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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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Recognition of HIV-infected cells by the Gen2.2 plasmacytoid-like cell line.a. Gen2.2 cells express HIV receptors and are sensitive to HIV infection. Surface expression levels of CD4, CXCR4 and CCR5 are shown on the upper left panel. Gen2.2 cells were then exposed to HIV particles, NL4-3 strain, at two doses (0.5 and 5 ng/0.1 ml Gag p24/106 cells). Viral replication was assessed by following the appearance of Gag+ cells (upper right panel) and Gag p24 release in supernatants (lower left panel). IFN production was measured in the same supernatants (lower right panels). Data are representative of 3 independent experiments. b. Schematic representation of the coculture system. HIV-1 infected T cells are mixed with Gen2.2 cells and levels of IFN released in supernatants are measured 12–22 h later. c. Sensing of HIV-infected cells by Gen2.2 and role of viral 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 22 h later (left panel). When stated, HIV-infected primary CD4+ cells were used as donors, instead of MT4C5 cells. Cell-free HIV virions (500 ng/ml p24) do not stimulate Gen2.2 cells. The efficiency of HIV capture by Gen2.2 was assessed by measuring the % of Gag+ cells by flow cytometry (right panel). Mean+sd of 10 independent experiments is shown, *p<0.05 (Kruskal-Wallis). d. Visualization of HIV capture by Gen2.2 cells. Gen2.2 cells were labeled with CFSE (green) and cocultured with MT4C5 infected with wild-type (HIV), ΔEnv and F522Y mutants. Cells were harvested after 1 h and stained for HIV Gag (red). Representative fields are shown.
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ppat-1001284-g003: Recognition of HIV-infected cells by the Gen2.2 plasmacytoid-like cell line.a. Gen2.2 cells express HIV receptors and are sensitive to HIV infection. Surface expression levels of CD4, CXCR4 and CCR5 are shown on the upper left panel. Gen2.2 cells were then exposed to HIV particles, NL4-3 strain, at two doses (0.5 and 5 ng/0.1 ml Gag p24/106 cells). Viral replication was assessed by following the appearance of Gag+ cells (upper right panel) and Gag p24 release in supernatants (lower left panel). IFN production was measured in the same supernatants (lower right panels). Data are representative of 3 independent experiments. b. Schematic representation of the coculture system. HIV-1 infected T cells are mixed with Gen2.2 cells and levels of IFN released in supernatants are measured 12–22 h later. c. Sensing of HIV-infected cells by Gen2.2 and role of viral 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 22 h later (left panel). When stated, HIV-infected primary CD4+ cells were used as donors, instead of MT4C5 cells. Cell-free HIV virions (500 ng/ml p24) do not stimulate Gen2.2 cells. The efficiency of HIV capture by Gen2.2 was assessed by measuring the % of Gag+ cells by flow cytometry (right panel). Mean+sd of 10 independent experiments is shown, *p<0.05 (Kruskal-Wallis). d. Visualization of HIV capture by Gen2.2 cells. Gen2.2 cells were labeled with CFSE (green) and cocultured with MT4C5 infected with wild-type (HIV), ΔEnv and F522Y mutants. Cells were harvested after 1 h and stained for HIV Gag (red). Representative fields are shown.

Mentions: We sought to use the Gen2.2 pDC-like cell line to dissect the pathways of recognition of HIV-infected cells. This cell line was derived from a patient with pDC leukemia, and possesses phenotypic and functional features of primary pDCs [46]. Gen2.2 cells express TLR7, TLR9 and other pDC markers. They produce IFN when exposed to FLUAV or other TLR agonists, although at lower levels than primary pDCs. Gen2.2 mature upon incubation with very high concentration of HIV particles [16]. These cells represent a useful tool as they can be transduced with lentiviral vectors encoding for shRNAs [47]. We first examined whether Gen2.2 cells are sensitive to HIV replication. Like primary pDCs [48], these cells express the HIV receptors CD4, CXCR4 and CCR5 (Fig. 3a). Gen2.2 cells efficiently replicated HIV, as assessed by measuring the appearance of Gag+ cells and viral release by Gag p24 ELISA in supernatants at different time points after infection (Fig. 3a). Viral release was particularly high, reaching 1600 ng/ml Gag p24 at day 8 pi. IFN was also detected in supernatants, but only at late time points (day 8–10 pi). IFN release reached 15 U/ml, when about 30-40 % of Gen2.2 where Gag+ (Fig. 3a). Gen2.2 cells thus replicate HIV-1 and produce IFN upon infection.


