Phenotypic heterogeneity of cerebrospinal fluid-derived HIV-specific and HLA-restricted cytotoxic T-cell clones

A variety of clinical syndromes, including AIDS and neurological disorders, may follow as a consequence of infection with the human immunodeficiency virus type 1 (HIV-1). It is not yet clear, however, to what extent the destruction of lymphocytes and neural cells associated with these conditions is caused by adverse immune responses to HIV-1 or how much is due to cytopathic effects of the virus itself. Here we document the existence of HLA-restricted, HIV-1-specific cytotoxic T lymphocytes in the cerebrospinal fluid of two AIDS patients manifesting neurologic disorders. These cytotoxic T lymphocytes showed dual specificity, recognizing target cells coated with purified HIV-1 envelope glycoprotein (gp 120) or inactivated HIV-1 in the context of HLA antigens. Cytotoxic T-cell clones derived from one of the AIDS patients revealed restriction specificities representing both HLA class I and HLA class II antigens. Considerable phenotypic heterogeneity was observed amongst these clones, some expressing conventional combinations of cytotoxic T-cell surface markers, and others displaying unusual phenotypes. The presence of HIV-specific cytotoxic T lymphocytes in AIDS patients, and in particular in their cerebrospinal fluid, suggests that these cytotoxic effectors may participate in the lymphoid cell and/or neurologic damage observed in such patients.     

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.     

T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV

Many viruses, including retroviruses, are characterized by their specific cell tropism. Lymphadenopathy-associated virus (LAV) is a human lymphotropic retrovirus isolated from patients with acquired immune deficiency syndrome (AIDS) or related syndromes, that displays selective tropism for a subset of T lymphocytes defined by the expression of a surface glycoprotein of relative molecular mass 62,000 (62K) termed T4 (refs 6-8). This glycoprotein delineates a subset of T lymphocytes with mainly helper/inducer functions, while T lymphocytes of the reciprocal subset express a glycoprotein termed T8, have mainly cytotoxic/suppressor activities, and are unable to replicate LAV. Such a tropism may be controlled at the genomic level by regulatory sequences, as described for the human T-cell leukaemia viruses HTLV-I and -II (refs 2, 3). Alternatively or concomitantly, productive cell infection may be controlled at the membrane level, requiring the interaction of a specific cellular receptor with the virus envelope, as demonstrated recently for Epstein-Barr virus (EBV). Therefore, we have investigated whether the T4 molecule itself is related to the receptor for LAV. We report here that preincubation of T4+ lymphocytes with three individual monoclonal antibodies directed at the T4 glycoprotein blocked cell infection by LAV. This blocking effect was specific, as other monoclonal antibodies--such as antibody to histocompatibility locus antigen (HLA) class II or anti-T-cell natural killer (TNK) target--directed at other surface structures strongly expressed on activated cultured T4+ cells, did not prevent LAV infection. Direct virus neutralization by monoclonal antibodies was also ruled out. These results strongly support the view that a surface molecule directly involved in cellular functions acts as, or is related to, the receptor for a human retrovirus.     

Human T-cell lymphotropic virus type-I antibodies in Falashas and other ethnic groups in Israel

Epidemiological studies of the human T-cell leukaemia/lymphoma virus type I (HTLV-I), a type-C retrovirus of the human T-lymphotropic virus family, have used serological surveys to identify population subgroups possessing a high prevalence of naturally occurring HTLV-I-specific antibodies. Studies carried out to delineate the global distribution of the virus have demonstrated natural antibodies to HTLV-I in the serum of healthy donors from specific geographical areas, and have defined viral endemic areas in Japan, the Caribbean basin, Africa and the southeastern United States. Such studies have suggested that the prevalence of HTLV-I antibodies is directly correlated with age, is associated with the clinical syndrome of adult T-cell lymphoma, and is associated with transmission from mother to child. A separate subtype of the human retrovirus, HTLV-II (refs 21, 22), has also been identified. The population of Israel in part comprises groups of immigrants of various ethnic and geographical origins. Because of this, and the fact that Israel has a highly developed public health system, we surmised that the ethnic groups in Israel could be used in a seroepidemiological survey of HTLV infection. The serological survey reported here demonstrates a high prevalence of HTLV-I antibodies in new immigrants from Ethiopia. This previously ethnically and geographically isolated group, the 'Black Jews' or 'Falashas', from the Gondar region in the northern rural highlands of Ethiopia, has the highest endemic rate of HTLV-I yet reported outside Japan.     

