Induction of HIV-1 Replication in Latently Infected CD4+ T Cells Using a Combination of Cytokines

Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1–infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4⁺ T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4⁺ T cells derived from HIV-infected individuals who are antiretroviral therapy–naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro. Given that an array of cytokines, including IL-6, TNF-α, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART.     

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4⁺ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4⁺ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4⁺ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-κB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.     

‘Self-Protection’ of Individual CD4+ T Cells against R5 HIV-1 Infection by the Synthesis of Anti-Viral CCR5 Ligands

It is well established that paracrine secretion of anti-viral CCR5 ligands by CD8⁺ and CD4⁺ T cells can block the infection of activated CD4⁺ T cells by R5 and dual-tropic isolates of HIV-1. By contrast, because CD4⁺ T cells can be infected by HIV-1 and at least some subsets secrete anti-viral CCR5 ligands, it is possible that these ligands protect against HIV-1 via autocrine as well as paracrine pathways. Here we use a model primary CD4⁺ T cell response in vitro to show that individual CD4⁺ T cells that secrete anti-viral CCR5 ligands are ‘self-protected’ against infection with R5 but not X4 strains of HIV-1. This protection is selective for CD4⁺ T cells that secrete anti-viral CCR5 ligands in that activated CD4⁺ T cells in the same cultures remain infectable with R5 HIV-1. These data are most consistent with an autocrine pathway of protection in this system and indicate a previously unappreciated selective pressure on the emergence of viral variants and CD4⁺ T cell phenotypes during HIV-1 infection.     

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4⁺ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8⁺ T cell clearance of CD4⁺ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4⁺ T cells. Moreover, DARTs mediated CD8⁺ T cell clearance of HIV from resting CD4⁺ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.     

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8⁺ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4⁺ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8⁺ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.     

Turnover of CD4+ and CD8+ T Lymphocytes in HIV-1 Infection as Measured by Ki-67 Antigen

We investigated CD4⁺ and CD8⁺ T cell turnover in both healthy and HIV-1–infected adults by measuring the nuclear antigen Ki-67 specific for cell proliferation. The mean growth fraction, corresponding to the expression of Ki-67, was 1.1% for CD4⁺ T cells and 1.0% in CD8⁺ T cells in healthy adults, and 6.5 and 4.3% in HIV-1–infected individuals, respectively. Analysis of CD45RA⁺ and CD45RO⁺ T cell subsets revealed a selective expansion of the CD8⁺ CD45RO⁺ subset in HIV-1–positive individuals. On the basis of the growth fraction, we derived the potential doubling time and the daily turnover of CD4⁺ and CD8⁺ T cells. In HIV-1–infected individuals, the mean potential doubling time of T cells was five times shorter than that of healthy adults. The mean daily turnover of CD4⁺ and CD8⁺ T cells in HIV-1–infected individuals was increased 2- and 6-fold, respectively, with more than 40-fold interindividual variation. In patients with <200 CD4⁺ counts, CD4⁺ turnover dropped markedly, whereas CD8⁺ turnover remained elevated. The large variations in CD4⁺ T cell turnover might be relevant to individual differences in disease progression.     

The Unenlarged Lymph Nodes of HIV-1–infected, Asymptomatic Patients with High CD4 T Cell Counts Are Sites for Virus Replication and CD4 T Cell Proliferation. The Impact of Highly Active Antiretroviral Therapy

The efficacy of triple drug therapy for HIV-1 infection encourages its early use to prevent damage to the immune system. We monitored the effects of such therapy on 12 patients with 14–75-mo histories of minimal disease, i.e., CD4⁺ counts constantly >500/μl and little or no lymph node enlargement. In this way, we could first determine the extent of viral replication and immunoarchitectural changes in unenlarged nodes early in disease, and second follow the response to triple therapy in plasma and lymphoid tissue in tandem. As is known for lymph nodes with more advanced disease, the germinal centers showed productively infected T cells, i.e., CD4⁺CD1a⁻CD68⁻ cells labeling intensely for HIV-1 RNA after in situ hybridization. The unenlarged nodes also showed extensive HIV-1 RNA retention on a well-preserved, follicular dendritic cell (FDC) network, and the follicles were abnormal. There were numerous CD8⁺ cells, many expressing TIA-1 granule antigen. Also, in contrast to normal follicles, CD4⁺ T cell proliferation was active, with marked increases in the number of cycling, Ki-67⁺CD4⁺CD45R0⁺ cells. After 28 d and 3 mo of therapy, productively infected T cells decreased dramatically and often were not apparent. The labeling of the FDC network for viral RNA also decreased, but not for gag protein. We conclude that HIV-1 replicates and accumulates in lymphoid organs before damage of the immune system, that at this stage of disease de novo production of T cells occurs in the lymphoid tissue, and that the infection is sensitive to triple drug therapy in both plasma and lymph nodes.     

