Characteristic Surface Markers Research Articles

Myeloid‐Derived Suppressor Cells Limit the Inflammation by Promoting T Lymphocyte Apoptosis in the Spinal Cord of a Murine Model of Multiple Sclerosis

Multiple Sclerosis (MS) is a demyelinating/inflammatory disease of the central nervous system. Relapsing-remitting MS is characterized by a relapsing phase with clinical symptoms and the production of inflammatory cell infiltrates, and a period of remission during which patients recover partially. Myeloid-derived suppressor cells (MDSCs) are immature cells capable of suppressing the inflammatory response through Arginase-I (Arg-I) activity, among other mechanisms. Here, we have identified Arg-I(+) -MDSCs in the spinal cord during experimental autoimmune encephalomyelitis (EAE), cells that were largely restricted to the demyelinating plaque and that always exhibited the characteristic MDSC surface markers Arg-I/CD11b/Gr-1/M-CSF1R. The presence and density of Arg-I(+) -cells, and the proportion of apoptotic but not proliferative T cells, were correlated with the EAE time course: peaked in parallel with the clinical score, decreased significantly during the remitting phase and completely disappeared during the chronic phase. Spinal cord-isolated MDSCs of EAE animals augmented the cell death when co-cultured with stimulated control splenic CD3 T cells. These data point to an important role for MDSCs in limiting inflammatory damage in MS, favoring the relative recovery in the remitting phase of the disease. Thus, the MDSC population should be considered as a potential therapeutic target to accelerate the recovery of MS patients.

Abstract P3-09-04: Exosome-Specific microRNA Signatures in Combination with Characteristic Surface Markers on the Circulating Exosomes Themselves Provide New Insights into the EMT

Abstract Background: Recent discoveries have established that cancer tumors exhibit distinct microRNA (miR) expression profiles compared to normal tissues. Detection of miRs in the peripheral blood of cancer patients is possible due to their high stability. This stability is attributed to encapsulation of the miRs inside microvesicles (MVs) where they are protected from endogenous RNase activity in circulation. The tumor cell-secreted MVs of primary interest are circulating exosomes, a subpopulation of MVs distinguished by their relatively small size: 40 to 100 nm in diameter. The isolation of circulating tumor-derived exosomes from the other cell-secreted MVs in peripheral blood is the critical barrier to successful development of a robust assay for cancer-specific miR signatures. We describe the development of such an assay to: (1) capture/isolate circulating exosomes based on characteristic surface markers that correlate with different cell types, and (2) characterize exosome-specific miR signatures based on these surface markers. Materials and Methods: We apply sequential ultracentrifugation to isolate cell-secreted MVs, followed by asymmetric flow field-flow fractionation to selectively separate exosomes based on their intrinsically small size and characteristic surface markers. Isolated exosomes are then screened for surface markers by selectively capturing them on microarrays printed with antibodies against a library of known cell-surface markers for breast cancer. For the assessment of exosome-specific miR signatures, we have devised an antibody microarray assay that also enables the in situ characterization of miR profiles of the captured exosomes locally by qRT-PCR analysis confined to subarrays of printed spots in 50-µl microwells of a custom-designed, multi-well microarray. Assay development has been carried out using two human cancer cell lines (MCF 7 and MDA MB 231) and a non-malignant cell line (MCF 10a) representing pre-malignant cells and cancer of the breast at different epithelial and mesenchymal states. Results: We have used light scattering and cryo-transmission electron microscopy to characterize the size, size distribution, and morphology of exosomes as a sub-population of the secreted MVs from each of the three cell lines. We have also screened for surface markers by selective capturing the exosome sub-populations derived from these cell lines on antibody microarrays, and correlated these surface markers with the miR content of MVs using qRT-PCR analysis. Discussion: Our results demonstrate that correlating the miR signatures of circulating exosomes with characteristic surface markers on these exosomes leads to robust distinctions between the three cell lines that could not be achieved using either analysis or characterization alone. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-04.

Stem cells in head and neck squamous cell carcinoma

The existence of a small subpopulation of tumourigenic cancer stem cells in the bulk of human head and neck squamous cancers (SCC) has been recognised in recent reports. This subpopulation has self-renewal properties and is responsible for the production of differentiated daughter cells that form the bulk of the tumour. Stem cells in head and neck SCC can be identified functionally using their self-renewal properties, or by their characteristic surface markers. As their resistance to contemporary cancer treatments may eventually lead to the failure of treatment there is an urgent need to better understand their biology with the ultimate goal of developing new diagnostic markers and curative cancer treatments.

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells

BackgroundThe growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Methodology/Principal FindingsHere, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Conclusion/SignificanceOur results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.

Antibody‐immobilized column for quick cell separation based on cell rolling

Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column.

