High-Efficiency Gene Transfer into Normal and Adenosine Deaminase-Deficient T Lymphocytes Is Mediated by Transduction on Recombinant Fibronectin Fragments

Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3⁺ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.     

RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication.

A key feature of the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection is the gradual loss of CD4-positive T cells. A number of gene therapy strategies have been designed with the intent of inhibiting HIV replication in mature T cells. As T cells are products of hematolymphoid differentiation, insertion of antiviral genes into hematopoietic stem cells could serve as a vehicle to confer long-term protection in progeny T cells derived from transduced stem cells. One such "cellular immunization" strategy utilizes the gene coding for the HIV-1 rev trans-dominant mutant protein RevM10 which has been demonstrated to inhibit HIV-1 replication in T-cell lines and in primary T cells. In this study, we used a Moloney murine leukemia virus-based retrovirus encoding a bicistronic message coexpressing RevM10 and the murine CD8-alpha' chain (Lyt2). This vector allows rapid selection of transgene-expressing cells as well as quantitation of transgene expression. We demonstrate that RevM10-transduced CD34-enriched hematopoietic progenitor-stem cells (HPSC) isolated from human umbilical cord blood or from granulocyte colony-stimulating factor-mobilized peripheral blood can give rise to mature thymocytes in the SCID-hu thymus/liver mouse model. The phenotypic distribution of HPSC-derived thymocytes is normal, and expression of the transgene can be detected by flow cytometric analysis. Moreover, we demonstrate that RevM10 can inhibit HIV replication in T cells derived from transduced HPSC after expansion in vitro. This is the first demonstration of anti-HIV efficacy in T cells derived from transduced human HPSC.     

Identification of a Human Immunodeficiency Virus Type 2 (HIV-2) Encapsidation Determinant and Transduction of Nondividing Human Cells by HIV-2-Based Lentivirus Vectors

Although previous lentivirus vector systems have used human immunodeficiency virus type 1 (HIV-1), HIV-2 is less pathogenic in humans and is amenable to pathogenicity testing in a primate model. In this study, an HIV-2 molecular clone that is infectious but apathogenic in macaques was used to first define cis-acting regions that can be deleted to prevent HIV-2 genomic encapsidation and replication without inhibiting viral gene expression. Lentivirus encapsidation determinants are complex and incompletely defined; for HIV-2, some deletions between the major 5′ splice donor and the gag open reading frame have been shown to minimally affect encapsidation and replication. We find that a larger deletion (61 to 75 nucleotides) abrogates encapsidation and replication but does not diminish mRNA expression. This deletion was incorporated into a replication-defective, envelope-pseudotyped, three-plasmid HIV-2 lentivirus vector system that supplies HIV-2 Gag/Pol and accessory proteins in trans from an HIV-2 packaging plasmid. The HIV-2 vectors efficiently transduced marker genes into human T and monocytoid cell lines and, in contrast to a murine leukemia virus-based vector, into growth-arrested HeLa cells and terminally differentiated human macrophages and NTN2 neurons. Vector DNA could be detected in HIV-2 vector-transduced nondividing CD34⁺ CD38⁻ human hematopoietic progenitor cells but not in those cells transduced with murine vectors. However, stable integration and expression of the reporter gene could not be detected in these hematopoietic progenitors, leaving open the question of the accessibility of these cells to stable lentivirus transduction.     

High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells.

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.     

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34⁺ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1⁺ lin⁻ primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.     

Transduction of CD34+ hematopoietic progenitor cells with an antitat gene protects T-cell and macrophage progeny from AIDS virus infection.

Transduction of hematopoietic stem cells with genes that inhibit human immunodeficiency virus (HIV) replication has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an autoregulated gene, antitat, to inhibit replication of simian immunodeficiency virus (SIV) and HIV type 1 (HIV-1) in hematopoietic cells derived from transduced progenitor cells. The antitat gene expresses an antiviral RNA encoding polymeric Tat activation response elements in combination with an antisense tat moiety under the control of the HIV-1 long terminal repeat. CD34+ hematopoietic progenitor cells were transduced with a retroviral vector containing the antitat gene and then cultured under conditions that support in vitro differentiation of T cells or macrophage-like cells. Rhesus macaque CD4+ T cells and macrophage-like cells derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV, reflecting a 2- to 3-log reduction in peak SIV replication compared with controls. Similarly, human CD4+ T cells derived from CD34+ cord blood cells transduced with antitat were also resistant to infection with HIV-1. No evidence for toxicity of the antitat gene was observed in any of five different lineages derived from transduced hematopoietic cells. These results demonstrate that a candidate therapeutic gene introduced into hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication following T-cell differentiation and support the potential use of the antitat gene for stem cell gene therapy.     

