Mechanism(s) promoting HIV-1 infection of primary unstimulated T lymphocytes in autologous B cell/T cell co-cultures

Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.     

Phenotypic analysis of complement receptor 2+ T lymphocytes: reduced expression on CD4+ cells in HIV-infected persons.

While expression of complement receptor 2 (CR2) (CD21) on some CD4+ cell lines renders them more susceptible to infection by complement-treated human immunodeficiency virus (HIV), coexpression of CR2 and CD4 on peripheral blood lymphocytes has not, until recently, been observed. Several recent studies, however, have found that human T lymphocytes express low levels of CR2. Additionally, complement treatment of HIV before addition to these cells has been reported to increase virus expression in peripheral blood lymphocyte cultures. These findings suggest that complement-mediated enhancement of infection of human T cells could occur in vivo and have prompted us to examine both the phenotypic properties of CD4+CR2+ T cells in healthy persons and the expression of CR2 on CD4+ lymphocytes during HIV infection. As was previously reported, we observed CR2 on a proportion (10-50%) of both CD8+ and CD4+ T cells. Approximately half of CD4+CR2+ cells expressed the memory cell markers CD45RO and CD29, 80% expressed the naive marker CD45RA, while 22% expressed CD25. These values were not substantially different from total CD4+ cells. Stimulation of lymphocytes with phytohaemagglutinin (PHA), OKT3 or calcium ionophore but not with phorbol myristate acetate (PMA) or interleukin-2 (IL-2) decreased expression of CR2 on CD4 cells by half over a 3-day culture period. The per cent of CD4+ cells expressing CR2 was significantly decreased in patients with asymptomatic and symptomatic HIV infection compared to uninfected control donors (P = 0.0001). In contrast, the decrease in CR2 expression was not observed with CD8+ lymphocytes from HIV-infected persons. These results confirm that CR2 is expressed on human T lymphocytes and suggest that a subset of CD4+ lymphocytes is selectively affected in HIV-infected individuals.     

Evidence for the Role of CR1 (CD35), in addition to CR2 (CD21), in Facilitating Infection of Human T Cells with Opsonized HIV

Complement activation by HIV results in the binding of C3 fragments to the gp160 complex and enhanced infection of C3 receptor-bearing target cells. We have studied complement-mediated enhancement of infection of the human CD4-positive T-cell line HPB-ALL which expresses the CR1 (CD35) and CR2 (CD21) receptors for C3. CR1 and CR2 are present on 15% and 40% of normal peripheral blood CD4-positive T lymphocytes respectively. Opsonization of the virus with complement resulted in a 3- to 10-fold enhancement of infection of HPB-ALL cells, as assessed by measuring the release of p24 antigen in culture supernatants throughout the culture period. Blockade of CR2 with cross-linked anti-CR2 monoclonal antibodies decreased infection to the level observed with unopsonized virus. Blocking CR1 reduced complement-mediated infection by 50–80%. Experiments using serum deficient in complement factor I demonstrated that CR1 mediates the interaction between opsonized virus and T cells in addition to its ability to serve as a cofactor for the cleavage of C3b into smaller fragments that interact with CR2. A requirement for CD4 in complement-mediated enhancement of infection was observed with HIV-1 Bru but not with HIV-1 RF. Thus, CR1 and CR2 contribute in an independent and complementary fashion to penetration of opsonized virus into complement receptor-expressing T cells. Involvement of CD4 in infection with opsonized virus depends on the viral strain.     