CD44's nuclear road trip

Lee et al. reveal how a characteristic surface marker for cancer stem cells triggers abnormal growth. The protein makes a dash for the nucleus, picking up a couple of accomplices, including the transcription activator STAT3, along the way. ![Figure][1] STAT3 (red) waits in the cytosol (left

Immunomodulatory properties of human periodontal ligament stem cells

Tissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell-based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non-cell contact dependent suppression of PBMNC proliferation in co-cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN-gamma partially suppressed PBMNC proliferation when compared to CMs without IFN-gamma stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF-beta1, hepatocyte growth factor (HGF) and indoleamine 2, 3-dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN-gamma. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667-676, 2009. (c) 2009 Wiley-Liss, Inc.

A Shortcut to Immortality: Rapid Reprogramming with Tissue Cells

A Shortcut to Immortality: Rapid Reprogramming with Tissue Cells

Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells

We have previously shown the simultaneous generation of CD73(+) mesenchymal stromal cells (MSCs) along with CD34(+) hematopoietic cells from human embryonic stem cells (ESCs) when they are cocultured with OP9 murine stromal cells. We investigated whether MSCs can be derived from human ESCs without coculturing with OP9 cells, and if such cells exhibit immunological properties similar to MSCs derived from adult human bone marrow (BM). Our starting populations were undifferentiated human ESCs cultured on Matrigel-coated plates without feeder cells. The differentiated fibroblast-looking cells were tested for expression of MSC markers and their potential for multilineage differentiation. We investigated surface expression of human leukocyte antigen (HLA) molecules on these MSCs before and after treatment with interferon-gamma (IFN-gamma). We also tested the proliferative response of T-lymphocytes toward MSCs and the effects of MSCs in mixed lymphocyte reaction (MLR) assays. We derived populations of MSCs from human ESCs with morphology, cell surface marker characteristics, and differentiation potential similar to adult BM-derived MSCs. Similar to BM-derived MSCs, human ESC-derived MSCs express cell surface HLA class I (HLA-ABC) but not HLA class II (HLA-DR) molecules. However, stimulation with IFN-gamma induced the expression of HLD-DR molecules. Human ESC-derived MSCs did not induce proliferation of T-lymphocytes when cocultured with peripheral blood mononuclear cells. Furthermore, ESC-derived MSCs suppressed proliferation of responder T-lymphocytes in MLR assays. MSCs can be derived from human ESCs without feeder cells. These human ESC-derived MSCs have cell surface markers, differentiation potentials, and immunological properties in vitro that are similar to adult BM-derived MSCs.

Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection

BackgroundHuman embryonic stem (hES) cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC) arise from CD34+ hematopoietic progenitor cells (HPCs) and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection.ResultsUndifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC) were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC). The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs.ConclusionPhenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.

α1β1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

Clonal Isolation and Characterization of Bone Marrow Stromal Cells from Patients with Osteoarthritis

The demand for treatment strategies for damaged musculoskeletal tissue is continuously growing, especially with the increasing number of older people with degenerative diseases of the skeletal system such as osteoarthritis (OA). Because depletion of multipotent cells has been implicated in degenerative joint diseases, cell-based therapies have been proposed for tissue regeneration, especially for cartilage repair. The aim of the present study is to focus on the possibility of deriving and expanding multipotential mesenchymal stem cells (MSCs) from bone marrow samples of patients with OA by characterizing MSCs at the single cell level. Single-cell clonal cultures were established in 96-well plates by limiting dilution of bone marrow stromal cells (BMSCs) from three patients with OA. Fourteen clones were established for subsequent characterization. There was a wide variation in cell doubling times, with the time taken to reach 20 population doublings ranging from 37 days to more than 100 days. The clones were grouped into fast-growing and slow-growing clones. All except one of the fast-growing stem cell clones were tripotential. However, the slow-growing clones showed limited differentiation potential and morphological changes associated with cellular senescence with extended duration in culture. Flow cytometric analysis indicated a strong need to investigate for novel cell-surface characteristic markers of BMSCs because there was no obvious difference in the expression of the selected characteristic BMSC cell surface markers CD29, CD44, CD90, CD105, and CD166 between fast-growing and slow-growing clones. This study has demonstrated the existence of a fast-growing multipotential MSC population from bone marrow samples of patients with OA. Therefore, despite a supposedly smaller stem cell compartment in these patients, we demonstrate here that they can still yield a potentially therapeutically useful source of syngeneic MSCs.

