Pathogenesis of primary R5 human immunodeficiency virus type 1 clones in SCID-hu mice

We studied the replication and cytopathicity in SCID-hu mice of R5 human immunodeficiency virus type 1 (HIV-1) biological clones from early and late stages of infection of three patients who never developed MT-2 cell syncytium-inducing (SI; R5X4 or X4) viruses. Several of the late-stage non-MT-2 cell syncytium-inducing (NSI; R5) viruses from these patients depleted human CD4(+) thymocytes from SCID-hu mice. Earlier clones from the same patients did not deplete CD4(+) thymocytes from SCID-hu mice as well as later clones. We studied three R5 HIV-1 clones from patient ACH142 in greater detail. Two of these clones were obtained prior to the onset of AIDS; the third was obtained following the AIDS diagnosis. In GHOST cell infection assays, all three ACH142 R5 HIV-1 clones could infect GHOST cells expressing CCR5 but not GHOST cells expressing any of nine other HIV coreceptors tested. Furthermore, these patient clones efficiently infected stimulated peripheral blood mononuclear cells from a normal donor but not those from a homozygous CCR5Delta32 individual. Statistical analyses of data obtained from infection of SCID-hu mice with patient ACH142 R5 clones revealed that only the AIDS-associated clone significantly depleted CD4(+) thymocytes from SCID-hu mice. This clone also replicated to higher levels in SCID-hu mice than the two earlier clones, and a significant correlation between viral replication and CD4(+) thymocyte depletion was observed. Our results indicate that an intrinsic property of AIDS-associated R5 patient clones causes their increased replication and cytopathic effects in SCID-hu mice and likely contributes to the development of AIDS in patients who harbor only R5 quasispecies of HIV-1.     

Rapid-high, syncytium-inducing isolates of human immunodeficiency virus type 1 induce cytopathicity in the human thymus of the SCID-hu mouse.

Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) disease is associated with an increased viral burden in the peripheral blood and a loss of circulating CD4+ T cells. HIV-1 isolates obtained prior to this stage of disease often have a "slow-low," non-syncytium-inducing (NSI) phenotype, whereas those obtained afterwards are often characterized as "rapid-high" and syncytium inducing (SI). Paired NSI and SI isolates from two different patients were inoculated into the human thymus implants of SCID-hu mice. The two slow-low, NSI isolates replicated to minimal levels in the grafts and did not induce thymocyte depletion. In contrast, the two SI isolates from the same patients showed high levels of viral replication and induced a marked degree of thymocyte depletion, accompanied by evidence of programmed cell death. These observations reveal a correlation between the replicative and cytopathic patterns of HIV-1 isolates in vitro and in the SCID-hu mouse in vivo and provide direct evidence that the biological phenotype of HIV-1 switch may be a causal and not a derivative correlate of HIV-1 disease progression.     

CXCR4 expression during lymphopoiesis: implications for human immunodeficiency virus type 1 infection of the thymus.

Human immunodeficiency virus type 1 (HIV-1) infection of the human thymus results in depletion of CD4-bearing thymocytes. This depletion is initially manifested in the immature CD4+/CD8+ thymocyte subset. To determine cellular factors involved in HIV infection in the thymus, we examined the expression of the recently identified viral coreceptor, CXCR4, on fresh human thymocytes and on human cells from SCID-hu (Thy/Liv) mice. CXCR4 is a member of the chemokine receptor family which is required along with CD4 for entry into the cell of syncytium-inducing (SI) HIV-1 strains. Our analyses show that CXCR4 expression is modulated during T-lymphoid differentiation such that immature thymocytes display an increased frequency and higher surface density of the coreceptor than do more mature cells. In addition, using an SI strain of HIV-1 which directs expression of a reporter protein on the surface of infected cells, we have found that the immature CD4+/CD8+ thymocytes that express the highest levels of both CD4 and CXCR4 are the cells that are preferentially infected and depleted by the virus in vitro. Thus, high levels of both primary receptor and coreceptor may allow efficient infection of the thymus by certain HIV-1 strains. This in part may explain the rapid disease progression seen in some HIV-infected children, where the thymus is actively involved in the production of new T lymphocytes.     

In vivo pathogenic properties of two clonal human immunodeficiency virus type 1 isolates.

