Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4⁺ lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34⁺ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.     

CD4+ T-Lymphocyte Depletion in Human Lymphoid Tissue Ex Vivo Is Not Induced by Noninfectious Human Immunodeficiency Virus Type 1 Virions

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4⁺ T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4⁺ T-cell depletion, consistent with mechanisms requiring productive infection.     

Differential Tropism and Replication Kinetics of Human Immunodeficiency Virus Type 1 Isolates in Thymocytes: Coreceptor Expression Allows Viral Entry, but Productive Infection of Distinct Subsets Is Determined at the Postentry Level

Human thymocytes are readily infected with human immunodeficiency virus type 1 (HIV-1) in vivo and in vitro. In this study, we found that the kinetics of replication and cytopathic effects of two molecular isolates, NL4-3 and JR-CSF, in postnatal thymocytes are best explained by the distribution of chemokine receptors used for viral entry. CXCR4 was expressed at high levels on most thymocytes, whereas CCR5 expression was restricted to only 0.1 to 2% of thymocytes. The difference in the amount of proviral DNA detected after infection of fresh thymocytes with NL4-3 or JR-CSF correlated with the levels of CXCR4 and CCR5 surface expression. Anti-CCR5 blocking studies showed that low levels of CCR5 were necessary and sufficient for JR-CSF entry in thymocytes. Interleukin-2 (IL-2), IL-4, and IL-7, cytokines normally present in the thymus, influenced the expression of CXCR4 and CCR5 on thymocytes and thus increased the infectivity and spread of both NL4-3 and JR-CSF in culture. NL4-3 was produced by both immature and mature thymocytes, whereas JR-CSF production was restricted to the mature CD1⁻/CD69⁺ population. Although CXCR4 and CCR5 distribution readily explained viral entry in mature CD69⁺ and immature CD69⁻ cells, and correlated with proviral DNA distribution, we found that viral production was favored in CD69⁺ cells. Therefore, while expression of CD4 and appropriate coreceptors are essential determinants of viral entry, factors related to activation and stage-specific maturation contribute to HIV-1 replication in thymocyte subsets. These results have direct implications for HIV-1 pathogenesis in pediatric patients.     

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.     

Loss of CD4+ T Cells in Human Immunodeficiency Virus Type 1-Infected Chimpanzees Is Associated with Increased Lymphocyte Apoptosis

Supportive evidence that apoptosis contributes to loss of CD4⁺ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected humans comes from an apparent lack of abnormal apoptosis in apathogenic lentivirus infections of nonhuman primates, including HIV-1 infection of chimpanzees. Two female chimpanzees were inoculated, one cervically and the other intravenously, with HIV-1 derived from the LAI/LAV-1b strain, which was isolated from a chimpanzee infected with the virus for 8 years. Within 6 weeks of infection, both recipient chimpanzees developed a progressive loss of CD4⁺ T cells which correlated with persistently high viral burdens and increased levels of CD4⁺ T-cell apoptosis both in vitro and in vivo. Lymph nodes from both animals also revealed evidence of immune hyperactivation. Intermediate levels of T-cell apoptosis in both peripheral blood and lymph nodes were seen in a third chimpanzee that had been infected with the LAI/LAV-1b strain for 9 years; this animal has maintained depressed CD4/CD8 T-cell ratios for the last 3 years. Similar analyses of cells from 4 uninfected animals and 10 other HIV-1-infected chimpanzees without loss of CD4⁺ cells revealed no difference in levels of apoptosis in these two control groups. These results demonstrate a correlation between immune hyperactivation, T-cell apoptosis, and chronic loss of CD4⁺ T cells in HIV-1-infected chimpanzees, providing additional evidence that apoptosis is an important factor in T-cell loss in AIDS. Furthermore, the results show that some HIV-1 strains are pathogenic for chimpanzees and that this species is not inherently resistant to HIV-1-induced disease.     

