Partial activation and induction of apoptosis in CD4(+) and CD8(+) T lymphocytes by conformationally authentic noninfectious human immunodeficiency virus type 1

Increased levels of apoptosis are seen in human immunodeficiency virus (HIV) infection, and this has been proposed as an important mechanism contributing to HIV pathogenesis. However, interpretation of in vitro studies aimed at understanding HIV-related apoptosis has been complicated by the use of high concentrations of recombinant proteins or by direct cytopathic effects of replicating virus. We have developed an inactivation procedure that destroys retroviral infectivity while preserving the structural and functional integrity of the HIV surface proteins. These noninfectious virions interact authentically with target cells, providing a powerful tool to dissect mechanisms of HIV pathogenesis that do or do not require viral replication. Noninfectious CXCR4-tropic HIV-1 virions, but not microvesicles, partially activated freshly isolated CD4(+) and CD8(+) peripheral blood mononuclear cell T lymphocytes to express FasL and Fas, but not CD69 or CD25 (interleukin-2 receptor alpha) and eventually die via apoptosis starting 4 to 6 days postexposure. These effects required conformationally intact virions, as heat-denatured virions or equivalent amounts of recombinant gp120 did not induce apoptosis. The maximal apoptotic effect was dependent on major histocompatibility complex (MHC) class II proteins being present on the virion, but was not MHC restricted. The results suggest that the immunopathogenesis of HIV infection may not depend solely on direct cytopathic effects of HIV replication, but that effects due to noninfectious HIV-1 virions may also contribute importantly.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.     

Insertion of host-derived costimulatory molecules CD80 (B7.1) and CD86 (B7.2) into human immunodeficiency virus type 1 affects the virus life cycle

Human immunodeficiency virus type 1 (HIV-1) carries virus-encoded and host-derived proteins. Recent advances in the functional characterization of host molecules inserted into mature virus particles have revealed that HIV-1 biology is influenced by the acquisition of host cell membrane components. The CD28/B7 receptor/ligand system is considered one of the fundamental elements of the normal immune response. Two major cell types that harbor HIV-1 in vivo, i.e., monocytes/macrophages and CD4+ T cells, express the costimulatory molecules CD80 (B7.1) and CD86 (B7.2). We investigated whether CD80 and CD86 are efficiently acquired by HIV-1, and if so, whether these host-encoded molecules can contribute to the virus life cycle. Here we provide the first evidence that the insertion of CD80 and CD86 into HIV-1 increases virus infectivity by facilitating the attachment and entry process due to interactions with their two natural ligands, CD28 and CTLA-4. Moreover, we demonstrate that NF-kappaB is induced by CD80- and CD86-bearing virions when they are combined with the engagement of the T-cell receptor/CD3 complex, an event that is inhibited upon surface expression of CTLA-4. Finally, both CD80 and CD86 were found to be efficiently incorporated into R5- and X4-tropic field strains of HIV-1 expanded in cytokine-treated macrophages. Thus, besides direct interactions between the virus envelope glycoproteins and cell surface constituents, such as CD4 and some specific chemokine coreceptors, HIV-1 may attach to target cells via interactions between cell-derived molecules incorporated into virions and their natural ligands. These findings support the theory that HIV-1-associated host proteins alter virus-host dynamics.     

Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus

Infection with the HIV type 1 (HIV-1) can result both in depletion of CD4(+) T cells and in the generation of dysfunctional CD8(+) T cells. In HIV-1-infected children, repopulation of the peripheral T cell pool is mediated by the thymus, which is itself susceptible to HIV-1 infection. Previous work has shown that MHC class I (MHC I) molecules are strongly up-regulated as result of IFN-alpha secretion in the HIV-1-infected thymus. We demonstrate in this study that increased MHC I up-regulation on thymic epithelial cells and double-positive CD3(-/int)CD4(+)CD8(+) thymocytes correlates with the generation of mature single-positive CD4(-)CD8(+) thymocytes that have low expression of CD8. Treatment of HIV-1-infected thymus with highly active antiretroviral therapy normalizes MHC I expression and surface CD8 expression on such CD4(-)CD8(+) thymocytes. In pediatric patients with possible HIV-1 infection of the thymus, a low CD3 percentage in the peripheral circulation is also associated with a CD8(low) phenotype on circulating CD3(+)CD8(+) T cells. Furthermore, CD8(low) peripheral T cells from these HIV-1(+) pediatric patients are less responsive to stimulation by Ags from CMV. These data indicate that IFN-alpha-mediated MHC I up-regulation on thymic epithelial cells may lead to high avidity interactions with developing double-positive thymocytes and drive the selection of dysfunctional CD3(+)CD8(low) T cells. We suggest that this HIV-1-initiated selection process may contribute to the generation of dysfunctional CD8(+) T cells in HIV-1-infected patients.     

