Human Immunodeficiency Virus Persistence and Production in T-Cell Development

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 Gag_HIV-1 proteins (KC57-FITC). The phenotype of Gag_HIV-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.

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-1_JR-CSF-infected implants) and (B) at 19 days postinfection (X4 HIV-1_NL4-3).

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%.

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-1_JR-CSF (A), R5 HIV-1_NFN-SX (B), and X4 HIV-1_NL4-3 (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.