molecule containing an MAM domain, is expressed by a specific subset of neurons, including layer II/III projection neurons, in the mouse neocortex. To investigate the function of Mdga1 in corticogenesis, we generated Mdga1-deficient mice and backcrossed them to obtain a congenic background. Gross anatomy of the Mdga1-deficient brain at postnatal day (P) 14 showed no obvious phenotype. However, the migration of Mdga1-mutant neurons to the superficial cortical plate was clearly delayed. Most Mdga1-mutant neurons reached the lower portion of the upper cortical layer by embryonic day 18.5 and stayed there through P0. By P7, the location of the mutant cells was the same as wild-type. The location of Cux2-expressing upper-layer neurons in the cortical plate was largely unaffected. These observations indicated that Mdga1 is involved in the migration and positioning of a subset of cortical neurons and suggested that the radial migration of upper-layer neurons might be differentially regulated.

Abstract 2380: Carcinoembryonic antigen (CEA) directly activates endothelial cells, thereby inducing (tumor)-angiogenesis

Abstract Angiogenesis has become a major target in treatment of a varietly of cancers, whereby the major angiogenic growth factor VEGF is currently in focus for therapeutic interventions. Nevertheless, the use of VEGF-targeting drugs has been shown to be of limited efficacy, which might lie in the fact that tumor angiogenesis is not only induced by VEGF, but also by a variety of other factors. In this study, we report that the human carcinoembryonic antigen (CEA), a GPI-anchored cell surface adhesion molecule, which is shedded by 70% of human cancers, directly binds to endothelial cells, thereby inducing angiogenic endothelial cell behavior in vitro and in vivo: Overexpressing CEA in endogenous negative renal cell cancer (RCC) cell line or in the sarcoma cell line MethA led to an enhanced tumor angiogenesis and an increase in tumor growth in vivo, effects which were reverted by downregulation of the CEA receptor in endothelial cells via a lentiviral shRNA construct. Furthermore, targeting CEA by CEA mimotope immunized BALB/c mice, led to a breakdown In tumor vascularization, which was reflected by retarded tumor growth. We found that CEA, by binding to its receptor, led to an increase in integrin activation state, leading to enhanced endothelial cell adhesion, followed by amplified adhesion-induced signal transduction, which involved c-src-activation, focal adhesion kinase (FAK) and ERK phosphorylation, as well as PI3kinase/Akt activation. As a consequence, pro-angiogenic endothelial cell behavior, such as migration and invasion or proliferation were induced. Whenever integrin beta-3 were blocked, CEA was effect less in endothelial cells. These results show that the tumor marker CEA might bear hitherto undescribed biological significance in promoting tumor-angiogenesis. Thus, CEA-mediated signaling may offer a potential novel therapeutic target for the treatment of cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2380.

Aberrantly expressed CEACAM6 is involved in the signaling leading to apoptosis of acute lymphoblastic leukemia cells

The aberrant expression of myeloid antigens on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon. So far, there have been no reports of a functional consequence of this aberrant expression. The granulocytic marker carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c) is a GPI-anchored molecule that is reported to be the most frequently aberrantly expressed myeloid marker in ALL with a strong correlation with genotype. We mimicked CEACAM6 signaling in ALL cells by cross-linking with anti-CEACAM6 antibody. Next, we measured a response to CEACAM6 signaling by integrin subunits expression, integrin ligand binding, phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt, and p38 mitogen-activated protein kinase (MAPK) and apoptosis by flow cytometry. Following CEACAM6 cross-linking in ALL cells, we detected Erk1/2, Akt, and p38 MAPK phosphorylation and integrin upregulation, as well as enhanced binding of integrin ligands (vascular cell adhesion molecule-1 [VCAM-1] and intercellular cell adhesion molecule-1 [ICAM-1]). However, CEACAM6 signaling resulted in an increase in apoptosis, unlike other GPI-anchored molecules, such as CD24. The present study is the first to demonstrate the functional consequences of CEACAM6 cross-linking in B-cell precursor ALL cells.

Trypanosoma cruzi and its gp83 ligand modulate host thrombospondin 1 and laminin γ‐1 gene networks to recruit trypanosomes in the early infection process

Trypanosoma cruzi, the protozoan that causes Chagas heart disease, uses the extracellular matrix to facilitate infection of human cells. However, the mechanisms underlying this process are unknown. Here we show that T. cruzi gp83 ligand, a GPI‐anchored molecule that the parasite uses to attach to host cells, is cleaved by the trypanosome PLC. The cleaved gp83 signals the host cell to increase laminin γ‐1 expression to facilitate the initial recruitment of trypanosomes to the extracellular matrix. Then, the recruited trypanosomes up‐regulate the expression of thrombospondin 1 (TSP1), which also enhances the initial infection process. The over‐expression of laminin γ‐1 and TSP1 in cells enhances trypanosome infection whereas silencing of laminin γ‐1 and TSP1 significantly reduces the trypanosome infection. We have determined the laminin γ‐1 and TSP1 specific gene networks that interact within the human protein interactome during the early infection process. We conclude that T. cruzi uses different strategies to manipulate the extracellular matrix to facilitate the initial infection process (Supported by NIH grants AI080580‐03, AI007281‐21 and AI083925‐01).

Expression of human FcγRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding

The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcγRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcγRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcγRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcγRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcγRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcγRIII and the cell lines were shown to have slightly higher levels of receptor than FcγRIII-positive peripheral blood mononuclear cells. Because the affinity of FcγRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcγRIIIa-158V–antibody interaction is 3- to 4-fold stronger that the interaction with FcγRIIIa-158F. Overall, these robust assays should be valuable for batch-testing clinical material as well as providing tools for improving the design of therapeutic IgG.