Fig 1.
Development of functional human CD34+ HPCs in BM of humanized mice.
(A) Representative dot plots showing distribution of human early CD34+CD38- and intermediate CD34+CD38+ HPCs in BM of humanized mice. Numbers indicate percentages of CD34+CD38- and CD34+CD38+ HPCs (gated on hCD45+mCD45-VLD-Lin-CD34+ cells). Data are representative of two independent experiments with mice reconstituted with three human donors. (B) Pooled data indicating numbers of colonies that developed from human CD34+ HPCs from BM of humanized mice and human fetal livers (n = 6). Total colonies, CFU-GM, colony-forming unit-granulocyte, macrophage; CFU-E, colony-forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte. (C) Representative histograms showing proliferation of CD34+ HPCs in vivo through BrdU labeling in humanized mice. PBS mouse samples were used as controls (upper panels). Numbers indicate percentages of BrdU+ HPCs within CD34+ HPC subsets. (D) Pooled data indicate percentages of BrdU+ CD34+ HPC subsets in BM of humanized mice (n = 5). (B, D) Data are shown as the mean ± s.e.m.
Fig 2.
Chronic HIV-1 infection preferentially depletes early CD34+CD38- HPCs in humanized mice in vivo.
(A) Representative histograms showing p24 expression on CD3+ T cells and CD34+ HPCs from BM of humanized mice with chronic HIV-1 infection. Numbers indicate percentages of p24-expressing CD3+ T cells and CD34+ HPCs. Data are representative of three independent experiments with mice reconstituted with two to three donors. (B) Representative dot plots showing chronic HIV-1 infection depleting early CD34+CD38- HPCs from BM of humanized mice. Numbers indicate percentages of early CD38-CD34+ and intermediate CD34+CD38+ HPCs. (C) Pooled data indicating mean percentages of early CD34+CD38- HPCs and intermediate CD34+CD38+ HPCs in BM of humanized mice with mock and HIV-1 infection shown in the stacked bar graph. P values are shown. (D-E) Pooled data indicating absolute cell numbers of early CD34+CD38- HPCs (D) and intermediate CD34+CD38+ HPCs (E) in BM of humanized mice with mock (n = 7) or chronic HIV-1 infection (n = 5). (F) Representative dot plots showing chronic HIV-1 infection depleting early CD34+CD38- HPCs from BM of HIV-1-infected patients compared to that of healthy control (HC) donors. Numbers indicate percentages of early CD38-CD34+ and intermediate CD34+CD38+ HPCs. (G-H) Pooled data indicating percentages of early CD34+CD38- HPCs (G) and intermediate CD34+CD38+ HPCs (H) in BM of HC donors (n = 6) and HIV-1-infected patients (n = 5). (D, E, G, H) Data are shown as the mean ± s.e.m. *P < 0.05 and ***P < 0.001 (two-tailed unpaired Student’s t-test).
Fig 3.
Chronic HIV-1 infection impairs proliferation of early CD34+CD38- HPCs in vivo of humanized mice.
(A) Representative histograms showing BrdU labeling of CD34+ HPC subsets from BM of humanized mice. Numbers indicate percentages of BrdU+ within the total CD34+ HPCs, early CD34+CD38- HPCs and intermediate CD34+CD38+ HPCs. (B-E) Pooled data indicating percentages of BrdU+ cells within early CD34+CD38- HPCs (B) and intermediate CD34+CD38+ HPCs (C); and absolute cell numbers of BrdU+ early CD34+CD38- HPCs (D) and intermediate CD34+CD38+ HPCs (E) in BM of mock (n = 4) and HIV-1-infected (n = 6) humanized mice. Data are shown as the mean ± s.e.m. *P < 0.05 (two-tailed unpaired Student’s t-test).
Fig 4.
Chronic HIV-1 infection impairs colony-forming activity of CD34+ HPCs in BM of humanized mice.
Pooled data indicate the number of colonies that developed from human CD34+ HPCs of mock (n = 4) and HIV-1-infected (n = 5) mice. Data are shown as the mean and s.e.m. *P < 0.05 (two-tailed unpaired Student’s t-test).
Fig 5.
Depletion of pDCs during chronic HIV-1 infection rescues early CD34+CD38- HPCs in humanized mice.
(A) Representative dot plots showing the recovery of early CD34+CD38- HPCs in BM of chronic HIV-1-infected humanized mice with pDC depletion. Numbers indicate percentages of early CD34+CD38- HPCs and intermediate CD34+CD38+ HPCs in various groups of mice. (B and C) Summary data of percentages (B) and absolute cell numbers (C) of early CD34+CD38- HPCs and intermediate CD34+CD38+ HPCs from BM in mock (n = 11), HIV-1-infected (n = 12) and HIV-1-infected humanized mice with pDC depletion (n = 13). Each dot represents one mouse. *P < 0.05 and ***P < 0.001 (two-tailed unpaired Student’s t-test). (D) Summary data of CFU that developed from CD34+ HPCs of mock-infected mice (n = 4), HIV-1-infected mice (n = 5) and HIV-1-infected mice with pDC depletion (n = 4). *P < 0.05 (two-tailed unpaired Student’s t-test). Error bars, s.e.m
Fig 6.
Depletion of pDCs reverses the dysregulated gene expression profile of human CD34+ HPCs affected by HIV-1 infection.
Humanized mice were infected with HIV-1 JR-CSF and treated with 15B or control IgG at 11 weeks post-infection and terminated at 21 weeks post-infection. huCD45+Lin-CD34+ cells from BM of mock, HIV-1/IgG or HIV-1/15B treated mice were purified by flow cytometry (> 95% purity). Total mRNA were isolated and used for the cDNA microarray assay. (A) Heat map showing the relative expression of 6108 significantly up- or down-regulated genes indicated by the color bars in huCD45+Lin-CD34+ cells from HIV-1-infected over mock-infected samples (green, suppressed genes; red, induced genes. Fold change ≥ 2). (B) Table showing numbers of genes significantly up-regulated and down-regulated in humanized mice with HIV-1 infection treated with control IgG or 15B antibody for pDC depletion. (C) Pathway maps representing a set of signaling and metabolic maps. Sorting was performed for statistically significant maps. Experimental data are represented as green (JR-CSF + IgG) and red (JR-CSF + 15B) histograms. The height of the histogram corresponds to the relative expression of particular pathways. (D) Fold changes of 88 genes from hematopoietic cell lineage in HIV-1-infected mice treated with control IgG or 15B antibody for pDC depletion in relation to mock controls. (E) Expression levels of genes associated with HPC proliferation, differentiation and cell death in huCD45+Lin-CD34+ cells are indicated by the heat-map color bars (green, suppressed genes; red, induced genes. Fold change ≥ 2). (F) Summary data of the expression of HPC-associated genes, including CD38, HES1, Notch-1 and AML-1 in huCD45+Lin-CD34+ cells from various groups of mice (n = 4 per group). Data are shown as the mean ± s.e.m. *P < 0.05 (two-tailed unpaired Student’s t-test).