Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) Vα transcripts (Vα1 and Vα2) and one TCR Vβ transcript (Vβ13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR Vα1Vβ13 combination specifically recognized both the HLA-A2+ and HLA-DR1+ target cells, whereas the Vα2Vβ13 combination did not result in a TCR on the cell surface. Thus a single TCRαβ complex can have dual specificity, recognizing both a peptide in the context of HLA class I as well as a peptide in the context of HLA class II. Transactivation of T cells by an unrelated antigen in the context of HLA class II may evoke an HLA class I-specific T cell response. We propose that this finding may have major implications for immunotherapeutic interventions and insight into the development of autoimmune diseases.

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

Cell-surface receptors for retroviruses and implications for gene transfer.

Retroviruses can utilize a variety of cell-surface proteins for binding and entry into cells, and the cloning of several of these viral receptors has allowed refinement of models to explain retrovirus tropism. A single receptor appears to be necessary and sufficient for entry of many retroviruses, but exceptions to this simple model are accumulating. For example, HIV requires two proteins for cell entry, neither of which alone is sufficient; 10A1 murine leukemia virus can enter cells by using either of two distinct receptors; two retroviruses can use different receptors in some cells but use the same receptor for entry into other cells; and posttranslational protein modifications and secreted factors can dramatically influence virus entry. These findings greatly complicate the rules governing retrovirus tropism. The mechanism underlying retrovirus evolution to use many receptors for cell entry is not clear, although some evidence supports a mutational model for the evolution of new receptor specificities. Further study of factors that govern retrovirus entry into cells are important for achieving high-efficiency gene transduction to specific cells and for the design of retroviral vectors to target additional receptors for cell entry.

Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.

Thrombopoietic potential and serial repopulating ability of murine hematopoietic stem cells constitutively expressing interleukin 11.

Based on transplantation studies with bone marrow cultured under various conditions, a role of interleukin 11 (IL-11) in the self-renewal and/or the differentiation commitment of hematopoietic stem cells has been indicated. To better evaluate the in vivo effects of IL-11 on stem/progenitor cell biology, lethally irradiated mice were serially transplanted with bone marrow cells transduced with a defective retrovirus, termed MSCV-mIL-11, carrying the murine IL-11 (mIL-11) cDNA and the bacterial neomycin phosphotransferase (neo) gene. High serum levels (i.e., > 1 ng/ml) of mIL-11 in all (20/20) primary and 86% (12/14) of secondary long-term reconstituted mice, as well as 86% (12/14) of tertiary recipients examined at 6 weeks posttransplant, demonstrated persistence of vector expression subsequent to transduction of bone marrow precursors functionally definable as totipotent hematopoietic stem cells. In agreement with results obtained with human IL-11 in other myeloablation models, ectopic mIL-11 expression accelerated recovery of platelets, neutrophils, and, to some extent, total leukocytes while preferentially increasing peripheral platelet counts in fully reconstituted mice. When analyzed 5 months posttransplant, tertiary MSCV-mIL-11 recipients had a significantly greater percentage of G418-resistant colony-forming cells in their bone marrow compared with control MSCV animals. Collectively, these data show that persistent stimulation of platelet production by IL-11 is not detrimental to stem cell repopulating ability; rather, they suggest that IL-11 expression in vivo may have resulted in enhanced maintenance of the most primitive hematopoietic stem cell compartment. The prolonged expression achieved by the MSCV retroviral vector, despite the presence of a selectable marker, contrasts with the frequent transcriptional extinction observed with other retroviral vectors carrying two genes. These findings have potentially important implications for clinical bone marrow transplantation and gene therapy of the hematopoietic system.

Host range restrictions of oncogenes: myc genes transform avian but not mammalian cells and mht/raf genes transform mammalian but not avian cells.

