Reconstitution of the Female Reproductive Tract of Humanized BLT Mice with Human Hematopoietic Cells

<p>Immunohistochemical analysis of the vagina, ectocervix, endocervix, and uterus of female BLT mice for the presence of human hematopoietic lineages (brown cells) (bars indicate 25 μm). Robust reconstitution with cells relevant to HIV-1 infection, including human T cells, monocyte/macrophages, and dendritic cells, was observed in each compartment of the FRT of humanized BLT mice, demonstrating the efficient repopulation of these important mucosal sites.</p

Systemic CD4⁺ T Cell Loss Resulting from Intravaginal HIV-1 Infection in Humanized BLT Mice

<div><p>(A) Comparison of the levels of CD4⁺ or CD8⁺ human T cells in the indicated tissues in representative BLT mice that were either naive, HIV-1 infected, or that received FTC/TDF for pre-exposure prophylaxis prior to exposure to HIV-1. Note the HIV-1 induced reduction in the double-positive CD4⁺CD8⁺ thymocytes.</p> <p>(B and C) Box plots depicting the levels of CD4⁺ (B) or CD8⁺ (C) T cells in the indicated tissues for naive (green), HIV-1 infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. In these plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Data from naive, HIV-1-, or FTC/TDF-treated plus HIV-1–exposed mice were not collected on the same day. Naive (<i>n</i> = 5), HIV-1 infected (<i>n</i> = 4), and FTC/TDF + HIV-1 (<i>n</i> = 3). Flow cytometry gating for this figure was performed as described for <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050016#pmed-0050016-g002" target="_blank">Figure 2</a>.</p> <p>BM, bone marrow; LN, lymph node; PB, peripheral blood; Thymic Org., implanted thymic organoid.</p></div

Pre-exposure Prophylaxis Prevents Intravaginal HIV-1 Transmission in Humanized BLT Mice

<div><p>(A) Kaplan-Meier plot of the time course to plasma antigenemia conversion following intravaginal HIV-1 exposure in BLT mice with or without the 7-d pre-exposure regimen of FTC/TDF (administered once daily starting 48 h prior to intravaginal inoculation).</p> <p>(B) Plasma from the seven infected BLT mice and the five FTC/TDF + HIV-1 mice was tested for the presence of HIV-1 RNA. Data presented depict the initial positive viral RNA value for each mouse examined. The dashed line indicates the limit of detection for this assay.</p> <p>(C–E) Shown are the levels of human CD4⁺ (orange squares) and CD8⁺ (blue circles) T cells in peripheral blood as well as the levels of HIV p24 antigenemia (black triangles) in plasma for (C) naive control (<i>n</i> = 6), (D) HIV-1 infected (<i>n</i> = 7), and (E) pre-exposure FTC/TDF-treated animals (<i>n</i> = 5). In (D), note that in seven of eight tested BLT mice, a single exposure to HIV-1 led to intravaginal transmission and an initial drop, with subsequent stabilization, in the levels of peripheral blood CD4⁺ human T cells. In contrast, no changes were observed in either the naive control (C) or BLT mice that received FTC/TDF for pre-exposure prophylaxis (E). In (D) and (E), day 0 is the day of inoculation and is indicated by an arrow. Gating strategy for flow cytometric analysis: live cells → human CD45 → human CD3 → human CD4 or CD8.</p> <p>(F) Immunohistochemical staining for human CD4⁺ cells within the vagina of a representative FTC/TDF-treated mouse demonstrating the continued presence of CD4⁺ T cells in this tissue (left, bar indicates 50 μm; right, bar indicates 12.5 μm; box indicates region magnified in subsequent image).</p></div

Changes in CD4⁺CCR5⁺ and CD8⁺CCR5⁺ Human T Cell Levels Resulting from HIV-1 Infection

