Dendritic cells infected with vpr-positive human immunodeficiency virus type 1 induce CD8+ T-cell apoptosis via upregulation of tumor necrosis factor alpha

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) plays a crucial role in viral replication and pathogenesis by inducing cell cycle arrest, apoptosis, translocation of preintegration complex, potentiation of glucocorticoid action, impairment of dendritic cell (DC) maturation, and T-cell activation. Recent studies involving the direct effects of Vpr on DCs and T cells indicated that HIV-1 containing Vpr selectively impairs phenotypic maturation, cytokine network, and antigen presentation in DCs and dysregulates costimulatory molecules and cytokine production in T cells. Here, we have further investigated the indirect effect of HIV-1 Vpr(+) virus-infected DCs on the bystander CD8(+) T-cell population. Our results indicate that HIV-1 Vpr(+) virus-infected DCs dysregulate CD8(+) T-cell proliferation and induce apoptosis. Vpr-containing virus-infected DC-mediated CD8(+) T-cell killing occurred in part through enhanced tumor necrosis factor alpha production by infected DCs and subsequent induction of death receptor signaling and activation of the caspase 8-dependent pathway in CD8(+) T cells. Collectively, these results provide evidence that Vpr could be one of the important contributors to the host immune escape by HIV-1 through its ability to dysregulate both directly and indirectly the DC biology and T-cell functions.     

Suppression of human immunodeficiency virus replication by CD8+ cells from infected and uninfected chimpanzees.

Over a 4-year period, infectious human immunodeficiency virus type 1 (HIV-1) has been recovered from cultured peripheral blood mononuclear cells (PBMC) of virus-infected animals only intermittently and at relatively low titers. In examining the possible mechanism for this observation, CD4+ cells or CD8+ cells were removed by panning from the PMBC before culture. A dramatic increase in frequency of HIV-1 recovery as well as in the level of virus replication was observed in the CD4+ cell-enriched or CD8+ cell-depleted cultures of PBMC from 3/3 infected animals. Moreover, addition of purified CD8+ effector cells from all 6 HIV-infected and 5/10 uninfected animals to an equal number of HIV-1 acutely infected purified CD4+ target cells resulted in 75-100% suppression of virus production. CD8+ cells from 3 additional uninfected animals caused delayed replication kinetics and moderate to low suppression of peak virus production. These findings contrast with the previously recognized absence of this HIV-1-suppressing activity of CD8+ cells from seronegative humans. This CD8+ cell-mediated suppression of viral replication could help explain the natural resistance of chimpanzees to HIV-induced disease.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Differential CD4+ versus CD8+ T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates

Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4⁺ and CD8⁺ T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4⁺ T-cell response (NYVAC). Remarkably, vector-induced differences in CD4⁺/CD8⁺ T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4⁺ T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4⁺ T-cell responses showed efficacies similar to those with stronger CD8⁺ T-cell responses.     

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.     

A recombinant Sendai virus is controlled by CD4+ effector T cells responding to a secreted human immunodeficiency virus type 1 envelope glycoprotein

The importance of antigen-specific CD4(+) helper T cells in virus infections is well recognized, but their possible role as direct mediators of virus clearance is less well characterized. Here we describe a recombinant Sendai virus strategy for probing the effector role(s) of CD4(+) T cells. Mice were vaccinated with DNA and vaccinia virus recombinant vectors encoding a secreted human immunodeficiency virus type 1 (HIV-1) envelope protein and then challenged with a Sendai virus carrying a homologous HIV-1 envelope gene. The primed mice showed (i) prompt homing of numerous envelope-primed CD4(+) T cell populations to the virus-infected lung, (ii) substantial production of gamma interferon, and interleukin-2 (IL-2), IL-4, and IL-5 in that site, and (iii) significantly reduced pulmonary viral load. The challenge experiments were repeated with immunoglobulin(-/-) microMT mice in the presence or absence of CD8(+) and/or CD4(+) T cells. These selectively immunodeficient mice were protected by primed CD4(+) T cells in the absence of antibody or CD8(+) T cells. Together, these results highlight the role of CD4(+) T cells as direct effectors in vivo and, because this protocol gives such a potent response, identify an outstanding experimental model for further dissecting CD4(+) T-cell-mediated immunity in the lung.     

Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI.

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.     

A candidate HIV/AIDS vaccine (MVA-B) lacking vaccinia virus gene C6L enhances memory HIV-1-specific T-cell responses

The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4+ and CD8+ T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8+ T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8+ T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8+ T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.     

Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.     

Exposure of normal monocyte-derived dendritic cells to human immunodeficiency virus type-1 particles leads to the induction of apoptosis in co-cultured CD4+ as well as CD8+ T cells

The depletion of immune T cells by human immunodeficiency virus type-1 (HIV-1) infection is a major mechanism involved in the pathogenesis of AIDS. Here, we examined a possible effector function of blood monocyte-derived dendritic cells (DCs) to induce apoptosis in bystander CD4+ and CD8+ T cells. The DCs were generated by culturing monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs exposed to HIV-1 particles were co-cultured with healthy donor-derived blood T cells at a ratio of 1:20. Analyses by percent cell mortality, staining with propidium iodide and reactivity with Annexin V revealed the induction of apoptosis in both CD4+ and CD8+ target T cells. Further, this apoptosis occurred without stimulation with mitogens when the cell cycle of target T cells shifted from G0 to G1, probably due to the mitogenic effect of the DCs. Thus, induction of apoptosis in both CD4+ and CD8+ T cells occurred via interaction with DCs adsorbed with HIV-1 particles.     

Generation of specific Th1 and CD8+ T-cell responses by immunization with mouse CD8+ dendritic cells loaded with HIV-1 viral lysate or envelope glycoproteins

Immunization with antigen-pulsed dendritic cells (DCs) can be used to elicit optimal immune responses. We developed the SRDC cell line, with a morphology, phenotype and activity similar to mouse splenic CD4(-)CD8alpha(+)CD205(+)CD11b(-) dendritic cells, which induce a polarized Th1 immune response. We evaluated the ability of SRDCs pulsed with HIV-1 viral lysate, oligomeric soluble gp140 or capsid p24 to induce specific antibody and T-cell responses in CBA/J mice. Immunization with all loaded SRDCs elicited antibody responses against the antigens tested. However, only HIV-1 viral lysate and gp140-pulsed SRDCs elicited specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate the value of well characterized DC lines for optimizing the antigen-loading mixture, according to the DC population targeted. Our data suggest that splenic DCs pulsed with complex antigens, such as HIV-1 viral lysate or oligomeric soluble gp140, could be used as vaccines, eliciting strong primary Th1-polarized and humoral immune responses against HIV proteins in vivo.     

Multiple specificities in the murine CD4+ and CD8+ T-cell response to dengue virus.

The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection.     

Infiltration by CD4+ and CD8+ lymphocytes in bursa of chickens infected with Infectious Bursal Disease Virus (IBDV): strain-specific differences

In order to investigate if there is any definite correlation between the degree of T-cell response in the bursa of Fabricius (BF) and the virulence of Infectious Bursal Disease (IBD) virus strains, chickens were infected with strains of different virulence i.e. mild (Lukert strain), intermediate (Georgia strain) or invasive intermediate (IV-95 strain). At various times post-inoculation, bursal samples were collected to study virus specific histopathological lesions, the distribution of viral antigen and the extent of T-cell infiltration in the bursa. Most severe bursal lesions were induced by IV-95 strain (the invasive intermediate strain), whereas Lukert, the mild strain caused the least severe lesions. The number of virus positive cells in the bursa was highest in chickens infected with IV-95 strain. Substantial infiltration of CD4+ and CD8+ T-cells in the bursal follicles of virus-infected groups was observed from 4 d.p.i. onwards. The magnitude of T-cell response was more in the birds infected with intermediate (Georgia) or invasive intermediate strains of virus than chickens inoculated with mild (Lukert) strain, even when 10-fold higher doses of the inoculums were used. PHA responses to peripheral lymphocytes were found suppressed in all the groups of chickens only transiently. The results indicate that the magnitude of T-cell responses in BF during IBDV infection is influenced more by the virulence of virus strain rather than the quantum of viral load in BF. Over all these studies may have implications in understanding the role of T-cells in pathogenesis and immunity in IBD.     

