Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection.
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ABSTRACT:
SUBMITTER: Sacha JB
PROVIDER: S-EPMC2687996 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To study CD4(+)T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4(+)T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8(+)T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1β, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4(+)T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4(+)effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4(+)T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4(+)T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4(+)T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model.
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Project description:Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8+ T cell response during Mtb infection, CD8+ T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages. We derived CD8+ T cell lines that recognized the Mtb immunodominant epitope TB10.44-11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. While the CD4+ T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8+ T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8+ T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8+ T cells. CD8+ T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8+ T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner. As TB10.4 elicits a dominant CD8+ T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8+ T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8+ T cell response may explain why CD8+ T cells make a disproportionately small contribution to host defense compared to CD4+ T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.
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Project description:SIV DNA can be detected in lymphoid tissue-resident macrophages of chronically SIV-infected Asian macaques. These macrophages also contain evidence of recently phagocytosed SIV-infected CD4+ T cells. Here, we examine whether these macrophages contain replication-competent virus, whether viral DNA can be detected in tissue-resident macrophages from antiretroviral (ARV) therapy-treated animals and humans, and how the viral sequences amplified from macrophages and contemporaneous CD4+ T cells compare. In ARV-naive animals, we find that lymphoid tissue-resident macrophages contain replication-competent virus if they also contain viral DNA in ARV-naive Asian macaques. The genetic sequence of the virus within these macrophages is similar to those within CD4+ T cells from the same anatomic sites. In ARV-treated animals, we find that viral DNA can be amplified from lymphoid tissue-resident macrophages of SIV-infected Asian macaques that were treated with ARVs for at least 5 months, but we could not detect replication-competent virus from macrophages of animals treated with ARVs. Finally, we could not detect viral DNA in alveolar macrophages from HIV-infected individuals who received ARVs for 3 years and had undetectable viral loads. These data demonstrate that macrophages can contain replication-competent virus, but may not represent a significant reservoir for HIV in vivo.
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Project description:ObjectiveHIV-associated CNS dysfunction is a significant problem among people with HIV (PWH), who now live longer due to viral suppression from combined anti-retroviral therapy (ART). Over the course of infection, HIV generates toxic viral proteins and induces inflammatory cytokines that have toxic effects on neurons in the CNS. Among these viral proteins, HIV Nef has been found in neurons of postmortem brain specimens from PWH. However, the source of Nef and its impact on neuronal cell homeostasis are still elusive.Methods and resultsHere, in using a simian immunodeficiency virus (SIV) infected rhesus macaque model of neuroHIV, we find SIV Nef reactivity in the frontal cortex, hippocampus and cerebellum of SIV-infected animals using immunohistochemistry (IHC). Interestingly, SIV-infected macaques treated with ART also showed frequent Nef positive cells in the cerebellum and hippocampus. Using dual quantitative RNAscope and IHC, we observed cells that were positive for Nef, but were not for SIV RNA, suggesting that Nef protein is present in cells that are not actively infected with SIV. Using cell specific markers, we observed Nef protein in microglia/macrophages and astrocytes. Importantly, we also identified a number of NeuN-positive neurons, which are not permissive to SIV infection, but contained Nef protein. Further characterization of Nef-positive neurons showed caspase 3 activation, indicating late stage apoptosis in the CNS neurons.ConclusionsOur results suggest that regardless of ART status, Nef is expressed in the brain of SIV infected macaques and may contribute to neurological complications seen in PWH.
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Project description:The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4(+) T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4(+) T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.
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Project description:It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8(+) (2-7 pool-specific responses) and CD4(+) (5-19 pool-specific responses) T-cell responses in IFN-? ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8(+) T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.
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Project description:Despite incredible scientific efforts, there is no cure for HIV infection. While antiretroviral treatment (ART) can help control the virus and prevent transmission, it cannot eradicate HIV from viral reservoirs established before the initiation of therapy. Further, HIV-infected individuals reliably exhibit viral rebound when ART is interrupted, suggesting that the host immune response fails to control viral replication in persistent reservoirs. Therapeutic vaccines are one current approach to improving antiviral host immune responses and enhance long term virus control. In the present study, we used an integrase defective lentiviral vector (IDLV) expressing SIV-Gag to boost anti-Gag specific immune responses in macaques chronically infected with the tier-2 SHIV-1157(QNE)Y173H. A single immunization with IDLV-SIV-Gag induced durable (>20 weeks) virus control in 55% of the vaccinated macaques, correlating with an increased magnitude of SIV-Gag specific CD8+ T-cell responses. IDLV-based therapeutic vaccines are therefore an effective approach to improve virus specific CD8+ T-cell responses and mediate virus control.
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