We have investigated the in vivo pathogenic properties of two molecularly cloned strains of human immunodeficiency virus type 1 (HIV-1), HIV-1NL4-3 and HIV-1JR-CSF, in human fetal thymus/liver implants in severe combined immunodeficient mice. Studies comparing their in vivo replication kinetics and abilities to induce CD4+ thymocyte depletion were performed. HIV-1NL4-3 replicated in vivo with faster kinetics and induced greater levels of CD4+ thymocyte depletion than did HIV-1JR-CSF. These results demonstrate that different viral isolates have different pathogenic properties in this system. In the SCID-hu model, this pathogenesis most likely occurs in the absence of an immune response. Therefore, we investigated whether the absence of immune selection resulted in extensive genetic variation and the generation of viral quasispecies. To this end, DNA corresponding to the fourth variable domain region of the viral envelope gp120 protein recovered from biopsy samples at 6 weeks postinfection was sequenced. Little genetic variation was noted in either HIV-1JR-CSF- or HIV-1NL4-3-infected implants. The mutation levels demonstrated in both viral strains were more reflective of the acute rather than the chronic phase of HIV-1 infection in humans. These results suggest that the SCID-hu mouse model can be used to study the in vivo pathogenicity of different HIV-1 isolates in the absence of host immune selective pressures.     

CCR5- and CXCR4-Utilizing Strains of Human Immunodeficiency Virus Type 1 Exhibit Differential Tropism and Pathogenesis In Vivo

CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.     

CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue

A rapid decline in T-cell counts and the progression to AIDS is often associated with a switch from CCR5-tropic (R5) HIV-1 to CXCR4-tropic (X4) HIV-1 or R5/X4 HIV-1 variants. Experimental infection with R5 HIV-1 causes less T-cell depletion than infection with X4 or R5/X4 variants in T-cell cultures, in ex vivo infected human lymphoid tissue and in SCID/hu mice, despite similar replication levels. Experimental genetic changes in those sequences in gp120 that transform R5 HIV-1 variants into otherwise isogenic X4 viruses make them highly cytopathic. Thus, it is now believed that R5 variants are less cytopathic for T cells than are X4 variants. However, it is not known why CCR5-mediated HIV-1 infection does not lead to a massive CD4+ T-cell depletion, as occurs in CXCR4-mediated HIV-1 infection. Here we demonstrate that R5 HIV-1 isolates are indeed highly cytopathic, but only for CCR5+/CD4+ T cells. Because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T-cell count. These results may explain why the clinical stage of HIV disease correlates with viral tropism.     

Human immunodeficiency virus type 1 (HIV-1) non-B subtypes are similar to HIV-1 subtype B in that coreceptor specificity is a determinant of cytopathicity in human lymphoid tissue infected ex vivo

We sought to determine the relationship between virus-mediated CD4(+) T-lymphocyte cytopathicity and viral coreceptor preference among various human immunodeficiency virus type 1 (HIV-1) subtypes in an ex vivo-infected human lymphoid tissue model. Our data show that all R5 HIV-1 infections resulted in mild depletion of CD4(+) T lymphocytes, whereas all X4 HIV-1 infections caused severe depletion of CD4(+) T lymphocytes regardless of their subtype origin. Thus, at least for the viruses within subtypes A, B, C, and E that were tested, coreceptor specificity is a critical factor that determines the ability of HIV-1 to deplete CD4(+) T cells in human lymphoid tissue infected ex vivo.     

Differential pathogenesis of primary CCR5-using human immunodeficiency virus type 1 isolates in ex vivo human lymphoid tissue

In the course of human immunodeficiency virus (HIV) disease, CCR5-utilizing HIV type 1 (HIV-1) variants (R5), which typically transmit infection and dominate its early stages, persist in approximately half of the infected individuals (nonswitch virus patients), while in the other half (switch virus patients), viruses using CXCR4 (X4 or R5X4) emerge, leading to rapid disease progression. Here, we used a system of ex vivo tonsillar tissue to compare the pathogeneses of sequential primary R5 HIV-1 isolates from patients in these two categories. The absolute replicative capacities of HIV-1 isolates seemed to be controlled by tissue factors. In contrast, the replication level hierarchy among sequential isolates and the levels of CCR5(+) CD4(+) T-cell depletion caused by the R5 isolates seemed to be controlled by viral factors. R5 viruses isolated from nonswitch virus patients depleted more target cells than R5 viruses isolated from switch virus patients. The high depletion of CCR5(+) cells by HIV-1 isolates from nonswitch virus patients may explain the steady decline of CD4(+) T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value reflecting whether, in an infected individual, X4 HIV-1 will eventually dominate.     

