Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.     

CD8+ T cells from HIV-1-infected individuals inhibit acute infection by human and primate immunodeficiency viruses.

T lymphocytes expressing the CD8 surface antigen block HIV replication in CD4+ peripheral blood cells from HIV-infected individuals. We report here that CD4+ cells from HIV seronegative donors, when infected in vitro with HIV, also do not replicate virus when cocultured with CD8+ T cells from HIV-infected individuals. CD8+ cells from HIV-uninfected donors did not show this effect on virus replication. HLA-restriction of the antiviral response was not observed, and virus-containing cells were not eliminated from culture. The antiviral activity was broadly cross-reactive, as CD8+ cells from individuals infected only with HIV-1 suppressed the replication of diverse strains of HIV-1 and HIV-2, as well as the simian immunodeficiency virus. This ability of CD8+ cells to control HIV replication could play an important role in the maintenance of an asymptomatic state in HIV-infected individuals.     

Human immunodeficiency virus type 1 (HIV-1) antigen secretion by latently infected resting CD4+ T lymphocytes from HIV-1-infected individuals

In resting CD4(+) T lymphocytes harboring human immunodeficiency virus type 1 (HIV-1), replication-competent virus persists in patients responding to highly active antiretroviral therapy (HAART). This small latent reservoir represents between 10(3) and 10(7) cells per patient. However, the efficiency of HIV-1 DNA-positive resting CD4(+) T cells in converting to HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) after in vitro CD4(+)-T-cell polyclonal stimulation has not been satisfactorily evaluated. By using an HIV-1-antigen enzyme-linked immunospot assay, 8 HIV-1-Ag-SCs per 10(6) CD4(+) resting T cells were quantified in 25 patients with a plasma viral load of <20 copies/ml, whereas 379 were enumerated in 10 viremic patients. In parallel, 369 and 1,238 copies of HIV-1 DNA per 10(6) CD4(+) T cells were enumerated in the two groups of patients, respectively. Only a minority of latently HIV-1 DNA-infected CD4(+) T cells could be stimulated in vitro to become HIV-1-Ag-SCs, particularly in aviremic patients. The difference between the number of HIV-1 immunospots in viremic versus aviremic patients could be explained by HIV-1 unintegrated viral DNA that gave additional HIV-1-Ag-SCs after in vitro CD4(+)-T-cell polyclonal stimulation. The ELISPOT approach to targeting the HIV-1-Ag-SCs could be a useful method for identifying latently HIV-1-infected CD4(+) T cells carrying replication-competent HIV-1 in patients responding to HAART.     

Patterns of cytokine production in human immunodeficiency virus type 1 (HIV-1)-specific human CD8+ T cells after stimulation with HIV-1-infected CD4+ T cells

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.     

HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression

The role of HIV-1-specific CD4+ T-cell responses in controlling HIV-1 infection remains unclear. Previous work has suggested that such cells are eliminated in the early stages of infection in most subjects, and thus cannot substantially contribute to host defense against HIV-1. Here, using flow cytometric detection of antigen-induced intracellular cytokines, we show that significant frequencies of gag specific, T-helper-1 CD4+ memory T cells are detectable in most subjects with active/progressive HIV-1 infection (median frequency, 0.12% of memory subset; range, 0-0.66%). Median frequencies of these cells were considerably higher in nonprogressive HIV-1 disease (0.40%), but there was substantial overlap between the two groups (range of nonprogressors, 0.10-1.7%). Continuous HIV-1 suppression with anti-retroviral therapy was associated with a time-dependent reduction in median frequencies of gag-specific CD4+ memory T cells: 0.08% in subjects treated for 4-24 weeks, and 0.03% in subjects treated for 47-112 weeks. Thus, functional HIV-1-specific CD4+ T cells are commonly available for support of anti-HIV-1 effector responses in active disease, but their decline with anti-retroviral therapy indicates that immunologic participation in long-term HIV-1 control will probably require effective vaccination strategies.     

Selective decrease of CD26 expression in T cells from HIV-1-infected individuals.

