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Innate sensing of HIV-infected cells.

Lepelley A, Louis S, Sourisseau M, Law HK, Pothlichet J, Schilte C, Chaperot L, Plumas J, Randall RE, Si-Tahar M, Mammano F, Albert ML, Schwartz O - PLoS Pathog. (2011)

Bottom Line: In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing.In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells.Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Virus and Immunity Unit, URA CNRS 3015, Paris, France.

ABSTRACT
Cell-free HIV-1 virions are poor stimulators of type I interferon (IFN) production. We examined here how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs) and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. Therefore, detection of HIV-infected lymphocytes occurs through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

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Related in: MedlinePlus

Sensing of HIV-infected lymphocytes by hematopoïetic cells.a. Schematic representation of the experimental system. HIV-1 infected T cells are mixed with PBMCs, and levels of IFN released in supernatants are measured 12-24h later. b. IFN release in cocultures of HIV-infected T cells with PBMCs. MT4C5 lymphoblastoid T cells (left panel) or primary CD4+ T lymphocytes (right panel) were used as donors. Whole PBMCs, PBMCs depleted of pDCs (PBMCs-pDCs), and purified pDCs where used as target cells. Targets were either left non stimulated (NS), cocultivated with non-infected (NI) or HIV-infected (NL4-3 strain) MT4C5 cells (MT4C5-HIV), exposed to cell-free HIV virions (free HIV) or Influenza virus (FLUAV). IFN production in supernatants was measured in a bioassay, after 12 h of culture. Donor to target cell ratio were 1∶2 for PBMCs and PBMCs-pDCs, and 2∶1 for pDCs. c. Dose response analysis of IFN release. Whole PBMCs were incubated with MT4C5 cells displaying increasing % of infected (Gag+) cells. IFN production was measured after 24 h of coculture (orange histrograms). The levels of living MT4C5 cells at the beginning of the coculture, determined by flow cytometry (forward and side scatters gating), are shown in blue. d. Sensing of cells infected with various HIV isolates by PBMCs. MT4C5, infected with NL4-3 (NL), NLAD8 (AD8) and with two primary isolates (BON and 132W) were cocultivated with PBMCs and IFN was measured 24 h later. With all isolates, about 25% of infected MT4C5 cells expressed Gag before coculture. e. Role of HIV envelope glycoproteins. MT4C5, infected with wild-type (HIV), Env-deleted (ΔEnv), or a non-fusogenic Env mutant (HIVF522Y), and with similar levels of Gag+ cells, were cocultivated with PBMCs and IFN was measured 12 h later (left panel). IFN levels are expressed as the % of the signal obtained with FLUAV. MT4C5 viability was similar with WT, ΔEnv, and F522Y HIV at the beginning of the coculture. The efficiency of HIV capture by PBMCs was assessed by measuring the % of Gag+ PBMCs by flow cytometry (right panel). Mean+sd of 3-5 independent experiments is shown, * p<0.05 (Kruskal-Wallis).
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ppat-1001284-g001: Sensing of HIV-infected lymphocytes by hematopoïetic cells.a. Schematic representation of the experimental system. HIV-1 infected T cells are mixed with PBMCs, and levels of IFN released in supernatants are measured 12-24h later. b. IFN release in cocultures of HIV-infected T cells with PBMCs. MT4C5 lymphoblastoid T cells (left panel) or primary CD4+ T lymphocytes (right panel) were used as donors. Whole PBMCs, PBMCs depleted of pDCs (PBMCs-pDCs), and purified pDCs where used as target cells. Targets were either left non stimulated (NS), cocultivated with non-infected (NI) or HIV-infected (NL4-3 strain) MT4C5 cells (MT4C5-HIV), exposed to cell-free HIV virions (free HIV) or Influenza virus (FLUAV). IFN production in supernatants was measured in a bioassay, after 12 h of culture. Donor to target cell ratio were 1∶2 for PBMCs and PBMCs-pDCs, and 2∶1 for pDCs. c. Dose response analysis of IFN release. Whole PBMCs were incubated with MT4C5 cells displaying increasing % of infected (Gag+) cells. IFN production was measured after 24 h of coculture (orange histrograms). The levels of living MT4C5 cells at the beginning of the coculture, determined by flow cytometry (forward and side scatters gating), are shown in blue. d. Sensing of cells infected with various HIV isolates by PBMCs. MT4C5, infected with NL4-3 (NL), NLAD8 (AD8) and with two primary isolates (BON and 132W) were cocultivated with PBMCs and IFN was measured 24 h later. With all isolates, about 25% of infected MT4C5 cells expressed Gag before coculture. e. Role of HIV envelope glycoproteins. MT4C5, infected with wild-type (HIV), Env-deleted (ΔEnv), or a non-fusogenic Env mutant (HIVF522Y), and with similar levels of Gag+ cells, were cocultivated with PBMCs and IFN was measured 12 h later (left panel). IFN levels are expressed as the % of the signal obtained with FLUAV. MT4C5 viability was similar with WT, ΔEnv, and F522Y HIV at the beginning of the coculture. The efficiency of HIV capture by PBMCs was assessed by measuring the % of Gag+ PBMCs by flow cytometry (right panel). Mean+sd of 3-5 independent experiments is shown, * p<0.05 (Kruskal-Wallis).