Human immunodeficiency virus type 1 Nef-mediated downregulation of CD4 correlates with Nef enhancement of viral pathogenesis

The nef gene products encoded by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus type 1 (SIV-1) increase viral loads in infected hosts and accelerate clinical progression to AIDS. Nef exhibits a spectrum of biological activities, including the ability to downregulate surface expression of CD4 and major histocompatibility complex (MHC) class I antigens, to alter the state of T-cell activation, and to enhance the infectivity of viral particles. To determine which of these in vitro functions most closely correlates with the pathogenic effects of Nef in vivo, we constructed recombinant HIV-1 NL4-3 viruses carrying mutations within the nef gene that selectively impair these functions. These mutant viruses were evaluated for pathogenic potential in severe combined immunodeficiency (SCID) mice implanted with human fetal thymus and liver (SCID-hu Thy/Liv mice), in which virus-mediated depletion of thymocytes is known to be Nef dependent. Disruption of the polyproline type II helix (Pxx)4 within Nef (required for binding of Hck and p21-activated kinase-like kinases, downregulation of MHC class I, and enhancement of HIV-1 infectivity in vitro but dispensable for CD4 downregulation) did not impair thymocyte depletion in virus-infected Thy/Liv human thymus implants. Conversely, three separate point mutations in Nef that compromised its ability to downregulate CD4 attenuated thymocyte depletion while not diminishing viral replication. These findings indicate that the functional ability of Nef to downregulate CD4 and not MHC class I downregulation, Hck or PAK binding, or (Pxx)4-associated enhancement of infectivity most closely correlates with Nef-mediated enhancement of HIV-1 pathogenicity in vivo. Nef-mediated CD4 downregulation merits consideration as a new target for the development of small-molecule inhibitors.     

In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells

The destruction of the immune system by progressive loss of CD4 T cells is the hallmark of AIDS. CCR5-dependent (R5) human immunodeficiency virus type 1 (HIV-1) isolates predominate in the early, asymptomatic stages of HIV-1 infection, while CXCR4-dependent (X4) isolates typically emerge at later stages, frequently coinciding with a rapid decline in CD4 T cells. Lymphocyte killing in vivo primarily occurs through apoptosis, but the importance of apoptosis of HIV-1-infected cells relative to apoptosis of uninfected bystander cells is controversial. Here we show that in human lymphoid tissues ex vivo, apoptosis of uninfected bystander CD4 T cells is a major mechanism of lymphocyte depletion caused by X4 HIV-1 strains but is only a minor mechanism of depletion by R5 strains. Further, X4 HIV-1-induced bystander apoptosis requires the interaction of the viral envelope glycoprotein gp120 with the CXCR4 coreceptor on CD4 T cells. These results emphasize the contribution of bystander apoptosis to HIV-1 cytotoxicity and suggest that in association with a coreceptor switch in HIV disease, T-cell killing evolves from an infection-restricted stage to generalized toxicity that involves a high degree of bystander apoptosis.     

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.     

Fusion-induced apoptosis contributes to thymocyte depletion by a pathogenic human immunodeficiency virus type 1 envelope in the human thymus

The mechanisms of CD4(+) T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection remain incompletely characterized. Of particular importance is how CD4(+) T cells are depleted within the lymphoid organs, including the lymph nodes and thymus. Herein we characterize the pathogenic mechanisms of an envelope from a rapid progressor (R3A Env) in the NL4-3 backbone (NL4-R3A) which is able to efficiently replicate and deplete CD4(+) thymocytes in the human fetal-thymus organ culture (HF-TOC). We demonstrate that uninterrupted replication is required for continual thymocyte depletion. During depletion, NL4-R3A induces an increase in thymocytes which uptake 7AAD, a marker of cell death, and which express active caspase-3, a marker of apoptosis. While 7AAD uptake is observed predominantly in uninfected thymocytes (p24(-)), active caspase-3 is expressed in both infected (p24(+)) and uninfected thymocytes (p24(-)). When added to HF-TOC with ongoing infection, the protease inhibitor saquinavir efficiently suppresses NL4-R3A replication. In contrast, the fusion inhibitors T20 and C34 allow for sustained HIV-1 production. Interestingly, T20 and C34 effectively prevent thymocyte depletion in spite of this sustained replication. Apoptosis of both p24(-) and p24(+) thymocytes appears to be envelope fusion dependent, as T20, but not saquinavir, is capable of reducing thymocyte apoptosis. Together, our data support a model whereby pathogenic envelope-dependent fusion contributes to thymocyte depletion in HIV-1-infected thymus, correlated with induction of apoptosis in both p24(+) and p24(-) thymocytes.     