HIV-specific cytotoxic T lymphocytes in seropositive individuals

Virus-specific cytotoxic T lymphocytes (CTL) which kill virus-infected cells are thought to be a major host defence against viral infections. Here we report the existence of human immunodeficiency virus (HIV)-specific CTL in persons infected with this virus, the aetiological agent of AIDS (acquired immunodeficiency syndrome). Recombinant HIV-vaccinia viruses were used to express HIV antigens in B-cell lines established from subjects seropositive for HIV and seronegative controls. Circulating lymphocytes capable of killing HIV env-expressing autologous B cells were detected in eight of eight seropositive subjects; in addition, at least three seropositive subjects demonstrated gag-specific cytotoxic responses. No HIV-specific cytotoxicity was observed in seronegative subjects. Selective inhibition of the env-specific cytotoxicity by a CD3-specific monoclonal antibody indicates that the effectors are T cells. This demonstration of a cytotoxic T-cell immune response to HIV in infected individuals should prove useful in investigating the immunopathogenesis of HIV infection further and in evaluating AIDS vaccine strategies.     

Cellular immune responses to HIV

The cellular immune response to the human immunodeficiency virus, mediated by T lymphocytes, seems strong but fails to control the infection completely. In most virus infections, T cells either eliminate the virus or suppress it indefinitely as a harmless, persisting infection. But the human immunodeficiency virus undermines this control by infecting key immune cells, thereby impairing the response of both the infected CD4+ T cells and the uninfected CD8+ T cells. The failure of the latter to function efficiently facilitates the escape of virus from immune control and the collapse of the whole immune system.     

Productive dual infection of human CD4+ T lymphocytes by HIV-1 and HHV-6

Although infection by HIV-1 has been implicated as the primary cause of AIDS and related disorders, cofactorial mechanisms may be involved in the pathogenesis of the disease. For example, several viruses commonly detected in AIDS patients and capable of transactivating the long terminal repeat of HIV-1, such as herpesviruses, papovaviruses, adenoviruses and HTLV-I have been suggested as potential cofactors. Another candidate is human herpesvirus-6 (HHV-6, originally designated human B-lymphotropic virus), which has not only been identified in most patients with AIDS by virus isolation, DNA amplification techniques and serological analysis, but is also predominantly tropic and cytopathic in vitro for CD4+ T lymphocytes. Here we demonstrate that HHV-6 and HIV-1 can productively co-infect individual human CD4+ T lymphocytes, resulting in accelerated HIV-1 expression and cellular death. We also present evidence that HHV-6 transactivates the HIV-1 long terminal repeat (LTR). These observations indicate that HHV-6 might contribute directly or indirectly to the depletion of CD4+ T cells in AIDS.     

Different H-2 subregions influence immunization against retrovirus and immunosuppression

Friend murine leukaemia virus complex (FV) causes an immunosuppressive retrovirus-induced disease. In certain mouse strains, FV shows striking similarities to human immunodeficiency virus (HIV) infection in man in that infected mice have severe T-cell immunosuppression but also develop virus-neutralizing antibodies incapable of eliminating infected cells. Previously we noted the influence of mouse major histocompatibility complex (H-2) genes on both FV-induced immunosuppression and on ability to protect mice against FV by immunizing with a vaccinia-Friend murine leukaemia helper virus (F-MuLV) envelope (env) recombinant virus. Here we show that different subregions of H-2 are involved in susceptibility to virus-induced immunosuppression (H-2D subregion) and protective immunization with a recombinant vaccinia virus (H-2K or I-A subregions). Thus, susceptibility to virus-induced immunosuppression does not preclude protection by vaccinia-Friend immunization. The mechanism of protection seems to involve priming of immune T cells, and not initial induction of neutralizing antibodies or cytotoxic T lymphocytes (CTL) (ref.2). Subsequent virus challenge generates a secondary response, resulting in appearance of IgG antibodies and CTL. In human HIV infection there could also be host genetic influences on elements of disease pathogenesis, such as immunosuppression, and on the success of T-cell priming by potential protective vaccines.     

AIDS virus-specific cytotoxic T lymphocytes in lung disorders

Human immunodeficiency virus (HIV) is implicated in the development of AIDS (acquired immune deficiency syndrome). HIV infection leads to the generation of HIV-specific thymus-derived (T) lymphocytes in humans and apes. We describe an experimental system permitting the quantitative and systematic analysis of HIV-specific cytotoxic T lymphocytes (CTL). Functional, HIV-specific CTL are obtained by broncho-alveolar lavage (BAL) from the lungs of seropositive patients with lymphocytic alveolitis. These alveolar CTL: (1) recognize and kill HIV-infected alveolar macrophages in vitro under autologous, but not heterologous, conditions; (2) correspond to standard CTL as they express the CD3 and CD8 surface markers, but not the CD4 marker; and (3) are restricted by class I HLA transplantation antigens in their cytotoxic activities. We propose the hypothesis that interactions between HIV-specific CTL and infected macrophages induce major inflammatory reactions in seropositive patients.     