Human immunodeficiency virus type 1 induces apoptosis in CD4(+) but not in CD8(+) T cells in ex vivo-infected human lymphoid tissue

Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4(+) T cells along with death and/or dysfunction of CD8(+) T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4(+) and CD8(+) T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4(+) T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8(+) T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4(+) T cells, neither viral isolate induced apoptosis in CD8(+) T cells. Moreover, in both infected and control tissues we found similar numbers of CD8(+) T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3-anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4(+) T cells, the death of CD8(+) T cells apparently requires additional factors.     

HIV-1 infection ex vivo accelerates measles virus infection by upregulating signaling lymphocytic activation molecule (SLAM) in CD4+ T cells

Measles virus (MV) infection in children harboring human immunodeficiency virus type 1 (HIV-1) is often fatal, even in the presence of neutralizing antibodies; however, the underlying mechanisms are unclear. Therefore, the aim of the present study was to examine the interaction between HIV-1 and wild-type MV (MVwt) or an MV vaccine strain (MVvac) during dual infection. The results showed that the frequencies of MVwt- and MVvac-infected CD4(+) T cells within the resting peripheral blood mononuclear cells (PBMCs) were increased 3- to 4-fold after HIV-1 infection, and this was associated with a marked upregulation of signaling lymphocytic activation molecule (SLAM) expression on CD4(+) T cells but not on CD8(+) T cells. SLAM upregulation was induced by infection with a replication-competent HIV-1 isolate comprising both the X4 and R5 types and to a lesser extent by a pseudotyped HIV-1 infection. Notably, SLAM upregulation was observed in HIV-infected as well as -uninfected CD4(+) T cells and was abrogated by the removal of HLA-DR(+) cells from the PBMC culture. Furthermore, SLAM upregulation did not occur in uninfected PBMCs cultured together with HIV-infected PBMCs in compartments separated by a permeable membrane, indicating that no soluble factors were involved. Rather, CD4(+) T cell activation mediated through direct contact with dendritic cells via leukocyte function-associated molecule 1 (LFA-1)/intercellular adhesion molecule 1 (ICAM-1) and LFA-3/CD2 was critical. Thus, HIV-1 infection induces a high level of SLAM expression on CD4(+) T cells, which may enhance their susceptibility to MV and exacerbate measles in coinfected individuals.     

Multifunctional human immunodeficiency virus (HIV) gag-specific CD8+ T-cell responses in rectal mucosa and peripheral blood mononuclear cells during chronic HIV type 1 infection

The intestinal tract is a lymphocyte-rich site that undergoes severe depletion of memory CD4(+) T cells within days of simian immunodeficiency virus or human immunodeficiency virus type 1 (HIV-1) infection. An ensuing influx of virus-specific CD8(+) T cells, which persist throughout the chronic phase of infection, has also been documented in the gastrointestinal tract. However, little is known of the functionality of these effector cells or their relationship to the disease course. In this study, we measured CD8(+) T-cell responses to HIV-1 peptides in paired rectal and blood samples from chronically infected patients. In both blood and rectum, there was an immunodominant CD8(+) T-cell response to HIV Gag compared to Pol and Env (P < 0.01). In contrast, cytomegalovirus pp65 peptides elicited gamma interferon (IFN-gamma) secretion strongly in peripheral blood mononuclear cells (PBMC) but weakly in rectal CD8(+) T cells (P = 0.015). Upon stimulation with HIV peptides, CD8(+) T cells from both sites were capable of mounting complex responses including degranulation (CD107 expression) and IFN-gamma and tumor necrosis factor alpha (TNF-alpha) production. In rectal tissue, CD107 release was frequently coupled with production of IFN-gamma or TNF-alpha. In patients not on antiretroviral therapy, the magnitude of Gag-specific responses, as a percentage of CD8(+) T cells, was greater in the rectal mucosa than in PBMC (P = 0.054); however, the breakdown of responding cells into specific functional categories was similar in both sites. These findings demonstrate that rectal CD8(+) T cells are capable of robust and varied HIV-1-specific responses and therefore likely play an active role in eliminating infected cells during chronic infection.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.     

Isolation and characterization of macaque dendritic cells from CD34(+) bone marrow progenitors.

