IMMUNOLOGY
ORIGINAL ARTICLE
Inhibition of R5-tropic HIV type-1 replication in CD4+ natural killer T cells by cd T lymphocytes Kyoko Omi, Masumi Shimizu, Eri Watanabe, Jiro Matsumura, Chizuno Takaku, Eiji Shinya and Hidemi Takahashi Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan
doi:10.1111/imm.12221 Received 24 July 2013; revised 15 November 2013; accepted 19 November 2013. Correspondence: Hidemi Takahashi, Department of Microbiology and Immunology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Email: [email protected] Senior author: Hidemi Takahashi
Summary After the development of highly active anti-retroviral therapy, it became clear that the majority of emergent HIV-1 is macrophage-tropic and infects CD4+, CCR5-expressing cells (R5-tropic). There are three distinct cell populations, R5-tropic, HIV-1-susceptible CD4+ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue-associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4+ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4+ T cells derived from circulating PBMCs, and that R5-tropic HIV-1 expands efficiently in the CD4+ NKT cells. Moreover, when PBMCs depleted of CD8a+ cells were stimulated in the presence of a-galactosylceramide (aGalCer) and R5-tropic HIV-1 [NL(AD8)], the production of HIV-1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8b+ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8aa+ but not CD8ab+ cells may have the ability to inhibit R5-tropic HIV-1 replication in CD4+ NKT cells. Here, we show that co-culturing R5-tropic HIV-1-infected CD4+ NKT cells with CD8aa+ cd T cells, in particular Vc1Vd1 cells, but not with CD8aa+ NKT cells or CD8aa+ dendritic cells, inhibits HIV-1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1a and 1b and RANTES. Collectively, these results indicate the importance of CD8aa+ cd T cells in the control of R5-tropic HIV-1 replication and persistence in CD4+ NKT cells. Keywords: CD4+ natural killer T cells; CD8aa+ cells; HIV-1 p24; R5-tropic HIV-1; viral replication; cd T cells.
Introduction Although the use of a combination of anti-retroviral drugs, called highly active anti-retroviral treatment (HAART),1 has dramatically reduced HIV-1-associated mortality, patients are never completely free of HIV-1 infection and must undergo HAART for life. The entry of HIV-1 into target CD4+ cells requires the cellular expression of two distinct chemokine receptors, CXCR4 and CCR5.2,3 CXCR4 is used for the entry of T-cell-tropic HIV-1 strains (X4-tropic),4 while CCR5 is used for the entry of macrophage-tropic HIV-1 (R5-tropic).5,6 R5-tropic HIV-1 is usually found early in the course of infection, whereas X4-tropic HIV-1 is observed most often in patients who have advanced to AIDS.7 As HAART has been widely used for the treatment of HIV-1, R5-tropic HIV-1 has become the most prevalent strain, and so 596
controlling the R5-tropic HIV-1-infected cells is necessary to clear the persistent infection. In the conventional CD4+ T cells observed mainly in the circulating blood, CXCR4 is predominantly expressed on resting, naive T-cell subsets, whereas CCR5 is almost exclusively expressed by activated memory T-cell subsets.8 Hence, only primed, conventional memory CD4+ T cells are susceptible to R5-tropic HIV-1 strains. In contrast, human type-I natural killer T (NKT) cells expressing an invariant pair of T-cell receptors (TCRs) (Va24 and Vb11) and elicited from peripheral blood mononuclear cells (PBMCs) by incubation with a-galactosylceramide (a-GalCer)9 express very high levels of CCR5 and low levels of CXCR4. Hence, CD4+ NKT cells are highly susceptible to R5-tropic HIV-1 strains and seem to be the main reservoir and major spreaders of R5-tropic HIV-1.10 Indeed, it has been reported that R5-tropic HIV-1 replicates more ª 2013 John Wiley & Sons Ltd, Immunology, 141, 596–608
