Production of phagocytosis-inducing factor and expression of 4B4 antigen by cloned human T cells before and after transformation with HTLV-I

The characteristics of 4 T-cell clones, each capable of producing phagocytosis-inducing factor (PIF), were compared before and after transformation with human T-lymphotropic virus Type 1 (HTLV-I). Before transformation, the four clones produced PIF transiently after stimulation with antigen or mitogen and expressed the phenotype T3(CD3)+, T4(CD4)+, T8(CD8)-, 4B4+, and 2H4-; the three clones that could be studied also expressed the OKT17 marker. After transformation, the cells expressed the same phenotypic markers, except for two clones that lost the CD3 antigen. The clones that were available for study before and after transformation also expressed the antigen detected by the monoclonal antibody 5/9. In addition, all clones secreted PIF constitutively after transformation. These characteristics of the four transformed T-cell clones closely resembled those of three long-term HTLV-I-transformed T-cell lines, HUT-102, C5/MJ, and MT-2, which also produced PIF constitutively and expressed the CD4 and 4B4, but not 2H4, markers. In addition, two other HTLV-I-transformed lines generated in the present study produced PIF constitutively. Since all nine HTLV-I transformed cell lines and all four untransformed clones secreted PIF, and since our previous studies have shown that only approximately 20% of CD4+ peripheral blood lymphocytes secrete PIF, these results suggest that HTLV-I may preferentially transform PIF-secreting CD4+ lymphocytes. The predominant 4B4+, 5/9+, 2H4- phenotype (characteristic of antigen-responsive T cells) of the untransformed and transformed clones as well as the long-term HTLV-I-transformed lines also suggests that the subset of CD4+ lymphocytes that proliferates in response to soluble antigen may be especially susceptible to transformation with this virus.     

Virions released from cells transfected with a molecular clone of human T-cell leukemia virus type I give rise to primary and secondary infections of T cells.

The ability of molecular clones of human T-cell leukemia virus type I (HTLV-I) to direct the synthesis of infectious virions has not previously been demonstrated. An HTLV-I provirus originating from an adult T-cell leukemia patient was cloned into a plasmid vector and is designated pCS-HTLV. This molecular clone was shown to direct the synthesis of viral mRNA and proteins in transiently transfected cells; in addition, virus structural proteins were released into the culture medium. Viral proteins were assembled into virions that sedimented at a buoyant density characteristic of retrovirus particles and whose morphology was verified by electron microscopy. Virions concentrated from transiently transfected cell supernatants were incubated with primary cord blood lymphocytes or with transformed T-cell lines to establish that these particles were infectious. Expression of spliced, viral mRNAs in the T-cell cultures after both primary and secondary infections with cell-free virus revealed that pCS-HTLV encodes an infectious provirus.     

Role of Cell Surface Glycosaminoglycans of Human T Cells in Human Immunodeficiency Virus Type-1 (HIV-1) Infection

To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV-1 infection in human T cells, HIV-1 binding and infection were determined after treatment of T-cell lines and CD4 + T cells from normal peripheral blood mononuclear cells (PBMC) with GAG-degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV-1/IIIB to MT-4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage-tropic strain HIV-1/SF162 than that of the T-cell line-tropic strain HIV-1/IIIB. The binding of HIV-1/SF162 was about 100-fold less dependent on cell surface HS than HIV-1/IIIB. Human HTLV-I positive T-cell lines expressed more HS than HTLV-I negative T-cell lines or normal CD4 + T cells when stained with anti-HS mAbs against either native or heparitinase-treated HS. With the exception of endo-β-galactosidase (endo-β-gal), GAG-degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV-1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV-1 infection by facilitating HIV-1/IIIB binding to MT-4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV-1 binding inhibition, other GAG-like molecules may also be involved.     

Infection of human natural killer (NK) cells with replication-defective human T cell leukemia virus type I provirus. Increased proliferative capacity and prolonged survival of functionally competent NK cells.