Naïve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis

In vitro evidence suggests that memory CD4⁺ cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA⁺ CD4⁺ (naïve) and CD45RO⁺ CD4⁺ (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4⁺ cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4⁺ subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the β-chemokines RANTES, MIP-1α, and MIP-1β upon stimulation. Neutralization of these β-chemokines rendered memory CD4⁺ cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and β-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4⁺ T cells serve as the targets for early rounds of HIV-1 replication.     

Determinant in Human Immunodeficiency Virus Type 1 for Efficient Replication under Cytokine-Induced CD4+ T-Helper 1 (Th1)- and Th2-Type Conditions

Cytokines are potent stimuli for CD4⁺-T-cell differentiation. Among them, interleukin-12 (IL-12) and IL-4 induce naive CD4⁺ T cells to become T-helper 1 (Th1) or Th2 cells, respectively. In this study we found that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains replicated more efficiently in IL-12-induced Th1-type cultures derived from normal CD4⁺ T cells than did T-cell-line-tropic (T-tropic) strains. In contrast, T-tropic strains preferentially infected IL-4-induced Th2-type cultures derived from the same donor CD4⁺ T cells. Additional studies using chimeric viruses demonstrated that the V3 region of HIV-1 gp120 was the principal determinant for efficiency of replication. Cell fusion analysis showed that cells expressing envelope protein from a T-tropic strain effectively fused with IL-4-induced Th2-type culture cells. Flow cytometric analysis showed that the level of CCR5 expression was higher on IL-12-induced Th1-type culture cells, whereas CXCR4 was highly expressed on IL-4-induced Th2-type culture cells, although a low level of CXCR4 expression was observed on IL-12-induced Th1-type culture cells. These results indicate that HIV-1 isolates exhibit differences in the ability to infect CD4⁺-T-cell subsets such as Th1 or Th2 cells and that this difference may partly correlate with the expression of particular chemokine receptors on these cells. The findings suggest that immunological conditions are one of the factors responsible for inducing selection of HIV-1 strains.     

In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses,Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

Background

The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission.

Methodology

The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited.

Findings

Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4⁺ and CD8⁺ T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4⁺ T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4⁺ T cell depletion which renders the newly activated HIV-specific CD4⁺ T cells prime targets for elimination.

Conclusion

Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.     

Strong Ability of Nef-Specific CD4+ Cytotoxic T Cells To Suppress Human Immunodeficiency Virus Type 1 (HIV-1) Replication in HIV-1-Infected CD4+ T Cells and Macrophages