Inhibition of R5-HIV-1 production in NKT cells by cd T cells efficiently in CD4+ NKT cells than in conventional CD4+ T cells and demonstrated that CD4+ NKT cells could produce 10-fold to 20-fold more virus particles than Staphylococcus enterotoxin B-activated conventional CD4+ T cells.8 Therefore, in addition to modern HAART, the inhibition of R5-tropic HIV-1 replication within CD4+ NKT cells will provide a new strategy for the control of HIV-1 infection. CD8+ T lymphocytes have been reported to block HIV-1 replication in CD4+ peripheral blood cells from HIV-1-infected individuals.11 Additionally, HIV-1 does not replicate in CD4+ cells from seronegative donors when these cells are co-cultured with CD8+ T cells from HIV-1-infected individuals in an HLA-unrestricted manner without elimination of HIV-1-infecting cells.12 The cell non-cytotoxic antiviral response of these CD8+ cells becomes evident during the acute stage of HIV-1 infection,13 when R5-tropic viruses are the predominant form and CD4+ NKT cells are the preferred targets. These results suggest that certain CD8+ cells suppress R5-tropic HIV-1 replication within the CD4+ NKT cells during the acute stage of infection. Therefore, in vitro depletion of CD8+ cells from PBMCs containing R5-tropic HIV-1-infected NKT cells may enhance viral replication and expansion and provide a clue to identify functional CD8+ cells, which can inhibit R5-tropic HIV-1 replication in HIV-1-infected NKT cells. In the present study, on the basis of these findings, we incubated PBMCs that had been previously depleted of either CD8a+ or CD8b+ cells with a-GalCer in the presence of R5-tropic HIV-1 NL(AD8)14 and measured the HIV-1 p24 antigen concentration in the culture supernatant. As expected, the amount of NL(AD8) p24 antigen was markedly inhibited in the presence of CD8+ cells. The concentration of p24 antigen was significantly higher following depletion of CD8a+ cells, whereas the production of p24 antigen was still strongly inhibited after depletion of CD8b+ cells. These findings suggest that CD8a+ but not CD8b+ cells inhibit R5-tropic HIV-1 production in CD4+ NKT cells. CD8a+ cells, including CD8aa+ dendritic cells (DCs), CD8aa+ NKT cells and CD8aa+ cd T cells in the innate arm of the immune system, express CD8aa on their surface, whereas CD8b+ cells such as CD8ab+ cytotoxic T lymphocytes (CTLs) in the acquired arm of the immune system, express CD8ab. Here, we show that innate CD8aa+ cd T cells are able to suppress R5-tropic HIV-1 production in infected NKT cells and propose the importance of cd T cells, in particular Vc1Vd1 cells, in the control of R5-tropic HIV-1 replication and persistence in CD4+ NKT cells.
Materials and methods Induction of NKT cells and cd T cells from human PBMCs Human PBMCs were separated from the buffy coats of healthy donors using Ficoll-Hypaque (Amershamª 2013 John Wiley & Sons Ltd, Immunology, 141, 596–608
Pharmacia Biotech, Uppsala, Sweden) and cultured in complete T-cell medium (CTM)15 composed of RPMI1640 (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Hyclone, Logan, UT), 5 mM HEPES buffer (Gibco BRL, Grand Island, NY), 100 U/ml penicillin (Gibco-BRL), 100 lg/ml streptomycin (Gibco-BRL), 2 mM L-glutamine (Gibco-BRL), 2 mM sodium pyruvate (Gibco-BRL), 2 mM non-essential amino acids (Gibco-BRL), 2 mM modified vitamins (Gibco-BRL), and 05 lM 2mercaptoethanol (Gibco-BRL). For the induction of NKT cells and Vc2Vd2 T cells, either 20 ng/ml a-GalCer (KRN7000 purchased from Funakoshi Co., Ltd, Tokyo, Japan) or 5 lM of risedronate (kindly provided by Ajinomonto Pharmaceutical Co. Ltd., Tokyo, Japan), respectively, was added to 2 9 106 PBMCs in 1 ml CTM. After 7 days, half of the medium was replaced with fresh CTM containing 20 U/ml interleukin-2 (IL-2; Shionogi & Co. Ltd., Osaka, Japan) and no a-GalCer or risedronate. The cells were cultured for another 7 days, with the medium replaced again on days 3 and 6. Vc1Vd1 T cells were sorted from freshly obtained PBMCs. This study was approved by the Review Board of the Nippon Medical School and all human participants gave written informed consent.
