Antigen stimulation induces HIV envelope gp120-specific CD4(+) T cells to secrete CCR5 ligands and suppress HIV infection

CD4(+) T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4(+) T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4(+) T cells elicited production of high amounts of CCR5 chemokines MIP-1alpha (CCL3), MIP-1beta (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-gamma or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4(+) T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1beta and IFN-gamma rapidly declined. Thus, vaccines that trigger HIV-specific CD4(+) T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4(+) T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.     

Differential susceptibility to monomeric HIV gp120-mediated apoptosis in antigen-activated CD4+ T cell populations

To support the hypothesis that indirect mechanisms mediated by viral products like the HIV envelope glycoprotein gp120 could be responsible for T lymphocyte depletion in HIV infection, we developed a system in which the impairment of T cell functions could be investigated in vitro. In particular, we characterized the conditions that allow T lymphocytes repeatedly stimulated with an antigen to be sensitive or resistant to gp120-mediated apoptotic signals. To achieve this goal, a panel of antigen-specific CD4⁺ T cell clones and primary CD4⁺ T lymphocytes were treated for 2 and 18 h with saturating amounts of monomeric gp120 (without cross-linking with specific antibodies) and antigen-driven T cell proliferation and apoptosis were analyzed. We show that monomeric gp120 induces apoptosis only in T lymphocytes repeatedly stimulated with the antigen, that primary T lymphocytes are resistant to programmed cell death mediated by monomeric gp120, but are sensitive to anti-CD4 antibodies, and that gp120-mediated apoptosis is dependent on the period of time between the binding of gp120 to CD4 and the encounter with antigen. To investigate the different susceptibility to gp120 induced apoptosis of primary CD4⁺ and T cell clones further, the number of membrane CD4 molecules and their affinity for gp120, together with Bcl-2 and Fas expression, were studied. Our data suggest that a down-modulation of membrane CD4 together with high expression of the Bcl-2 gene and protein characterizes the susceptibility to apoptosis of gp120-treated cells. In conclusion, our results define the phenotypic features of T cells susceptible to HIV gp120-induced apoptosis and demonstrate that the same clonotype, depending on the activation state, may present a differential sensitivity to apoptosis induction.     

Interactions of CCR5 and CXCR4 with CD4 and gp120 in human blood monocyte-derived dendritic cells

Dendritic cells (DC) and macrophages play an important role in the generation of immune responses and transmission of HIV infection. It has been recently found that, in the presence of gp120, CD4 can be efficiently coimmunoprecipitated by anti-CXCR4 antibodies from lymphocytes and monocytes but not from blood monocyte-derived macrophages. The gp120-CD4-CXCR4 complex formation paralleled the ability for these cell types to support X4 (LAV) HIV-1 envelope glycoprotein (Env)-mediated fusion. Here we report that, unlike macrophages but similar to lymphocytes and monocytes, human blood monocyte-derived DC allow efficient complex formation among the HIV-1 coreceptor CXCR4, the primary receptor CD4, and the Env gp120 (LAV) which parallels their fusion ability with cells expressing HIV-1 Env (LAV). In addition, DC behaved similarly to macrophages, lymphocytes, and monocytes in their ability to support formation of complexes between CD4 and the other major HIV-1 coreceptor CCR5 even in the absence of gp120 as demonstrated by CD4 coimmunoprecipitation with anti-CCR5 antibodies. Further, the amount of gp120-CD4-CXCR4 (or CCR5) complexes was proportional to the extent of cell fusion mediated by the HIV-1 Env (LAV or JRFL, respectively). These results demonstrate that of all the major types of host cells important for HIV-1 infection, the first central stage in the entry mechanism, the formation of gp120-CD4-coreceptor complexes, is not impaired except for the formation of the gp120-CD4-CXCR4 complex in macrophages. Therefore, for most CD4+ target cells restraint(s) on productive HIV-1 infection appears to occur at stages of the virus life cycle subsequent to the gp120-CD4-coreceptor complex formation.     