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)

Recognition of HIV-infected cells by the Gen2.2 plasmacytoid-like cell line.a. Gen2.2 cells express HIV receptors and are sensitive to HIV infection. Surface expression levels of CD4, CXCR4 and CCR5 are shown on the upper left panel. Gen2.2 cells were then exposed to HIV particles, NL4-3 strain, at two doses (0.5 and 5 ng/0.1 ml Gag p24/106 cells). Viral replication was assessed by following the appearance of Gag+ cells (upper right panel) and Gag p24 release in supernatants (lower left panel). IFN production was measured in the same supernatants (lower right panels). Data are representative of 3 independent experiments. b. Schematic representation of the coculture system. HIV-1 infected T cells are mixed with Gen2.2 cells and levels of IFN released in supernatants are measured 12–22 h later. c. Sensing of HIV-infected cells by Gen2.2 and role of viral 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 22 h later (left panel). When stated, HIV-infected primary CD4+ cells were used as donors, instead of MT4C5 cells. Cell-free HIV virions (500 ng/ml p24) do not stimulate Gen2.2 cells. The efficiency of HIV capture by Gen2.2 was assessed by measuring the % of Gag+ cells by flow cytometry (right panel). Mean+sd of 10 independent experiments is shown, *p<0.05 (Kruskal-Wallis). d. Visualization of HIV capture by Gen2.2 cells. Gen2.2 cells were labeled with CFSE (green) and cocultured with MT4C5 infected with wild-type (HIV), ΔEnv and F522Y mutants. Cells were harvested after 1 h and stained for HIV Gag (red). Representative fields are shown.
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ppat-1001284-g003: Recognition of HIV-infected cells by the Gen2.2 plasmacytoid-like cell line.a. Gen2.2 cells express HIV receptors and are sensitive to HIV infection. Surface expression levels of CD4, CXCR4 and CCR5 are shown on the upper left panel. Gen2.2 cells were then exposed to HIV particles, NL4-3 strain, at two doses (0.5 and 5 ng/0.1 ml Gag p24/106 cells). Viral replication was assessed by following the appearance of Gag+ cells (upper right panel) and Gag p24 release in supernatants (lower left panel). IFN production was measured in the same supernatants (lower right panels). Data are representative of 3 independent experiments. b. Schematic representation of the coculture system. HIV-1 infected T cells are mixed with Gen2.2 cells and levels of IFN released in supernatants are measured 12–22 h later. c. Sensing of HIV-infected cells by Gen2.2 and role of viral 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 22 h later (left panel). When stated, HIV-infected primary CD4+ cells were used as donors, instead of MT4C5 cells. Cell-free HIV virions (500 ng/ml p24) do not stimulate Gen2.2 cells. The efficiency of HIV capture by Gen2.2 was assessed by measuring the % of Gag+ cells by flow cytometry (right panel). Mean+sd of 10 independent experiments is shown, *p<0.05 (Kruskal-Wallis). d. Visualization of HIV capture by Gen2.2 cells. Gen2.2 cells were labeled with CFSE (green) and cocultured with MT4C5 infected with wild-type (HIV), ΔEnv and F522Y mutants. Cells were harvested after 1 h and stained for HIV Gag (red). Representative fields are shown.
Mentions: We sought to use the Gen2.2 pDC-like cell line to dissect the pathways of recognition of HIV-infected cells. This cell line was derived from a patient with pDC leukemia, and possesses phenotypic and functional features of primary pDCs [46]. Gen2.2 cells express TLR7, TLR9 and other pDC markers. They produce IFN when exposed to FLUAV or other TLR agonists, although at lower levels than primary pDCs. Gen2.2 mature upon incubation with very high concentration of HIV particles [16]. These cells represent a useful tool as they can be transduced with lentiviral vectors encoding for shRNAs [47]. We first examined whether Gen2.2 cells are sensitive to HIV replication. Like primary pDCs [48], these cells express the HIV receptors CD4, CXCR4 and CCR5 (Fig. 3a). Gen2.2 cells efficiently replicated HIV, as assessed by measuring the appearance of Gag+ cells and viral release by Gag p24 ELISA in supernatants at different time points after infection (Fig. 3a). Viral release was particularly high, reaching 1600 ng/ml Gag p24 at day 8 pi. IFN was also detected in supernatants, but only at late time points (day 8–10 pi). IFN release reached 15 U/ml, when about 30-40 % of Gen2.2 where Gag+ (Fig. 3a). Gen2.2 cells thus replicate HIV-1 and produce IFN upon infection.

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