CD83: an update on functions and prospects of the maturation marker of dendritic cells

CD83 is one of the most characteristic cell surface markers for fully matured dendritic cells (DCs). In their function as antigen presenting cells they induce T-cell mediated immune responses. In this review we provide an overview on well described and proposed functions of this molecule as well as on very recent insights and new hypothesis. Already the CD83 messenger RNA processing differs remarkably from the processing of other cellular mRNAs: instead of the usual TAP mRNA export pathway, the CD83 mRNA is exported by the specific CRM1-mediated pathway, utilized only by a minority of cellular mRNAs. On the protein level, two different isoforms of CD83 exist: a membrane-bound and a soluble form. The isoforms are generated by different subsets of cells, including DCs, T-cells and B-cells, and also differ in their biological function. While the membrane-bound CD83 is of immune stimulatory capacity, activates T-cells and is important for the generation of thymocytes, the soluble CD83 has the opposite effect and has an immune inhibitory capacity. Due to its immune inhibitory function, CD83 has great potential for treatment of autoimmune diseases, for organ transplantations, and for immunotherapy, just to name a few examples. Moreover, some viruses prevent recognition by the host's immune system by specifically targeting CD83 surface expression.

Simultaneous generation of CD34 + primitive hematopoietic cells and CD73 + mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells

Human embryonic stem cells (hESCs) have been shown to generate CD34(+) primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73(+) multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34(+) hematopoietic cells and CD73(+) mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73(+) cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34(+) cells are generated. We cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34(+) and CD73(+) cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34(+) cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73(+) cells generated using in vitro differentiation assays. We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34(+) hematopoietic and CD73(+) MSC generation follows a similar pattern. We sorted the CD34(+) cells and showed that they can generate hematopoietic progenitor colonies. Starting with cocultured cells on day 8, and through an enrichment procedure, we also could generate a pure population of MSCs. These hESC-derived MSCs had typical morphological and cell surface marker characteristics of adult bone marrow-derived MSCs, and could be differentiated toward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs. OP9 cells when cocultured with hESCs support simultaneous generation of CD34(+) primitive hematopoietic cells and CD73(+) MSCs from hESCs.

Labelling of human mesenchymal stem cells with indium-111 for SPECT imaging: effect on cell proliferation and differentiation

Stem cell therapy seems to be a new treatment option within cardiac diseases to improve myocardial perfusion and function. However, the delivery and traceability of the cells represent a problem. Radioactive labelling with 111In could be a method for tracking mesenchymal stem cells (MSCs). However, 111In could influence the viability and differentiation capacity of MSCs, which would limit its use. Therefore, the aim of this study was to evaluate the influence of 111In labelling in doses relevant for SPECT imaging in humans on the viability and differentiation capacity of human MSCs. Human MSCs isolated from bone marrow were incubated with 111In-tropolone (15-800 Bq/cell). The labelling efficiency was approximately 25% with 30 Bq/cell 111In. The MSC doubling time was 1.04+/-0.1 days and was not influenced by 111In within the range 15-260 Bq/cell. Using 30 Bq 111In/cell it was possible to label MSCs to a level relevant for clinical scintigraphic use. With this dose, 111In had no effect on characteristic surface and intracellular markers of cultured MSCs analysed both by flow cytometry and by real-time polymerase chain reaction. Further, the labelled MSCs differentiated towards endothelial cells and formed vascular structures. It is possible to label human MSCs with 111In for scintigraphic tracking of stem cells delivered to the heart in clinical trials without affecting the viability and differentiation capacity of the MSCs. This creates an important tool for the control of stem cell delivery and dose response in clinical cardiovascular trials.

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy

BackgroundMany novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS.ResultsUndifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation.ConclusionPhenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection. Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated.

1046. Derivation of Functionally Normal Macrophages from Human Embryonic Stem (hES) Cells for HIV-1 Gene Therapy

Human embryonic stem cells (hES cells) and their derivatives can be utilized for many promising novel therapies. As a first step towards developing stem cell based gene therapy for AIDS using embryonic stem cells, here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages. Normal and lentiviral vector transduced hES cells were differentiated into CD34 hematopoietic progenitor cells by culturing on S17 mouse bone marrow stromal cell layers. The hES-CD34 cells were cultured in cytokine media that promotes macrophage differentiation. Our results have shown differentiation of hES-CD34 cells into phenotypically normal macrophages that displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR. Studies evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation suggested that these macrophages are also functionally normal. When challenged with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells that expressed the transgene GFP also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. These results demonstrated for the first time that functionally normal mature macrophages can be obtained from hES cells and that they can be used in infectious disease research. Now it is possible to test the utility of hES-CD34 cells for anti-HIV gene transduction and evaluation of the differentiated cells such as macrophages for anti- viral resistance in a gene therapy setting. Human embryonic stem cells (hES cells) and their derivatives can be utilized for many promising novel therapies. As a first step towards developing stem cell based gene therapy for AIDS using embryonic stem cells, here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages. Normal and lentiviral vector transduced hES cells were differentiated into CD34 hematopoietic progenitor cells by culturing on S17 mouse bone marrow stromal cell layers. The hES-CD34 cells were cultured in cytokine media that promotes macrophage differentiation. Our results have shown differentiation of hES-CD34 cells into phenotypically normal macrophages that displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR. Studies evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation suggested that these macrophages are also functionally normal. When challenged with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells that expressed the transgene GFP also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. These results demonstrated for the first time that functionally normal mature macrophages can be obtained from hES cells and that they can be used in infectious disease research. Now it is possible to test the utility of hES-CD34 cells for anti-HIV gene transduction and evaluation of the differentiated cells such as macrophages for anti- viral resistance in a gene therapy setting.

