Human immunodeficiency virus-1 env impairs Fc receptor-mediated phagocytosis via a cyclic adenosine monophosphate-dependent mechanism.

Human immunodeficiency virus (HIV)-1 expression in mononuclear phagocytes is associated with multiple functional defects, including phagocytosis. To assess Fcgamma receptor (FcgammaR) function in cells expressing HIV-1, human promonocytic cells (U937) acutely or chronically infected with HIV-1, or stably transfected with a noninfectious reverse transcriptase (RT) defective HIV-1 provirus (Deltapol), were treated with phorbol 12-myristate 13-acetate for 48 hours and tested for their ability to ingest sheep erythrocytes coated with IgG (E-IgG). HIV-1-infected or transfected U937 cells ingested 50% to 65% fewer E-IgG than controls despite normal surface expression of FcgammaRs. HIV-1 specifically impaired FcgammaR-mediated phagocytosis, as ingestion of complement-coated erythrocytes was unaffected. U937 cells transfected with an env deficient mutant of HIV-1 ingested E-IgG normally, suggesting that the expression of HIV-1 env was required for HIV-1 to inhibit FcgammaR-mediated phagocytosis. Expression of HIV-1 in U937 cells was associated with an increased accumulation of intracellular cyclic adenosine monophosphate (cAMP); addition of the adenylate cyclase inhibitor 2',5'-dideoxyadenosine to these cells decreased intracellular cAMP levels to that of controls and restored FcgammaR-mediated phagocytosis. Addition of either interferon (IFN)-gamma or an inhibitor of cAMP-dependent protein kinase A (KT 5720) to HIV-1-transfected U937 cells also restored FcgammaR-mediated phagocytosis. Expression of HIV-1 induces a specific defect of FcgammaR function in mononuclear phagocytes that correlates with increased levels of cAMP, and can be corrected by pharmacologic manipulation.

TAT-mediated transcellular activation of HIV-1 long terminal repeat directed gene expression by HIV-1-infected peripheral blood mononuclear cells.

We have previously shown evidence of Tat-mediated transcellular activation of the HIV-1 long terminal repeat (LTR) in vitro by using a laboratory-adapted strain of HIV-1. To examine the biologic significance of this observation, we asked whether primary PBMCs from HIV-1-infected individuals will transactivate the HIV-1 LTR transcellularly in suitable indicator cells. In cultures of PBMCs isolated from HIV-1-infected patients at various clinical stages, with either HIV-1 LTR-transfected Jurkat T cells or nonfusigenic HIV-1 LTR-transfected murine fibroblasts, transcellular activation was readily detected. Transactivation of the LTR in cocultured cells with HIV-1-infected PBMCs is detectable 1 to 2 wk before the onset of significant virus production and at ratios as low as 1 infected cell to 10(6) surrounding cells. Addition of the Tat inhibitor RO5-3335 substantially decreases transcellular activation, even at low concentrations (0.01 microM) that do not affect virus levels. In contrast, addition of the antiretroviral agent zidovudine has no effect on transcellular activation. These data suggest that Tat-mediated transcellular activation of the HIV-1 LTR occurs independently of cellular infection, and provides a mechanism that can promote the spread of HIV-1 in susceptible cell populations.

Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in T lymphocytes requires CD4-gp120 binding.

Cells expressing human immunodeficiency virus type 1 (HIV-1) tat can transactivate the HIV-1 long terminal repeat (LTR) in cocultured T lymphocytes. In this report, we describe the molecular requirements for transcellular activation of the LTR in Jurkat cells. An analysis with deletion mutants and blocking antibodies demonstrated a requirement for env expression in addition to tat expression for transcellular activation to occur. The results suggest that the transient association of CD4 and gp120 in cocultured cells is required for tat-mediated transcellular activation. The events that follow CD4-gp120 binding in transactivation, however, do not require the gp120-neutralizing domain, in contrast to HIV-mediated fusion and infection. The consequences of this interaction on cellular function are currently under investigation.

Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in cocultured lymphocytes.