In Vivo Pathogenesis of a Human Immunodeficiency Virus Type 1 Reporter Virus

Our understanding of human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis is hampered by the inability to detect HIV-1 gene expression in infected viable cells. In this report, we describe two HIV-1 reporter constructs that are replication competent and cytopathic in vivo. These constructs contain DNA regions of two different lengths that bear the cDNA for the murine heat-stable antigen in the vpr region of a CXCR4-tropic virus. We used the SCID-hu mouse model and these reporter viruses to perform detailed kinetic studies of HIV-1 infection of human thymocytes in vivo. We document that the CD4⁺/CD8⁺ thymocytes are the first to express virus and that this subset demonstrates the most rapid and extensive HIV-1-induced cell depletion. Following depletion of this subset, subsequent virus expression occurs predominantly in phenotypically CD4⁻ cells, suggesting that CD4 down-regulation occurs in HIV-1-infected thymocytes in vivo. These results demonstrate the utility of these HIV-1 reporter constructs to monitor HIV pathogenesis in vitro and in vivo.     

Lentiviral vectors encoding human MUC1-specific,MHC-unrestricted single-chain TCR and a fusion suicide gene: potential for universal and safe cancer immunotherapy

MUC1 tumor antigen is a target for immunotherapy of most human adenocarcinomas and some hematological malignancies. Expression of a MUC1-specific, MHC-unrestricted single-chain T cell receptor (scTCR) on cells of both innate and adaptive immune system through reconstitution of lethally irradiated mice by retroviral vector-transduced bone marrow cells, had been shown to effectively control the growth of MUC1⁺ tumors independent of their MHC type, suggesting that this receptor is a good candidate for broadly applicable gene therapy/immunotherapy. However, the translational application of this immuno-gene therapy modality was discouraged by the progressive transgene silencing in reconstituted T and B cells, as well as the potential of tumorogenesis intrinsic to oncoretroviral vectors. To overcome these problems and facilitate the future clinical use of this receptor, we have constructed a panel of novel self-inactivating lentiviral vectors (LVs) which harbor two independent internal promoters, one driving expression of the scTCR gene and the other of a fusion suicide gene, the HSV-TK–EGFP fusion gene, allowing the transduced cells to be destroyable by the pro-drug ganciclovir. Despite the large size of insert, these vectors were efficiently packaged into high titer virus that transferred the expression of transgene in both T cell lines and primary T cells. Sustained expression was maintained in a T cell line for over 4 months in vitro, suggesting its efficient resistance to transgene silencing. Both scTCR and HSV-TK–EGFP genes were functional in the transduced cells, as evidenced by their specific recognition of MUC1⁺ tumors and efficient eradication by ganciclovir.     

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.     

HIV-1 Induces DCIR Expression in CD4+ T Cells

The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4⁺ T cells found in the synovial tissue from rheumatoid arthritis (RA) patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4⁺ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4⁺ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons) and cells acutely infected in vitro (seen in both virus-infected and uninfected cells). Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4⁺ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals) and -independent intrinsic apoptotic pathways (involving the death effector AIF). Finally, we demonstrate that the higher surface expression of DCIR in CD4⁺ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4⁺ T cells, a process that might promote virus dissemination throughout the infected organism.     

Transient gene expression mediated by integrase-defective retroviral vectors

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.     

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.     

Locus control region of the human CD2 gene in a lentivirus vector confers position-independent transgene expression

Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the beta-globin gene locus or the CD2 gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various cis-acting elements such as LCRs.     

Retroviral vector targeting to human immunodeficiency virus type 1-infected cells by receptor pseudotyping

We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell-tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression.     