The decrease of CD4+ cells in AIDS patients is widely documented, although the selective loss within different subsets of CD4+ cells and the mechanisms involved in this phenomenon are controversial. In the present report we have analyzed the proliferative response to Ag and mitogen of peripheral blood T lymphocytes from HIV-infected individuals, the phenotype profile of CD26+ and CD26- subset of cells and their infectivity by the HIV. The expression of CD26 Ag, either in CD4+ or CD8+ cells, was clearly diminished in all the patients tested. On the other hand, the expression of CD29 seems not to be affected, nevertheless T cells from these patients were unable to generate a proliferative response against soluble Ag. In 11 out of 13 patients, polymerase chain reaction studies demonstrated that the CD26- subset of CD4+ cells was the main reservoir for HIV-1 in infected individuals and HIV-1 virus preferentially infected in vitro CD4+/CD26- subpopulation. This capacity for preferential infectivity, together with the selective loss of cells expressing CD26 Ag, helps to explain the progressive impairment in the immune system of these patients and sheds new light on our understanding of the AIDS pathophysiology.     

CD8+ T lymphocyte control of HIV replication in cultured CD4+ cells varies among infected individuals

Production of the human immunodeficiency virus (HIV) by cultured peripheral blood mononuclear cells (PMC) from many seropositive individuals is inhibited by the presence of CD8+ T lymphocytes. In a study of 10 subjects, high levels of virus replication could be detected in cultures of purified CD4+ cells, but not in unseparated PMC. Addition of highly purified, autologous CD8+ cells to the enriched CD4+ cells resulted in a dose-dependent inhibition of HIV growth and revealed that for some individuals, even low numbers of CD8+ cells can prevent replication of the virus. The data also indicated that culturing enriched CD4+ cells could greatly enhance detection of infectious virus in blood specimens and demonstrated that the CD4+ molecule is expressed on infected T cells isolated directly from the peripheral blood.     

Polymorphisms of CUL5 are associated with CD4+ T cell loss in HIV-1 infected individuals

Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (Apobec3) antiretroviral factors cause hypermutation of proviral DNA leading to degradation or replication-incompetent HIV-1. However, HIV-1 viral infectivity factor (Vif) suppresses Apobec3 activity through the Cullin 5-Elongin B-Elongin C E3 ubiquitin ligase complex. We examined the effect of genetic polymorphisms in the CUL5 gene (encoding Cullin 5 protein) on AIDS disease progression in five HIV-1 longitudinal cohorts. A total of 12 single nucleotide polymorphisms (SNPs) spanning 93 kb in the CUL5 locus were genotyped and their haplotypes inferred. A phylogenetic network analysis revealed that CUL5 haplotypes were grouped into two clusters of evolutionarily related haplotypes. Cox survival analysis and mixed effects models were used to assess time to AIDS outcomes and CD4(+) T cell trajectories, respectively. Relative to cluster I haplotypes, the collective cluster II haplotypes were associated with more rapid CD4(+) T cell loss (relative hazards [RH] = 1.47 and p = 0.009), in a dose-dependent fashion. This effect was mainly attributable to a single cluster II haplotype (Hap10) (RH = 2.49 and p = 0.00001), possibly due to differential nuclear protein-binding efficiencies of a Hap10-specifying SNP as indicated by a gel shift assay. Consistent effects were observed for CD4(+) T cell counts and HIV-1 viral load trajectories over time. The findings of both functional and genetic epidemiologic consequences of CUL5 polymorphism on CD4(+) T cell and HIV-1 levels point to a role for Cullin 5 in HIV-1 pathogenesis and suggest interference with the Vif-Cullin 5 pathway as a possible anti-HIV-1 therapeutic strategy.     

The soluble viral glycoprotein of vesicular stomatitis virus efficiently sensitizes target cells for lysis by CD4+ T lymphocytes

The soluble glycoprotein Gs of vesicular stomatitis virus (VSV), at approximately 10(4) molecules per cell, sensitized target cells for lysis by clones of CD4+ cytolytic T lymphocytes (CTL). In addition to lysis, the clones responded by proliferation and interleukin-2 release. Targets sensitized by Gs competed effectively with VSV-infected cells for recognition. Immune cytolysis by these CD4+ CTLs was restricted by class II major histocompatibility complex (MHC) antigens and was specific to VSV. The specific class II MHC antigen which was restricting for each clone remained the same whether the targets were sensitized by infection with VSV or by exogenously added soluble antigen. Sensitization by Gs appeared to require prior processing because the antigen-presenting cells that were fixed prior to exposure to Gs failed to be recognized by the CTL clones. The high efficiency of this uptake and processing was suggested by the inability of Gs at concentrations up to 10(7) per cell to block superinfection by VSV or to effect the RNA-synthetic machinery of uninfected cells. Also, Gs failed to hemolyze sheep erythrocytes when there was hemolysis by virions or an amino-terminal peptide of the VSV glycoprotein. Extrapolation of these results to viral diseases was possible because soluble viral glycoproteins were naturally synthesized during many viral infections and class II MHC antigens were inducible in cells of nonlymphoid origin. Therefore, CD4+ CTLs may be important participants in increasing virus-induced pathology, especially among adjacent uninfected cells.     

