<i>In vivo</i> R5 HIV-1 infection in hNOJ mice.

<p>hNOJ mice were challenged intravenously with HIV-1_NL-AD8-D and divided into two groups: naïve-rich hNOJ (IR+) mice at 10 wk post-transplantation (<i>n</i> = 7) and memory-rich hNOJ (IR−) mice at ≥12 wk post-transplantation (<i>n</i> = 8), based on the percentage of each individual CD4⁺ T cell subsets at pre-challenge. (A) Weekly analysis of the plasma viral load. Individual hNOJ (IR−) mice are denoted by different colors in this and in the following figures. (B) The plasma viral load at 1 wk post-challenge. Data are plotted individually along with the mean (black lines). Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01) between hNOJ (IR+) mice (<i>n</i> = 7) and hNOJ (IR−) mice in which the plasma viral load was detectable (>5000 VL, <i>n</i> = 6) or all hNOJ (IR−) mice (<i>n</i> = 8) were determined by the Mann-Whitney U test. (C) The absolute number of CD4⁺ T cells in the peripheral blood at pre-challenge [hNOJ (IR+) mice; <i>n</i> = 7 and hNOJ (IR−) mice; <i>n</i> = 8]. Each CD4⁺ T cell subset (Naïve, CM, and EM) was defined as outlined in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053495#pone-0053495-g006" target="_blank">Figure 6</a>. Data are plotted individually along with the mean (black lines). Significant differences (^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by the Mann-Whitney U test. (D) The peak plasma viral load during 5 wk post-challenge [hNOJ (IR+) mice; <i>n</i> = 6 and hNOJ (IR−) mice; <i>n</i> = 7]. Data are plotted individually along with the mean (black lines). Significant differences (^**<i>P</i><0.01) were determined by the Mann-Whitney U test.</p

HIV-1 co-receptors, CCR5 and CXCR4, expression profiles and <i>ex vivo</i> R5 HIV-1 infectivity of CD4⁺ T Cells in hNOJ mice.

<p>(A) Changes in the percentage of CCR5⁺ (left) and CXCR4⁺ (right) cells within the peripheral blood CD4⁺ T cell population isolated from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 18 and <i>n</i> = 6, respectively). Data are expressed as the mean ± SD. (B) Percentage of CCR5⁺ and CXCR4⁺ cells within the naïve, CM, EM_early, and EM_int/late subsets of peripheral blood CD4⁺ T cells isolated from hNOJ mice at 16 wk post-transplantation (<i>n</i> = 18 and <i>n</i> = 6, respectively) and from humans (<i>n</i> = 5). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^***<i>P</i><0.001) were determined by two-way factorial ANOVA with the Bonferroni multiple comparison test. (C, D, E) Fusion assay using R5 HIV-1 and CD4⁺ T cells. Splenic CD4⁺ T cells from hNOJ mice at ≥17 wk post-transplantation (<i>n</i> = 4; three hNOJ (IR+) mice and one hNOJ (IR−) mouse) or peripheral blood CD4⁺ T cells from humans (<i>n</i> = 5) were infected <i>ex vivo</i> with HIV-1_{NL-AD8-D-BlaM-Vpr}. (C) Naïve, CM, and EM subsets of CD4⁺ T cells (gated on CD3⁺CD4⁺CD8⁻) were defined as CD45RA⁺CCR7⁺, CD45RA⁻CCR7⁺, and CD45RA⁻CCR7⁻, respectively, by flow cytometry. (D) Percentage of R5 HIV-1 fusion cells within the total CD4⁺ T cell population and within the naïve, CM, and EM subsets in hNOJ mice and humans. Individual data points are plotted. The black lines represent the mean. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01) were determined by the unpaired <i>t</i> test. (E) Relative ratio of R5 HIV-1 fusion among the naïve, CM, and EM subsets from hNOJ mice and humans. The level of R5 HIV-1 fusion in each of the CD4⁺ T cell subsets relative to that in the corresponding CM subset. Data are expressed as the mean ± SD. Significant differences (^***<i>P</i><0.001) were determined by Tukey’s multiple comparison test.</p

The number of mice used in the present study.

<p>The number of mice used in the present study.</p

Possible occurrence of HSP of CD4⁺ T Cells in hNOJ mice.

