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MICROBIAL IMMUNOLOGY

Human Immunodeficiency Virus Persistence and Production in T-Cell Development

Kevin B. Gurney, Christel H. Uittenbogaart
Kevin B. Gurney
1Department of Microbiology, Immunology, and Molecular Genetics
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Christel H. Uittenbogaart
1Department of Microbiology, Immunology, and Molecular Genetics
2Department of Pediatrics, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, David E. Geffen School of Medicine at UCLA, Los Angeles, California
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  • For correspondence: uittenbo@ucla.edu
DOI: 10.1128/CVI.00184-06
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  • FIG. 1.
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    FIG. 1.

    Thymocyte development scheme. (A) Combinations of antibodies were used to identify thymocytes at five distinct stages of T-cell development. Stage I, CD69− thymocytes; stage I-P (a subset of stage I in which thymocytes express CD71), CD69− thymocytes; stage II, CD69+ cells that have not yet acquired CD27 expression; stage III, CD69+ CD27+ thymocytes that have not yet acquired CD45RA expression; stage IV, CD27+ thymocytes expressing CD45RA. Within stage I, the first circle represents the CD34+ thymic immigrant. The second circle represents the immature CD4 single-positive thymocyte. The third and fourth circles represent CD3− CD4+ CD8+ double-positive (DP) thymocytes and CD3+/low DP thymocytes, respectively. The fifth circle (stages II/III) represents CD69+ positively selected thymocytes in transition to the next stage of either CD4 single-positive or CD8 single-positive thymocytes. The last stage (stage IV) represents the CD3+/hi CD45RA+ thymic emigrant. (B) Cell cycle analysis was determined for total and CD3− CD71+ thymocytes by a combination of cell surface staining with antibodies to CD3 and CD71 and 7-AAD. Histograms were drawn for DNA content (7-AAD) in ungated thymocytes (left panel) and CD3− CD71+ gated thymocytes (right panel). The proportions of the thymocytes in the G0/G1 stages and S/G2/M stages of the cell cycle were calculated by Modfit software.

  • FIG. 2.
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    FIG. 2.

    X4 HIV-1 replicates in immature thymocytes, while R5 HIV-1 replicates in mature thymocytes in HIV-1-infected SCID-hu mice. Thymocytes were obtained from HIV-1-infected implants in SCID-hu mice at 19 days (NL4-3) and 26 days (JR-CSF) postinfection. The cells were surface stained with antibodies to CD3, CD71, CD69, CD27, and CD45RA combined with intracellular staining for GagHIV-1 proteins (KC57-FITC). The phenotype of GagHIV-1-expressing cells was determined by gating on KC57+ cells. The percentages of positive cells in each quadrant are indicated. Isotype controls were used to set the cursors. Results for one representative staining of two thymic implants at these time points are shown.

  • FIG. 3.
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    FIG. 3.

    R5 and X4 HIV-1 proviral burdens in thymocyte subsets at different stages of maturation. Thymocyte subsets were obtained from HIV-1-infected implants from SCID-hu mice by immunomagnetic bead selection. Quantitative TaqMan PCR for HIV-1 proviral burden relative to that of the human beta-globin gene was performed and calculated based on interpolation from standard curves. The level of HIV-1 per 100 cell equivalents for each implant in each subset is shown (open circles) (five to six data points in each subset). Asterisks indicate statistically significant differences. (A) Proviral burdens at 4 and 7 weeks postinfection (R5 HIV-1JR-CSF-infected implants) and (B) at 19 days postinfection (X4 HIV-1NL4-3).

  • FIG. 4.
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    FIG. 4.

    HIV-1 coreceptor expression in thymocytes at five developmental stages. CCR5 and CXCR4 expression at stages I-P to IV of thymocyte development was determined by four-color flow cytometric analysis using combinations of monoclonal antibodies to CCR5-PE, CXCR4-PE, CD3-FITC and -APC, CD45RA-APC, CD27-FITC, CD69-TC, and CD71-FITC in five separate experiments as described in Materials and Methods. Asterisks indicate statistically significant differences. As only small numbers of thymocytes express CCR5 and almost all thymocytes express CXCR4, CCR5 data are expressed in percentages and CXCR4 data as mean fluorescence intensities. (A) The mean percent CCR5+ cells in each subset was as follows: for subset I-P, 0.95% ± 0.6%; for subset I, 0.08% ± 0.07%; for subset II, 0.16% ± 0.06%; for subset III, 2.5% ± 0.8%; and for subset IV, 1% ± 0.9%. (B) Since CXCR4 is expressed in each of these subsets, we measured the mean fluorescent intensity (or geometric mean) of CXCR4 for each subset relative to that for subset I-P, which consistently expressed the highest levels of CXCR4 in all five experiments. Subset I-P, 100%; subset I, 32% ± 9%; subset II, 14% ± 9%; subset III, 23% ± 17%; and subset IV, 39% ± 28%.