Antibodies, flow cytometric analysis and cell sorting FITC-labelled mouse anti-human Va24 and anti-human Vb11, as well as phycoerythrin-labelled mouse antihuman Va24, CD8b, Vd1, NKG2D and KC57 (p24) monoclonal antibodies (mAbs) were purchased from Beckman Counter Inc. (La Brea, CA). Phycoerythrinlabelled mouse anti-human CXCR4 and anti-human CD11c, as well as FITC-labelled mouse anti-human CD3 mAbs were purchased from BD Biosciences (Mountain View, CA). Phycoerythrin-labelled mouse anti-human CCR5 and Vd2, phycoerythrin-Cy7-labelled mouse antihuman CD4 and allophycocyanin-labelled mouse antihuman CD8a and MHC class I-related chain A/B (MICA/ MICB) mAbs were purchased from Biolegend (San Diego, CA). After incubation with the relevant mAbs at 4° for 30 min, cells were washed and re-suspended in PBS with 2% FCS and 001 M sodium azide (PBS-based medium) for analysis using a FACSCanto II (BD Biosciences) and FLOWJO software (TreeStar, Ashland, OR). For intracellular staining of p24, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) at 4° for 20 min. After washing twice with perm/Wash solution (BD Biosciences), cells were incubated with anti-human mAb to p24 at 4° for 30 min. A Zenon Mouse IgG Labeling Kit (Molecular Probes, Eugene, OR) was used to stain Vd1. In some experiments, the stained cells were sorted with a FACS Aria II (BD Bioscience) and DIVA software to obtain populations of CD4+ Va24+, 597
K. Omi et al. CD8a+ CD8b Va24+, CD4 CD8b Va24+, CD8a + CD8b + Va24 , CD8a + CD8b V24a , CD8a + + + + CD8b Vd1 , CD8a CD8b Vd1 , CD8a CD8b Vd2 + and CD8a CD8b Vd2 + T cells.
Depletion of cells from PBMCs For the depletion of CD8a+ cells, PBMCs were incubated with a mouse anti-human CD8a IgG mAb (OKT8) purchased from the American Type Culture Collection (Manassas, VA) for 30 min at 4° and washed three times to remove free mAb. The labelled cells were then incubated with magnetic beads conjugated to anti-mouse IgG (Dynabeads Pan Mouse IgG; DYNAL BIOTECH, Oslo, Norway) for an additional 30 min at 4°, and the CD8a+ cells were removed with a magnetic device (Perspective Biosystems, Framingham, MA) following the manufacturer’s instructions. CD8b+ cells, Vd1+ cells or Vd2+ cells were depleted by a similar procedure using a mouse antihuman CD8b IgG mAb (2ST8.5H7) obtained from Immunotech (Marseille, France), a mouse anti-human Vd1 mAb (R9.12) (Beckman Counter Inc.) or a mouse antihuman Vd2 (B9) mAb (Biolegend).
Measurement of p24 antigen by ELISA Infectious NL(AD8) virus particles14 were obtained by transfection of 293T cells with pNL(AD8) (a gift from Malcolm A. Martin, National Institutes of Health– National Institute of Allergy and Infectious Diseases) using polyethylenimine (25 000 molecular weight; SigmaAldrich, St Louis, MO). The quantitative analysis of p24 gag protein in the culture supernatant was performed as previously described.16 Briefly, Nunc-Immuno Plates (Thermo Scientific, Roskilde, Denmark) were coated with an anti-HIV-1 p24 mAb (183-H12-5C),17 and the samples were incubated in the plates for 2 hr. After washing, biotinylated human anti-HIV immunoglobulin was added to detect p24. HIV-1 p25/24 Gag protein (National Institutes of Health AIDS Research and Reference Reagent Program)18 was used as a standard. +
+
+
+
Stimulation of intact, CD8a , CD8b , Vd1 or Vd2 cell-depleted PBMCs with a-GalCer in the presence of NL(AD8)
Intact, CD8a+, CD8b+, Vd1+ or Vd2+ cell-depleted PBMCs (1 9 105) were cultured in 200 ll of CTM containing 20 ng/ml a-GalCer and R5-tropic NL(AD8) HIV-1 at a multiplicity of infection of 01. On days 7, 14, 17 or 20 after the cultures were initiated, 100 ll culture supernatants were collected and stored at – 80° until the measurement of p24 antigen. The cultured cells were harvested, stained with the relevant antibodies and analysed with a FACSCanto II as described above. 598
