Article Figures & Data
Figures
- FIGURE 1.
Percentages of human cells in spleen. Human cells in spleens of uninfected and HIV-1–infected mice were determined by FACS at the time of sacrifice. Individual numbers (percentages) per animal, ratio of CD4/CD8, cells and medians are shown. The p values between groups analyzed by nonparametric Mann-Whitney U test are shown.
- FIGURE 2.
Peripheral blood profiles in HIV-1–infected humanized NSG mice. To determine the dynamics of viral replication and immune cell numbers in peripheral blood, mice were bled biweekly starting 3 wk after HIV-1 infection. Only three animals were frequently bled; because facial vein bleeding with short intervals was affecting animal behavior, we did not follow the same procedure for all of the animals.
- FIGURE 3.
Profile of IFN-γ and IL-2 cytokine-secreting human cells in mouse spleens at 5 wk postinfection. FACS plots are shown from three infected mice and one uninfected mouse. The numbers of cells producing cytokines in response to in vitro stimulation by HIV-1 gag and envelope peptide pools were determined by ICS, and the results were compared with those in nonstimulated cells. Splenocytes were enriched for human CD45+ cells using the magnetic bead separation method and stimulated with peptide pools for 18 h with brefeldin A included during the last 6 h. CD8+ (blue) and CD4+ (red) T cells producing IFN-γ and IL-2 are shown, and the numbers represent percentages of cells positive for each cytokine. The hierarchy followed for gating was CD45 → CD3 → CD8 or CD4 → IFN-γ or IL-2. IFN-γ and IL-2 double-positive cells shown are from respective gates indicated by arrows.
- FIGURE 4.
Effect of CD8+ cell depletion on peripheral viral load and T cell numbers. A, Two uninfected/CD8+ cell-depleted (upper panels, m306 and m303) and two HIV-1–infected (m304-305) are shown. Dynamics of viral load in peripheral blood were determined by the Amplicor kit, and the detection limit was 1750 copies per milliliter. The numbers of human CD4+ and CD8+ T cells were analyzed by FACS, and the percentages shown are from human CD45 gated cells. CD8+ cell depletion was performed at 5 wk postinfection. B, Representative flow cytometric plots from #305 showing human CD4+ and CD8+ cells in peripheral blood before and after 1 and 3 wk of depletion. The inset at 3 wk postdepletion shows CD3+CD8+ cells reappearing in circulation. Similar recovery of CD3+CD8+ cells in spleen collected at the end point (3 wk after depletion) is also shown. All of the animals were reconstituted with the same sample of cord blood-derived HSCs.
- FIGURE 5.
Summary of changes in peripheral blood viral load in HIV-1–infected animals following CD8+ cell depletion. A, In HIV-1–infected control animals, viral load was analyzed at different time-points starting at 2 wk postinfection (black squares, n = 10). Plasma from the animals depleted 2 wk postinfection were analyzed for viral load at 2 wk (bleed) and 4 wk (sacrifice) postinfection (open squares, n = 4). In animals that were depleted 5–7 wk postinfection, viral load was analyzed until sacrifice (i.e., 8–9 wk postinfection) (open circles, n = 7). Dashed line represents the assay detection limit of <1750 viral copies per milliliter. The level of significance was *p < 0.05 as determined by Wilcoxon test. Data are presented as geometrical means and standard deviations. B and C, Values are compared with that of nondepleted HIV-1–infected animals at 4 wk postinfection (B) and 7–9 wk postinfection (C) time points for statistical analyses, and the level of significance was determined by Mann-Whitney U test. Data are presented for individual mice as geometrical means.
- FIGURE 6.
Immunohistochemical staining analyses of lymphoid tissue in CD8+ cell depletion. Immunohistochemical staining of cervical lymph nodes from uninfected and HIV-1–infected/CD8+ cell-depleted animals. Serial sections of paraffin-embedded tissue were stained for human cell markers, CD8, HIV-1 p24, CD79α, HLA-DR, and Ki-67. Images were captured under an objective lens at original magnification ×10. Lymph nodes from HIV-1–infected animals were analyzed at 2 wk following CD8+ cell depletion performed at 4 wk postinfection. Commonly seen HIV-1 p24 Ag-positive cells and syncytia, altered B cell distribution, and increased activation (HLA-DR) were detected.
- FIGURE 7.