Mentions: We first examined whether PBMCs and pDCs produce IFN when they encounter HIV-1. We compared the efficiency of recognition of infected cells and cell-free virions. We used as donor cells the MT4C5 lymphoid line (a derivative of MT4 cells expressing CCR5), which can be infected by CXCR4-tropic (X4) or CCR5-tropic (R5) HIV strains. Targets were fresh PBMCs isolated from healthy donors. Typically, pDCs, identified as BDCA4/BDCA2 double positive cells, represented 0.2 to 1 % of whole PBMCs (Fig. S1a). We also used as targets PBMCs depleted of pDCs (after removal of >80% of pDCs), or purified pDCs (corresponding to >85% of purity based on BDCA2/BDCA4+ double labeling) (Fig. S1a). Influenza virus (FLUAV, 40 HAU/mL), a well-characterized IFN inducer [35] was used as a positive control (Fig. 1b).


Innate sensing of HIV-infected cells.

Lepelley A, Louis S, Sourisseau M, Law HK, Pothlichet J, Schilte C, Chaperot L, Plumas J, Randall RE, Si-Tahar M, Mammano F, Albert ML, Schwartz O - PLoS Pathog. (2011)

Sensing of HIV-infected lymphocytes by hematopoïetic cells.a. Schematic representation of the experimental system. HIV-1 infected T cells are mixed with PBMCs, and levels of IFN released in supernatants are measured 12-24h later. b. IFN release in cocultures of HIV-infected T cells with PBMCs. MT4C5 lymphoblastoid T cells (left panel) or primary CD4+ T lymphocytes (right panel) were used as donors. Whole PBMCs, PBMCs depleted of pDCs (PBMCs-pDCs), and purified pDCs where used as target cells. Targets were either left non stimulated (NS), cocultivated with non-infected (NI) or HIV-infected (NL4-3 strain) MT4C5 cells (MT4C5-HIV), exposed to cell-free HIV virions (free HIV) or Influenza virus (FLUAV). IFN production in supernatants was measured in a bioassay, after 12 h of culture. Donor to target cell ratio were 1∶2 for PBMCs and PBMCs-pDCs, and 2∶1 for pDCs. c. Dose response analysis of IFN release. Whole PBMCs were incubated with MT4C5 cells displaying increasing % of infected (Gag+) cells. IFN production was measured after 24 h of coculture (orange histrograms). The levels of living MT4C5 cells at the beginning of the coculture, determined by flow cytometry (forward and side scatters gating), are shown in blue. d. Sensing of cells infected with various HIV isolates by PBMCs. MT4C5, infected with NL4-3 (NL), NLAD8 (AD8) and with two primary isolates (BON and 132W) were cocultivated with PBMCs and IFN was measured 24 h later. With all isolates, about 25% of infected MT4C5 cells expressed Gag before coculture. e. Role of HIV envelope glycoproteins. MT4C5, infected with wild-type (HIV), Env-deleted (ΔEnv), or a non-fusogenic Env mutant (HIVF522Y), and with similar levels of Gag+ cells, were cocultivated with PBMCs and IFN was measured 12 h later (left panel). IFN levels are expressed as the % of the signal obtained with FLUAV. MT4C5 viability was similar with WT, ΔEnv, and F522Y HIV at the beginning of the coculture. The efficiency of HIV capture by PBMCs was assessed by measuring the % of Gag+ PBMCs by flow cytometry (right panel). Mean+sd of 3-5 independent experiments is shown, * p<0.05 (Kruskal-Wallis).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040675&req=5