Inefficient formation of a complex among CXCR4, CD4 and gp120 in U937 clones resistant to X4 gp120-gp41-mediated fusion

Certain subclones (designated as minus clones) of the promonocytic U937 cell line do not support efficient infection and fusion mediated by T cell line adapted (TCLA) X4 HIV-1 gp120-gp41 (Env) although the CXCR4 and CD4 concentrations at their surfaces are similar to those at the surfaces of clones susceptible to HIV-1 entry (plus clones) (H. Moriuchi et al., J. Virol. 71, 9664-9671, 1997). To test the hypothesis that inefficient formation of gp120-CD4-CXCR4 complexes could contribute to the mechanism of resistance to Env-mediated fusion in the minus clones, we incubated plus and minus cells with HIV-1 LAI gp120 and coimmunoprecipitated CD4 by using anti-CXCR4 antibodies. The gp120 induced inefficient coimmunoprecipitation of CD4 in the minus clones but not in the plus ones. Overexpression of CD4 resulted in significant restoration of the minus clones' susceptibility to fusion in parallel with an increase in the amount of the gp120-CD4-CXCR4 complexes. These results not only suggest that the resistance to TCLA X4 HIV-1 entry in the U937 minus clones is due to the inability of these cells to efficiently form complexes among CD4, gp120, and CXCR4, but also provide a direct evidence for the correlation between fusion and the cell surface concentration of the complexes among CXCR4, CD4, and gp120. These data and similar recent observations in macrophages suggest that inefficient complex formation among CXCR4, CD4, and gp120 could be a general mechanism of cell resistance to gp120-gp41-mediated fusion and a major determinant of HIV-1 evolution in vivo.     

Cell-surface-expressed HIV-1 envelope induces the death of CD4 T cells during GP41-mediated hemifusion-like events

Cells expressing the HIV-1 envelope glycoprotein complex (gp120/gp41, Env) induce the death of target cells either after cell-to-cell fusion or after cell-to-cell contact in a fusion-independent fashion. Here, we demonstrate that Env-induced death of single cells (including primary CD4 T cells) required gp120 and gp41 function. The gp41 peptide C34, which blocked syncytium formation, completely inhibited the death of single target cells by specifically acting on gp41 function. Moreover, Env-induced single cell death was exclusively observed in CD4 cells and was associated with specific gp41-mediated transfer of lipids from the membrane of Env-expressing cells to the target cell but not with detectable cytoplasm mixing (complete fusion). We conclude that after gp120 function, gp41 mediates close cell-to-cell contacts, thereby triggering cell death in single uninfected cells in the absence of detectable cell-to-cell fusion.     

Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4⁺ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4⁺ T cell attrition in vivo.     

CD4-independent T cells impair TCR triggering of CD4-dependent T cells: a putative mechanism for T cell affinity maturation

In vivo, T cells expressing low-affinity TCR predominate in primary, but not in secondary responses, a process referred to as T cell affinity maturation. Using CD4 dependence as a measure of the avidity of the interaction between the allospecific TCR and the alloantigen, we show that a similar process occurs in mixed lymphocyte cultures in vitro. Moreover, in coculture experiments high-avidity (CD4-independent) T cell clones inhibited the TCR internalization of low-avidity (CD4-dependent) T cell clones, whereas low-avidity T cell clones had no such effect on high-avidity T cell clones. The extent of inhibition of TCR internalization was dependent on both the avidity of the competing clone and the number of competing cells. Thus, there was a cell dose- and avidity-dependent effect on TCR internalization, an early parameter in T cell activation. These results suggest that low- and high-avidity T cell clones compete for the availability of antigen-presenting cells and that this favors the selective outgrowth of high-avidity T cell clones.     