Multiple HLA class I-dependent cytotoxicities constitute the "non-HLA-restricted" response in infectious mononucleosis

Primary Epstein-Barr virus (EBV) infection, when manifest as infectious mononucleosis (IM), induces a broad-ranging and apparently non-HLA-restricted cytotoxic response whose nature has not been resolved. In the present experiments the ability to cryo-preserve IM mononuclear cell preparations, after depletion of CD16+ natural killer cells, has allowed detailed analysis of the response on appropriately constructed target cell panels. The results show that IM effector preparations are polyclonal with separate HLA class I antigen-dependent reactivities against the autologous EBV-transformed lymphoblastoid cell line (LCL) and particular HLA class I-mismatched LCLs. The autologous LCL-directed response shows the hallmarks of immunologically specific T cell cytotoxicity; only EBV+ B cell blasts are recognized and the interaction can be blocked by monoclonal antibodies to CD3 and CD8 on the effector cell surface and to HLA class I antigens on the target cell. Such findings demonstrate, for the first time, that the primary cytotoxic response to EBV infection includes a virus-specific HLA-restricted component like that found in the T cell memory of persistently infected individuals. Separate components of the response are preferentially active against some (but not all) HLA-mismatched LCLs, the pattern of reactivity being distinct for each individual IM patient and reproducible on repeated testing. Monoclonal antibody blocking experiments show that these HLA-mismatched interactions also involve CD3 and CD8 antigens on the effector cell and HLA class I antigens on the target cell. Where tested, such lysis affected both EBV+ and EBV- B cell blasts from the relevant HLA-mismatched donors. We postulate that a widespread primary infection of the B cell system by EBV leads to a generalized expansion not just of the virus-specific response but also of other T cell responses coincidentally active at the time. The unusual activity of IM effector preparations against HLA-mismatched LCLs arises from fortuitous cross-recognition of allogeneic cells by immunologically specific cytotoxic T cell clones coincidentally expanded in vivo alongside the EBV-specific response.     

HIV-1 induced generation of C5a attracts immature dendriticcells and promotes infection of autologous T cells

For the recruitment of dendritic cells (DC) to the site of infection, DC express several sensors for danger signals, such as receptors for C5a. This anaphylatoxin is generated upon complement activation. As HIV-1 triggers the complement cascade, we determined whether C5a is generated by the virus and tested the functional activity of C5a in migration and infection assays. The immature (i)DC responded in migration assays to recombinant C5a and native C5a, which was generated in situ upon activation of the complement system by HIV-1. In combined migration and infection assays, a C5a-dependent enhancement of HIV-1 infection in DC-T cell cocultures was observed. These results indicate that HIV induces generation of C5a and thereby attracts iDC, which in turn promote the productive infection of autologous primary T cells.     

T cell-mediated cytotoxic immune responsiveness of chimeric mice bearing a thymus graft fully allogeneic to the graft of lymphoid stem cells

Fully allogeneic, chimeric mice were established by adult thymectomy of (A x B) F1 animals, grafting parental A-type thymus under the kidney capsula, followed by lethal (900 rd) irradiation and reconstitution with B parental-type bone marrow cells treated with xenogeneic anti-T cell antiserum plus complement. Following in vivo sensitization with inactivated Sendai virus (SV) suspensions, no virus-specific T cells could be detected within the spleen cells of the mice. Upon stimulation with third-party allogenic cells in a primary mixed lymphocyte culture, spleen cells of all animals generated alloreactive cytotoxic T lymphocytes (CLT). More interestingly, upon secondary in vitro stimulation with inactivated SV-conjugated B-type stimulator cells, B-type-restricted, virus-specific CTL were inducible in each case. Upon restimulation with SV-conjugated A-type stimulator cells, A being the H-2 type of the grafted thymus, T cells of some but no all mice generated A-type-restricted, virus-specific CTL. The data suggest that in allogeneic, chimeric mice virus-specific CTL can be induced. Moreover, virus-specific CTL, restricted to the H-2 type of the lymphoid stem cell inoculum, are more readily inducible than those restricted to the H-2 type of the allogeneic thymus.     