A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4⁺ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4⁺ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4⁺ T cells. The CD4⁺ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4⁺ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4⁺ T cells from an HLA-DRB1*0803⁺ donor. In addition, these Nef-specific cytotoxic CD4⁺ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4⁺ T cells in vitro. Nef187-203-specific cytotoxic CD4⁺ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803⁺ donors, respectively. These results suggest that HIV-1-specific CD4⁺ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.Human immunodeficiency virus (HIV)-specific CD8⁺ cytotoxic T cells (CTLs) play a central role in the control of HIV type 1 (HIV-1) during acute and chronic phases of an HIV-1 infection (5, 29, 34). However, HIV-1 escapes from the immune surveillance of CD8⁺ CTLs by mechanisms such as mutations of immunodominant CTL epitopes and downregulation of major histocompatibility complex class I (MHC-I) molecules on the infected cells (9, 11, 12, 49). Therefore, most HIV-1-infected patients without highly active antiretroviral therapy (HAART) develop AIDS eventually.HIV-1-specific CD4⁺ T cells also play an important role in host immune responses against HIV-1 infections. An inverse association of CD4⁺ T-cell responses with viral load in chronically HIV-1-infected patients was documented in a series of earlier studies (8, 36, 39, 41, 48), although the causal relationship between them still remains unclear (23). Classically, CD4⁺ T cells help the expansion of CD8⁺ CTLs by producing growth factors such as interleukin-2 (IL-2) or by their CD40 ligand interaction with antigen-processing cells and CD8⁺ CTLs. In addition, CD4⁺ T cells provide activation of macrophages, which can professionally maintain CD8⁺ T-cell memory (17). On the other hand, the direct ability of virus-specific cytotoxic CD4⁺ T cells (CD4⁺ CTLs) to kill target cells has been widely observed in human virus infections such as those by human cytomegalovirus, Epstein-Barr virus (EBV), hepatitis B virus, Dengue virus, and HIV-1 (2, 4, 10, 19, 30, 31, 38, 50). Furthermore, one study showed that mouse CD4⁺ T cells specific for lymphocytic choriomeningitis virus have cytotoxic activity in vivo (25). These results, taken together, indicate that a subset of effector CD4⁺ T cells develops cytolytic activity in response to virus infections.HIV-1-specific CD4⁺ CTLs were found to be prevalent in HIV-1 infections, as Gag-specific cytotoxic CD4⁺ T cells were detected directly ex vivo among peripheral blood mononuclear cells (PBMCs) from an HIV-1-infected long-term nonprogressor (31). Other studies showed that up to 50% of the CD4⁺ T cells in some HIV-1-infected donors can exhibit a clear cytolytic potential, in contrast to the fact that healthy individuals display few of these cells (3, 4). These studies indicate the real existence of CD4⁺ CTLs in HIV-1 infections.The roles of CD4⁺ CTLs in the control of an HIV-1 infection have not been widely explored. It is known that Gag-specific CD4⁺ CTLs can suppress HIV-1 replication in a human T-cell leukemia virus type 1-immortalized CD4⁺ T-cell line (31). However, the functions of CD4⁺ T cells specific for other HIV-1 antigens remain unclear. On the other hand, the abilities of CD4⁺ CTLs to suppress HIV-1 replication in infected macrophages and CD4⁺ T cells may be different, as in the case of CD8⁺ CTLs for HIV-1-infected macrophages (17). In this study, we identified Nef-specific CD4⁺ T cells and investigated their ability to kill HIV-1 R5 virus-infected macrophages and HIV-1 X4 virus-infected CD4⁺ T cells and to suppress HIV-1 replication in the infected macrophages and CD4⁺ T cells. The results obtained in the present study show for the first time the ability of HIV-1-specific CD4⁺ CTLs to suppress HIV-1 replication in natural host cells, i.e., macrophages and CD4⁺ T cells.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Preservation of FoxP3+ regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors correlates with low CD4+ T-cell activation

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4⁺ T-cell counts and control viremia to levels that are below the limit of detection of current assays. The mechanisms involved in long-term control of viremia have not been fully elucidated. CD4⁺ CD25⁺ regulatory T cells (Tregs) downmodulate chronic inflammation by suppressing the activation and proliferation of effector lymphocytes. We found that while Tregs were functional in ES and patients on highly active antiretroviral therapy (HAART), ES maintained high levels of Tregs in peripheral blood mononuclear cells whereas patients on HAART had evidence of Treg depletion. We also demonstrated that Tregs can serve as reservoirs for HIV-1 in vivo. These data suggest that both direct infection by HIV-1 and tissue redistribution are possible explanations for declining FoxP3⁺ Tregs in progressive HIV-1 infection. Furthermore, the maintenance of Tregs may be one mechanism associated with the nonprogressive nature of HIV-1 infection in ES.     

Dendritic Cells Transmit Human Immunodeficiency Virus Type 1 to Monocytes and Monocyte-Derived Macrophages

Previous studies have shown that human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DC) to replicate and spread among CD4⁺ T cells. To explain the predominance of non-syncytium-inducing (NSI) over syncytium-inducing (SI) strains during the initial viremia of HIV, we investigated the ability of blood monocyte (Mo)-derived DC to transmit HIV-1 to CD4⁺ cells of the monocytoid lineage. First, we demonstrate that in our system, DC are able to transmit NSI strains, but not SI strains, of HIV-1 to fresh blood Mo and to Mo-derived macrophages (MDM). To establish a productive infection, a 10-fold-lower amount of virus was necessary for DC-mediated transmission of HIV-1 to Mo than in case of cell-free infection. Second, immature CD83⁻ DC (imDC) transmit virus to Mo and MDM with higher efficacy compared to mature CD83⁺ DC (maDC); this finding is in contrast to data previously obtained with CD4⁺ T cells. Third, maturation from imDC to maDC efficiently silenced expression of β₂-integrins CD11b, CD11c, and CD18 by maDC. Moreover, monoclonal antibody against CD18 inhibited transmission of HIV-1 from imDC to Mo. We propose that the adhesion molecules of the CD11/CD18 family, involved in cell-cell interactions of DC with the microenvironment, may play a major role in imDC-mediated HIV-1 infection of Mo and MDM.     