Constrained use of CCR5 on CD4+ lymphocytes by R5X4 HIV-1: Efficiency of Env-CCR5 interactions and low CCR5 expression determine a range of restricted CCR5-mediated entry

R5X4 HIV-1 has impaired utilization of CCR5 on primary CD4+ lymphocytes but the mechanisms responsible are not well defined. Using a panel of diverse R5X4 Envs we identified a spectrum of CCR5 use on CD4+ lymphocytes. Greater lymphocyte CCR5 use correlated with relative resistance to CCR5 mAbs and small molecule antagonists. Increasing CCR5 expression on lymphocytes increased the proportion of entry mediated by CCR5 for all R5X4 isolates except 89.6. In cell lines with regulated CCR5 expression, strains with greater lymphocyte CCR5 use better exploited limiting levels of CCR5. Introduction of an R306S mutation in the 89.6 V3 domain enhanced its utilization of CCR5 at low levels and switched its preference to CCR5 for lymphocyte entry. Thus, the degree to which R5X4 HIV-1 use primary lymphocyte CCR5 is determined by low CCR5 expression coupled with variations in the efficiency of Env-CCR5 interactions, which is in part governed by V3 sequences.     

Fluticasone propionate increases CD4CD25 T regulatory cell suppression of allergen-stimulated CD4CD25 T cells by an IL-10-dependent mechanism

BACKGROUND: Corticosteroids are the most effective anti-inflammatory therapy for allergic diseases, and these drugs inhibit TH2 T-cell activation. We previously reported that CD4+CD25+ T cells from atopic donors suppressed allergen-stimulated T cells less than those from nonatopic donors. OBJECTIVE: We sought to determine the effect of fluticasone propionate (FP) on allergen-stimulated CD4+CD25- T cells and on the suppressive ability of CD4+CD25+ T cells. METHODS: CD4+CD25+ and CD4+CD25- T cells were separated from peripheral blood of atopic and nonatopic volunteers and cultured alone or mixed in the presence of allergen. Effects of FP were assessed by means of addition to cultures or preincubation with CD4+CD25+ T cells. RESULTS: FP inhibited allergen-stimulated proliferation of CD4+CD25- T cells in a dose-dependent manner. Preincubation of CD4+CD25+ T cells in FP increased subsequent suppressive activity of these cells in allergen-stimulated cultures with CD4+CD25- T cells. This effect was seen when cells were obtained from both nonatopic and atopic donors but was less for cells obtained from atopic individuals. Prior exposure of CD4+CD25+ T cells to FP also increased subsequent IL-10 production by these cells when stimulated with allergen, and addition of anti-IL-10 antibody reversed the steroid-induced enhancement of suppression in mixed cultures. CONCLUSION: Increased suppression by CD4+CD25+ T cells might play a role in anti-inflammatory effects of corticosteroids in asthma and allergic diseases.     

HIV-1 gp120/V3-derived epitopes promote activation-induced cell death to superantigen-stimulated CD4+/CD45RO+ T cells

The third hypervirable (V3) domain of the HIV-1 envelope glycoprotein gp120 has been implicated in HIV pathogenesis via co-receptor usage of chemokine receptors CCR5 and CXCR4. As the protagonist cell populations in the asymptomatic phase of HIV-1 infection are infected macrophages and effector/memory (CD45RO+) CD4+ T cells that express CCR5, we established an in vitro model using human primary monocyte-derived macrophages and lymphocytes to investigate the role of V3 in affecting antigen presentation. We used staphylococcal enterotoxin A (SEA) as a superantigen at a low concentration of 1ng/ml, to activate na?ve CD4+ T cells. Exposure of cells to SEA and lipoV3-liposomes increased the percentage of CD4+/CD45RO+/CCR5+ T cell population as compared to cells treated with SEA and plain liposomes. A consequent decrease of the percentage of CD4+/CD45RO+/CXCR4+ subset was observed. The V3-mediated activation was competitively inhibited by soluble V3-derived peptides with higher cationic charge. V3 enhanced also apoptosis as demonstrated by flow cytometry and intracellular calcium ion assays. These results reinforce the postulation that V3 alters the antigen presentation function itself, independent of specific antigens, thus leading to an enhanced activation-induced cell death (AICD) of responding T cells.     