Frequency and function of HIV-specific CD8(+) T cells

The virus-specific CD8(+) T cell responses of 27 HIV-infected patients were studied, including a unique cohort of long term nonprogressors (LTNP) with normal CD4(+) T cell counts, low levels of plasma viral RNA, strong proliferative responses to HIV antigens and an over-representation of the HLA B*5701 class I allele. The frequencies of CD8(+) T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular interferon-gamma (IFN-gamma) in response to HIV-vaccinia recombinant infected autologous B cells. Very high frequencies (1.4-22%) of circulating CD8(+) T cells were found to be HIV-specific and were not only found in LTNP with reduced plasma virus. No correlation was evident between the frequency of HIV-specific CD8(+) T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to Gag-Pol gene products. Although similar frequencies of Gag peptide-specific CD8(+) T cells were found in LTNP and progressors by either intracellular IFN-gamma or MHC class I tetramer staining, the breadth of these responses was greater in patients with progressive HIV infection compared with the LTNP group. The frequency of CD8(+) T cells specific for a single peptide was not representative of an individual patient's total HIV-specific CD8(+) T cell response. These data demonstrate that high numbers of HIV-specific CD8(+) T cells exist even in patients with high level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8(+) T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive infection.     

B lymphocytes in lymph nodes and peripheral blood are important for binding immune complexes containing HIV-1

We investigated the interaction of HIV immune complexes (HIV IC) with mononuclear cells from lymph nodes and blood. While antibody alone did not affect binding of HIV IC to mononuclear cells, antibody plus complement increased binding by as much as 10-fold and complement alone also increased binding slightly. Most of the increased binding of HIV IC to mononuclear cells was blocked by heat-inactivation of complement and by OKB7 monoclonal antibody, indicating that virus binding was to CR2 on B cells. A similar pattern of antibody and complement dependence for binding of HIV IC was observed with two model systems; Raji and Arent B-cell lines. Most of the HIV IC that bound to lymph node cells were not internalized, but remained on the cell surface and were gradually released. However, even after 48 hr some HIV IC could be detected bound to cells. Under certain conditions, HIV IC were infectious for T cells if bound to B cells but not infectious if added directly to T cells. Additionally, HIV IC bound to B cells led to higher virus replication. These studies show that B lymphocytes from blood and lymph nodes can transfer infectious HIV IC to T cells.     

CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4.

Peripheral blood and tissue mononuclear phagocytes serve as major viral reservoirs in HIV-infected individuals. We investigated the role of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) in mediating productive infection with complement-opsonized HIV-1 and HIV-2 of cultured normal human peripheral blood monocytes, the promonocytic cell line THP-1, the monocytic cell line Mono Mac 6 and the glial cell line U251-MG. Cells were infected with the HTLV-IIIB strain of HIV-1 or the LAV-2 strain of HIV-2 that had been preopsonized with fresh human normal HIV seronegative serum. Productive infection was assessed by syncytia formation, the MTT cytotoxicity assay and/or release of p24 antigen in culture supernatants. Using suboptimal amounts of virus to infect the cells, we observed a higher and earlier productive infection of the cells with complement-opsonized HIV than with unopsonized virus. The enhancing effect of complement was totally suppressed by blocking CR1 or CR3 function with F(ab)'2 fragments of anti-receptor MoAbs; while blocking of the LFA-1 antigen had no effect. The infection of monocytic cells with complement-opsonized virus occurred independently of CD4 since it was not inhibited by F(ab)'2 fragments of a MoAb against the gp120 binding site of CD4 and since infection also occurred with Mono Mac 6 and U251-MG cells, which lack expression of the CD4 antigen and of CD4 mRNA. These observations suggest that complement may mediate productive infection of cells of the monocytic lineage with 'lymphocytotropic' HIV strains independently of CD4.     

MHC class II-deficient mice allow functional human CD4+ T-cell development

Humanized mouse models have been developed to study cell-mediated immune responses to human pathogens in vivo. How immunocompetent human T cells are selected in a murine thymus in such humanized mice remains poorly explored. To gain insights into this mechanism, we investigated the differentiation of human immune compartments in mouse MHC class II-deficient immune-compromised mice (humanized Ab0 mice). We observed a strong reduction in human CD4⁺ T-cell development but despite this reduction Ab0 mice had no disadvantage during Epstein–Barr virus (EBV) infection. Viral loads were equally well controlled in humanized Ab0 mice compared to humanized NSG mice, and improved T-cell recognition of autologous EBV-transformed B cells was observed, especially with respect to cytotoxicity. MHC class II blocking experiments with CD4⁺ T cells from humanized Ab0 mice demonstrated MHC class II restriction of lymphoblastoid cell line recognition. These findings suggest that a small number of CD4⁺ T cells in humanized mice can be solely selected on human MHC class II molecules, presumably expressed by reconstituted human immune cells, leading to improved effector functions.     