Rapid memory CD8+ T-lymphocyte induction through priming with recombinant Mycobacterium smegmatis

The most promising vaccine strategies for the induction of cytotoxic-T-lymphocyte responses have been heterologous prime/boost regimens employing a plasmid DNA prime and a live recombinant-vector boost. The priming immunogen in these regimens must elicit antigen-specific memory CD8+ T lymphocytes that will expand following the boosting immunization. Because plasmid DNA immunogens are expensive and their immunogenicity has proven disappointing in human clinical trials, we have been exploring novel priming immunogens that might be used in heterologous immunization regimens. Here we show that priming with a prototype recombinant Mycobacterium smegmatis strain expressing human immunodeficiency virus type 1 (HIV-1) gp120-elicited CD4+ T lymphocytes with a functional profile of helper cells as well as a CD8+ T-lymphocyte population. These CD8+ T lymphocytes rapidly differentiated to memory cells, defined on the basis of their cytokine profile and expression of CD62L and CD27. Moreover, these recombinant-mycobacterium-induced T lymphocytes rapidly expanded following boosting with a recombinant adenovirus expressing HIV-1 Env to gp120-specific CD8+ T lymphocytes. This work demonstrates a remarkable skewing of recombinant-mycobacterium-induced T lymphocytes to durable antigen-specific memory CD8+ T cells and suggests that such immunogens might be used as priming vectors in prime/boost vaccination regimens for the induction of cellular immune responses.     

Vaccines based on novel adeno-associated virus vectors elicit aberrant CD8+ T-cell responses in mice

We recently discovered an expanded family of adeno-associated viruses (AAVs) that show promise as improved gene therapy vectors. In this study we evaluated the potential of vectors based on several of these novel AAVs as vaccine carriers for human immunodeficiency virus type 1 Gag. Studies with mice indicated that vectors based on AAV type 7 (AAV7), AAV8, and AAV9 demonstrate improved immunogenicity in terms of Gag CD8(+) T-cell and Gag antibody responses. The quality of these antigen-specific responses was evaluated in detail for AAV2/8 vectors and compared to results with an adenovirus vector expressing Gag (AdC7). AAV2/8 produced a vibrant CD8(+) T-cell effector response characterized by coexpression of gamma interferon and tumor necrosis factor alpha as well as in vivo cytolytic activity. No CD8(+) T-cell response generated by any of the AAVs was effectively boosted with AdC7, a result consistent with the finding of a relative lack of cells expressing interleukin-2 (IL-2) or a central memory phenotype at 3 months after the prime. The primary response to an AdC7 vaccine differed from that generated by AAVs in that the peak effector response evolved into populations of Gag-specific T cells expressing high levels of cytokines, including IL-2, and with effector memory and central memory phenotypes. A number of mechanisms could be considered to explain the aberrant activation of CD8(+) T cells by AAV, including insufficient inflammatory responses, CD4 help, and/or chronic antigen expression and T-cell exhaustion. Interestingly, the B-cell response to AAV-encoded Gag was quite vibrant and easily boosted with AdC7.     

Reduced hepatitis B virus (HBV)-specific CD4+ T-cell responses in human immunodeficiency virus type 1-HBV-coinfected individuals receiving HBV-active antiretroviral therapy

Functional hepatitis B virus (HBV)-specific T cells are significantly diminished in individuals chronically infected with HBV compared to individuals with self-limiting HBV infection or those on anti-HBV therapy. In individuals infected with human immunodeficiency virus type 1 (HIV-1), coinfection with HBV is associated with an increased risk of worsening liver function following antiviral therapy and of more rapid HBV disease progression. Total HBV-specific T-cell responses in subjects with diverse genetic backgrounds were characterized by using a library of 15-mer peptides overlapping by 11 amino acids and spanning all HBV proteins. The magnitude and breadth of CD4(+) and CD8(+) T-cell responses to HBV in peripheral blood were examined by flow cytometry to detect gamma interferon production following stimulation with HBV peptide pools. Chronic HBV carriers (n = 34) were studied, including individuals never treated for HBV infection (n = 7), HBV-infected individuals receiving anti-HBV therapy (n = 13), and HIV-1-HBV-coinfected individuals receiving anti-HBV therapy (n = 14). CD4(+) and CD8(+) HBV-specific T-cell responses were more frequently detected and the CD8(+) T-cell responses were of greater magnitude and breadth in subjects on anti-HBV treatment than in untreated chronic HBV carriers. There was a significant inverse correlation between detection of a HBV-specific T-cell response and HBV viral load. HBV-specific CD4(+) and CD8(+) T-cell responses were significantly (fivefold) reduced compared with HIV-specific responses. Although, the frequency and breadth of HBV-specific CD8(+) T-cell responses were comparable in the monoinfected and HIV-1-HBV-coinfected groups, HBV-specific CD4(+) T-cell responses were significantly reduced in HIV-1-HBV-coinfected individuals. Therefore, HIV-1 infection has a significant and specific effect on HBV-specific T-cell immunity.