Distinct Mechanisms Trigger Apoptosis in Human Immunodeficiency Virus Type 1-Infected and in Uninfected Bystander T Lymphocytes

Apoptosis is a main feature of AIDS pathogenesis and is thought to play a role in the progressive decrease of CD4⁺ T lymphocytes in infected individuals. To determine whether apoptosis occurs in infected and/or in uninfected peripheral blood T lymphocytes, we have used a recombinant human immunodeficiency virus type 1 (HIV-1) infectious clone expressing the green fluorescent protein (GFP). Using flow cytometry, we have determined the incidence of apoptosis by either terminal transferase dUTP nick end labeling or annexin-V assays in different cell subpopulations, i.e., in CD4⁺ or CD8⁺ T cells that were GFP positive or negative. After HIV-1 infection of purified peripheral blood lymphocytes, we observed that apoptosis occurred mostly in infected CD4⁺ peripheral blood lymphocytes. Remarkably, the presence of monocyte-derived macrophages in the culture increased dramatically the apoptosis of uninfected bystander T lymphocytes, while apoptosis in HIV-infected T lymphocytes was not changed. We therefore demonstrate that HIV-induced apoptosis results from at least two distinct mechanisms: (i) direct apoptosis in HIV-infected CD4⁺ T lymphocytes and (ii) indirect apoptosis in uninfected T cells mediated by antigen-presenting cells.     

Human Immunodeficiency Virus Type 1 Escapes from Interleukin-2-Producing CD4+ T-Cell Responses without High-Frequency Fixation of Mutations