Infection of CD4+ NKT cells and co-culture with effector cells Sorted CD4+ Va24+ NKT cells were stimulated with 2 lg/ml phytohaemagglutinin (PHA) in CTM containing 20 U/ml IL-2 for 3 days. Subsequently, the PHA-stimulated CD4+ Va24+ NKT cells were washed to remove free PHA, and 1 9 105 to 2 9 105 cells were infected with NL(AD8) HIV-1 at a multiplicity of infection of 01 for 2 hr at 37° in the presence of 30 lg/ml DEAE-Dextran (Sigma-Aldrich). After the NL(AD8)-infected NKT cells were washed three times with RPMI-1640 containing 2% FCS, they were further cultured in fresh medium containing 20 U/ml IL-2. Then, 2 9 104 NL(AD8)-infected NKT cells were cultured with 2 9 104 sorted various effectors, such as CD8a+ CD8b Va24+, CD4 CD8a Va24+, CD8a+ CD8b+ Va24 , CD8a+ CD8b Va24 , CD8a+ CD8b Vd1+ and CD8a+ CD8b Vd2+, in 200 ll of medium containing 20 U/ml IL-2 in round-bottom 96well plates. On day 4 after the co-cultures were initiated, 100 ll of culture supernatant was collected from each well and stored at 80° until the measurement of p24 antigen or chemokines. To quantify the amount of chemokines secreted in the medium, CCL3 (MIP-1a) ELISA kit (R&D Systems, Minneapolis, MN), CCL4 [macrophage inflammatory protein-1b (MIP-1b)] ELISA kit (R&D Systems), and CCL5 (RANTES) ELISA kit (R&D Systems) were used following the manufacturer’s instructions. In some experiments, 20 ll/ml anti-human CCR3 mAb (clone 61828) (R&D Systems), anti-human CCR4 mAb (clone 205410) (R&D Systems), anti-human CCR5 antibody (clone 45531) (R&D Systems), anti-human MICA/MICB mAb (clone 6D4) (Biolegend), and/or antihuman CD314 (NKG2D) mAb (clone 1D11) (Biolegend) were added to the culture medium when the co-cultures were initiated. NKT cells infected with NL(AD8) HIV-1 as described above were also cultured in medium containing 20 U/ml IL-2 for 4 days and stained with the relevant antibodies and analysed with a FACSCanto II.
Statistical analysis The results were analysed by Student’s t-test, and the results are presented as the mean standard deviation (SD). Differences of P-values of
ORIGINAL ARTICLE
Inhibition of R5-tropic HIV type-1 replication in CD4+ natural killer T cells by cd T lymphocytes Kyoko Omi, Masumi Shimizu, Eri Watanabe, Jiro Matsumura, Chizuno Takaku, Eiji Shinya and Hidemi Takahashi Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan
doi:10.1111/imm.12221 Received 24 July 2013; revised 15 November 2013; accepted 19 November 2013. Correspondence: Hidemi Takahashi, Department of Microbiology and Immunology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Email: [email protected] Senior author: Hidemi Takahashi
Summary After the development of highly active anti-retroviral therapy, it became clear that the majority of emergent HIV-1 is macrophage-tropic and infects CD4+, CCR5-expressing cells (R5-tropic). There are three distinct cell populations, R5-tropic, HIV-1-susceptible CD4+ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue-associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4+ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4+ T cells derived from circulating PBMCs, and that R5-tropic HIV-1 expands efficiently in the CD4+ NKT cells. Moreover, when PBMCs depleted of CD8a+ cells were stimulated in the presence of a-galactosylceramide (aGalCer) and R5-tropic HIV-1 [NL(AD8)], the production of HIV-1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8b+ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8aa+ but not CD8ab+ cells may have the ability to inhibit R5-tropic HIV-1 replication in CD4+ NKT cells. Here, we show that co-culturing R5-tropic HIV-1-infected CD4+ NKT cells with CD8aa+ cd T cells, in particular Vc1Vd1 cells, but not with CD8aa+ NKT cells or CD8aa+ dendritic cells, inhibits HIV-1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1a and 1b and RANTES. Collectively, these results indicate the importance of CD8aa+ cd T cells in the control of R5-tropic HIV-1 replication and persistence in CD4+ NKT cells. Keywords: CD4+ natural killer T cells; CD8aa+ cells; HIV-1 p24; R5-tropic HIV-1; viral replication; cd T cells.