One of the unexplained aspects of the progression of AIDS is that immunological abnormalities are detectable before CD4+ T-helper cell depletion occurs (A.R. Gruters, F.G. Terpstra, R. De Jong, C.J.M. Van Noesel, R.A.W. Van Lier, and F. Miedema, Eur. J. Immunol. 20:1039-1044, 1990; F. Miedema, A.J. Chantal-Petit, F.G. Terpstra, J.K.M.E. Schattenkerk, F. de Wolf, B.J.M. Al, M. Roos, J.M.A. Lang, S.A. Danner, J. Goudsmit, and P.T.A. Schellekens, J. Clin. Invest. 82:1908-1914, 1988; G.M. Shearer, D.C. Bernstein, K.S. Tung, C.S. Via, R. Redfield, S.Z. Salahuddin, and R.C. Gallo, J. Immunol. 137:2514-2521, 1986). In this report, we describe a mechanism by which human immunodeficiency virus type 1 (HIV-1)-infected cells can influence neighboring HIV-1-infected T lymphocytes and uninfected T cells as well. We have examined the interaction of T-cell and macrophage cell lines that are transfected with HIV-1 DNA by using cocultured lymphocytes. The HIV-1 constructs we used lack a functional pol gene and therefore do not produce infectious virus. Cocultivation results in the transcellular activation of the HIV long terminal repeat in the cocultured T cells. This transcellular activation is evident in as little as 3 h of cocultivation, at ratios of HIV-expressing cells to target cells as low as 1:1,000, and is dependent on the Tat-responsive element. The demonstration that a small number of HIV-expressing cells can affect a large number of uninfected bystander cells in a short period of time suggests a mechanism by which global immune dysfunction can precede the high prevalence of infected cells.

Common double- and single-stranded DNA binding factor for a sterol regulatory element.

A cis-acting element necessary for sterol regulation, SRE-1, has previously been identified in the promoters of the low density lipoprotein receptor, hydroxymethylglutaryl (HMG)-CoA reductase, and HMG-CoA synthase genes. In this report we describe a nuclear factor, SRE-BF, isolated from Chinese hamster ovary nuclear extracts, that binds to the SRE-1 octanucleotide sequence. In addition to sequence-specific binding to SRE-1, as indicated by competition analysis with double-stranded DNA fragments, single-stranded oligomer DNA sequences also compete for binding in a sequence-specific fashion. Photochemical cross-linking experiments suggest that a common protein factor, with apparent molecular mass of 45-49 kDa, recognizes both single-stranded and double-stranded SRE-1. The binding specificity of SRE-BF to single-stranded SRE-1 closely correlates with the reported in vivo ability of SRE-1 to direct sterol responsiveness of transcription.

The ability of Ia and H-2Kk-bearing membranes to replace the antigen-presenting cell in an H-2Kk allogeneic cytotoxic T cell response.

Induction of an allogeneic cytotoxic T lymphocyte (CTL) response is dependent, in part, on uptake and processing of the class I alloantigen by antigen-presenting cells and subsequent Ia-restricted recognition of the alloantigen by helper T cells, resulting in lymphokine production. The nature of the antigen-processing event has been investigated using reconstituted membranes to replace the antigen-presenting cells in the generation of a secondary allogeneic CTL response. Membranes were isolated from an Iad-positive antigen presenting B cell lymphoma (D2N), detergent solubilized and then reconstituted together with affinity-purified H-2Kk antigen in the presence of protease inhibitors. These reconstituted vesicles, containing both syngeneic Ia and alloantigen, were able to induce the helper T cell arm of the CTL response in cultures depleted of antigen-presenting cells. A variety of control experiments provided strong evidence that the helper T cells recognized the H-2Kk, probably in its native form, in an Ia-restricted manner on the vesicles, while the pre-CTL can directly recognize H-2Kk. Recognition was only effective if both the Ia and alloantigen were inserted into the same membrane bilayer. The results strongly suggest that the obligatory antigen processing event required for helper T cell recognition of alloantigen is simply the insertion of the alloantigen into the same membrane bilayer as the syngeneic Ia restricting element.

Characterization of a Q subregion gene in the murine major histocompatibility complex.

We have used restriction enzyme digests, Southern blot analysis, and gene transfer experiments to identify a class I gene in the Q subregion of the murine major histocompatibility complex. By comparisons of class I genes from Q congeneic strains, five restriction fragment length polymorphisms were identified. Further studies of mutant (Qa-2-) and wild-type (Qa-2+) BALB/c sublines indicated that at least part of the structural or regulatory gene controlling a Q subregion antigen resides on a 3.7-kilobase Xba I DNA fragment and is absent in all tested Qa-2- strains. The spontaneously occurring Qa-2- BALB/cBy mutant appears to have an extensive deletion in this region. The identity of this gene was confirmed by gene transfer experiments as well as by the use of a single-copy probe.