We have developed a method for isolating and characterizing pigtailed macaque dendritic cells (DCs) generated from CD34(+) bone marrow (BM) progenitors based on methods previously developed for isolating human DCs. Macaque DCs displayed a characteristic morphology and were potent stimulators of allogeneic T cell proliferation. They expressed a set of DC-associated markers, such as MHC class II, CD1a, CD4, CD11a, CD40, CD58, CD80, CD83, CD86, and CXCR4. Macaque DCs, as well as peripheral blood CD4(+) T cells, were highly susceptible to HIV-2 infection, as detected by DNA-PCR. The expression of HIV-2 in macaque DCs was downregulated by treatment with the beta-chemokine RANTES. Macaque DCs will be useful for defining the in vivo role of DCs in HIV pathogenesis and for optimizing and testing peptide-DC vaccines or tolerizing regimens.     

Regulation of LFA-1 activity through cytoskeleton remodeling and signaling components modulates the efficiency of HIV type-1 entry in activated CD4+ T lymphocytes

Besides interactions between the viral envelope glycoproteins with cell surface receptors, interactions between cell-derived molecules incorporated onto virions and their ligand could also modulate HIV type-1 (HIV-1) entry inside CD4(+) T lymphocytes. Although incorporation of host ICAM-1 within HIV-1 increases both virus attachment and fusion, the precise mechanism through which this phenomenon is occurring is still unclear. We demonstrate in this study that activation of primary human CD4(+) T lymphocytes increases LFA-1 affinity and avidity states, two events promoting the early events of the HIV-1 replication cycle through interactions between virus-embedded host ICAM-1 and LFA-1 clusters. Confocal analyses suggest that HIV-1 is concentrated in microdomains rich in LFA-1 clusters that also contain CD4 and CXCR4 molecules. Experiments performed with specific inhibitors revealed that entry of HIV-1 in activated CD4(+) T cells is regulated by LFA-1-dependent ZAP70, phospholipase Cgamma1, and calpain enzymatic activities. By using laboratory and clinical strains of HIV-1 produced in primary human cells, we demonstrate the importance of the LFA-1 activation state and cluster formation in the initial step of the virus life cycle. Overall, these data provide new insights into the complex molecular events involved in HIV-1 binding and entry.     

CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA

Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific T-cell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of antigen-presenting cells (APC). The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell. In contrast, CTLA-4 (CD152) is a distinct T-cell receptor that, upon binding to B7 molecules, sends an inhibitory signal to T cell activation. Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells. Recently, functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice, and the authors concluded that CD86 could be more important for initiating T-cell responses, while CD80 could be more significant for maintaining these immune responses. In this study, we directly compared the role of CD80 and CD86 in initiating and maintaining proliferation of resting CD4(+) T cells in an in vitro mode system that allowed to provide the first signal-to-effector cells through the use of suboptimal doses of PHA and the second costimulatory signal through cells expressing CD80 or CD86, but not any other costimulatory molecules. Using this experimental system we demonstrate that the CD80 and CD86 molecules can substitute for each other in the initial activation of resting CD4(+) T cells and in the maintenance of their proliferative response.     

CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses

We compared splenic DC activation during infection with either the Th2 response-inducing parasite Schistosoma mansoni or with the Th1 response-inducing parasite Toxoplasma gondii. CD8alpha(+) DC from schistosome-infected mice exhibited a 2- to 3-fold increase in the expression of MHC class II, CD80, and CD40 (but not CD86) compared with DC from uninfected control animals, while CD8alpha(-) DC exhibited a 2- to 3-fold increase in the expression of MHC class II and CD80 and no alteration, compared with DC from uninfected mice, in the expression of CD86 or CD40. Intracellular staining revealed that DC did not produce IL-12 during infection with S. mansoni. In contrast, infection with T. gondii resulted in a more pronounced increase in the expression of activation-associated molecules (MHC class II, CD80, CD86, and CD40) on both CD8alpha(-) and CD8alpha(+) splenic DC and promoted elevated IL-12 production by DC. Analysis of MHC class I and of additional costimulatory molecules (ICOSL, ICAM-1, OX40L, 4-1BBL, and B7-DC) revealed a generally similar pattern, with greater indication of activation in T. gondii-infected mice compared with S. mansoni-infected animals. Strikingly, the activation of DC observed during infection with either parasite was not apparent in DC from infected CD154(-/-) mice, indicating that CD40/CD154 interactions are essential for maintaining DC activation during infection regardless of whether the outcome is a Th1 or a Th2 response. However, the ability of this activation pathway to induce IL-12 production by DC is restrained in S. mansoni-infected, but not T. gondii-infected, mice by Ag-responsive CD11c(-) cells.     