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4⁺ T-cell responses has been associated with the immunological control of HIV-1 replication; however, the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4⁺ T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells, each of the individuals in the present study exhibited progressive disease, with one individual showing rapid progression. In this rapid progressor, three IL-2-producing HIV-1 Gag-specific CD4⁺ T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER, REPRGSDIAGT, and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to >1 year postinfection, and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDRFYKT epitope sequence, FRDYVDQFYKT, was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4⁺ T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost, and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus, our data support that IL-2-producing HIV-1-specific CD4⁺ T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4⁺ T cells rather than to the fixation of escape mutations at high frequencies.In the typical course of acute human immunodeficiency virus type 1 (HIV-1) infection an initial burst of high-level viremia is reduced by at least 100-fold to a set point level (11, 12). This precipitous drop in viral load is suggestive of a partially effective host immune response to primary HIV-1 infection. Several lines of evidence support an important role for CD8⁺ T cells in suppressing HIV-1 replication in acute infection: principally, the decline in HIV-1 viremia is temporally associated with the emergence of an HIV-1-specific CD8⁺ T-cell response, and the in vivo depletion of CD8⁺ T cells in simian immunodeficiency virus-infected macaques consistently results in elevated viral loads (7, 24, 30). Consistent with the application of effective immune pressure, it has been well established that HIV-1- and simian immunodeficiency virus-specific CD8⁺ T cells drive the emergence and fixation of escape mutations in the epitopes that they target (1, 3, 8, 18, 31, 33, 34). This evidence has contributed to the prioritization of vaccine candidates that elicit potent HIV-1-specific CD8⁺ T-cell responses.The role of CD4⁺ T-cell responses in the response to acute HIV-1 infection is less clear. There is compelling evidence that CD4⁺ T-cell help may be critical for the establishment of a qualitatively and quantitatively robust CD8⁺ T-cell memory pool for persistent virus infections (4, 9, 17, 37, 39). Furthermore, an important role for CD4⁺ help in maintaining an effective CD8⁺ T-cell response has been established in the lymphocytic choriomeningitis virus model of chronic viral infection (28, 45). Evidence in support of a role for the CD4⁺ T-cell response to HIV-1 infection in suppressing viral replication is derived from studies which demonstrated that a CD4⁺ T-cell response characterized by vigorous proliferation and production of interleukin-2 (IL-2) is associated with control of viremia (6, 35). It has further been demonstrated that the functional defect of CD8⁺ T cells observed in chronic HIV-1 infection can be induced in vitro by the depletion of CD4⁺ T cells or the addition of IL-2-neutralizing antibodies and can be corrected in vivo by vaccine-mediated augmentation of HIV-1-specific CD4⁺ T-cell responses (26). These observations have suggested that an IL-2-producing response may be necessary for controlling viremia. However, in the majority of HIV-1-infected individuals, a qualitative impairment of the HIV-1-specific CD4⁺ T-cell response occurs early after infection, resulting in the loss of proliferative capacity as well as the ability to produce IL-2 (43). This impairment correlates well with levels of antigen and viremia (29). The relationship between viral control and the presence of IL-2-producing HIV-specific CD4⁺ T-cell responses must be interpreted with caution, however, as the causal relationship between these two factors is unclear. The maintenance of an IL-2-producing HIV-1-specific CD4⁺ T-cell proliferative response could simply be the result of control of viremia achieved through another means, rather than causal in the association. Therapeutic administration of IL-2 to chronically infected individuals failed to reveal any clinical benefit, perhaps supporting that IL-2 is a marker, rather than a driver, of immunological control (25). However, it is unclear whether the systemic administration of IL-2 effectively substitutes for the targeted production of IL-2 by HIV-1-specific CD4⁺ T cells.The fixation of escape mutations in CD4⁺ T-cell epitopes during acute infection would provide direct evidence that CD4⁺ T cells apply immunological pressure against HIV-1. Harcourt et al. identified epitopes targeted by proliferative CD4⁺ T-cell responses in chronically infected individuals and sequenced these epitopes from proviral DNA at multiple time points (16). Variations in these epitope sequences were observed over time, and a minority of these variants failed to stimulate CD4⁺ T-cell lines raised against the index peptide. This study indicated the potential for HIV-1 virus to escape within proviral populations. However, the observation that the majority of emergent variants were still able to stimulate CD4⁺ T-cell responses argues against potent selective pressure for escape mutants (16). A second study examined gamma interferon (IFN-γ)-producing CD4⁺ T-cell responses and contemporaneous circulating virus epitopes in a cohort of chronically infected, untreated, HIV-1-infected individuals. A lack of intrapatient variability within CD4⁺ T-cell epitopes was observed in this study, and while two of four subjects exhibited epitope sequences that differed from the consensus HIV-1 sequence, there was a trend to greater sequence variability outside of epitopic regions, arguing against potent immune pressure (23). These studies support that HIV-1-specific CD4⁺ T-cell responses fail to exert potent selective pressure against cognate epitopes in chronic infection; however, it is difficult to determine whether or not the observed epitopic variations are indicative of relatively weak selective pressures. Since the overall cellular immune response to HIV-1 infection is particularly robust and effective during the acute phase of infection, we examined the kinetics of the HIV-1-specific IL-2-secreting CD4⁺ T-cell-mediated immune response during acute/early HIV-1 infection and studied the effects of this response on circulating plasma viruses.     

Virological consequences of early events following cell-cell contact between human immunodeficiency virus type 1-infected and uninfected CD4+ cells

Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4⁺ cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4⁺ cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4⁺ lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4⁺ cell can occur through different mechanisms, leading to distinguishable virological outcomes.     