Downregulation of CCR5 on activated CD4 T cells in HIV-infected Indians

BACKGROUND: HIV infection in India is unique as it occurs predominantly by CCR5-utilizing isolates that exhibit no co-receptor switch. OBJECTIVES: To study HIV-1 co-receptor dynamics on T cells and monocytes following viral infection. STUDY DESIGN: HIV co-receptor expression was evaluated by flow cytometry on various cell subsets in HIV-infected Indians and in vitro in human peripheral blood mononuclear cells infected with CCR5- or CXCR4-utilizing HIV-1. Transfection of the T cell line CEM-CCR5 (which expresses CD4, CCR5 and CXCR4) with HIV-1 Nef or Vpu expression vectors, or treatment with recombinant soluble gp120 from CCR5- and CXCR4-tropic HIV-1, was carried out to determine their effects on co-receptor expression. RESULTS: Indian HIV patients had fewer CD4(+)CCR5(+) T cells and CCR5-expressing activated CD4(+) T cells, but higher CXCR4-expressing activated CD4(+) T cells compared with controls. Expression of CCR5 was not different on monocytes in HIV patients as compared to controls. The CCR5 downregulation on T cells was HIV infection specific and was governed by the co-receptor-utilization phenotype of the virus. The Nef and soluble gp120 proteins induced CCR5 downregulation, the latter in a co-receptor-utilization phenotype specific manner. CONCLUSIONS: The HIV-1 co-receptor dynamics in Indian patients is distinct from western patients and depends upon the virus surface protein. We propose this to be a viral survival strategy.     

Enhanced replication of R5 HIV-1 over X4 HIV-1 in CD4(+)CCR5(+)CXCR4(+) T cells

To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.     

HIV pathogenesis: gp120-antibody complexes bind CD4 and kill T4 cells--immunotoxin therapy should prevent the progression of HIV to AIDS.

Current models of the role of human immunodeficiency virus (HIV) in the pathogenesis of acquired immune deficiency syndrome (AIDS) are inadequate and inconsistent with the literature. This article reviews a wide range of AIDS research and proposes the first model of HIV pathogenesis that is entirely consistent with the literature. This model is based on antibody-gp120 complex crosslinking of CD4 on the T4 cells. Previously unexplained observations embraced by this model include early qualitative defects in the immune system, changes in cytokine expression, 'bystander' T4 cell death, and the apparent discrepancy between the low rate of T4 cell infection and the near-complete elimination of T4 cells in AIDS. A new class of drugs based on this model is detailed. These drugs should disrupt the pathway leading to AIDS and leave an HIV infection indefinitely asymptomatic. These drugs are designed to be readily modified, so the treatment is immune to HIV's notorious mutation-based drug resistance.     

A strategy simplifying site-directed mutagenesis in the CD4-binding region of HIV gp 120

Summary By site-directed mutagenesis, two unique restriction sites, surrounding the CD4-binding DNA region of HIV gp 120, were created. The mutations do not cause amino acid substitutions but generate a DNA fragment, which is stable when cloned into M13 for further mutagenesis and is also easy to reclone.     

Non-covalent complexes of HIV gp120 with CD4 and/or mAbs enhance activation of gp120-specific T clones and provide intermolecular help for anti-CD4 antibody production