<div><p>(A) Comparison of the levels of human CD4⁺CCR5⁺ T cells in the indicated tissues in a representative naive BLT mouse, an HIV-1–infected, and an HIV-1–exposed BLT mouse that received FTC/TDF for pre-exposure prophylaxis. Liver and lung were the examined tissues with the greatest constitutive CCR5 expression, and they both showed significant loss of CD4⁺CCR5⁺ T cells due to HIV-1 infection.</p> <p>(B) Box plot depicting the levels of CD8⁺CCR5⁺ T cells in the indicated tissues for naive (green), HIV-1 infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice.</p> <p>(C) Comparison of the levels of human CD8⁺CCR5⁺ T cells in the indicated tissues in representative naive, HIV-1 infected and FTC/TDF treated BLT mice. All tissues examined showed increases in CD8⁺CCR5⁺ T cells resulting from HIV-1 infection of BLT mice.</p> <p>(D) Box plot depicting the levels of CD8⁺CCR5⁺ T cells in the indicated tissues for naive (green), HIV-1–infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. In the box plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Naive (<i>n</i> = 5), HIV-1 infected (<i>n</i> = 4), and FTC/TDF + HIV-1 (<i>n</i> = 3). Gating strategy for flow cytometric analysis: live cells → human CD45 → human CD3 → human CD4 or CD8 → CCR5.</p> <p>BM, bone marrow; LN, lymph node; PB, peripheral blood; Thymic Org., implanted thymic organoid.</p></div

Pre-exposure FTC/TDF Treatment Resulted in Complete Protection of Humanized BLT Mice from Intravaginal HIV-1 Transmission

<div><p>(A) Box plot depicting real-time PCR levels of HIV-1 viral DNA in the indicated tissues for HIV-1–infected (white) and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. (Viral DNA copies per million CD4⁺ T cells shown.) HIV-1 infected (<i>n</i> = 2) and FTC/TDF + HIV-1 (<i>n</i> = 4).</p> <p>(B) Box plot depicting virus rescue results from HIV-1–infected (white) and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. Virus rescue data expressed as pg/ml of p24 per 1 × 10⁵ CD4⁺ T cells cocultured with PHA/IL2-activated peripheral blood lymphocyte from a seronegative donor. HIV-1 infected (<i>n</i> = 2) and FTC/TDF + HIV-1 (<i>n</i> = 4). In the box plots, the middle line indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Dashed lines indicate the limit of detection for each assay.</p> <p>(C) In situ hybridization analysis to determine the presence of productively HIV-1–infected cells in the indicated tissues from HIV-1–infected or FTC/TDF-treated BLT mice (bars indicate 50 μm). Note the lack of HIV-1 in the BLT mice that received pre-exposure prophylaxis with FTC/TDF. HIV-1 infected (<i>n</i> = 4) and FTC/TDF + HIV-1 (<i>n</i> = 3).</p> <p>BM, bone marrow; LPL, lamina propria lymphocytes; Thymic Org., implanted thymic organoid; SI, small intestine.</p></div

Loss of CCR5⁺ and Effector Memory CD4⁺ T Cells from GALT during HIV-1 Infection

<div><p>(A) Comparison of the levels of CD4⁺ or CD8⁺ human T cells in the GALT of representative naive and HIV-1–infected mice.</p> <p>(B) Box plot depicting the levels of GALT CD4⁺ T cells for naive (green) and HIV-1–infected (white) BLT mice.</p> <p>(C) Comparison of the levels of human CD4⁺CCR5⁺ T cells in the GALT of naive and HIV-1–infected BLT mice. Significantly fewer GALT CD4⁺ T cells had detectable CCR5 expression levels following HIV-1 infection.</p> <p>(D) Box plot depicting the levels of CD4⁺CCR5⁺ T cells in the GALT for naive (green) and HIV-1–infected (white) BLT mice.</p> <p>(E and F) Comparison of the levels of human CD4⁺ effector memory T cells in the small intestine intra-epithelial (E) and lamina propria (F) lymphocyte compartments of representative naive and HIV-1–infected BLT mice. HIV-1–infected BLT mice have statistically fewer effector memory CD4⁺ T cells present in their small intestines.</p> <p>(G) Box plot depicting the levels of CD45RA^negCD27^neg effector memory T cells in the small intestine of naive (green) and HIV-1–infected (white) BLT mice. In the box plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Naive (<i>n</i> = 4), HIV-1 infected (<i>n</i> = 4). Gating strategy for flow cytometric analysis: live cells → human CD45 → human CD3 → human CD4 → CCR5, CD27, or CD45RA.</p> <p>IEL, intraepithelial lymphocytes; LI large intestine; LPL, lamina propria lymphocytes; SI, small intestine.</p></div