Introduction Although the use of a combination of anti-retroviral drugs, called highly active anti-retroviral treatment (HAART),1 has dramatically reduced HIV-1-associated mortality, patients are never completely free of HIV-1 infection and must undergo HAART for life. The entry of HIV-1 into target CD4+ cells requires the cellular expression of two distinct chemokine receptors, CXCR4 and CCR5.2,3 CXCR4 is used for the entry of T-cell-tropic HIV-1 strains (X4-tropic),4 while CCR5 is used for the entry of macrophage-tropic HIV-1 (R5-tropic).5,6 R5-tropic HIV-1 is usually found early in the course of infection, whereas X4-tropic HIV-1 is observed most often in patients who have advanced to AIDS.7 As HAART has been widely used for the treatment of HIV-1, R5-tropic HIV-1 has become the most prevalent strain, and so 596
controlling the R5-tropic HIV-1-infected cells is necessary to clear the persistent infection. In the conventional CD4+ T cells observed mainly in the circulating blood, CXCR4 is predominantly expressed on resting, naive T-cell subsets, whereas CCR5 is almost exclusively expressed by activated memory T-cell subsets.8 Hence, only primed, conventional memory CD4+ T cells are susceptible to R5-tropic HIV-1 strains. In contrast, human type-I natural killer T (NKT) cells expressing an invariant pair of T-cell receptors (TCRs) (Va24 and Vb11) and elicited from peripheral blood mononuclear cells (PBMCs) by incubation with a-galactosylceramide (a-GalCer)9 express very high levels of CCR5 and low levels of CXCR4. Hence, CD4+ NKT cells are highly susceptible to R5-tropic HIV-1 strains and seem to be the main reservoir and major spreaders of R5-tropic HIV-1.10 Indeed, it has been reported that R5-tropic HIV-1 replicates more ª 2013 John Wiley & Sons Ltd, Immunology, 141, 596–608
Inhibition of R5-HIV-1 production in NKT cells by cd T cells efficiently in CD4+ NKT cells than in conventional CD4+ T cells and demonstrated that CD4+ NKT cells could produce 10-fold to 20-fold more virus particles than Staphylococcus enterotoxin B-activated conventional CD4+ T cells.8 Therefore, in addition to modern HAART, the inhibition of R5-tropic HIV-1 replication within CD4+ NKT cells will provide a new strategy for the control of HIV-1 infection. CD8+ T lymphocytes have been reported to block HIV-1 replication in CD4+ peripheral blood cells from HIV-1-infected individuals.11 Additionally, HIV-1 does not replicate in CD4+ cells from seronegative donors when these cells are co-cultured with CD8+ T cells from HIV-1-infected individuals in an HLA-unrestricted manner without elimination of HIV-1-infecting cells.12 The cell non-cytotoxic antiviral response of these CD8+ cells becomes evident during the acute stage of HIV-1 infection,13 when R5-tropic viruses are the predominant form and CD4+ NKT cells are the preferred targets. These results suggest that certain CD8+ cells suppress R5-tropic HIV-1 replication within the CD4+ NKT cells during the acute stage of infection. Therefore, in vitro depletion of CD8+ cells from PBMCs containing R5-tropic HIV-1-infected NKT cells may enhance viral replication and expansion and provide a clue to identify functional CD8+ cells, which can inhibit R5-tropic HIV-1 replication in HIV-1-infected NKT cells. In the present study, on the basis of these findings, we incubated PBMCs that had been previously depleted of either CD8a+ or CD8b+ cells with a-GalCer in the presence of R5-tropic HIV-1 NL(AD8)14 and measured the HIV-1 p24 antigen concentration in the culture supernatant. As expected, the amount of NL(AD8) p24 antigen was markedly inhibited in the presence of CD8+ cells. The concentration of p24 antigen was significantly higher following depletion of CD8a+ cells, whereas the production of p24 antigen was still strongly inhibited after depletion of CD8b+ cells. These findings suggest that CD8a+ but not CD8b+ cells inhibit R5-tropic HIV-1 production in CD4+ NKT cells. CD8a+ cells, including CD8aa+ dendritic cells (DCs), CD8aa+ NKT cells and CD8aa+ cd T cells in the innate arm of the immune system, express CD8aa on their surface, whereas CD8b+ cells such as CD8ab+ cytotoxic T lymphocytes (CTLs) in the acquired arm of the immune system, express CD8ab. Here, we show that innate CD8aa+ cd T cells are able to suppress R5-tropic HIV-1 production in infected NKT cells and propose the importance of cd T cells, in particular Vc1Vd1 cells, in the control of R5-tropic HIV-1 replication and persistence in CD4+ NKT cells.