Interleukin-7 in plasma correlates with CD4 T-cell depletion and may be associated with emergence of syncytium-inducing variants in human immunodeficiency virus type 1-positive individuals

Human immunodeficiency virus type 1 (HIV-1) primary infection is characterized by the use of CCR5 as a coreceptor for viral entry, which is associated with the non-syncytium-inducing (NSI) phenotype in lymphoid cells. Syncytium-inducing (SI) variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a coreceptor. The emergence of SI variants is accompanied by a rapid decrease in the number of T cells. However, it is unclear why SI variants emerge and what factors trigger the evolution of HIV from R5 to X4 variants. Interleukin-7 (IL-7), a cytokine produced by stromal cells of the thymus and bone marrow and by keratin, is known to play a key role in T-cell development. We evaluated IL-7 levels in plasma of healthy donors and HIV-positive patients and found significantly higher levels in HIV-positive patients. There was a negative correlation between circulating IL-7 levels and CD4(+) T-cell count in HIV-positive patients (r = -0.621; P < 0.001), suggesting that IL-7 may be involved in HIV-induced T-cell depletion and disease progression. IL-7 levels were higher in individuals who harbored SI variants and who had progressed to having CD4 cell counts of lower than 200 cells/microl than in individuals with NSI variants at a similar stage of disease. IL-7 induced T-cell proliferation and up-regulated CXCR4 expression in peripheral blood mononuclear cells in vitro. Taken together, our results suggest a role for IL-7 in the maintenance of T-cell regeneration and depletion by HIV in infected individuals and a possible relationship between IL-7 levels and the emergence of SI variants.     

TCR gamma delta+ and CD161+ thymocytes express HIV-1 in the SCID-hu mouse,potentially contributing to immune dysfunction in HIV infection

The vast diversity of the T cell repertoire renders the adaptive immune response capable of recognizing a broad spectrum of potential antigenic peptides. However, certain T cell rearrangements are conserved for recognition of specific pathogens, as is the case for TCRgammadelta cells. In addition, an immunoregulatory class of T cells expressing the NK receptor protein 1A (CD161) responds to nonpeptide Ags presented on the MHC-like CD1d molecule. The effect of HIV-1 infection on these specialized T cells in the thymus was studied using the SCID-hu mouse model. We were able to identify CD161-expressing CD3(+) cells but not the CD1d-restricted invariant Valpha24/Vbeta11/CD161(+) NK T cells in the thymus. A subset of TCRgammadelta cells and CD161-expressing thymocytes express CD4, CXCR4, and CCR5 during development in the thymus and are susceptible to HIV-1 infection. TCRgammadelta thymocytes were productively infectable by both X4 and R5 virus, and thymic HIV-1 infection induced depletion of CD4(+) TCRgammadelta cells. Similarly, CD4(+)CD161(+) thymocytes were depleted by thymic HIV-1 infection, leading to enrichment of CD4(-)CD161(+) thymocytes. Furthermore, compared with the general CD4-negative thymocyte population, CD4(-)CD161(+) NK T thymocytes exhibited as much as a 27-fold lower frequency of virus-expressing cells. We conclude that HIV-1 infection and/or disruption of cells important in both innate and acquired immunity may contribute to the overall immune dysfunction seen in HIV-1 disease.     