The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus

Acquired immune deficiency syndrome (AIDS) is characterized by opportunistic infections and by 'opportunistic neoplasms' (for example, Kaposi's sarcoma). Persistent generalized lymphadenopathy (PGL) is epidemiologically associated with AIDS, especially in male homosexuals. A subset of T lymphocytes positive for the CD4 antigen (also termed T4 antigen), is depleted in AIDS and PGL patients. A retrovirus found in T-cell cultures from these patients is strongly implicated in the aetiology of AIDS because of the high frequency of isolation and the prevalence of specific antibodies in the patients. Here we have detected cell-surface receptors for the AIDS retrovirus (human T-cell leukaemia virus-III (HTLV-III) and lymphadenopathy-associated virus-1 (LAV-1) isolates) by testing the susceptibility of cells to infection with pseudotypes of vesicular stomatitis virus bearing retroviral envelope antigens, and by the formation of multinucleated syncytia on mixing virus-producing cells with receptor-bearing cells. Receptors were present only on cells expressing CD4 antigen; among 155 monoclonal antibodies tested, each of the 14 anti-CD4 antibodies inhibited formation of syncytia and blocked pseudotypes. Productive infection of CD4+ cells with HTLV-III or LAV-1 markedly reduced cell-surface expression of CD4. In contrast, receptors for HTLV-I and HTLV-II were not restricted to CD4+ cells, were not blocked by anti-CD4 antibodies; cells productively infected with HTLV-I and HTLV-II expressed surface CD4. Hence, we conclude that the CD4 antigen is an essential and specific component of the receptor for the causative agent of AIDS.     

Construction and anti-tumor effects of recombinant fowlpox virus expressing Newcastle disease virus hemagglutinin-neuramidinase gene

Hemagglutinin-neuramidinase (HN), a Newcastle disease virus-derived protein, not only mediates receptor recognition but also possesses neuraminidase (NA) activity, the ability to cleave a component of those receptors, N-acetylneuraminic acid (NAcneu, sialic acid). It is known that this protein in mammalian species, including human beings, has interesting anti-neoplastic as well as immune stimulating properties. To explore the use of the HN gene in cancer gene therapy, we constructed a recombi-nant fowlpox virus expressing the HN protein (vFV-HN) and compared the anti-tumor activity of the recombinant virus with that of wild-type fowlpox virus (FPV) in vivo and in vitro. Here we found that although B16 cells were somewhat resistant to the basal cytotoxic effect of wild-type fowlpox virus, infection with vFV-HN caused a pronounced cytotoxic effect and, the survival of tumor-bearing mice immunized with vFV-HN was significantly increased compared with the survival of mice immunized with the FPV alone. Furthermore, the immunization of mice with vFV-HN elicited a B16 tumor-specific cytotoxic T lymphocyte (CTL) response and clonal expansion of both CD4 and CD8 T cell populations in vivo. In addition, T cells from lymph nodes of mice vaccinated with vFV-HN secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with vFV-HN may be a potential strategy for cancer gene therapy.     

Antigenic peptides recognized by T lymphocytes from AIDS viral envelope-immune humans

T-lymphocyte immunity is likely to be an important component of the immune defence against the AIDS virus, because helper T cells are necessary for the antibody response as well as the cytotoxic response. We have previously predicted two antigenic sites of the viral envelope protein gp120 likely to be recognized by T lymphocytes, based on their ability to fold as amphipathic helices, and have demonstrated that these are recognized by T cells of mice immunized with gp120 (ref. 1). A peptide corresponding to one of these sites can also be induce immunity in mice to the whole gp120 protein. Because many clinically healthy seropositive blood donors have already lost their T-cell proliferative response to specific antigen, we tested the response to these synthetic peptides of lymphocytes from 14 healthy human volunteers who had been immunized with a recombinant vaccinia virus containing the AIDS viral envelope gene and boosted with a recombinant fragment. Eight of the 14 responded to one peptide, and four to the other peptide, not included in the boost. These antigenic sites recognized by human T cells may be useful components of a vaccine against AIDS. We also found a correlation between boosting with antigen-antibody complexes (compared to free antigen) and higher stimulation indices, suggesting a more effective method of immunization.     

HTLV-III-neutralizing antibodies in patients with AIDS and AIDS-related complex

The isolation of the human T-cell leukaemia (lymphotropic) virus type III (HTLV-III or lymphadenopathy-associated virus) from cells of many patients with acquired immune deficiency syndrome (AIDS) presented the first evidence that the virus was the aetiological agent of the disease. Subsequent seroepidemiological studies have shown the presence of HTLV-III-specific antibodies in the serum of most patients with AIDS and AIDS-related complex (ARC), and in the serum of many individuals at risk for AIDS. Despite these extensive studies, there are no reports of protective effects of HTLV-III antibodies. In contrast, neutralizing antibodies specific for HTLV-I and -II have been identified previously. Therefore, we investigated whether HTLV-III-exposed individuals possess antibody activities capable of inhibiting viral infection. Here, we report that natural antibodies capable of neutralizing HTLV-III infection of H9 cells were detected in most adults AIDS and ARC patients but in no normal healthy heterosexual controls. Geometric mean antibody titres in ARC patients were double those in AIDS patients, and were even higher in two antibody-positive healthy homosexuals. This suggests that virus neutralizing antibodies may exert an in vivo protective effect. The presence of these antibodies indicates an immunological response to HTLV-III which potentially may be manipulated for therapeutic advantage. The methodology used here will be useful in monitoring future vaccine approaches.     