Decreased accessory cell function of macrophages after infection with human immunodeficiency virus type 1 in vitro

Peripheral blood monocytes from human immunodeficiency virus (HIV)-infected individuals or AIDS-related complex/AIDS patients ex vivo exhibit distinct alterations in some but not all immune functions. In studies presented here, monocytes from healthy donors were infected with HIV 1 in vitro and co-cultures with autologous uninfected T lymphocytes were set up. The monocyte/macrophage (M phi)-dependent T cell function was determined by measurement of proliferative and secretory [interleukin (IL)2, interferon-gamma] responses to lectin (phytohemagglutinin), mitogen (anti-CD3 monoclonal antibody), or recall antigen (tetanus toxoid, tuberculin). Accessory function of M phi was normal after HIV infection when optimal amounts (10%-20%) were added to the T lymphocytes. However, HIV infection of M phi significantly decreased T cell proliferative responses and secretion of IL2 when supplemented at limited dilution (0.5%-5%), although interferon-gamma production was not affected. Whereas the lipopolysaccharide-triggered M phi production of IL1 was not impaired by HIV 1 infection, there was a significant decrease in this response when anti-CD3 monoclonal antibody or tetanus toxoid were used to trigger the peripheral blood mononuclear cells. The impairment of proliferation of T lymphocytes in the presence of HIV 1-infected M phi could be overcome by addition of exogenous IL 1. Taken together, these data clearly show that the mononuclear phagocyte-dependent enhancement of stimulated T cell proliferation and lymphokine secretion is decreased when the restricted numbers of monocytes/M phi are HIV 1 infected. There are, therefore, two possible roles of M phi in HIV infection and progression to disease. First, as a reservoir and vehicle for dissemination of the virus, and second, as an immune cell whose essential functions are impaired by infection.     

Defining parameters for successful immunocytotherapy of persistent viral infection

Persistent infections with viruses such as HIV, Epstein-Barr virus, cytomelagovirus, and hepatitis B and C viruses continue to be major human health problems. Immunocytotherapy for persistent viral infections has proven successful in animal models but less effective in humans. While the requirement of antigen-specific CD8(+) T cells is known, the precise role of CD4(+) T cells as regards specific priming, numbers needed, and interaction with CD8(+) T cells is less clear. To address these issues, we used a mouse model of persistent virus infection in which adoptive transfer of T cells effectively purges virus from all tissues. We demonstrate that (1) inclusion of antigen-specific CD4(+) in addition to CD8(+) T cells is mandatory for efficient and long-term virus control. Neither naive nor CD4(+) T cells with specificity for a different virus are sufficient. (2) The minimal numbers of virus-specific T cells required for virus clearance from sera and tissues are 350,000 virus-specific CD8(+) and 7000 virus-specific CD4(+) T cells or approximately 5 x 10(7) CD8(+) and as few as 1 x 10(6) CD4(+) T cells per square meter of body surface area, a CD8:CD4 ratio of 50:1. (3) Production of interferon-gamma, obligatory for resolution of persistent infection, is dependent on the interaction of virus-specific CD4(+) and CD8(+) T cells. (4) Maintenance of CD8(+) T cell effector functions after adoptive transfer is directly proportional to the amount of cotransferred, virus-specific CD4(+) T cells.     