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of human cell types, but only T lymphocytes are efficiently immortalized after HTLV-I infection. This study reports an attempt to infect and to immortalize NK cells with HTLV-I. Co-cultivation of freshly isolated NK cells with a HTLV-I-producing T cell line did not result in NK cell infection. However, NK cells activated with an anti-CD16 mAb and co-cultivated with a HTLV-I-producing T cell line were reproducibly infected by HTLV-I. HTLV-I infection was documented in NK cell lines and clones by the detection of defective integrated provirus by both Southern blot and polymerase chain reaction analysis. Although HTLV-I-infected NK cells produced viral proteins, they did not produce infectious viral particles. HTLV-I-infected NK cells were phenotypically indistinguishable from their uninfected counterparts (CD16+, CD2+, CD56+, CD3-). They also retained the ability to mediate both natural and antibody-dependent cell cytotoxicity. The IL-2-dependent proliferation of HTLV-I-infected NK cells was significantly greater than that of uninfected NK cells. The doubling time of this infected population was reduced from 9 days to 3 days, and the overall survival of the culture in the absence of restimulation was extended from 5 wk to 18 wk. Unlike T lymphocytes, HTLV-I-infected NK cells were not immortal, implying a fundamental difference between these two lymphocyte populations.     

Transmission of human T-cell leukaemia virus type I into adult mice

Human T-cell leukaemia virus type I (HTLV-I)-transformed rabbit T-cells, F647a, were intraperitoneally injected into eight 10-week-old C3H/He and C3H/HeJ mice (1 x 10(7) F647a cells/mouse), respectively. Antibody titres against HTLV-I increased to a peak at 1-3 months after injection in both C3H/He and C3H/HeJ mice. At 12 months after injection, antibody titres of two of the eight C3H/HeJ mice became undetectable, whereas those of all the C3H/He mice still ranged from 1:10 to 1:40. Sera from both seropositive C3H/He and C3H/HeJ mice reacted with HTLV-I core proteins, but not with the env protein. HTLV-I proviral sequences were detected in two of eight C3H/He mice and three of the eight C3H/HeJ mice. These results suggest that HTLV-I is able to infect an adult mouse.     

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.     

Isolation of a novel simian T-cell lymphotropic virus from Pan paniscus that is distantly related to the human T-cell leukemia/lymphotropic virus types I and II.

An unusual serological profile against human T-cell leukemia/lymphotropic virus type I and II (HTLV-I and -II) proteins was reported in several human Pygmy tribes in Zaire and Cameroon with serum antibodies reactive with gp21 and p24. Here we describe a similar pattern of serum antibodies in a colony of captive pygmy chimpanzees and the isolation of a novel retrovirus, simian T-cell lymphotropic virus from Pan paniscus (STLVpan-p), from the peripheral blood mononuclear cells of several seropositive animals. Cocultures of peripheral blood mononuclear cells from three seropositive pygmy chimpanzees with human cord blood mononuclear cells led to the expression of an HTLV-I- and HTLV-II-related virus initially demonstrated by electron microscopy. Furthermore, several of these cocultures became immortalized T-cell lines expressing the CD4+ CD8+ DR+ phenotype of mature activated T cells. Southern blotting and DNA sequencing of a PCR fragment of viral DNA from these cell cultures demonstrated a distant evolutionary relationship of these viruses to HTLV-I and -II and distinct from the known STLV isolates. We designated this virus STLVpan-p. A genealogical analysis of the captive pygmy chimpanzees colony, originated from wild-caught animals, revealed a prevalence of seropositive offspring from infected mothers, as also observed with HTLVs. The presence in this old African Great Ape species of a virus which is genetically quite distinct from HTLV-I and -II could provide new insights in the phylogenesis of STLVs and HTLVs and be instrumental in the discovery of related human viruses.     

Transcriptional suppression of the human T-cell leukemia virus type I long terminal repeat occurs by an unconventional interaction of a CREB factor with the R region.

To analyze regulation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR), cell lines were generated from LTR-tax x LTR-beta-galactosidase (beta-Gal) doubly transgenic mouse fibroblastic tumors. The HTLV-I LTR directs expression of both the tax and lacZ genes, and Tax up-modulates both promoters in primary cells. However, once cells were transformed by tax, beta-Gal but not tax expression was suppressed. Supertransformation of these cells with v-src suppressed both beta-Gal and tax expression. This suppression was reversed by treatment with the tyrosine kinase inhibitor herbimycin A or protein kinase A inhibitor H8. Electrophoretic mobility shift assays demonstrated augmented binding in the R but not U3 region. This binding was competitively inhibited by a high-affinity CREB oligodeoxynucleotide and super-shifted with a specific CREB antibody. Treatment of cells with the cyclic AMP analog dibutyryl cyclic AMP also transiently increased the R region binding dramatically. In vitro DNase I footprint analysis identified a protein-binding sequence in the R region which corresponded with suppression. However, this target sequence lacked a conventional CREB-binding site. A 70.5-kDa DNA-binding protein was partially purified by affinity chromatography, along with a 49-kDa protein which reacted with CREB-specific sera. These data demonstrate that HTLV-I LTR suppression is associated with CREB factor binding in the R region, probably by direct interaction with a 70.5-kDa protein, and provide a novel mechanism for maintenance of viral latency.     

Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.

The viral infectivity factor gene vif of human immunodeficiency virus type 1 has been shown to affect the infectivity but not the production of virus particles. In this study, the effect of vif in the context of the HXB2 virus on virus replication in several CD4+ T-cell lines was investigated. vif was found to be required for replication in the CD4+ T-cell lines CEM and H9 as well as in peripheral blood T lymphocytes. vif was not required for replication in the SupT1, C8166, and Jurkat T-cell lines. The infectivity of vif-defective viruses depended on the cell type in which the virus was produced. In CEM cells, vif was required for production of virus capable of initiating infection in all cell lines studied. vif-defective virus produced by SupT1, C8166, and Jurkat cells and the monkey cell line COS-1 could initiate infection in multiple cell lines, including CEM and H9. These results suggest that vif can compensate for cellular factors required for production of infectious virus particles that are present in some cell lines such as SupT1, C8166, and Jurkat but are absent in others such as CEM and H9 as well as peripheral blood T lymphocytes. The effect of vif was not altered by deletion of the carboxyl terminus of gp41, a proposed target for vif (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). These studies demonstrate that vif enhances viral infectivity during virus production and also suggest that vif is likely to be important for natural infections.     

The nuclear 42-kDa phosphoprotein preferentially binds promoter-containing single-stranded DNA

The DNA-binding activity of a 42-kDa phosphoprotein from salivary gland cells and cultured epithelial cells of Chironomus tentans have been analyzed by the Southwestern technique. Both the salivary gland and the epithelial cell 42-kDa polypeptides were found to be single-stranded DNA-binding proteins. They bind to single-stranded promoter-containing restriction fragments including sequences from -204 to +74 from the ecdysterone controlled I-18C gene as well as sequences including the joint histone H2A/H2B promoters in a sequence selective manner. By contrast, the 42-kDa polypeptides show no significant binding to intragenic restriction fragments from +71 to +351 from the I-18C gene. Previous and present data taken together suggest that the 42-kDa protein has a general role in the regulation of protein coding genes.     

The relationship between group C adenovirus tumor antigen and the adenovirus single-strand DNA-binding protein

The group C adenoviruses code for a single-strand specific DNA-binding protein of molecular weight 72,000 daltons which is synthesized at early times after productive viral infection. Experiments were designed to determine whether this single-strand specific DNA-binding protein was expressed in adenovirus tumors and transformed cells.Two independently derived preparations of antisera from hamsters bearing group C adenovirus tumors were tested for antibody against the single-strand DNA-binding proteins. One antiserum contained antibodies that reacted with these DNA-binding proteins, while the second antiserum did not contain detectable levels of antibody. Five adenovirus type 2 transformed rat cell lines were tested for the presence of the single-strand specific DNA-binding proteins. Two of the five transformed cells expressed detectable levels of this protein. These results indicate that the group C adenovirus single-strand specific DNA-binding proteins are expressed in some, but not all, adenovirus tumors and transformed cell lines.Those transformed cell lines (type 2) containing a portion of the adenovirus genome designated by the Eco R-I-B restriction enzyme fragment express the single-strand specific DNA-binding proteins. Those cell lines missing this Eco R-I-B fragment do not contain this viral protein. Other experiments have located the structural gene of the single-strand specific DNA-binding protein in the Eco R-I-B DNA fragment, indicating that when this gene is present in a transformed cell, it is expressed.     

Detection of virus-specific T cells and CD8+ T-cell epitopes by acquisition of peptide-HLA-GFP complexes: analysis of T-cell phenotype and function in chronic viral infections

Antigen-specific CD8+ T cells acquire peptide-major histocompatibility complex (MHC) clusters through T-cell receptor (TCR)-mediated endocytosis after specific antigen stimulation. We generated an antigen-presenting cell (APC) expressing human leukocyte antigen (HLA)-A*201 coupled to the enhanced green fluorescent protein (GFP), which delivered GFP to an antigen-specific T cell when pulsed with antigenic peptide. We quantitatively identified human T-cell lymphotropic virus type I (HTLV-I) Tax(11-19) peptide-specific T-cell populations in peripheral blood mononuclear cells (PBMCs) from patients with HTLV-I-associated neurologic disease and defined a new CD8+ T-cell epitope in the HTLV-I envelope region. Acquisition of peptide-HLA-GFP complexes by antigen-specific T cells could distinguish, with respect to phenotype and perforin production, T cells from the chronic viral infections cytomegalovirus and HTLV-I. This approach will be a powerful tool in understanding the role of antigen-specific T-cell responses in health and disease.     