757. Engineered Expression of Novel TRIM5 Isoforms Inhibits HIV-1 Infection in CD34 Hematopoietic Stem Cell Derived Macrophages

Top of pageAbstract Species specific innate resistance against viral infections offers novel avenues for antiviral therapeutic and prophylactic approaches. The retroviral and lentiviral restriction factors Ref1 and Lv1 are variants of the tripartite motif protein, TRIM5a, a component of cytoplasmic bodies. TRIM5a severely restricts productive retroviral infections at the level of post-entry and pre-integration by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5arh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5arh via a lentiviral vector in a permissive cell culture line, Magi-CXC4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5arh transgene was stable introduced into CD34+ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5arh expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5arh were also found to be phenotypically and functionally normal expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHCII, and B7.1. Based on these promising results, we sought to further improve the TRIM5a inhibiting action by designing more novel constructs by modifying the native human isoform of TRIM5a. These modified isoforms have also been shown to be effective in conferring viral resistance. These results demonstrate that the species specific restriction factor TRIM5arh is effective in conferring HIV-1 resistance in a stem cell setting thus paving the way for its application in AIDS gene therapy.

TRIM5αrh expression restricts HIV-1 infection in lentiviral vector-transduced CD34+-cell-derived macrophages

Species-specific innate resistance against viral infections offers novel avenues for antiviral therapeutic and prophylactic approaches. The retroviral and lentiviral restriction factors Ref1 and Lv1 are variants of the tripartite motif protein TRIM5α, a component of cytoplasmic bodies. TRIM5α severely restricts productive retroviral infections at the postentry and preintegration steps by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5αrh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5αrh via a lentiviral vector in a permissive cell culture line, Magi-CXCR4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5αrh transgene was stably introduced into CD34+ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5αrh-expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5αrh were also found to be phenotypically and functionally normal, expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHC II, and B7.1. These results demonstrate that the species-specific restriction factor TRIM5αrh is effective in conferring HIV-1 resistance in a stem cell setting, thus paving the way for its application in AIDS gene therapy.

Sources of interferon-gamma (IFN- γ) in early immune response to Listeria monocytogenes

Early, innate production of interferon- γ (IFN- γ) is a critical step in immunological defense against certain pathogens such as intracellular bacteria (e.g. Listeria monocytogenes), viruses and fungi. While activated T cells and activated natural killer (NK) cells were initially thought to be the only relevant source of IFN- γ, macrophages (M Φ) and dendritic cells can also be stimulated to produce IFN- γ in vitro under certain conditions. However, a convincing analysis at single cell level of the source(s) of IFN- γ in the early immune response to an acute bacterial infection is still missing. In the light of controversial literature, the work presented here aimed to clarify the role of NK cells and other components of the innate cellular immune system in the early IFN- γ production, thereby avoiding in vitro artifacts whenever possible. Immunocompetent C57BL/6 (wild type (WT)) and T and B cell-deficient C57BL/6 rag-1 −/− (RAG) mice were infected intravenously with a pathogenic strain of L. monocytogenes. Leukocyte populations of spleen and liver were discriminated by characteristic surface markers and analyzed for intracellular interleukin (IL)-12 and IFN- γ using flow cytometry. These cells have not been restimulated in vitro nor sorted before analysis. In RAG mice, at least, a large NK1.1 + cell population produced IFN- γ 19 h p.i. No MHC class II + population co-expressed intracellular IFN- γ at this time point. For comparison with the immunocompetent situation, syngeneic WT mice were also infected and sacrificed 9, 19, and 29 h later. At 9 h p.i., the situation resembled that of uninfected mice. At 19 and 29 h p.i. it was again the NK1.1 + population that contained most of the IFN- γ-positive events. MHC II + CD19 − M Φ/dendritic cells and MHC II + CD19 + B cells did not co-express intracellular IFN- γ at these time points. CD3 + T cells were also found to contain intracellular IFN- γ; most were also CD8 + and some CD4 +. These results indicate that after infection of C57BL/6 mice with L. monocytogenes, NK1.1 + cells and, to a lesser extent, CD3 + cells are the prominent sources of innate IFN- γ. MHC II + cells do not play a significant role in the early IFN- γ production following an acute primary bacterial infection.