Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse

Infection with the human immunodeficiency virus (HIV) results in immunosuppression and depletion of circulating CD4+ T cells. Since the thymus is the primary organ in which T cells mature it is of interest to examine the effects of HIV infection in this tissue. HIV infection has been demonstrated in the thymuses of infected individuals and thymocytes have been previously demonstrated to be susceptible to HIV infection both in vivo, using the SCID-hu mouse, and in vitro. The present study sought to determine which subsets of thymocytes were infected in the SCID-hu mouse model and to evaluate HIV-related alterations in the thymic microenvironment. Using two different primary HIV isolates, infection was found in CD4+/CD8+ double positive thymocytes as well as in both the CD4+ and CD8+ single positive subsets of thymocytes. The kinetics of infection and resulting viral burden differed among the three thymocyte subsets and depended on which HIV isolate was used for infection. Thymic epithelial (TE) cells were also shown to endocytose virus and to often contain copious amounts of viral RNA in the cytoplasm by in situ hybridization, although productive infection of these cells could not be definitively shown. Furthermore, degenerating TE cells were observed even without detection of HIV in the degenerating cells. Two striking morphologic patterns of infection were seen, involving either predominantly thymocyte infection and depletion, or TE cell involvement with detectable cytoplasmic viral RNA and/or TE cell toxicity. Thus, a variety of cells in the human thymus is susceptible to HIV infection, and infection with HIV results in a marked disruption of the thymic microenvironment leading to depletion of thymocytes and degeneration of TE cells.     

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and viral entry into the central nervous system.     

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4(+) T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4(+) cells during gene therapy of AIDS.     

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.     

Critical role for CD4(+) T cells in controlling retrovirus replication and spread in persistently infected mice

Reactivations of persistent viral infections pose a significant medical problem in immunocompromised cancer, transplant, and AIDS patients, yet little is known about how persistent viral infections are immunologically controlled. Here we describe a mouse model for investigating the role of the immune response in controlling a persistent retroviral infection. We demonstrate that, following recovery from acute Friend virus infection, a small number of B cells evade immunological destruction and harbor persistent virus. In vivo depletions of T-cell subsets in persistently infected mice revealed a critical role for CD4(+) T cells in controlling virus replication, spread to the erythroid lineage, and induction of erythroleukemia. The CD4(+) T-cell effect was independent of CD8(+) T cells and in some cases was also independent of virus-neutralizing antibody responses. Thus, the CD4(+) T cells may have had a direct antiviral effect. These results may have relevance for human immunodeficiency virus (HIV) infections where loss of CD4(+) T cells is associated with an increase in HIV replication, reactivation of persistent viruses, and a high incidence of virus-associated cancers.     

Thymoma associated with CD4+ lymphopenia, cytomegalovirus infection, and Kaposi's sarcoma

A case of thymoma with associated opportunistic infections, CD4/CD8 T-lymphocyte imbalance, low CD4-positive T-lymphocyte counts and Kaposi's sarcoma (KS) without HIV infection is reported. Cytomegalovirus inclusions were identified in the nuclei of some KS spindle and endothelial cells. It is known that KS has a high prevalence in AIDS patients and has occasionally been associated with other causes of immunosuppression. In previous studies, coexisting KS and thymoma were related to myasthenia gravis, corticosteroid treatment and excess CD8-positive T-lymphocyte counts. More recently an imbalance between CD4 and CD8 positive T lymphocytes has been identified in association with thymoma. The present case suggests that there may be a relationship between thymoma, CD4-positive lymphopenia, and KS.     

Increased CD4-positive monocytes in HIV-infected haemophilic patients

The monocyte-macrophage system is known to play a central role in HIV infection, and expression of CD4 on the surface of monocytes/macrophages is important, since this molecule is a key factor for the entrance of HIV into susceptible cells. In this paper we evaluated the expression of CD4 in monocytes of haemophilic patients (He) who had been infected with HIV (HIV + He) through transfusion of contaminated plasma concentrates. Thirty seropositive patients (HIV + He), 10 seronegative He patients (HIV-He) and 20 voluntary normal blood donors were studied. Phenotypic evaluation of monocytes was performed by flow cytometry of peripheral blood stained with anti-CD45, -CD3, -CD4 and -CD14 monoclonal antibodies. The percentage of CD4 monocytes was increased in all HIV+ patients groups, but it was highest in those belonging to Groups III and IV A of the CDC classification. Furthermore, the median of fluorescence intensity of CD4+ monocytes from individual patients was shifted to the right, indicating expression of increased numbers of CD4 molecules on the cell membrane of monocytes. This could in turn favour HIV infection and viral persistence, facilitating in vivo dissemination of the virus.     