The host range of retroviral oncogenes is naturally limited by the host range of the retroviral vector. The question of whether the transforming host range of retroviral oncogenes is also restricted by the host species has not been directly addressed. Here we have tested in avian and murine host species the transforming host range of two retroviral onc genes, myc of avian carcinoma viruses MH2 and MC29 and mht/raf of avian carcinoma virus MH2 and murine sarcoma virus MSV 3611. Virus vector-mediated host restriction was bypassed by recombining viral oncogenes with retroviral vectors that can readily infect the host to be tested. It was found that, despite high expression, transforming function of retroviral myc genes is restricted to avian cells, and that of retroviral mht/raf genes is restricted to murine cells. Since retroviral oncogenes encode the same proteins as certain cellular genes, termed protooncogenes, our data must also be relevant to the oncogene hypothesis of cancer. According to this hypothesis, cancer is caused by mutation of protooncogenes. Because protooncogenes are conserved in evolution and are presumed to have conserved functions, the oncogene hypothesis assumes no host range restriction of transforming function. For example, mutated human proto-myc is postulated to cause Burkitt lymphoma, because avian retroviruses with myc genes cause cancer in birds. But there is no evidence that known mutated protooncogenes can transform human cells. The findings reported here indicate that host range restriction appears to be one of the reasons (in addition to insufficient transcriptional activation) why known, mutated protooncogenes lack transforming function in human cells.

Highly efficient germ-line transmission of proviral insertions in zebrafish.

An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximately 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.

Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.

Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein

TVA, the cellular receptor for subgroup A avian leukosis viruses (ALV-A) can mediate viral entry when expressed as a transmembrane protein or as a glycosylphosphatidylinositol-linked protein on the surfaces of transfected mammalian cells. To determine whether mammalian cells can be rendered susceptible to ALV-A infection by attaching a soluble form of TVA to their plasma membranes, the TVA-epidermal growth factor (EGF) fusion protein was generated. TVA-EGF is comprised of the extracellular domain of TVA linked to the mature form of human EGF. Flow cytometric analysis confirmed that TVA-EGF is a bifunctional reagent capable of binding simultaneously to cell surface EGF receptors and to an ALV-A surface envelope-Ig fusion protein. TVA-EGF prebound to transfected mouse fibroblasts expressing either wild-type or kinase-deficient human EGF receptors, rendered these cells highly susceptible to infection by ALV-A vectors. Viral infection was blocked specifically in the presence of a recombinant human EGF protein, demonstrating that the binding of TVA-EGF to EGF receptors was essential for infectivity. These studies have demonstrated that a soluble TVA-ligand fusion protein can mediate viral infection when attached to specific cell surfaces, suggesting an approach for targeting retroviral infection to specific cell types.

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4+ T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4+ cells during gene therapy of AIDS.

Tumor cells expressing a retroviral envelope escape immune rejection in vivo

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins—or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the “penetrance” of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.

Production of infectious recombinant Moloney murine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors.

We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney murine leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney murine leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.

Plasmoviruses: nonviral/viral vectors for gene therapy.

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

A genetic screen identifies cellular factors involved in retroviral -1 frameshifting.

To identify cellular factors that function in -1 ribosomal frameshifting, we have developed assays in the yeast Saccharomyces cerevisiae to screen for host mutants in which frameshifting is specifically affected. Expression vectors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of the lacZ gene or the CUP1 gene so that the reporters are in the -1 frame relative to the initiation codon. These vectors have been used to demonstrate that -1 frameshifting is recapitulated in yeast in response to retroviral mRNA signals. Using these reporters, we have isolated spontaneous host mutants in two complementation groups, ifs1 and ifs2, in which frameshifting is increased 2-fold. These mutants are also hypersensitive to antibiotics that target the 40S ribosomal subunit. We have cloned the IFS1 gene and shown that it encodes a previously undescribed protein of 1091 aa with clusters of acidic residues in the carboxyl-terminal region. Haploid cells lacking 82% of the IFS1 open reading frame are viable and phenotypically identical to ifs1-1 mutants. This approach could help identify potential targets for antiretroviral agents.