Thymectomized, irradiated, and bone marrow-reconstituted chimeras have normal cytolytic T lymphocyte precursors but a defect in lymphokine production.

A model system has been developed to study extrathymic T cell differentiation; mice have been thymectomized, lethally irradiated, and reconstituted with bone marrow cells depleted of Thy-1+ cells. After 8 wk, the spleen cells of these athymic, bone marrow-reconstituted chimeras contain Thy-1+ precytolytic T lymphocytes (CTL) that are able to respond to antigen only if supernatant from Con A-activated T cells is added to culture. The phenotype of these pre-CTL is similar to that of thymocytes, suggesting that they may be immature T cells. Initial evaluation of the CTL repertoire of these athymic mice demonstrated that the CTL generated to trinitrophenyl-modified syngeneic cells are H-2-restricted, and that the CTL generated to alloantigens have many of the cross-reactivities observed in normal mice but not in nude mice. In this report, we demonstrate a helper T cell defect in these thymectomized chimeras. These chimeras lack an Ly-1+ helper cell required for thymocytes to differentiate to CTL. Further studies revealed that when spleen cells from these thymectomized chimeras were stimulated with Con A, they produced normal levels of interleukin 2. However, these splenocytes were defective in the production of another factor needed for CTL differentiation.

Responses to the H-2Kba mutant involve recognition of syngeneic Ia molecules.

Conventional antigens appear to be recognized by T lymphocytes only when associated with major histocompatibility complex (MHC) antigens. Using antigen-specific proliferation as a model for helper T lymphocytes, it has been demonstrated that Ly1+T cells recognize antigen presented in association with syngeneic Ia molecules. In contrast to responses to conventional antigens, however, a large number of studies have suggested that the stimulation of alloreactive Ly1+T cells, and helper T cells specific for allogeneic cytotoxic T lymphocyte (CTL) responses, involve the direct recognition of Ia alloantigens. For the generation of optimal allogeneic CTL activity it has been proposed that Ly1+T cells recognize allo-Ia antigens directly and provide help to pre-CTLs that respond to allo-H-2K and/or D determinants. Thus, the B6.C.H-2bm1 mutant (bm1, formerly referred to as Hz1), which is believed to consist of a substitution of two amino acids in the H-2Kb antigen, has presented a paradox, for it can stimulate strong mixed lymphocyte culture (MLC), graft versus host and CTL responses by T cells of H-2b haplotype mice in the apparent absence of any alloantigenic differences in the I region. We now present evidence that the stimulation of proliferative and helper T cells by the mutant B6.C.H-2bm1 results from the H-2Kba antigen being recognized in the context of syngeneic Ia determinants. Thus responses to both conventional antigens and allogeneic MHC gene products may proceed via the recognition of antigen in the context of self Ia molecules.

Role of syngeneic Ia+ accessory cells in the generation of allospecific CTL responses.

Under conditions in which antigen dose is suboptimal, the recognition of Ia determinants is a necessary component of the generation of allospecific CTL responses. With monoclonal anti-Ia antibodies used as blocking reagents, it is demonstrated that the recognition of alloantigens may proceed via two pathways. Alloantigens can be recognized in the context of syngeneic Ia determinants in a similar fashion to conventional antigens. If, however, the Ia+ cells are removed from the responder population, the generation of such responses involves the recognition of allogeneic Ia determinants directly. A non-T, non-B, adherent accessory cell was identified as the critical syngeneic Ia+ cell required for CTL responses in these cultures.

Analysis of the two-signal requirement for precursor cytolytic T lymphocyte activation using H-2Kk in liposomes.

Activation of primed pre-cytolytic T lymphocytes (pCTL) requires two signals: recognition of antigen (signal 1) and interaction with a nonspecific helper factor (signal 2). The two signals necessary for generation of a secondary allogeneic CTL response have been analyzed using H-2Kk in liposomes as the stimulating antigen. Use of the liposomes allows the alloantigen to be separated from responder cells after a brief exposure. Thus, the requirements for effective delivery of each signal could be studied independently. A 12-hr exposure of pCTL to alloantigen was sufficient for optimum signal 1 delivery. pCTL recognition of the antigen occurs during this time, and no requirement for adherent cells could be demonstrated. The structure of the antigen-containing liposomes affects the efficiency of pCTL triggering. Factor(s) necessary for signal 2 could be provided by supernatants from mitogen-stimulated lymphocytes. Alternatively, it could be generated with alloantigen, providing that adherent cells were present. Optimum interaction of factor(s) with pCTL, i.e., optimum delivery of signal 2, occurred only if factor(s) was present at 12 to 24 hr after interaction of pCTL with alloantigen. The results suggest that alloantigen recognition triggers pCTL to synthesize and/or express receptors for the factor(s).