Virological consequences of early events following cell-cell contact between human immunodeficiency virus type 1-infected and uninfected CD4+ cells

Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4⁺ cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4⁺ cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4⁺ lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4⁺ cell can occur through different mechanisms, leading to distinguishable virological outcomes.     

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Background

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

Methods

A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.

Results

Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

Conclusion

These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.     

Gene transfer of human TCR in primary murine T cells is improved by pseudo-typing with amphotropic and ecotropic envelopes

BACKGROUND: T cell receptor (TCR) gene therapy represents an attractive anti-cancer treatment but requires further optimization of its efficacy and safety in clinically relevant models, such as those using a tumor antigen and TCR of human origin. Currently, however, there is no consensus as to what protocol is most optimal for retroviral human TCR gene transfer into primary murine T cells, most notably with respect to virus pseudo-type. METHODS: Primary murine T cells were transduced, expanded and subsequently tested for transgene expression, proliferation and antigen-specific function. To this end, murine leukemia virus (MLV) retroviruses were produced upon transfection of various packaging cells with genes encoding either green fluorescent protein (GFP) or TCRalphabeta specific for human melanoma antigen gp100(280-288) and the helper elements GAG/POL and ENV. Next to viral pseudotyping, the following parameters were studied: T cell densities; T cell activation; the amounts of IL-2 and the source of serum used to supplement medium. RESULTS: The pseudo-type of virus produced by packaging cells critically determines T cell transduction efficiencies. In fact, MLV-A and MLV-E pseudo-typed viruses derived from a co-culture of Phoenix-A and 293T cells resulted in T cell transduction efficiencies that were two-fold higher than those based on retroviruses expressing either VSV-G, GALV, MLV-A or MLV-E envelopes. In addition, T cell densities during transduction were inversely related to transduction efficiencies. Further optimization resulted in transduction efficiencies of over 90% for GFP, and 68% for both a murine and a human (i.e. murinized) TCR. Importantly, TCR-transduced T cells proliferate (i.e. showing a log increase in cell number in a few days) and show antigen-specific function. CONCLUSIONS: We set up a quick and versatile method to genetically modify primary murine T cells based on transient production of TCR-positive retroviruses, and show that retroviral gene transfer of a human TCR into primary murine T cells is critically improved by viral pseudo-typing with both MLV-A and MLV-E envelopes.     

CD4 coexpression regulates DC-SIGN-mediated transmission of human immunodeficiency virus type 1

Dendritic cells (DCs) potently stimulate the cell-cell transmission of human immunodeficiency virus type 1 (HIV-1). However, the mechanisms that underlie DC transmission of HIV-1 to CD4⁺ T cells are not fully understood. DC-SIGN, a C-type lectin, efficiently promotes HIV-1 trans infection. DC-SIGN is expressed in monocyte-derived DCs (MDDCs), macrophage subsets, activated B lymphocytes, and various mucosal tissues. MDDC-mediated HIV-1 transmission to CD4⁺ T cells involves DC-SIGN-dependent and -independent mechanisms. DC-SIGN transmission of HIV-1 depends on the donor cell type. HIV-1 Nef can upregulate DC-SIGN expression and promote DC-T-cell clustering and HIV-1 spread. Nef also downregulates CD4 expression; however, the effect of the CD4 downmodulation on DC-mediated HIV-1 transmission has not been examined. Here, we report that CD4 expression levels correlate with inefficient HIV-1 transmission by monocytic cells expressing DC-SIGN. Expression of CD4 on Raji B cells strongly impaired DC-SIGN-mediated HIV-1 transmission to T cells. By contrast, enhanced HIV-1 transmission was observed when CD4 molecules on MDDCs and DC-SIGN-CD4-expressing cell lines were blocked with specific antibodies. Coexpression of CD4 and DC-SIGN in Raji cells promoted the internalization and intracellular retention of HIV-1. Interestingly, internalized HIV-1 particles were sorted and confined to late endosomal compartments that were positive for CD63 and CD81. Furthermore, in HIV-1-infected MDDCs, significant downregulation of CD4 by Nef expression correlated with enhanced viral transmission. These results suggest that CD4, which is present at various levels in DC-SIGN-positive primary cells, is a key regulator of HIV-1 transmission.