Materials and methods Induction of NKT cells and cd T cells from human PBMCs Human PBMCs were separated from the buffy coats of healthy donors using Ficoll-Hypaque (Amershamª 2013 John Wiley & Sons Ltd, Immunology, 141, 596–608
Pharmacia Biotech, Uppsala, Sweden) and cultured in complete T-cell medium (CTM)15 composed of RPMI1640 (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Hyclone, Logan, UT), 5 mM HEPES buffer (Gibco BRL, Grand Island, NY), 100 U/ml penicillin (Gibco-BRL), 100 lg/ml streptomycin (Gibco-BRL), 2 mM L-glutamine (Gibco-BRL), 2 mM sodium pyruvate (Gibco-BRL), 2 mM non-essential amino acids (Gibco-BRL), 2 mM modified vitamins (Gibco-BRL), and 05 lM 2mercaptoethanol (Gibco-BRL). For the induction of NKT cells and Vc2Vd2 T cells, either 20 ng/ml a-GalCer (KRN7000 purchased from Funakoshi Co., Ltd, Tokyo, Japan) or 5 lM of risedronate (kindly provided by Ajinomonto Pharmaceutical Co. Ltd., Tokyo, Japan), respectively, was added to 2 9 106 PBMCs in 1 ml CTM. After 7 days, half of the medium was replaced with fresh CTM containing 20 U/ml interleukin-2 (IL-2; Shionogi & Co. Ltd., Osaka, Japan) and no a-GalCer or risedronate. The cells were cultured for another 7 days, with the medium replaced again on days 3 and 6. Vc1Vd1 T cells were sorted from freshly obtained PBMCs. This study was approved by the Review Board of the Nippon Medical School and all human participants gave written informed consent.
Antibodies, flow cytometric analysis and cell sorting FITC-labelled mouse anti-human Va24 and anti-human Vb11, as well as phycoerythrin-labelled mouse antihuman Va24, CD8b, Vd1, NKG2D and KC57 (p24) monoclonal antibodies (mAbs) were purchased from Beckman Counter Inc. (La Brea, CA). Phycoerythrinlabelled mouse anti-human CXCR4 and anti-human CD11c, as well as FITC-labelled mouse anti-human CD3 mAbs were purchased from BD Biosciences (Mountain View, CA). Phycoerythrin-labelled mouse anti-human CCR5 and Vd2, phycoerythrin-Cy7-labelled mouse antihuman CD4 and allophycocyanin-labelled mouse antihuman CD8a and MHC class I-related chain A/B (MICA/ MICB) mAbs were purchased from Biolegend (San Diego, CA). After incubation with the relevant mAbs at 4° for 30 min, cells were washed and re-suspended in PBS with 2% FCS and 001 M sodium azide (PBS-based medium) for analysis using a FACSCanto II (BD Biosciences) and FLOWJO software (TreeStar, Ashland, OR). For intracellular staining of p24, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) at 4° for 20 min. After washing twice with perm/Wash solution (BD Biosciences), cells were incubated with anti-human mAb to p24 at 4° for 30 min. A Zenon Mouse IgG Labeling Kit (Molecular Probes, Eugene, OR) was used to stain Vd1. In some experiments, the stained cells were sorted with a FACS Aria II (BD Bioscience) and DIVA software to obtain populations of CD4+ Va24+, 597
K. Omi et al. CD8a+ CD8b Va24+, CD4 CD8b Va24+, CD8a + CD8b + Va24 , CD8a + CD8b V24a , CD8a + + + + CD8b Vd1 , CD8a CD8b Vd1 , CD8a CD8b Vd2 + and CD8a CD8b Vd2 + T cells.
Depletion of cells from PBMCs For the depletion of CD8a+ cells, PBMCs were incubated with a mouse anti-human CD8a IgG mAb (OKT8) purchased from the American Type Culture Collection (Manassas, VA) for 30 min at 4° and washed three times to remove free mAb. The labelled cells were then incubated with magnetic beads conjugated to anti-mouse IgG (Dynabeads Pan Mouse IgG; DYNAL BIOTECH, Oslo, Norway) for an additional 30 min at 4°, and the CD8a+ cells were removed with a magnetic device (Perspective Biosystems, Framingham, MA) following the manufacturer’s instructions. CD8b+ cells, Vd1+ cells or Vd2+ cells were depleted by a similar procedure using a mouse antihuman CD8b IgG mAb (2ST8.5H7) obtained from Immunotech (Marseille, France), a mouse anti-human Vd1 mAb (R9.12) (Beckman Counter Inc.) or a mouse antihuman Vd2 (B9) mAb (Biolegend).