Dynamic Interaction between STLV-1 Proviral Load and T-Cell Response during Chronic Infection and after Immunosuppression in Non-Human Primates

We used mandrills (Mandrillus sphinx) naturally infected with simian T-cell leukemia virus type 1 (STLV-1) as a model for evaluating the influence of natural STLV-1 infection on the dynamics and evolution of the immune system during chronic infection. Furthermore, in order to evaluate the role of the immune system in controlling the infection during latency, we induced immunosuppression in the infected monkeys. We first showed that the STLV-1 proviral load was higher in males than in females and increased significantly with the duration of infection: mandrills infected for 10–6 years had a significantly higher proviral load than those infected for 2–4 years. Curiously, this observation was associated with a clear reduction in CD4+ T-cell number with age. We also found that the percentage of CD4⁺ T cells co-expressing the activation marker HLA-DR and the mean percentage of CD25⁺ in CD4⁺ and CD8⁺ T cells were significantly higher in infected than in uninfected animals. Furthermore, the STLV-1 proviral load correlated positively with T-cell activation but not with the frequency of T cells secreting interferon γ in response to Tax peptides. Lastly, we showed that, during immunosuppression in infected monkeys, the percentages of CD8⁺ T cells expressing HLA-DR⁺ and of CD4⁺ T cells expressing the proliferation marker Ki67 decreased significantly, although the percentage of CD8⁺ T cells expressing HLA-DR⁺ and Ki67 increased significantly by the end of treatment. Interestingly, the proviral load increased significantly after immunosuppression in the monkey with the highest load. Our study demonstrates that mandrills naturally infected with STLV-1 could be a suitable model for studying the relations between host and virus. Further studies are needed to determine whether the different compartments of the immune response during infection induce the long latency by controlling viral replication over time. Such studies would provide important information for the development of immune-based therapeutic strategies.     

Ex Vivo Bioactivity and HIV-1 Latency Reversal by Ingenol Dibenzoate and Panobinostat in Resting CD4+ T Cells from Aviremic Patients

The human immunodeficiency virus type 1 (HIV-1) latent reservoir in resting CD4⁺ T cells represents a major barrier to viral eradication. Small compounds capable of latency reversal have not demonstrated uniform responses across in vitro HIV-1 latency cell models. Characterizing compounds that demonstrate latency-reversing activity in resting CD4⁺ T cells from aviremic patients ex vivo will help inform pilot clinical trials aimed at HIV-1 eradication. We have optimized a rapid ex vivo assay using resting CD4⁺ T cells from aviremic HIV-1⁺ patients to evaluate both the bioactivity and latency-reversing potential of candidate latency-reversing agents (LRAs). Using this assay, we characterize the properties of two candidate compounds from promising LRA classes, ingenol 3,20-dibenzoate (a protein kinase C agonist) and panobinostat (a histone deacetylase inhibitor), in cells from HIV-1⁺ antiretroviral therapy (ART)-treated aviremic participants, including the effects on cellular activation and cytotoxicity. Ingenol induced viral release at levels similar to those of the positive control (CD3/28 receptor stimulation) in cells from a majority of participants and represents an exciting LRA candidate, as it combines a robust viral reactivation potential with a low toxicity profile. At concentrations that blocked histone deacetylation, panobinostat displayed a wide range of potency among participant samples and consistently induced significant levels of apoptosis. The protein kinase C agonist ingenol 3,20-dibenzoate demonstrated significant promise in a rapid ex vivo assay using resting CD4⁺ T cells from treated HIV-1-positive patients to measure latent HIV-1 reactivation.     

HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir

The persistence of latently infected, resting CD4+ T cells is considered to be a major obstacle in preventing the eradication of HIV-1 even in patients who have received effective antiviral therapy for an average duration of 5 years. Although previous studies have suggested that the latent HIV reservoir in the resting CD4+ T cell compartment is virologically quiescent in the absence of activating stimuli, evidence has been mounting to suggest that low levels of ongoing viral replication persist and in turn, prolong the overall half-life of HIV in patients receiving antiviral therapy. Here, we demonstrate the persistence of replication-competent virus in CD4+ T cells in a cohort of patients who had received uninterrupted antiviral therapy for up to 9.1 years that rendered them consistently aviremic throughout that time. Surprisingly, substantially higher levels of HIV proviral DNA were found in activated CD4+ T cells when compared with resting CD4+ T cells in the majority of patients we studied. Phylogenetic analyses revealed evidence for cross infection between the resting and activated CD4+ T cell compartments, suggesting that ongoing reactivation of latently infected, resting CD4+ T cells and spread of virus by activated CD4+ T cells may occur in these patients. Such events may allow continual replenishment of the CD4+ T cell reservoir and resetting of the half-life of the latently infected, resting CD4+ T cells despite prolonged periods of aviremia.     

HIV-1 Directly Kills CD4+ T Cells by a Fas-independent Mechanism

The mechanism by which HIV-1 induces CD4⁺ T cell death is not known. A fundamental issue is whether HIV-1 primarily induces direct killing of infected cells or indirectly causes death of uninfected bystander cells. This question was studied using a reporter virus system in which infected cells are marked with the cell surface protein placental alkaline phosphatase (PLAP). Infection by HIV-PLAP of peripheral blood mononuclear cells (PBMCs) and T cell lines leads to rapid depletion of CD4⁺ T cells and induction of apoptosis. The great majority of HIV-induced T cell death in vitro involves direct loss of infected cells rather than indirect effects on uninfected bystander cells. Because of its proposed role in HIV-induced cell death, we also examined the Fas (CD95/Apo1) pathway in killing of T cells by HIV-1. Infected PBMCs or CEM cells display no increase in surface Fas relative to uninfected cells. In addition, HIV-1 kills CEM and Jurkat T cells in the presence of a caspase inhibitor that completely blocks Fas-mediated apoptosis. HIV-1 also depletes CD4⁺ T cells in PBMCs from patients who have a genetically defective Fas pathway. These results suggest that HIV-1 induces direct apoptosis of infected cells and kills T cells by a Fas-independent mechanism.     

Disturbed CD4+ T Cell Homeostasis and In Vitro HIV-1 Susceptibility in Transgenic Mice Expressing T Cell Line–tropic HIV-1 Receptors

T cell line–tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4⁺ T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4⁺ T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4⁺ T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4⁺ T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4⁺ T cell decline in patients.     

Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy

Despite prolonged treatment with highly active antiretroviral therapy (HAART), infectious HIV-1 continues to replicate and to reside latently in resting memory CD4(+) T lymphocytes, creating a major obstacle to HIV-1 eradication. It is therefore not surprising to observe a prompt viral rebound after discontinuation of HAART. The nature of the rebounding virus, however, remains undefined. We now report on the genetic characterization of rebounding viruses in eight patients in whom plasma viremia was undetectable throughout about 3 years of HAART. Taking advantage of the extensive length polymorphism in HIV-1 env, we found that in five patients who did not show HIV-1 replication during treatment, the rebound virus was identical to those isolated from the latent reservoir. In three other patients, two of whom had been free of plasma viremia but had showed some residual viral replication, the rebound virus was genetically different from the latent reservoir virus, corresponding instead to minor viral variants detected during the course of treatment in lymphoid tissues. We conclude that in cases with apparent complete HIV-1 suppression by HAART, viral rebound after cessation of therapy could have originated from the activation of virus from the latent reservoir. In patients with incomplete suppression by chemotherapy, however, the viral rebound is likely triggered by ongoing, low-level replication of HIV-1, perhaps occurring in lymphoid tissues.     

Productive Infection of Neonatal CD8+ T Lymphocytes by HIV-1

CD8⁺ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8⁺ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell–tropic (T-tropic) HIV strains. Physiological activation of CD8-α/β⁺ CD4⁻ T cell receptor–α/β⁺ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1–activated cord blood CD8⁺ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8⁺ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.     