Two forms of the T-cell receptor gamma protein found on peripheral blood cytotoxic T lymphocytes

The T-cell receptor (TCR) gamma polypeptide is expressed associated with CD3 (T3) on the surface of normal human peripheral blood lymphocytes. These cells function as non-MHC-restricted cytotoxic T lymphocytes (CTL)and thus may play an important role in host immune defence. The TCR gamma polypeptide occurs as a dimer in at least two molecular forms based on the absence or presence of disulphide linkage. These forms use TCR gamma polypeptides with strikingly different peptide backbone sizes.     

Evolution and transmission of stable CTL escape mutations in HIV infection.

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother-child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression. In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design.     

Activated CD8 binding to class I protein mediated by the T-cell receptor results in signalling

The CD8 glycoprotein of T cells bind nonpolymorphic regions of class I major histocompatibility complex proteins on target cells and these interactions promote antigen recognition and signalling by the T-cell receptor. Studies using artificial membranes indicated that effective CD8/class I interaction is critical for response by alloantigen-specific cytotoxic T lymphocytes when class I protein is the only ligand on the antigen-bearing surface. But significant CD8-mediated binding of cytotoxic T lymphocytes to non-antigenic class I protein could not be detected in the absence of the alloantigen. These apparently contradictory findings indicate that CD8 binding to class I protein might be activated through the T-cell receptor and the results reported here demonstrate that this is the case. Treatment of cytotoxic T lymphocytes with soluble anti-T-cell receptor antibody activates adhesion of the cytotoxic T lymphocytes to class I, but not class II proteins. The specificity of this binding implies that it is mediated by CD8 and blocking by anti-CD8 antibodies confirmed this. Furthermore, binding of CD8 to class I protein resulted in generation of an additional signal(s) necessary to initiate response at low T-cell receptor occupancy levels.     

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.     

Trans-dominant inactivation of HTLV-I and HIV-1 gene expression by mutation of the HTLV-I Rex transactivator

The rex gene of the type I human T-cell leukaemia virus (HTLV-I) encodes a phosphorylated nuclear protein of relative molecular mass 27,000 which is required for viral replication. The Rex protein acts by promoting the cytoplasmic expression of the incompletely spliced viral messenger RNAs that encode the virion structural proteins. To identify the biologically important peptide domains within Rex, we introduced a series of mutations throughout its sequence. Two distinct classes of mutations lacking Rex biological activity were identified. One class corresponds to trans-dominant repressors as they inhibit the function of the wild-type Rex protein. The second class of mutants, in contrast, are recessive negative, rather than dominant negative, as they are not appropriately targeted to the cell nucleus. These results indicate the presence of at least two functionally distinct domains within the Rex protein, one involved in protein localization and a second involved in effector function. The trans-dominant Rex mutants may represent a promising new class of anti-viral agents.     

Strong xenogeneic HLA response in transgenic mice after introducing an alpha 3 domain into HLA B27.

The pronounced response by mouse T cells to the major histocompatibility complex (MHC) class I antigens of the same species is characterized by a relatively large fraction of responding cells. Responses to MHC class I allelles of other species are, however, generally much weaker. T lymphocytes are positively selected on thymic MHC antigens, resulting in a T-cell repertoire with strong alloreactivity. This has been explained in terms of a mouse T-cell repertoire that is not efficiently selected for recognition of HLA molecules owing to the absence of HLA in mice. Here we show that mice transgenic for HLA mount a T-cell response against allogeneic HLA that is no better than in normal mice. We decided instead to test whether the mouse accessory molecule Lyt-2 on cytotoxic T lymphocytes could interact efficiently with the alpha 3 domain of HLA. To do this, we replaced the alpha 3 domain of HLA-B27 by a murine alpha 3 domain in a gene construct used to produce transgenic mice, and then used the spleen cells from these mice to stimulate normal mouse T cells. Under these conditions cytotoxic T lymphocytes were generated with the same frequency against xenogeneic HLA-B27 determinants as against allogeneic mouse class I antigens. These findings indicate that the normally weak xeno-MHC response is due to the inefficient interaction of the murine Lyt-2 accessory molecule with HLA class I, and not to limitations of the mouse T-cell repertoire.