Thymus morphology in uninfected and HIV-1–infected/CD8+ cell-depleted animals. A, Representative sections of paraffin-embedded thymi tissues were stained for human cell markers. Thymi from one uninfected control mouse and two HIV-1–infected mice (collected at 1 wk after CD8+ depletion and 8 wk following virus infection) are shown. Top panels, original magnification ×20; all other panels, original magnification ×400. Control thymus was populated by CD45RO+, CD4/CD8 double-positive cells. A small portion of the cortical area was occupied by CD4 single-positive cells. In CD8+ cell-depleted thymi, the numbers of double-positive cells were reduced, and the proliferative activity was increased as detected by Ki-67 staining. HLA-DR–positive cells with dendritic cell morphology were present in all of the thymi along with claudin-3–positive mouse epithelial cells (data not shown). B, Representative FACS plots of thymic cells from an uninfected control and HIV-1–infected/CD8+ cell-depleted mice. Compared to the control thymus, CD8+ cell depletion resulted in a decrease in CD4/CD8 double-positive cells and a profound decrease in CD8+ cells.
Tables
Engraftment Parameters Groupsa Age (wk) Weeks Postinfectionb CD45+ (%, Spleen) IgM (μg/ml) IgG (μg/ml) HIV RNA Copies/mlc Uninfected control 26.5 30.4 103.4 208.0 n = 14 23–36 3.8–50.5 <3.1–962.0 <7.8–402.0 HIV-1–infected 29 10 28.8 168.0 93.5 114,800 n = 15 26–36 5–13 3.1–72.9 18.0–1019.6 <7.8–453.8 13,335–1,526,000 HIV-1–infected CD8+ cell-depleted 29 7 20.2 154.2 70.1 382,550 n = 12 25–33 4–9 3.1–52.6 48.4–523.0 <7.8–856.7 24,745 to >35,000,000 HIV-1–uninfected CD8+ cell-depleted 33 9.4 225.8 175.3 n = 7 29–35 3.1–80.6 123.4–1005.1 <7.8–576.3 ↵a End point data are shown as median and the range (minimal and maximal values).
↵b Period of HIV-1 infection before the animals were euthanized.
↵c Detection limit was 1750 copies/ml. Detection was performed using 20 μl plasma diluted to a total volume of 700 μl as a requirement for automated evaluation by Amplicor kit with detection limit of 50 copies/ml. Productive infection was confirmed by detection of HIV-1 p24-positive cells in lymphoid tissues.
- Table II. Dynamics of T cells and viral load in peripheral blood of HIV-1–infected mice with established infection with and without CD8+ cell depletion
CD8 (%)a CD4 (%) VL (Copies/ml) VL (log10) Mouse No. Before After Before After Δ (%) Before After Before After Δ CD8+ cell depleted animalsb 104 1.2 0 4.9 0.5 −89.8 114,800 454,500 5.06 5.66 0.60 108 5.6 0 10.1 2.7 −73.3 5,740 1,239,000 3.76 6.09 2.33 304 11.4 0.9 25.6 12.3 −51.9 245,350 1,225,000 5.39 6.09 0.70 305 33.2 0 45.9 11.6 −74.7 8,435,000 31,395,000 6.93 7.50 0.57 Nondepleted animalsc 348 2.2 nd 5.2 nd 187,250 75,250 5.27 4.88 −0.40 302 10.7 7.2 52.4 13.3 −74.6 8,050,000 990,500 6.91 6.00 −0.91 169 4.4 21.9 7.4 4.1 −45.0 205,450 455,000 5.31 5.66 0.35 149 25.2 17.4 15.2 1.0 −93.4 2,961,000 62,650 6.47 4.80 −1.67 159 35.7 19.1 12.7 7.7 −39.4 409,500 38,150 5.61 4.58 −1.03 pd 0.5 0.014 0.443 0.343 0.278 0.032 0.438 0.016 0.008 ↵a Percentage of human cells in peripheral blood at the time of viral load analysis.
↵b Mice were analyzed at 5–7 wk postinfection before CD8+ cell depletion and 2 wk after depletion.
↵c Data from nondepleted mice represent the first detected peak of viremia (before) and 2 wk after.
↵d p Values were determined by Mann-Whitney U test.
nd, no data.
Data Supplement
Files in this Data Supplement:
- Supplemental Table 1 (PDF, 32.8 Kb) - Characteristics of humanized animals (Table 1).
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