Mosaic HIV-1 Gag antigens can be processed and presented to human HIV-specific CD8+ T cells

Polyvalent mosaic HIV immunogens offer a potential solution for generating vaccines that can elicit immune responses against genetically diverse viruses. However, it is unclear whether key T cell epitopes can be processed and presented from these synthetic Ags and recognized by epitope-specific human T cells. In this study, we tested the ability of mosaic HIV immunogens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors to process and present major HIV clade B and clade C CD8 T cell epitopes in human cells. A bivalent mosaic vaccine expressing HIV Gag sequences was used to transduce PBMCs from 12 HIV-1-infected individuals from the United States and 10 HIV-1-infected individuals from South Africa; intracellular cytokine staining, together with tetramer staining, was used to assess the ability of mosaic Gag Ags to stimulate pre-existing memory responses compared with natural clade B and C vectors. Mosaic Gag Ags expressed all eight clade B epitopes tested in 12 United States subjects and all 5 clade C epitopes tested in 10 South African subjects. Overall, the magnitude of cytokine production induced by stimulation with mosaic Ags was comparable to clade B and clade C Ags tested, but the mosaic Ags elicited greater cross-clade recognition. Additionally, mosaic Ags induced HIV-specific CD4 T cell responses. Our studies demonstrate that mosaic Ags express major clade B and clade C viral T cell epitopes in human cells, as well as support the evaluation of mosaic HIV-1 vaccines in humans.     

Homeostatic proliferation fails to efficiently reactivate HIV-1 latently infected central memory CD4+ T cells

Homeostatic proliferation ensures the longevity of central memory T-cells by inducing cell proliferation in the absence of cellular differentiation or activation. This process is governed mainly by IL-7. Central memory T-cells can also be stimulated via engagement of the T-cell receptor, leading to cell proliferation but also activation and differentiation. Using an in vitro model of HIV-1 latency, we have examined in detail the effects of homeostatic proliferation on latently infected central memory T cells. We have also used antigenic stimulation via anti-CD3/anti-CD28 antibodies and established a comparison with a homeostatic proliferation stimulus, to evaluate potential differences in how either treatment affects the dynamics of latent virus populations. First, we show that homeostatic proliferation, as induced by a combination of IL-2 plus IL-7, leads to partial reactivation of latent HIV-1 but is unable to reduce the size of the reservoir in vitro. Second, latently infected cells are able to homeostatically proliferate in the absence of viral reactivation or cell differentiation. These results indicate that IL-2 plus IL-7 may induce a detrimental effect by favoring the maintenance of the latent HIV-1 reservoir. On the other hand, antigenic stimulation efficiently reactivated latent HIV-1 in cultured central memory cells and led to depletion of the latently infected cells via virus-induced cell death.     

HIV-1 Nef enhances dendritic cell-mediated viral transmission to CD4+ T cells and promotes T-cell activation

HIV-1 Nef enhances dendritic cell (DC)-mediated viral transmission to CD4(+) T cells, but the underlying mechanism is not fully understood. It is also unknown whether HIV-1 infected DCs play a role in activating CD4(+) T cells and enhancing DC-mediated viral transmission. Here we investigated the role of HIV-1 Nef in DC-mediated viral transmission and HIV-1 infection of primary CD4(+) T cells using wild-type HIV-1 and Nef-mutated viruses. We show that HIV-1 Nef facilitated DC-mediated viral transmission to activated CD4(+) T cells. HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+) T cells. However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+) T cells. Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+) T cells and in the activation and proliferation of resting CD4(+) T cells, which likely contribute to viral pathogenesis.     

Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodeficiency virus CD8+ and CD4+ T-cell responses from chronically infected individuals

Recombinant canarypox virus vectors containing human immunodeficiency virus type 1 (HIV-1) sequences are promising vaccine candidates, as they replicate poorly in human cells. However, when delivered intramuscularly the vaccines have induced inconsistent and in some cases transient antigen-specific cytotoxic T-cell (CTL) responses in seronegative volunteers. An attractive way to enhance these responses would be to target canarypox virus to professional antigen-presenting cells such as dendritic cells (DCs). We studied (i) the interaction between canarypox virus and DCs and (ii) the T-cell responses induced by DCs infected with canarypox virus vectors containing HIV-1 genes. Mature and not immature DCs resisted the cytopathic effects of canarypox virus and elicited strong effector CD8+ T-cell responses from chronically infected HIV+ individuals, e.g., cytolysis, and secretion of gamma interferon (IFN-gamma) and beta-chemokines. Furthermore, canarypox virus-infected DCs were >30-fold more efficient than monocytes and induced responses that were comparable to those induced by vaccinia virus vectors or peptides. Addition of exogenous cytokines was not necessary to elicit CD8+ effector cells, although the presence of CD4+ T cells was required for their expansion and maintenance. Most strikingly, canarypox virus-infected DCs were directly able to stimulate HIV-specific, IFN-gamma-secreting CD4 helper responses from bulk as well as purified CD4+ T cells. Therefore, these results suggest that targeting canarypox virus vectors to mature DCs could potentially elicit both anti-HIV CD8+ and CD4+ helper responses in vivo.     

Primary type II alveolar epithelial cells present microbial antigens to antigen-specific CD4+ T cells

Type II alveolar epithelial cells (AEC) can produce various antimicrobial and proinflammatory effector molecules. This, together with their abundance and strategic location, suggests a role in host defense against pulmonary pathogens. We report that murine type II AEC, like their human counterparts, express class II major histocompatibility complex (MHC). Using a murine model of pulmonary tuberculosis, we find that type II AEC become activated and have increased cell surface expression of class II MHC, CD54, and CD95 following infection. Type II AEC use the class II MHC pathway to process and present mycobacterial antigens to immune CD4+ T cells isolated from mice infected with Mycobacterium tuberculosis. Therefore, not only can type II AEC contribute to the pulmonary immunity by secreting chemokines that recruit inflammatory cells to the lung, but they can also serve as antigen-presenting cells. Although type II AEC are unlikely to prime na?ve T cells, their ability to present antigens to T cells demonstrates that they can participate in the effector phase of the immune response. This represents a novel role for type II AEC in the immunological response to pulmonary pathogens.     

C1.7 antigen expression on CD8+ T cells is activation dependent: increased proportion of C1.7+CD8+ T cells in HIV-1-infected patients with progressing disease.

The C1.7 Ag is a surface marker previously shown to be expressed on all NK cells and on a subset of CD8+ T cells. We report in this study that C1.7 Ag expression on peripheral blood-derived CD8+ T cells overlaps with activation markers S6F1high and CD29high and is reciprocally expressed with CD62L. C1.7 Ag expression can be induced in vitro on CD8+ T cells by anti-CD3 cross-linking, suggesting that C1.7 Ag is activation dependent. In contrast to NK cells, C1.7 Ag does not signal on CD8+ T cells, nor does it induce redirected lysis upon ligation. The proportion of C1.7 Ag+CD8+ T cells is increased in HIV-infected patients compared with healthy donors. In 69 HIV-infected patients, we observed a significant inverse correlation between the percentage of C1.7 Ag-expressing CD8+ T cells and the absolute CD4+ T cell count. Two-year clinical follow-up of patients with initial CD4+ T cell count of >400 cells/mm3 and a normal proportion of C1.7 Ag+CD8+ T cells revealed that these patients were clinically stable with minimal HIV-associated symptoms. In contrast, 10 of 12 patients with CD4+ T cell counts of >400 cells/mm3 and an elevated proportion of C1.7 Ag+CD8+ T cells were symptomatic. ANOVA analysis of patients indicates that C1.7 Ag is a better predictor of disease progression than CD4 count. Overall, our findings indicate that C1.7 Ag is the first described marker for activated/memory CD8+ T cells and a useful parameter for evaluating the level of CD8+ T cell activation in vivo.     

HIV-1-specific interleukin-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function

Functional defects in cytotoxic CD8(+) T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8(+) T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication without treatment. HIV-specific triggering of IL-21 by CD4(+) T cells was significantly enriched in these persons (P = 0.0007), while isolated loss of IL-21-secreting CD4(+) T cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21(+) CD4(+) T cells in acute infection resulted in lower viral set points (P = 0.002). Moreover, IL-21 production by CD4(+) T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8(+) T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8(+) T cells showed an enhanced ability to efficiently inhibit viral replication in vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21(+) CD4(+) T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance for vaccine design.