Activation of B lymphocytes by Epstein-Barr virus/CR2 receptor interaction

Epstein-Barr virus (EBV) infects and transforms human B lymphocytes. The virus receptor was shown to be identical to the complement receptor CR2. The consequences of EBV/CR2 receptor interaction on B lymphocyte activation were analyzed by infection of B cells with the transforming (B95-8) and the nontransforming (P3HR1) virus strains and with UV-inactivated B95-8 virus. Similar to mitogens and antibodies against surface IgM and CR2 receptor, transforming and nontransforming EBV induced the release of leukocyte migration inhibitory factor (LIF). LIF production occurred early after infection and was not affected by irradiation of the B95-8 virus with doses of UV-light which prevented the expression of viral functions. B lymphocytes infected with UV-inactivated virus responded to T cell-derived B cell growth factors. The results demonstrate that binding of EBV to the CR2 receptor activates the B cells. Progression of the infected cells in the activation pathway required helper cell-derived factors or signals provided by the intact viral genome. The nontransforming P3HR1 virus induced a low level of RNA synthesis and increase of cell size and major histocompatibility complex class I antigen expression. Only the transforming virus induced expression of EBV nuclear antigens and cellular DNA synthesis.     

Impaired Antibody Responses but Normal Proliferation of Specific CD4+ T Cells in Mice Lacking Complement Receptors 1 and 2

Severely impaired Ab responses are seen in animals lacking C (complement) factors C2, C3 or C4 as well as CR1/2 (C receptors 1 and 2). The molecular mechanism behind this phenomenon is not understood. One possibility is that C-containing immune complexes are endocytosed via CR2 on B cells and presented to specific CD4⁺ T cells, which would then proliferate and provide efficient help to specific B cells. In vitro , B cells can endocytose immune complexes via CR1/2 and present the Ag to T cells. Whether absence of this Ag presenting function in Cr2^−/− mice (mice lacking CR1/2) explains their low Ab response is unclear. To address this question, Cr2^−/− and wild type mice were transferred with OVA-specific T cells, obtained from the DO11.10 strain which has a transgenic TCR recognizing an OVA peptide. The animals were subsequently immunized with sheep red blood cells (SRBC) conjugated to OVA. Interestingly, proliferation of the OVA-specific T cells was normal in Cr2^−/− mice, although their Ab response to both SRBC and OVA was severely impaired. These observations suggest that the impaired Ab response in Cr2^−/− mice cannot be explained by a lack of appropriate induction of T cell help.     

Functional activity in vivo of effector T cell populations. III. Protection against Moloney murine sarcoma virus (M-MSV)-induced tumors in T cell deficient mice by the adoptive transfer of a M-MSV-specific cytolytic T lymphocyte clone

The functional activity of Moloney murine sarcoma virus (M-MSV)-specific T lymphocytes in vivo was assayed by the i.v. injection of virus-specific T lymphocytes into T cell-deficient "B mice". Virus-specific T lymphocytes generated in mixed lymphocyte tumor cell cultures were transferred i.v. into syngeneic "B mice" injected simultaneously at a distant site with the virus. These experiments indicated that a low dose (1 X 10(6) cultured cells) of infused lymphocytes can afford protection. To define the T lymphocyte subpopulation which was active, Lyt-2+ lymphocytes were selected by "panning" on plastic petri dishes coated with anti-Lyt-2 monoclonal antibody, and Lyt-2- lymphocytes selected by treatment with anti-Lyt-2 monoclonal antibody and complement. The results indicated that a Lyt-2+ lymphocyte-enriched population was more efficient in conferring protection against M-MSV-induced tumors. To investigate if cytolytic T lymphocytes (CTL) alone had a protective effect, a M-MSV-specific CTL clone was transferred in the same model system. The results demonstrated that a M-MSV-specific CTL clone prevented M-MSV-induced tumor growth and also induced the destruction of syngeneic Moloney murine leukemia virus (M-MuLV)-induced MBL-2 leukemic cells in the peritoneal cavity. However, the cell dose required to obtain protection using a CTL clone was higher than that which was effective when mixed lymphocyte tumor cell culture cells were used. To assess the ability of the transferred cells to home and to repopulate the lymphoid organs of the "B mice", the frequency of virus-specific CTL precursors in the spleen was evaluated by limiting dilution analysis. The results indicated that lymphocytes from mixed lymphocyte tumor cell cultures can be recovered from the spleens of "B mice" injected i.v. 25 days earlier. On the contrary, following the transfer of an active CTL clone, a very low frequency (less than 1/200,000 cells) of virus-specific CTL precursors was present in the spleens of recipient animals. The same M-MSV-specific CTL clone did not yield protection against M-MSV-induced tumors or MBL-2 leukemic cells when injected i.v. into M-MuLV tolerant mice.     