The ‘dangerous liaison’ between CD4 and gp120 thatoffers the first entry opportunity to HIV may also provoke perturbationsof the immune control of the host with far-reaching immunopathologicalconsequences. We wondered whether a mechanism of intermolecularhelp (T help across the gap of a non-covalent bond, in contrastto the interamolecular help of carrier to hapten) could breakself-tolerance and be the cause of the frequent anti-CD4 autoantibodiesfound in AIDS patients. To determine whether this hypothesisdeserves further testing, we designed a series of in vitro andin vivo experiments of increasing complexity, focused on thepresentation of gp120 to specific T cells by antigen presentingcells (APC) exposed to the envelope protein in the form of non-covalentcomplexes. Bi-molecular complexes were constructed by allowinggp120 or gp160 to bind specific human mAbs. Tri-molecular complexeswere constructed by introducing CD4 as an intermediate ligandbetween gp120 and mouse mAbs specific for CD4. In all casesthe use of complexes did enhance the immunogenic capacity ofsubstimulatory doses of gp120 or gp160 by facilitating uptakeby APC via Fc receptor and consequent presentation to specifichuman T cell clones. Finally, help for the production in vivoof anti-CD4 antibodies was obtained from T lymphocytes specificfor gp120 when CD4-primed memory B cells were pulsed with CD4complexed with gp120, thus demonstrating in the mouse the entirecycle of intermolecular help via non-covalent interaction, andsetting the stage for future experiments on self-tolerance breakagein a human molecular context.     

CCL3/MIP-1alpha is pro-inflammatory in murine T cell-mediated hepatitis by recruiting CCR1-expressing CD4(+) T cells to the liver

T cell-mediated hepatitis is associated with significant morbidity and mortality worldwide. Levels of C-C chemokine ligand 3/macrophage inflammatory protein-1alpha (CCL3/MIP-1alpha) are elevated in the serum of patients with T cell-mediated liver diseases, but its role is not fully understood. Con A-induced hepatitis is a murine liver-specific inflammation mediated by activated T cells and is driven by an up-regulation of the hepatic expression of IFN-gamma. In this study, we have used CCL3/MIP-1alpha gene-deficient mice to examine the role of CCL3/MIP-1alpha in the pathogenesis of Con A-induced hepatitis. We demonstrate a novel pro-inflammatory role for CCL3/MIP-1alpha since CCL3/MIP-1alpha deficiency significantly attenuated hepatic injury, both biochemically and histologically. Moreover, the recruitment of CCR1-expressing CD4(+) T cells to the liver after Con A treatment was strikingly attenuated by CCL3/MIP-1alpha deficiency. Correspondingly, hepatic IFN-gamma produced by the recruited CD4(+) T cells was significantly reduced by CCL3/MIP-1alpha deficiency during Con A-induced hepatitis. Furthermore, treatment of mice with a dual CCR1/CCR5 peptide antagonist, methionylated RANTES, also markedly reduced hepatic injury and decreased the numbers of CD4(+) T cells within the liver producing IFN-gamma during Con A-induced hepatitis. These findings demonstrate that blockade of the CCL3/MIP-1alpha-CCR1 pathway may represent a novel therapeutic target for treating T cell-mediated liver diseases.     

Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.     

Induction of FOXP3-expressing regulatory CD4pos T cells by human mature autologous dendritic cells

Current literature suggests that T cells recognizing antigen on mature dendritic cells (DC) differentiate into effector T cells whereas tolerance is induced when antigen is presented by immature DC. We investigated the consequences of the interactions between immature or lipopolysaccharide-matured DC and CD4(pos) T lymphocytes in absence of foreign antigen. While immature DC did not induce significant CD4(pos) T cell activation, we observed that a significant fraction of CD4(pos) T cells cultured with mature autologous DC displayed phenotypic features of activation and produced IL-2, IFN-gamma, IL-10 and TGF-beta. Furthermore, CD4(pos) T lymphocytes primed by mature, but not immature, autologous DC acquired regulatory properties. Indeed, when added to an allogeneic mixed leukocyte reaction, they suppressed the response of alloreactive T lymphocytes to the priming DC while responses to third-party stimulators were spared. The generation of CD4(pos) T cells with regulatory function by autologous stimulation did not require the presence of natural CD4(pos)CD25(pos) regulatory T cells. In addition, the acquisition of regulatory function by CD4(pos)CD25(neg) T cells stimulated by autologous mature DC was accompanied by the induction of FOXP3 expression. Our data suggest that during inflammatory conditions, presentation of self antigens by mature DC to autologous T lymphocytes could contribute to the generation of regulatory mechanisms.