  • FIG. 5.
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    FIG. 5.

    HIV-1 entry in thymocyte subsets at different stages of maturation. Postnatal thymocytes were separated into individual subsets by magnetic beads and infected in vitro. Eighteen hours postinfection, quantitative PCR was performed to measure the number of initial reverse transcription products (R-U5), and the cell numbers were determined by beta-globin quantitative PCR. Values were calculated based on interpolation from a standard curve, and HIV-1 copy numbers were normalized to 1,000-cell equivalents from each subset after infection by R5 HIV-1JR-CSF (A), R5 HIV-1NFN-SX (B), and X4 HIV-1NL4-3 (C). The mean viral-copy numbers and standard deviations for X4 and R5 HIV-1 DNA based on triplicate measurements in one representative experiment out of three are shown. Asterisks indicate statistically significant differences calculated based on data from three independent experiments.

  • FIG. 6.
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    FIG. 6.

    Levels of HIV-1 reverse transcription are similar in thymocyte subsets at different stages of maturation. Postnatal thymocytes were separated into individual subsets by magnetic beads and infected in vitro. Eighteen hours postinfection, quantitative PCR was performed to measure the numbers of initial and full reverse transcription products (R-U5) and full reverse transcripts only (LTR-gag). The percent completion of reverse transcription for each subset was calculated by dividing the number of full-length transcripts by the number of total transcripts (initial and full). Values were calculated based on interpolation from a standard curve. (A) R5 HIV-1NFN-SX-infected thymocytes. (B) X4 HIV-1NL4-3-infected thymocytes. The means and standard deviations for triplicate measurements from one representative experiment out of three are shown.

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  • TABLE 1.

    Frequencies of X4 HIV-1 (NL4-3) and R5 HIV-1 (JR-CSF) expression in thymocyte subsets in SCID-hu micea

    VirusWkFrequency of virus expression in indicated thymocyte subset
    Subset I-PSubset ISubset IISubset IIISubset IV
    X4 HIV-1 (NL4-3)2.50.080.040.030.020.01
    2.50.60.30.130.160.2
    428.818.25.455.5
    427.517.94.35.36.5
    424.818.85.37.28.7
    52618.824ND1.5
    52310.74.5ND4.5
    R5 HIV-1 (JR-CSF)3.50.50.30.43.12
    3.50.60.30.42.45.1
    40.50.30.62.72.7
    40.70.61.13.13.6
    40.50.61.02.72.3
    40.70.20.42.62.4
    41.20.20.32.22.1
    42.30.3121.4
    71.00.51.1ND1.1
    72.10.92.3ND1.6
    73.21.11.4ND1.3
    70.40.20.6ND1.8
    • ↵ a Thymocytes were isolated from HIV-1-infected implants from 2.5 to 7 weeks postinfection. The cells were surface stained with antibodies to CD3, CD71, CD69, CD27, and CD45RA combined with intracellular staining for the GagHIV-1 protein (KC57-FITC). Each specific subset was gated electronically, and the frequencies of GagHIV-1 (KC57+)-expressing cells in the subsets were determined. ND, not determined.

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Human Immunodeficiency Virus Persistence and Production in T-Cell Development
Kevin B. Gurney, Christel H. Uittenbogaart
Clinical and Vaccine Immunology Nov 2006, 13 (11) 1237-1245; DOI: 10.1128/CVI.00184-06

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Human Immunodeficiency Virus Persistence and Production in T-Cell Development
Kevin B. Gurney, Christel H. Uittenbogaart
Clinical and Vaccine Immunology Nov 2006, 13 (11) 1237-1245; DOI: 10.1128/CVI.00184-06
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KEYWORDS

Cell Differentiation
HIV-1
T-Lymphocytes
Virus Replication

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