Changes in chemokines and chemokine receptor expression on tonsillar B cells upon Epstein–Barr virus infection

Chemokines and chemokine receptors are likely to play important roles in the pathogenesis of Epstein–Barr virus (EBV) -associated disease. The primary EBV infection occurs in the oropharynx where the virus infects mainly tonsillar B cells. We have previously shown that CXCR4 expression on tonsillar B cells is modulated by EBV. Here, CXCR5 and CCR7 expression, which is important for migration into lymphoid tissue, was followed for 14 days after EBV infection of tonsillar B cells. Early after infection (2 days) there were only minor changes in CXCR5 and CCR7 expression. However, at day 7 the expression of CXCR5, as well as of CCR7, was decreased and by day 14 these molecules were no longer present at the cell surface. Furthermore, EBV infection affects the chemotactic response to CXCL13 and CCL21 (the ligands for CXCR5 and CCR7, respectively) with a reduction of ligand-induced migration at day 2. Using gene expression profiling, we identified an additional set of chemokines and chemokine receptors that were changed upon EBV infection in comparison with non-infected tonsillar B cells. In particular, messenger RNA expression for CCR9 and the complement receptor C5AR1 was increased. Both receptors mediate homing to mucosal tissue. The alterations of the expression of these molecules may lead to retention of EBV-infected tonsillar B cells in the interfollicular region of the tonsil.     

Enhanced replication of R5 HIV-1 over X4 HIV-1 in CD4(+)CCR5(+)CXCR4(+) T cells

To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.     

Understanding and exploiting dendritic cells in human immunodeficiency virus infection using the nonhuman primate model

Dendritic cells (DC) are pivotal cells in the innate immune system. Recent interest in the role of DC in human immunodeficiency virus (HIV) pathogenesis has increased with the finding that both myeloid (mDC) and plasmacytoid DC (pDC) are lost from blood during infection, associated with progression to disease. DC are also being studied intensively for their capacity to stimulate robust virus-specific immunity as vaccines. Here we discuss our work in these contrasting fields of DC biology using the rhesus macaque nonhuman primate model. We focus on studies of DC dynamics in lymphoid tissues during pathogenic simian immunodeficiency virus (SIV) infection, DC trafficking in health and disease, DC-based vaccination and the use of autologous virus as antigen for stimulation of virus-specific T cells.     

Stimulatory and suppressive effects of infection of dendritic cells with HIV-1.

Two effects of HIV infection on human dendritic cells (DC) in vitro have been examined. The first was the stimulation of primary responses to HIV antigens in autologous lymphocytes from normal donors. When DC were exposed to HIV (10(4) TCID/10(5) cells) for up to 24 h before addition to autologous lymphocytes, a marked primary proliferative response to the virus was observed. No proliferative response was seen when the period of pre-exposure of DC to virus was extended. Cytotoxic T cells specific for HIV-infected target cells developed in stimulated cultures. The second effect of HIV infection of DC was to block responses to other antigens, such as alloantigens and the recall antigens tetanus toxoid and influenza virus. This inhibitory effect was only evident when the DC were exposed to HIV for longer than 24 h before being added to cultures. These in vitro studies suggest that infection of DC can produce both stimulatory and inhibitory responses in lymphocytes. Such effects operating through DC might underlie in vivo activity of HIV both in stimulating the proliferation of lymphocytes (e.g., in persistent generalised lymphadenopathy) and in the development of immunosuppression.