Increased rate of HIV-1 entry and its cytopathic effect in CD4+/CXCR4+ T cells expressing relatively high levels of CD26

The role of the T-cell activation antigen CD26 was evaluated in viral entry and infection of CD4(+)/CXCR4(+) cells by the lymphotropic HIV-1 Lai isolate. For this purpose, CEM T cells, which are permissive to HIV infection and express low levels of CD26, were used to establish by transfection four groups of cell clones expressing either low, high, and very high levels of CD26, or expressing the anti-sense RNA of CD26. Entry was monitored by the detection of proviral DNA synthesis and the kinetics of virus production, whereas the cytopathic effect was demonstrated by the occurrence of apoptosis. HIV entry and infection were consistently accelerated by at least 24 to 48 h in clones expressing high levels of CD26 compared to the parental cells or to the clones expressing low levels of CD26. Interestingly, infection of clones expressing very high levels of CD26 was not accelerated and showed a kinetics of infection similar to that of low CD26 expressing clones. Moreover, HIV infection was significantly reduced in the clones expressing CD26 anti-sense RNA. In the different clones, apoptosis was dependent on the severity of virus infection and occurred after the accumulation of HIV envelope glycoproteins. Our results demonstrate that with equivalently expressed levels of CD4 and CXCR4 in cell lines established from CEM cells, relatively high levels of CD26 contribute to an increased rate of HIV entry, infection, and apoptosis. Furthermore, they point out that overexpression of CD26 in a given cell line may lead to a negative effect on HIV infection. Consequently, CD26 appears to regulate HIV entry and apoptosis, processes which are critical for viral pathogenesis.     

Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.     

Rabbit cells expressing human CD4 and human CCR5 are highly permissive for human immunodeficiency virus type 1 infection

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.     

Coexpression of CD4 and CD8 antigens and the appearance of HLA-DR and CD25 receptors on lymphocytes as markers of immune activation in patients with HIV infection

Results of quantitative determinations of peripheral blood lymphocytes presenting CD4 or simultaneously CD4 and CD8 antigens as well as HLA-DR and CD25 antigens in patients with different clinical stages of CD4+ and CD4+CD8+ cells were observed. The fall of CD4+CD8+ lymphocytes percentage was slight in the initial stages of infection but more sharp during the full-blown AIDS. The percentage of HLA-DR positive cells was higher in seropositive patients in comparison with controls and increased with the progression of HIV infection into AIDS. The CD25 expression was also higher in HIV-infected patients, however, it decreased in patients with late stages of AIDS. The observed disturbances could be caused by the direct destruction by replicating HIV or by severe abnormalities of immune system function.     

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.     

Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.     

CD4+ CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway

Ligation of the protein tyrosine phosphatase CD45 on both mature and immature T cells modulates the amplitude of TCR-mediated signals. In this work, we have evaluated the consequences of CD45 ligation on immature T cells, in the absence of TCR engagement. Cross-linking of CD45 on thymocytes by mAbs led to the induction of cellular death, characterized by a reduction in mitochondrial membrane potential (delta psi(m)), production of reactive oxygen species, loss in membrane asymmetry, exposure of phosphatidylserine residues, and incorporation of vital dyes. In sharp contrast to most stimuli causing thymocyte death, CD45 cross-linking did not lead to DNA degradation. Cell death was not blocked by Bcl-2 overexpression or treatment with caspase inhibitor. However, death was inhibited by the addition of scavengers of reactive oxygen species. We also established that susceptibility to CD45-mediated death is acquired during the transition of early CD4- CD8- TCR- T cell precursors into CD4+ CD8+ TCR- thymocytes and is increased with further acquisition of surface TCR on these cells. Moreover, mature thymocytes were much less sensitive to CD45 cross-linking than CD4+ CD8+ cells. We propose that during T cell development, CD45 ligation could induce the death of those immature thymocytes that do not fulfill the requirements for positive selection.     

Spontaneous and ligand-induced trafficking of CXC-chemokine receptor 4

A chimeric protein consisting of CXC-chemokine receptor 4 (CXCR4) and the green fluorescent protein (GFP) was used for studying receptor localization and trafficking in real time in stably transduced HeLa, U-937, CEM, and NIH/3T3 cells. CXCR4-GFP was fully active as a co-receptor in mediating human immunodeficiency virus (HIV) entry. Both CXCR4 and CXCR4-GFP were found to undergo significant spontaneous endocytosis. Only 51.5 +/- 7.8% of receptor molecules were found on the plasma membrane in CD4-positive cells, 43.9 +/- 8.5% were found in CD4-negative HeLa cells, 75.6 +/- 9.7% were found in U-937 cells, 72.5 +/- 7.9 were found in CEM cells, and almost none were found in in NIH/3T3 cells. Stromal cell-derived factor-1alpha induced rapid endocytosis of cell surface receptor molecules. A significant part of CXCR4 was targeted to lysosomes upon binding of the ligands, and recycling of internalized CXCR4 was not efficient. Only about 30% of receptor molecules recycled back to the cell surface in HeLa cells, 5% recycled in U937, and 10% recycled in CEM cells, suggesting that the protective effect of chemokines against HIV infection can be attributed not only to competition for binding but also to depletion of the co-receptor molecules from the cell surface. Envelope glycoprotein gp120 of syncytia-inducing/lymphocyte tropic HIV-1 strains induced rapid internalization of CXCR4 in both CD4-negative and CD4-positive cells, suggesting that gp120 is a high affinity ligand of CXCR4.     

Peripheral blood-derived CD34+ progenitor cells: CXC chemokine receptor 4 and CC chemokine receptor 5 expression and infection by HIV

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.     

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.     

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.