In vitro generation of helper T cells and suppressor T cells that regulate the cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells.

Helper T cells and suppressor T cells have been generated in vitro that regulate the cytolytic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified syngeneic cells. B6D2F1 helper cells generated to TNP-modified parental (P1) cells augment the CTL response to those P1-TNP-modified antigens but not to P2-TNP-modified antigens. The generation of these helper T cells requires the presence of splenic adherent cells and these helper T cells are radioresistant. A soluble factor can be obtained from the helper T cell cultures that can also augment the CTL response. The suppressor T cells generated in culture do not demonstrate the specificity observed with the helper T cells; however, they are antigen-dependent in their induction. Whether helper or suppressor activity is obtained depends upon the length of time cells are cultured in vitro.

Cellular interactions in the generation of cytolytic T lymphocyte responses. Analysis of the helper T cell pathway.

Murine splenic lymphocytes exhibit a requirement for helper T cells for the induction of a cytolytic T lymphocyte response to suboptimal doses of allogeneic cells, membranes from allogeneic cells, or purified H-2 antigen in liposomes. The conditions where a requirement for help is apparent are the same conditions where a dependence on splenic adherent cells (SAC) has been demonstrated (Weinberger, O. et al., PROC. Natl. Acad, Sci, USA 1980. 77: 6091). Help can be provided in the form of primed, radioresistant, Ly-1+ spleen or lymph node cells or helper factor (interleukin 2, IL-2). A factor generated from phytohemagglutinin-stimulated human lymphocytes, when added to culture in the presence of antigen, bypassed the requirement for Ia+ SAC and helper cells. IL-2 reconstituted the response to H-2K(k) in liposomes in cultures depleted of SAC, strongly suggesting that the helper cell must see antigen re-expressed by an antigen-presenting cell, whereas the prekiller does not. IL-2 could be generated by culturing Ly-1+ murine spleen cells with H-2K(k) pulsed on SAC.

Antigen-presenting cell function in induction of helper T cells for cytotoxic T-lymphocyte responses: evidence for antigen processing.

We demonstrate that splenic adherent cells (SACs) play an active role in the presentation of H-2Kk antigen for an alloreactive cytotoxic T-lymphocyte (CTL) response. If antigen is incubated with SACs for 12 hr, they will provide maximal stimulation and present the antigen in the context of their Ia molecules. UV irradiation of these SACs, prior to the 12-hr incubation with H-2Kk antigen, abrogates this stimulatory capacity. Macrophage-bound antigen is not sufficient for stimulation of a response; a second signal is required as well, that, in our system, is provided by phorbol myristic acetate. The SACs are involved in the activation of helper T cells; however, they are not required for presentation of antigen to the precytotoxic T-lymphocyte, which requires two signals for activation, one provided by antigen and the other by a T-cell-derived helper factor.

Cellular interactions in the generation of cytolytic T lymphocyte responses: role of Ia-positive splenic adherent cells in presentation in H-2 antigen.

Splenic adherent cells are required for generation of both primary and, at limiting antigen dose, secondary allogeneic responses of cytolytic T lymphocytes to intact stimulator cells. Secondary responses to purified allogeneic plasma membranes or purified H-2Kk antigens in liposomes are also dependent upon splenic adherent cells. Generation of these responses requires the presence of an Ia-positive. Thy 1,2-negative, radiation-resistant cell in the splenic adherent cell population that is adherent to glass, plastic, and nylon wool. Stimulation of cytolytic T lymphocyte precursors with purified H-2Kk alloantigen bound to Ia+ splenic adherent cells is 10-20 times more efficient than stimulation with antigen added directly to culture. Furthermore, a marked decrease in the response of cytolytic T lymphocytes to liposomes was observed when antiserum against Iad specific for the Ia of the responder cells was added to culture. These results demonstrate that, for purified proteins of the major histocompatibility complex, antigen presentation by Ia+ splenic adherent cells plays a role in the generation of a cytolytic T lymphocyte response.