Analysis of human T-lymphotrophic virus sequences in multiple sclerosis tissue

Several observations suggest that retroviral infection is involved in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). First, lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III), the agent of acquired immune deficiency syndrome (AIDS), has been shown to be neurotropic in man. Second, the genetic organization of the lentivirus visna, which causes a chronic demyelinating disease of sheep, closely resembles that of LAV/HTLV-III. Recently, Koprowski and colleagues reported that MS is associated both with raised levels of circulating antibodies to HTLV-I and with the presence of HTLV-I-specific RNA within cell lines derived from the cerebrospinal fluid (CSF). Here we report that no HTLV-I-like or LAV/HTLV-III-like sequences can be detected, by in situ hybridization, in central nervous system (CNS) tissues from MS patients, and that nonspecific HTLV-I-like signal in peripheral blood mononuclear cells or in CSF cell lines is characteristic of MS. Furthermore, enzyme-linked immunosorbent assay (ELISA) analysis of circulating and CSF antibodies for HTLV-I reactivity fails to distinguish between MS and control groups.     

An HTLV-III peptide produced by recombinant DNA is immunoreactive with sera from patients with AIDS

Human T-cell lymphotropic retrovirus type III (HTLV-III), also called lymphadenopathy-associated virus (LAV), has been identified as the aetiological agent of acquired immune deficiency syndrome (AIDS). The sera of most patients with AIDS or AIDS-related complexes, and of asymptomatic individuals infected with HTLV-III, contain antibodies against antigens of HTLV-III. The characterization of these antibodies and their corresponding viral antigens is important not only for understanding immunity against HTLV-III and the pathology of AIDS, but also for the development of diagnostic methods and preventive vaccine for AIDS. Following the successful establishment of a long-term T-cell line permissive for HTLV-III replication, large quantities of virus have been produced, facilitating the purification of viral proteins and the development of mouse monoclonal antibodies against several viral antigens. More recently, the structure of HTLV-III proviral DNA has been elucidated. We now report the production, by genetic engineering methods, of a peptide encoded by a gene segment of HTLV-III. A 1.1-kilobase (kb) EcoRI DNA segment from an isolate of HTLV-III was inserted into a lpp and lac promoter-coupled expression vector, pIN-III-ompA. Escherichia coli transformants of this plasmid produced a peptide of relative molecular mass (Mr) 15,000 (15K) which was strongly immunoreactive with anti-HTLV-III antibodies present in sera from AIDS patients. Lysates of the clones expressing this 15K peptide inhibited the reactivity of the p31 virion protein with AIDS sera, suggesting that it is a fragment of the viral p31 protein. The peptide reacted with sera from all 20 AIDS patients but none of the 8 normal controls tested. These results suggest that the peptide may be useful for detecting anti-HTLV-III antibodies in blood samples.     

Common site of integration of HTLV in cells of three patients with mature T-cell leukaemia-lymphoma

Human T-cell leukaemia-lymphoma virus (HTLV) was first isolated in the United States from a patient with an aggressive form of cutaneous T-cell lymphoma (CTCL) and was later found associated with clusters of adult T-cell leukaemia-lymphomas (ATL) in various parts of the world, including Japan and the Caribbean. Leukaemic cells of the HTLV-positive patients seem to be clonal expansions of single infected cells since the provirus(es) are found at the same sites in a given patient. In avian leukosis virus-induced B-cell lymphomas, the provirus very frequently integrates at several discrete sites in a common domain near the cellular gene, c-myc, that it activates and it has been speculated that the same would hold true for other chronic leukaemia viruses. We report here that cultured cells from two US patients with CTCL and fresh leukaemia cells of a Japanese patient with ATL contained an HTLV provirus integrated at the same site. In addition, a cord blood T-lymphocyte cell line established by co-cultivation with one of the two HTLV-positive CTCL cell lines also contained HTLV provirus contiguous with the same flanking cellular sequences. Ten other HTLV-positive cell samples did not show integration of HTLV at this site, suggesting that there is more than one discrete site of HTLV integration in tumour cells.     

