Long-range mapping of Mis-2, a common provirus integration site identified in murine leukemia virus-induced thymomas and located 160 kilobase pairs downstream of Myb.

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in mice or rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens most frequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirus insertions, recognized as common provirus integration sites, have been instrumental in identifying novel cellular genes involved in tumor formation. We have searched for new common provirus integration sites in Moloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of these tumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%) percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstream of myb. The Mis-2 region may contain a novel gene involved in tumor development.     

Identification of a new common provirus integration site in gross passage A murine leukemia virus-induced mouse thymoma DNA.

The Gross passage A murine leukemia virus (MuLV) induced T-cell leukemia of clonal (or oligoclonal) origin in inoculated mice. To study the role of the integrated proviruses in these tumor cells, we cloned several newly integrated proviruses (with their flanking cellular sequences) from a single tumor in procaryotic vectors. With each of the five clones obtained, a probe was prepared from the cellular sequences flanking the provirus. With one such probe (SS8), we screened several Gross passage A MuLV-induced SIM.S mouse tumor DNAs and found that, in 11 of 40 tumors, a provirus was integrated into a common region designated Gin-1. A 26-kilobase-pair sequence of Gin-1 was cloned from two lambda libraries, and a restriction map was derived. All proviruses were integrated as a cluster in the same orientation within a 5-kilobase-pair region of Gin-1, and most of them had a recombinant structure of the mink cell focus-forming virus type. The frequency of Gin-1 occupancy by provirus was much lower in thymoma induced by other strains of MuLV in other mouse strains. Using somatic-cell hybrid DNAs, we mapped Gin-1 on mouse chromosome 19. Gin-1 was not homologous to 16 known oncogenes and was distinct from the other common regions for provirus integration previously described. Therefore, Gin-1 appears to represent a new common provirus integration region. The integration of a provirus within Gin-1 might be an important event leading to T-cell transformation, and the Gin-1 region might harbor sequences which are involved in tumor development.     

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.     

Massive Depletion of Bovine Leukemia Virus Proviral Clones Located in Genomic Transcriptionally Active Sites during Primary Infection

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.     

Insertional mutagenesis of the Abelson murine leukemia virus genome: identification of mutants with altered kinase activity and defective transformation ability.

A library of Abelson murine leukemia virus (A-MuLV) proviral DNAs with 12- or 6-base-pair (bp) insertional mutations was constructed. The 29 mutations characterized spanned the entire protein-coding region of the provirus. We tested the effects of these mutations both on the kinase activity of the gag-abl fusion protein encoded by the provirus and on the ability of the provirus to transform NIH 3T3 fibroblasts. To simplify assessment of the mutant kinases, we expressed the A-MuLV-encoded kinase in the bacterial expression vector pATH2, resulting in production of a trpE-gag-abl fusion protein in Escherichia coli. We used an immunoprecipitation kinase assay to measure both autophosphorylation and artificial substrate phosphorylation by the mutant kinases. To assay transformation ability of the mutant proviruses, we transfected NIH 3T3 fibroblasts with the mutants and with helper virus (Moloney MuLV) by the DEAE-dextran method. Our analysis of these A-MuLV insertional mutants allows the division of the protein-coding region of the provirus into four domains: domain A (proviral bp 1068 to 1685), in which insertions have no effect on the bacterially expressed kinase, but diminish both kinase activity and transformation efficiency in fibroblasts; domain B (bp 1750 to 2078), in which insertions have no effect on the provirus; domain C (bp 2181 to 2878), the critical kinase domain, in which 12-bp or even 6-bp insertions completely inactivate the A-MuLV kinase and result in transformation-defective proviruses; and domain D (bp 2956 to 4610), the large C-terminal domain in which mutations are silent.     

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.     

IL-4 potentiates the IL-2-dependent proliferation of mouse cytotoxic T cells

In addition to the regulation of B lymphocyte growth and differentiation, the cytokine IL-4 (BSF-1) exerts effects on T lymphocytes and other bone marrow-derived lineages. We show here that recombinant mouse IL-4 synergizes with low levels of IL-2 to increase the yield of cytotoxic activity in a primary MLR, and the proliferation of both cloned IL-2-dependent CTL lines and cells obtained from a primary MLR. IL-4 did not induce the proliferation of any of several cloned CTL cell lines on its own. It also did not replace IL-2 in stimulating the growth or reactivation of quiescent, antigen-dependent CTL clones. However, IL-4 was synergistic with IL-2 after reactivation of the quiescent cells with antigen plus IL-2. Enhancement by IL-4 of the IL-2-driven proliferation of an antigen-independent line was blocked by the addition of anti-IL-4 monoclonal antibody. Although incubation of the CTL clones with IL-4 or with IL-2 plus IL-4 induced a transient increase in the expression of the mRNA encoding the 55 kDa IL-2 receptor, no change in the number or affinity of IL-2 receptors because of IL-4 was detected. This suggests that IL-4 does not potentiate the IL-2 response by altering IL-2 receptor levels. Instead, we propose that the synergistic effect of IL-4 is mediated by a different signalling mechanism from that used by IL-2.     

IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.     

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.     

Growth of AKR T cell leukemia lymphoblasts in medium containing interleukin 2 (IL-2)

Long-term cloned mouse leukemic T cell lines were established in vitro using interleukin-2 (IL-2) conditioned media. The cell lines were tested for retention of both antigenic expression and tumorigenicity, as well as for growth characteristics. Several important findings resulted from these studies. A reliable method was developed for consistent success in the culturing and cloning of leukemic T cell lines. Cultured cells from IL-2-dependent lines were found to retain their original histocompatibility and differentiation antigen characteristics for up to 2 yr. Mortality patterns, comparing long-term cloned leukemic T cell lines with fresh AKR leukemia cells, showed that the cloned cells had greater tumorigenicity. An especially interesting finding was that cell lines established both from different mice and from a single organ of an individual mouse were heterogeneous with respect to antigenic makeup, cell growth kinetics, and tumorigenicity. Finally, because of their dependence on IL-2, the cloned tumor cell lines provided excellent index cells to quantify IL-2 activity.     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.     

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.     

IL-2 secretion by soluble antigen-reactive human T-cell clones

Human tetanus toxoid specific T-cell lines and clones capable of producing IL-2 were established. IL-2 production occurred only when the antigen-specific T cells were cultured with both tetanus toxoid antigen and an autologous, irradiated adherent cell population. The T-cell lines and clones remained strictly dependent on exogenous IL-2 for proliferation at all other times. Phenotypic characterization with monoclonal antibodies recognizing T-cell subsets revealed that the antigen-specific lines and clones bore predominantly OKT3 and OKT4 markers with essentially no OKT8 positive cells present. T-cell clones which were demonstrated to secrete IL-2 activity could also partially deplete media of IL-2 if cultured in the absence of soluble antigen and irradiated adherent cells.