Human Immunodeficiency Virus Type 1 Strains R5 and X4 Induce Different Pathogenic Effects in hu-PBL-SCID Mice, Depending on the State of Activation/Differentiation of Human Target Cells at the Time of Primary Infection

In a previous study, we had found that the extent of T-cell dysfunctions induced by a T-tropic strain of human immunodeficiency virus type 1 (HIV-1) in SCID mice reconstituted with human peripheral blood lymphocytes (hu-PBLs) (hu-PBL-SCID mice) was related to the in vivo state of activation of the human lymphocytes. In this article, we compared the effect of infection of hu-PBL-SCID mice with either T-tropic (X4) or M-tropic (R5) strains of HIV-1 by performing virus inoculation at either 2 h or 2 weeks after the hu-PBL transfer, when the human T cells exhibited a marked activation state or a predominant memory phenotype, respectively. A comparable level of infection was found when hu-PBL-SCID mice were challenged with either the SF162 R5 or the IIIB X4 strain of HIV at 2 h postreconstitution, while at 2 weeks, the R5 virus infection resulted in a higher level of HIV replication than the X4 virus. The R5 strain induced a marked human CD4(+) T-cell depletion along with a drop in levels of human immunoglobulin M in serum and release of soluble factors at both infection times, while the X4 virus induced severe immune dysfunctions only at 2 h. Of interest, injection of hu-PBLs into SCID mice resulted in a marked up-regulation of CCR5 on human CD4(+) T cells. The percentage of CXCR4(+) cells did not change after transplantation, even though a significant decrease in antigen expression was observed. Comparative experiments with two molecular clones of HIV-1 (X4 SF2 and R5 SF162) and two envelope recombinant viruses generated from these viruses showed that R5 viruses (SF162 and the chimeric env-SF162-SF2) caused an extensive depletion of human CD4(+) T cells in SCID mice at both 2 h and 2 weeks after reconstitution, while the X4 viruses (SF2 and the chimeric env-SF2-SF162) induced CD4 T-cell depletion only when infection was performed at the 2-h reconstitution time. These results emphasize the importance of the state of activation/differentiation of human CD4(+) T cells and gp120-coreceptor interactions at the time of primary infection in determining HIV-1 pathogenicity in the hu-PBL-SCID mouse model.     

Purification of a modified form of bovine antithrombin III as an HIV-1 CD8+ T-cell antiviral factor

CD8(+) T-cells secrete soluble factor(s) capable of inhibiting both R5- and X4-tropic strains of human immunodeficiency virus type 1 (HIV-1). CCR5 chemokine ligands, released from activated CD8(+) T-cells, contribute to the antiviral activity of these cells. These CC-chemokines, however, do not account for all CD8(+) T-cell antiviral factor(s) (CAF) released from these cells, particularly because the elusive CAF can inhibit the replication of X4 HIV-1 strains that use CXCR4 and not CCR5 as a coreceptor. Here we demonstrate that activated CD8(+) T-cells of HIV-1-seropositive individuals modify serum bovine antithrombin III into an HIV-1 inhibitory factor capable of suppressing the replication of X4 HIV-1. These data indicate that antithrombin III may play a role in the progression of HIV-1 disease.     

Impact of cytokines on replication in the thymus of primary human immunodeficiency virus type 1 isolates from infants

Early infection of the thymus with the human immunodeficiency virus (HIV) may explain the more rapid disease progression among children infected in utero than in children infected intrapartum. Therefore, we analyzed infection of thymocytes in vitro by HIV type 1 primary isolates, obtained at or near birth, from 10 children with different disease outcomes. HIV isolates able to replicate in the thymus and impact thymopoiesis were present in all infants, regardless of the timing of viral transmission and the rate of disease progression. Isolates from newborns utilized CCR5, CXCR4, or both chemokine receptors to enter thymocytes. Viral expression was observed in discrete thymocyte subsets postinfection with HIV isolates using CXCR4 (X4) and isolates using CCR5 (R5), despite the wider distribution of CXCR4 in the thymus. In contrast to previous findings, the X4 primary isolates were not more cytopathic for thymocytes than were the R5 isolates. The cytokines interleukin-2 (IL-2), IL-4, and IL-7 increased HIV replication in the thymus by inducing differentiation and expansion of mature CD27(+) thymocytes expressing CXCR4 or CCR5. IL-2 and IL-4 together increased expression of CXCR4 and CCR5 in this population, whereas IL-4 and IL-7 increased CXCR4 but not CCR5 expression. IL-2 plus IL-4 increased the viral production of all pediatric isolates, but IL-4 and IL-7 had a significantly higher impact on the replication of X4 isolates compared to R5 isolates. Our studies suggest that coreceptor use by HIV primary isolates is important but is not the sole determinant of HIV pathogenesis in the thymus.     

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.