Unique cell lines harbouring both Epstein-Barr virus and adult T-cell leukaemia virus, established from leukaemia patients

Members of three distinct classes of animal virus have been associated with naturally occurring neoplasias in man: Epstein--Barr virus (EBV), a DNA virus belonging to herpesvirus group, papillomavirus, and a novel human RNA (retro) virus, human T-cell leukemia virus or adult T-cell leukaemia (ATL) virus. We have established seven continuous cell lines from ATL patients and 0.1-7% of these cells consistently express ATL-specific antigens (ATLA). EBV-associated nuclear antigen (EBNA) is also found in more than 90% of these cells. We have cloned cells from two of these lines and show here that both ATLA and EBNA were present in the same B-cell clone carrying surface immunoglobulin (sIg).     

Cell lines producing human T-cell lymphoma virus show altered HLA expression

Human T-cell leukaemia/lymphoma virus (HTLV) can be identified in fresh and cultured T-lymphocytes from patients with adult T-cell malignancies. HLA typing of the peripheral blood lymphocytes and cultured cell lines from the patient from which the virus was originally isolated suggested the expression of additional HLA-A and -B locus antigens on the HTLV positive cultured T-cells that were not present on the EBV transformed B-cell line or on the peripheral blood lymphocytes. Peripheral blood lymphocytes (PBLs) and T-cell lines established from patients and cord blood lymphocytes, infected with virus by co-culture with T-cell lines, were typed for HLA antigens with alloantisera and in addition tested for reactivity with a monoclonal antibody (4D12) which recognizes a polymorphic HLA class-I antigen. In all HTLV positive cells, with demonstrable provirus replication, altered HLA alloantigen expression was observed. This may be explained by the observations reported in the accompanying paper which shows homology between the envelope gene region of HTLV and the region of an HLA-B locus gene which codes for the extracellular portion of a class I histocompatibility antigen.     

Epstein-Barr virus-positive Burkitt's lymphoma cells not recognized by virus-specific T-cell surveillance

The pathogenesis of Epstein-Barr (EB) virus-positive Burkitt's lymphoma (BL) appears to involve the combined actions of virus-induced B-cell proliferation, and a rare chromosomal translocation juxtaposing c-myc and immunoglobulin gene loci in a single B cell; holoendemic malarial infection in some way facilitates the oncogenic process. Outgrowth of the EB virus-positive tumour suggests either breakdown or evasion of those immune controls, in particular cytotoxic T-cell responses against the virus-induced lymphocyte-detected membrane antigen LYDMA, which limit virus-infected B-cell numbers in healthy virus carriers. Immunosuppression, such as that which malarial infection may induce, cannot itself be a sufficient explanation in this regard since our studies have identified a number of BL patients who retain detectable LYDMA-specific T-cell surveillance. The present work shows that in many cases of virus-associated BL, the emerging malignant clone is insensitive to such surveillance. Several EB virus-positive BL cell lines, recently established in vitro and expressing the class I histocompatibility locus antigens (HLAs) which restrict cytotoxic T-cell function, were not killed by HLA-matched LYDMA-specific effector populations in assays where the EB virus-positive lymphoblastoid cell line (LCL), derived from normal B cells of the same patient, sustained high levels of lysis.     

Molecular cloning and polymorphism of the human immune deficiency virus type 2

We recently reported the isolation of a novel retrovirus, the human immune deficiency virus type 2 (HIV-2, previously named LAV-2), from patients with acquired immune deficiency syndrome (AIDS) originating from West Africa. This virus is related to HIV-1, the causative agent of the AIDS epidemic now spreading in Central and East Africa, as well as the USA and Europe (see ref. 3 for review) both by its morphology and by its tropism and in vitro cytopathic effect on CD4 (T4) positive cell lines and lymphocytes. But preliminary hybridization experiments indicated that there are substantiated differences between the sequences of the two genomes. Furthermore, the proteins of HIV-1 and HIV-2 have different sizes and their serological cross-reactivity is restricted to the major core protein, as the envelope glycoproteins of HIV-2 are not immunoprecipitated by HIV-1-positive sera. We now report the molecular cloning of the complete 9.5-kilobase (kb) genome of HIV-2, the observation of restriction site polymorphism between different isolates, and a preliminary analysis of the relationship of HIV-2 with other human and simian retroviruses.     