ppat-1001284-g001: Sensing of HIV-infected lymphocytes by hematopoïetic cells.a. Schematic representation of the experimental system. HIV-1 infected T cells are mixed with PBMCs, and levels of IFN released in supernatants are measured 12-24h later. b. IFN release in cocultures of HIV-infected T cells with PBMCs. MT4C5 lymphoblastoid T cells (left panel) or primary CD4+ T lymphocytes (right panel) were used as donors. Whole PBMCs, PBMCs depleted of pDCs (PBMCs-pDCs), and purified pDCs where used as target cells. Targets were either left non stimulated (NS), cocultivated with non-infected (NI) or HIV-infected (NL4-3 strain) MT4C5 cells (MT4C5-HIV), exposed to cell-free HIV virions (free HIV) or Influenza virus (FLUAV). IFN production in supernatants was measured in a bioassay, after 12 h of culture. Donor to target cell ratio were 1∶2 for PBMCs and PBMCs-pDCs, and 2∶1 for pDCs. c. Dose response analysis of IFN release. Whole PBMCs were incubated with MT4C5 cells displaying increasing % of infected (Gag+) cells. IFN production was measured after 24 h of coculture (orange histrograms). The levels of living MT4C5 cells at the beginning of the coculture, determined by flow cytometry (forward and side scatters gating), are shown in blue. d. Sensing of cells infected with various HIV isolates by PBMCs. MT4C5, infected with NL4-3 (NL), NLAD8 (AD8) and with two primary isolates (BON and 132W) were cocultivated with PBMCs and IFN was measured 24 h later. With all isolates, about 25% of infected MT4C5 cells expressed Gag before coculture. e. Role of HIV envelope glycoproteins. MT4C5, infected with wild-type (HIV), Env-deleted (ΔEnv), or a non-fusogenic Env mutant (HIVF522Y), and with similar levels of Gag+ cells, were cocultivated with PBMCs and IFN was measured 12 h later (left panel). IFN levels are expressed as the % of the signal obtained with FLUAV. MT4C5 viability was similar with WT, ΔEnv, and F522Y HIV at the beginning of the coculture. The efficiency of HIV capture by PBMCs was assessed by measuring the % of Gag+ PBMCs by flow cytometry (right panel). Mean+sd of 3-5 independent experiments is shown, * p<0.05 (Kruskal-Wallis).
Mentions: We first examined whether PBMCs and pDCs produce IFN when they encounter HIV-1. We compared the efficiency of recognition of infected cells and cell-free virions. We used as donor cells the MT4C5 lymphoid line (a derivative of MT4 cells expressing CCR5), which can be infected by CXCR4-tropic (X4) or CCR5-tropic (R5) HIV strains. Targets were fresh PBMCs isolated from healthy donors. Typically, pDCs, identified as BDCA4/BDCA2 double positive cells, represented 0.2 to 1 % of whole PBMCs (Fig. S1a). We also used as targets PBMCs depleted of pDCs (after removal of >80% of pDCs), or purified pDCs (corresponding to >85% of purity based on BDCA2/BDCA4+ double labeling) (Fig. S1a). Influenza virus (FLUAV, 40 HAU/mL), a well-characterized IFN inducer [35] was used as a positive control (Fig. 1b).

Bottom Line: In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing.In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells.Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Virus and Immunity Unit, URA CNRS 3015, Paris, France.

ABSTRACT
Cell-free HIV-1 virions are poor stimulators of type I interferon (IFN) production. We examined here how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs) and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. Therefore, detection of HIV-infected lymphocytes occurs through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

Show MeSH
Related in: MedlinePlus