Unstimulated primary CD4+ T cells from HIV-1-positive elite suppressors are fully susceptible to HIV-1 entry and productive infection

Elite controllers or suppressors (ES) are a group of HIV-1-infected individuals who maintain viral loads below the limit of detection of commercial assays for many years. The mechanisms responsible for this remarkable control are under intense study, with the hope of developing therapeutic vaccines effective against HIV-1. In this study, we addressed the question of the intrinsic susceptibility of ES CD4(+) T cells to infection. While we and others have previously shown that CD4(+) T cells from ES can be infected by HIV-1 isolates in vitro, these studies were confounded by exogenous activation and in vitro culture of CD4(+) T cells prior to infection. In order to avoid the changes in chemokine receptor expression that have been associated with such exogenous activation, we infected purified CD4(+) T cells directly after isolation from the peripheral blood of ES, viremic patients, and uninfected donors. We utilized a green fluorescent protein (GFP)-expressing proviral construct pseudotyped with CCR5-tropic or CXCR4-tropic envelope to compare viral entry using a fluorescence resonance energy transfer-based, single-round virus-cell fusion assay. The frequency of productive infection was also compared by assessing GFP expression. CD4(+) T cells from ES were as susceptible as or more susceptible than cells from viremic patients and uninfected donors to HIV-1 entry and productive infection. The results of this physiological study strongly suggest that differences in HIV-1 entry and infection of CD4(+) T cells alone cannot explain the elite control of viral replication.     

Quantitation of HLA class II protein incorporated into human immunodeficiency type 1 virions purified by anti-CD45 immunoaffinity depletion of microvesicles

Among the many host cell-derived proteins found in human immunodeficiency virus type 1 (HIV-1), HLA class II (HLA-II) appears to be selectively incorporated onto virions and may contribute to mechanisms of indirect imunopathogenesis in HIV infection and AIDS. However, the amount of HLA-II on the surface of HIV-1 particles has not been reliably determined due to contamination of virus preparations by microvesicles containing host cell proteins, including HLA-II. Even rigorous sucrose density centrifugation is unable to completely separate HIV-1 from microvesicles. CD45, a leukocyte integral membrane protein, is found on microvesicles, yet appears to be excluded from HIV-1 particles. Exploiting this observation, we have developed a CD45-based immunoaffinity depletion method for removing CD45-containing microvesicles that yields highly purified preparations of virions. Examination of CD45-depleted HIV-1(MN) by high-pressure liquid chromatography, protein sequencing, and amino acid analyses determined a molar ratio of HLA-II to Gag of 0.04 to 0.05 in the purified virions, corresponding to an estimated average of 50 to 63 native HLA-II complexes (i.e., a dimer of alpha and beta heterodimers) per virion. These values are approximately 5- to 10-fold lower than those previously determined for other virion preparations that contained microvesicles. Our observations demonstrate the utility of CD45 immunoaffinity-based approaches for producing highly purified retrovirus preparations for applications that would benefit from the use of virus that is essentially free of microvesicles.     

Differential regulation of the Let-7 family of microRNAs in CD4+ T cells alters IL-10 expression

MicroRNAs (miRNAs) are ～22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1-infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4(+) T cells from uninfected healthy controls, chronic HIV-1-infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4(+) T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4(+) T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4(+) T cells and provide the virus with an important survival advantage by manipulating the host immune response.     

Human immunodeficiency virus type 1 Vpu protein interacts with CD74 and modulates major histocompatibility complex class II presentation

The human immunodeficiency virus type 1 (HIV-1) Vpu accessory protein is a transmembrane protein that down regulates CD4 expression and promotes the release of new virions. We screened a human leukocyte-specific yeast two-hybrid expression library to discover novel Vpu-interacting cellular proteins. The major histocompatibility complex class II (MHC II) invariant chain, also called Ii or CD74, was found to be one such protein. We show direct binding of Vpu and CD74 by using a yeast two-hybrid assay and coimmunoprecipitation from HIV-1-infected cells. The cytoplasmic region of Vpu was found to interact with the 30-amino-acid cytoplasmic tail of CD74. Human monocytic U937 cells infected with wild-type or Vpu-defective HIV-1 and transfected cells showed that Vpu down modulated the surface expression of mature MHC II molecules. The reduction in cell surface mature MHC II molecules correlated with decreased antigen presentation to T cells in culture. Thus, the Vpu protein also contributes to viral persistence by attenuating immune responses during HIV infection. This report further exemplifies the rich diversity and redundancy shown by HIV in immune evasion.     

Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases

Homozygosity for the naturally occurring Delta32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted approximately 50% of CCR5 alleles in a pool of primary human CD4(+) T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4(+) T cells had lower viral loads and higher CD4(+) T-cell counts than mice engrafted with wild-type CD4(+) T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4(+) T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN-modified autologous CD4(+) T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.