Transformation of NIH 3T3 cells by a human c-sis cDNA clone

The mechanism of leukaemogenic transformation by human T-cell leukaemia/lymphoma virus (HTLV), a retrovirus implicated in the aetiology of certain adult T-cell leukaemias and lymphomas, is unknown but is conceivably associated with the expression of the cellular analogues of retroviral oncogenes. The HUT-102 cell line, derived from a cutaneous T-cell lymphoma and infected with HTLV, expresses several cellular oncogenes. It is unusual among haemopoietic cell lines in that one of these is c-sis, the gene from which the oncogene v-sis of the simian sarcoma virus was derived, and perhaps the gene for platelet-derived growth factor (PDGF). To explore the possible role of c-sis expression in HTLV-induced disease, we have obtained cDNA clones of c-sis from HUT-102 cells. Here we describe two such clones and report that one of them transforms NIH-3T3 cells. This is the first example of transformation of NIH-3T3 cells by a human onc gene other than c-ras or Blym, as well as the first demonstration of transformation by a human cDNA clone.     

Neutralization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients

Human T-lymphotropic virus type III (LAV, HTLV-III) is aetiologically linked to acquired immune deficiency syndrome (AIDS) and persistent general lymphadenopathy (PGL). Specific radioimmunoassays (RIA), enzyme-linked assays, immunofluorescence assays (IFA) and immunoblotting techniques are being used widely to detect serum antibodies to HTLV-III in infected patients and in those at risk of infection. However, these assays do not functionally identify those antibodies that neutralize the infectivity of the virus. We have used three methods of titrating serum neutralizing factors: inhibition of syncytium induction, neutralization of envelope pseudotypes of vesicular stomatitis virus (VSV) and reduction of infectivity of HTLV-III for a cell line permissive to virus replication. We report here that sera from subjects in various disease categories possess only low-level neutralizing activity, even when antibodies to viral membrane antigens are present in high titre. Envelope pseudotypes prepared from four HTLV-III isolates made in three different countries are equally sensitive to neutralization by positive sera, including sera from patients yielding two of the virus isolates.     

Accessory cell-dependent selection of specific T-cell functions

Activation of many T-cell functions depends on their interaction with antigen-presenting accessory cells which express I region associated (Ia) products. Cells expressing accessory cell function include those of the monocyte/macrophage lineage and dendritic cells. More recently, B cells and a number of tumour cell lines of macrophage or B-cell origin were shown to act as accessory cells in certain assays. We showed previously that normal peritoneal exudate macrophages (PEC) induced both T-cell proliferation as well as T-cell help, whereas various Ia+ tumour lines of macrophage or B-cell origin, although stimulating antigen-specific T-cell proliferation, did not significantly activate T-cell help. We report here that during the initial T-cell activation in vitro accessory cells (PEC or Ia+ tumour cells) select particular T cells to express previously determined functions. Moreover, some tumour cell lines induce suppressor T cells which inhibit helper activity.     

A survey of human leukaemias for sequences of a human retrovirus

Human T-cell leukaemia-lymphoma virus (HTLV) is an exogenous human retrovirus distinct from all known animal retroviruses. HTLV is closely linked to a subtype of adult T-cell malignancies and except for isolated cases, has not been found associated with any other form of leukaemia, lymphoma or other cancers (see refs 1, 2 for review). HTLV can be transmitted to cord blood T lymphocytes in vitro and the infected cells exhibit characteristics of transformed neoplastic T cells. We have recently cloned DNA sequences derived from approximately 1 kilobase (kb) of the 5' and 3' termini of the HTLV genome, as well as a 4-5-kb defective HTLV provirus flanked by cellular sequences. The availability of these probes has enabled us to carry out a limited survey of different fresh or cultured cells from patients of different lymphoid and myeloid malignancies for HTLV-related DNA sequences. The results presented here show that cells from all Japanese patients with adult T-cell leukaemia and several patients with various mature T-cell malignancies from elsewhere contained one or more copies of a highly conserved HTLV genome. The infected cells are of clonal origin. Fresh cells from 1 of the 10 myeloid leukaemic patients contained exogenous DNA sequences distantly related to HTLV.     