Costimulation of Naive CD8+ Lymphocytes Induces CD4 Expression and Allows Human Immunodeficiency Virus Type 1 Infection

Human immunodeficiency virus type 1 (HIV-1) infection requires cell surface expression of CD4. Costimulation of CD8⁺/CD4⁻ T lymphocytes by anti-CD3 and anti-CD28 antibodies or by allogeneic dendritic cells induced expression of CD4 and rendered these CD8 cells susceptible to HIV-1 infection. Naive CD45RA⁺ cells responded with greater expression of CD4 than did CD45RO⁺ cells. CD8⁺ lymphocytes derived from fetal or newborn sources exhibited a greater tendency to express CD4, consistent with their naive states. This mechanism of infection suggests HIV-induced perturbation of the CD8 arm of the immune response and could explain the generally rapid disease progression seen in HIV-infected children.     

Changes in Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Responsible for the Pathogenicity of a Multiply Passaged Simian-Human Immunodeficiency Virus (SHIV-HXBc2)

In vivo passage of a poorly replicating, nonpathogenic simian-human immunodeficiency virus (SHIV-HXBc2) generated an efficiently replicating virus, KU-1, that caused rapid CD4⁺ T-lymphocyte depletion and AIDS-like illness in monkeys (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L.-J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189–3197, 1996). The env gene of the KU-1 virus was used to create a molecularly cloned virus, SHIV-HXBc2P 3.2, that differed from a nonpathogenic SHIV-HXBc2 virus in only 12 envelope glycoprotein residues. SHIV-HXBc2P 3.2 replicated efficiently and caused rapid and persistent CD4⁺ T-lymphocyte depletion in inoculated rhesus macaques. Compared with the envelope glycoproteins of the parental SHIV-HXBc2, the SHIV-HXBc2P 3.2 envelope glycoproteins supported more efficient infection of rhesus monkey peripheral blood mononuclear cells. Both the parental SHIV-HXBc2 and the pathogenic SHIV-HXBc2P 3.2 used CXCR4 but none of the other seven transmembrane segment receptors tested as a second receptor. Compared with the parental virus, viruses with the SHIV-HXBc2P 3.2 envelope glycoproteins were more resistant to neutralization by soluble CD4 and antibodies. Thus, changes in the envelope glycoproteins account for the ability of the passaged virus to deplete CD4⁺ T lymphocytes rapidly and specify increased replicative capacity and resistance to neutralization.     

Human Lymphoblastoid CD4+ T Cells Become Permissive to Macrophage-Tropic Strains of Human Immunodeficiency Virus Type 1 after Passage into Severe Combined Immunodeficient Mice through In Vivo Upregulation of CCR5: In Vivo Dynamics of CD4+ T-Cell Differentiation in Pathogenesis of AIDS

In this article, we show that passage in SCID mice rendered a human CD4⁺ T-cell line (CEM cells) highly susceptible to infection by macrophage-tropic (M-tropic) strains and primary clinical isolates of human immunodeficiency virus type 1 (HIV-1). This in vivo-acquired permissiveness of CEM cells was associated with the induction of a CD45RO⁺ phenotype as well as of some β-chemokine receptors. Regulated upon activation, normal T-cell expressed and secreted chemokine entirely inhibited the ability of M-tropic HIV-1 strains to infect these cells. These findings may lead to new approaches in investigating in vivo the capacity of different HIV strains to exploit chemokine receptors in relation to the dynamics of the activation and/or differentiation state of human CD4⁺ T cells.     

Endogenous Production of β-Chemokines by CD4+, but Not CD8+, T-Cell Clones Correlates with the Clinical State of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and May Be Responsible for Blocking Infection with NonSyncytium-Inducing HIV-1 In Vitro

Recent studies have demonstrated that the β-chemokines RANTES, MIP-1α, and MIP-1β suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of β-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global β-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of β-chemokines in nonprogressors and AIDS patients by examination of β-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4⁺ and CD8⁺ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4⁺ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4⁺ clones from nonprogressors and CD8⁺ clones from AIDS patients secreted high levels of RANTES, MIP1α, and MIP-1β. In contrast, CD4⁺ clones from AIDS patients produced no RANTES and little or no MIP-1α or MIP-1β. The infection of CD4⁺ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to β-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4⁺ clones from nonprogressors could be partially reversed by treatment with anti-β-chemokine antibodies. These results indicate that CD4⁺ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including β-chemokines, and that β-chemokine production by CD4⁺, but not CD8⁺, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.