Infected Cell Protein (ICP)47 Enhances Herpes Simplex Virus Neurovirulence by Blocking the CD8+ T Cell Response

The herpes simplex virus (HSV) infected cell protein (ICP)47 blocks CD8⁺ T cell recognition of infected cells by inhibiting the transporter associated with antigen presentation (TAP). In vivo, HSV-1 replicates in two distinct tissues: in epithelial mucosa or epidermis, where the virus enters sensory neurons; and in the peripheral and central nervous system, where acute and subsequently latent infections occur. Here, we show that an HSV-1 ICP47⁻ mutant is less neurovirulent than wild-type HSV-1 in mice, but replicates normally in epithelial tissues. The reduced neurovirulence of the ICP47⁻ mutant was due to a protective CD8⁺ T cell response. When compared with wild-type virus, the ICP47⁻ mutant expressed reduced neurovirulence in immunologically normal mice, and T cell–deficient nude mice after reconstitution with CD8⁺ T cells. However, the ICP47⁻ mutant exhibited normal neurovirulence in mice that were acutely depleted of CD8⁺ T cells, and in nude mice that were not reconstituted, or were reconstituted with CD4⁺ T cells. In contrast, CD8⁺ T cell depletion did not increase the neurovirulence of an unrelated, attenuated HSV-1 glycoprotein (g)E⁻ mutant. ICP47 is the first viral protein shown to influence neurovirulence by inhibiting CD8⁺ T cell protection.     

Immediate Early and Early Lytic Cycle Proteins Are Frequent Targets of the Epstein-Barr Virus–induced Cytotoxic T Cell Response

Epstein-Barr virus (EBV), a human γ-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8⁺ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8⁺ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBVreplicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in α- and β-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of γ-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.     

HIV-1 Induces Cytotoxic T Lymphocytes in the Cervix of Infected Women

Although T lymphocytes are present in the genital mucosa, their function in sexually transmitted diseases is unproven. To determine if cervical T cells mediate HIV-specific cytolysis, mononuclear cells in cytobrush specimens from HIV-1-infected women were stimulated in vitro with antigen. Resultant cell lines lysed autologous targets expressing HIV-1 proteins in 12/19 (63%) subjects, and these responses were detected intermittently on repeated visits. All 8 subjects with blood CD4⁺ counts 500 cells/μl had HIV-1-specific cervical CTL, whereas only 4/11 with counts <500 cells/μl had detectable responses (P = 0.008). Class II MHC– restricted CD4⁺ CTL clones lysed targets expressing Env gp41 or infected with HIV-1. Class I MHC-restricted CD8⁺ clones recognized HIV-1 Gag- or Pol-expressing targets, and the epitopes were mapped to within 9–20 amino acids. Comparisons of intra-individual cervical and blood CTL specificities indicate that epitopes recognized by CTL in the cervix were commonly recognized in the blood. These studies provide the first definitive evidence for an MHC-restricted effector function in human cervical lymphocytes.     

Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4⁺ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4⁺ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4⁺ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4⁺ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4⁺ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4⁺ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4⁺ T cells during HIV-1 infection.     

Preferential coreceptor utilization and cytopathicity by dual-tropic HIV-1 in human lymphoid tissue ex vivo

Many HIV-1 isolates at the late stage of disease are capable of using both CXCR4 and CCR5 in transfected cell lines, and are thus termed dual-tropic. Here we asked whether these dual-tropic variants also use both coreceptors for productive infection in a natural human lymphoid tissue microenvironment, and whether use of a particular coreceptor is associated with viral cytopathicity. We used 3 cloned dual-tropic HIV-1 variants, 89.6 and its chimeras 89-v345.SF and 89-v345.FL, which use both CCR5 and CXCR4 in transfected cell lines. In human lymphoid tissue ex vivo, one variant preferentially used CCR5, another preferentially used CXCR4, and a third appeared to be a true dual-tropic variant. The 2 latter variants severely depleted CD4⁺ T cells, whereas cytopathicity of the virus that used CCR5 only in lymphoid tissue was mild and confined to CCR5⁺/CD4⁺ T cells. Thus, (a) HIV-1 coreceptor usage in vitro cannot be unconditionally extrapolated to natural microenvironment of human lymphoid tissue; (b) dual-tropic viruses are not homogeneous in their coreceptor usage in lymphoid tissue, but probably comprise a continuum between the 2 polar variants that use CXCR4 or CCR5 exclusively; and (c) cytopathicity toward the general CD4⁺ T cell population in lymphoid tissue is associated with the use of CXCR4.