A novel human lymphokine that inhibits haematopoietic progenitor cell proliferation

T lymphocytes in culture synthesize and secrete a variety of factors that activate and guide the differentiation, replication and maturation of haematopoietic cells in vitro. Malignant T-cell lines as well as T-cell hybridomas producing several of these factors have been established. We report here a factor produced by a human cell line that exerts a potent inhibitory effect on the growth of bone marrow progenitor cells. The properties of this factor, which we have termed colony-inhibiting lymphokine ( CIL ), differ from other inhibitors of haematopoietic progenitor cell proliferation, but resemble those of a T-cell-derived factor causally linked with some cases of severe aplastic anaemia in humans. Sensitivity of cells to this factor appears to correlate positively with expression of HLA-DR surface antigens.     

Transformation of mononuclear phagocytes in vivo and malignant histiocytosis caused by a novel murine spleen focus-forming virus

The study of retrovirus-induced leukaemias in mice is a powerful tool for the elucidation of the normal regulation of the haematopoietic system. The acute murine spleen focus-forming viruses (SFFV) can be classified according to the haematopoietic lineage on which they exert their effects in the adult mouse. Here we report a new SFFV isolate, the AF-1 virus, with the novel ability to transform cells of the mononuclear phagocyte lineage. The virus was isolated from sarcomas that were induced on passage of a cloned Friend helper virus (F-MuLV, 643/22F) in newborn BALB/c mice. We have cloned the transforming defective subunit of the AF-1 viral complex in NRK cells and isolated several subclones. Analysis of the proviral genome in two non-producer cell clones reveals that AF-1 virus contains Harvey v-ras-specific sequences (Fig. 1). Thus, AF-1 virus is closely related to Harvey murine sarcoma virus (Ha-MSV), and is, at present, the only tool by which permanent cell lines can be obtained from mononuclear phagocytes in the mouse.     

Mature murine B lymphocytes immortalized by Kirsten sarcoma virus

Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus.     

Antibody production to the nucleocapsid and envelope of the hepatitis B virus primed by a single synthetic T cell site

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) can induce antibody responses via both a T-cell dependent and a T-cell independent pathway and is highly immunogenic during infection. We have examined the T-cell determinants of the antigen and find that HBcAg-specific helper T cells (TH) can help B cells produce antibody against envelope (HBsAg) antigens as well as HBcAg, even though these antigens are found on separate molecules. We have also been able to prime helper T cells with synthetic T-cell epitopes of HBcAg; helper cells primed with a single synthetic epitope can induce B cells to produce antibody that reacts with multiple HBsAg epitopes. One problem with the development of an HBV vaccine is that some vaccinees and patients do not respond to HBsAg directly; our results indicate that this problem can be circumvented using the response to HBcAg.     

Rejection of transplantable AKR leukaemia cells following MHC DNA-mediated cell transformation

Major histocompatibility complex (MHC) class I molecules can function as specific target antigens in T-cell-mediated cytotoxity. In addition, T cells can kill target cells through non-MHC antigens, for example, virally infected cells, if the target and effector cells express the same MHC class I antigens. Consequently, quantitative and/or qualitative variations in the expression of the H-2/HLA antigens on the target cells could interfere with MHC-restricted immune reactions. We have reported that the AKR leukaemia cell line K36.16, a subline of K36 (ref. 3), on which the H-2Kk antigen cannot be detected, is resistant to T-cell lysis and grows very easily in AKR mice. Other AKR tumour cell lines, like 369, which have a relatively large amount of H-2Kk on their surface, are easily killed by T cells in vitro and require a much larger inoculum to grow in vivo. Monoclonal antibodies against H-2Kk, but not against H-2Dk, prevented the killing by T cells. This suggests that some tumour cells grow in vivo because tumour-associated antigen(s) cannot be recognized efficiently by the host's immune system, due to the absence of MHC molecules which would function as restriction elements for T-cell cytotoxicity. We have tested this hypothesis by introducing the H-2Kk gene into the H-2Kk-deficient AKR tumour cell line K36.16 and have now demonstrated directly the biological relevance of H-2Kk antigen expression in the regulation of the in vivo growth of this tumour cell line.     

Identification of a protein encoded by the vpu gene of HIV-1

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture. The complexity of virus-host cell interaction is reflected in the complex organization of the viral genome. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3' orf proteins, as well as a protein of unknown function from the open reading frame called R. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV) that do not have the capacity to encode a similar protein.