Measurement of p24 antigen by ELISA Infectious NL(AD8) virus particles14 were obtained by transfection of 293T cells with pNL(AD8) (a gift from Malcolm A. Martin, National Institutes of Health– National Institute of Allergy and Infectious Diseases) using polyethylenimine (25 000 molecular weight; SigmaAldrich, St Louis, MO). The quantitative analysis of p24 gag protein in the culture supernatant was performed as previously described.16 Briefly, Nunc-Immuno Plates (Thermo Scientific, Roskilde, Denmark) were coated with an anti-HIV-1 p24 mAb (183-H12-5C),17 and the samples were incubated in the plates for 2 hr. After washing, biotinylated human anti-HIV immunoglobulin was added to detect p24. HIV-1 p25/24 Gag protein (National Institutes of Health AIDS Research and Reference Reagent Program)18 was used as a standard. +
+
+
+
Stimulation of intact, CD8a , CD8b , Vd1 or Vd2 cell-depleted PBMCs with a-GalCer in the presence of NL(AD8)
Intact, CD8a+, CD8b+, Vd1+ or Vd2+ cell-depleted PBMCs (1 9 105) were cultured in 200 ll of CTM containing 20 ng/ml a-GalCer and R5-tropic NL(AD8) HIV-1 at a multiplicity of infection of 01. On days 7, 14, 17 or 20 after the cultures were initiated, 100 ll culture supernatants were collected and stored at – 80° until the measurement of p24 antigen. The cultured cells were harvested, stained with the relevant antibodies and analysed with a FACSCanto II as described above. 598
Infection of CD4+ NKT cells and co-culture with effector cells Sorted CD4+ Va24+ NKT cells were stimulated with 2 lg/ml phytohaemagglutinin (PHA) in CTM containing 20 U/ml IL-2 for 3 days. Subsequently, the PHA-stimulated CD4+ Va24+ NKT cells were washed to remove free PHA, and 1 9 105 to 2 9 105 cells were infected with NL(AD8) HIV-1 at a multiplicity of infection of 01 for 2 hr at 37° in the presence of 30 lg/ml DEAE-Dextran (Sigma-Aldrich). After the NL(AD8)-infected NKT cells were washed three times with RPMI-1640 containing 2% FCS, they were further cultured in fresh medium containing 20 U/ml IL-2. Then, 2 9 104 NL(AD8)-infected NKT cells were cultured with 2 9 104 sorted various effectors, such as CD8a+ CD8b Va24+, CD4 CD8a Va24+, CD8a+ CD8b+ Va24 , CD8a+ CD8b Va24 , CD8a+ CD8b Vd1+ and CD8a+ CD8b Vd2+, in 200 ll of medium containing 20 U/ml IL-2 in round-bottom 96well plates. On day 4 after the co-cultures were initiated, 100 ll of culture supernatant was collected from each well and stored at 80° until the measurement of p24 antigen or chemokines. To quantify the amount of chemokines secreted in the medium, CCL3 (MIP-1a) ELISA kit (R&D Systems, Minneapolis, MN), CCL4 [macrophage inflammatory protein-1b (MIP-1b)] ELISA kit (R&D Systems), and CCL5 (RANTES) ELISA kit (R&D Systems) were used following the manufacturer’s instructions. In some experiments, 20 ll/ml anti-human CCR3 mAb (clone 61828) (R&D Systems), anti-human CCR4 mAb (clone 205410) (R&D Systems), anti-human CCR5 antibody (clone 45531) (R&D Systems), anti-human MICA/MICB mAb (clone 6D4) (Biolegend), and/or antihuman CD314 (NKG2D) mAb (clone 1D11) (Biolegend) were added to the culture medium when the co-cultures were initiated. NKT cells infected with NL(AD8) HIV-1 as described above were also cultured in medium containing 20 U/ml IL-2 for 4 days and stained with the relevant antibodies and analysed with a FACSCanto II.
Statistical analysis The results were analysed by Student’s t-test, and the results are presented as the mean standard deviation (SD). Differences of P-values of