Analysis of Tax-expressing cell lines generated from HTLV-I tax-transgenic mice: correlation between c-myc overexpression and neoplastic potential

Human T-cell leukemia/lymphotropic virus type I (HTLV-I) infection causes a variety of human diseases, including adult T-cell leukemia/lymphoma. The viral transactivator Tax has been implicated as a key factor in the HTLV-I-induced transformation pathway. To investigate the components of this pathway, we derived fibroblast-like cell lines, designated T6 and T9, from tail biopsies of tax-transgenic C57BL/6 mice that do not develop tumors. Phenotypic characterization of T6 and T9 cells and T6-derived subclones revealed that they differ in their abilities to form foci in vitro and tumors in vivo. The observed differences in the levels of Tax expression did not correlate with their degree of neoplastic potential. However, a control cell line derived from a nontransgenic C57BL/6 mouse did not form foci in vitro or tumors in vivo, indicating that Tax was required for the transformation process. Results of Northern analyses showed that the T9 cells and the highly malignant derivatives of T6 cells expressed elevated levels of c-myc mRNA. These findings suggest that progression of the tax-transgenic cells toward a more malignant phenotype might involve c-myc deregulation.     

Tax responsiveness of the GM-CSF promoter is mediated by mitogen-inducible sequences other than kappa B.

The mechanism by which the human T-cell leukemia viruses type I and II (HTLV-I and -II) transform T cells is unknown, but the nonstructural Tax protein that these viruses produce is known to be essential for viral replication and to have the capacity to trans-activate cellular gene expression. The HTLV-I and -II Tax proteins have been shown to activate the promoter of both the human and mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in mature T-cell lines. T-cell-specific Tax-responsive sequences were previously localized to the 90-bp region extending from base pairs -53 to +37 in the human GM-CSF promoter. In this study, a series of site-directed and deletion mutations were created in the human GM-CSF promoter, which was linked to the chloramphenicol acetyltransferase (CAT) gene, and the constructs were assayed for their response to Tax by using a Tax-expressing plasmid in transient cotransfection assays. The results demonstrated that both copies of the repeated sequence CATTA (A/T), located between base pairs -48 and -36, are required for Tax responsiveness in T cells and that these sequences bind nuclear factors present in T cells. The Tax-responsiveness of other sequences located 5' of base pair -53 was also examined, including an NF-kappa B consensus sequence and the CK1, CK2, and GC-rich regions identified in both the mouse and human GM-CSF promoters. These sequences did not have Tax-responsive regulatory activity when they were examined in the context of the intact human GM-CSF promoter in T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minimal T-cell-stimulatory sequences and spectrum of HLA restriction of immunodominant CD4+ T-cell epitopes within hepatitis C virus NS3 and NS4 proteins

The hepatitis C virus (HCV)-specific CD4+ T-cell response against nonstructural proteins is strongly associated with successful viral clearance during acute hepatitis C. To further develop these observations into peptide-based vaccines and clinical immunomonitoring tools like HLA class II tetramers, a detailed characterization of immunodominant CD4+ T-cell epitopes is required. We studied peripheral blood mononuclear cells from 20 patients with acute hepatitis C using 83 overlapping 20-mer peptides covering the NS3 helicase and NS4. Eight peptides were recognized by > or = 40% of patients, and specific CD4+ T-cell clones were obtained for seven of these and three additional, subdominant epitopes. Mapping of minimal stimulatory sequences defined epitopes of 8 to 13 amino acids in length, but optimal T-cell stimulation was observed with 10- to 15-mers. While some epitopes were presented by different HLA molecules, others were presented by only a single HLA class II molecule, which has implications for patient selection in clinical trials of peptide-based immunotherapies. In conclusion, using two different approaches we identified and characterized a set of CD4+ T-cell epitopes in the HCV NS3-NS4 region which are immunodominant in patients achieving transient or persistent viral control. This information allows the construction of a valuable panel of HCV-specific HLA class II tetramers for further study of CD4+ T-cell responses in chronic hepatitis C. The finding of immunodominant epitopes with very constrained HLA restriction has implications for patient selection in clinical trials of peptide-based immunotherapies.