<p>(A) Association between the percentage of hCD45⁺ cells within the PBMC population and that of CD34⁺ cells in the BM cells from hNOJ (IR+) and hNOJ (IR−) mice at ≥16 wk post-transplantation (<i>n</i> = 12 and <i>n</i> = 8, respectively). Spearman’s rank correlation coefficient was used for statistical analysis. (B) Percentage of Ki-67⁺ cells among naïve, CM, EM_early, and EM_int/late subsets of splenic CD4⁺ T cells from hNOJ (IR+) and hNOJ (IR−) mice at ≥16 wk post-transplantation (<i>n</i> = 6 and <i>n</i> = 6, respectively) and from human PBMCs (<i>n</i> = 10). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by Tukey’s multiple comparison test. (C and D) <i>Ex vivo</i> IFN-γ production by CD4⁺ T cells after stimulation with PMA/ionomycin. CD4⁺ T cells were prepared from the spleens of hNOJ (IR+) and hNOJ (IR−) mice at ≥16 wk post-transplantation or from human PBMCs. (C) Representative flow cytometry profiles showing the proportion of IFN-γ⁺ cells within each of the CD4⁺ T cell subsets from a hNOJ (IR+) mouse at 16 wk post-transplantation. (D) Cumulative data showing the percentage of IFN-γ⁺ cells within each of the CD4⁺ T cell subsets from hNOJ (IR+) and hNOJ (IR−) mice and humans (<i>n</i> = 3, <i>n</i> = 3, and <i>n</i> = 4, respectively). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by Tukey’s multiple comparison test.</p

Development of human hematopoietic cells in hNOJ mice.

<p>(A) Changes in the percentage of human CD45⁺ (hCD45⁺) cells within the PBMC population from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 22 and <i>n</i> = 13, respectively). Data are expressed as the mean ± SD. Significant differences (^**<i>P</i><0.01) were determined by the Mann-Whitney U test. (B) Percentage of human CD34⁺ cells within the BM cells isolated from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 5 and 6, respectively) at 8 wk post-transplantation. Data are expressed as the mean ± SD. Significant differences (^**<i>P</i><0.01) were determined by the Mann-Whitney U test. (C) Association between the percentage of hCD45⁺ cells within the PBMC population and that of CD34⁺ cells within the BM cells of hNOJ (IR+) and hNOJ (IR−) mice at 8 wk post-transplantation (11 plots from five hNOJ (IR+) and six hNOJ (IR−) mice). Spearman’s rank correlation coefficient was used for statistical analysis. (D, E) Changes in the percentage of human CD19⁺ B cells, CD14⁺ monocytes, CD4⁺ T cells (CD3⁺CD4⁺CD8⁻ cells), and CD8⁺ T cells (CD3⁺CD4⁻CD8⁺ cells) within the peripheral blood hCD45⁺ cell population (D) or total PBMC populstion (E) from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 22 and <i>n</i> = 13, respectively). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by the Mann-Whitney U test.</p

image_3_Introduction of Human Flt3-L and GM-CSF into Humanized Mice Enhances the Reconstitution and Maturation of Myeloid Dendritic Cells and the Development of Foxp3+CD4+ T Cells.PDF

<p>Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3^null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14⁻CD1c⁺ conventional DCs (cDCs) and CD14⁻CD141⁺ cDCs, in addition to CD14⁺ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123⁺ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c⁺ myeloid cells, CD141⁺ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3⁺ cells within splenic CD4⁺ T cells were significantly increased in the presence of GM-CSF. Foxp3⁺ T cells could be subdivided into two subpopulations, CD45RA⁻Foxp3^hi and CD45RA⁻Foxp3^lo T cells. Whereas CD45RA⁻Foxp3^hi T cells were increased only after treatment with GM-CSF alone, CD45RA⁻Foxp3^lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3⁺ T cells. The correlation analysis demonstrated that the development of these Foxp3⁺ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.</p

image_1_Introduction of Human Flt3-L and GM-CSF into Humanized Mice Enhances the Reconstitution and Maturation of Myeloid Dendritic Cells and the Development of Foxp3+CD4+ T Cells.PDF

<p>Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3^null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14⁻CD1c⁺ conventional DCs (cDCs) and CD14⁻CD141⁺ cDCs, in addition to CD14⁺ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123⁺ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c⁺ myeloid cells, CD141⁺ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3⁺ cells within splenic CD4⁺ T cells were significantly increased in the presence of GM-CSF. Foxp3⁺ T cells could be subdivided into two subpopulations, CD45RA⁻Foxp3^hi and CD45RA⁻Foxp3^lo T cells. Whereas CD45RA⁻Foxp3^hi T cells were increased only after treatment with GM-CSF alone, CD45RA⁻Foxp3^lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3⁺ T cells. The correlation analysis demonstrated that the development of these Foxp3⁺ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.</p

table_1_Introduction of Human Flt3-L and GM-CSF into Humanized Mice Enhances the Reconstitution and Maturation of Myeloid Dendritic Cells and the Development of Foxp3+CD4+ T Cells.PDF

<p>Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3^null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14⁻CD1c⁺ conventional DCs (cDCs) and CD14⁻CD141⁺ cDCs, in addition to CD14⁺ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123⁺ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c⁺ myeloid cells, CD141⁺ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3⁺ cells within splenic CD4⁺ T cells were significantly increased in the presence of GM-CSF. Foxp3⁺ T cells could be subdivided into two subpopulations, CD45RA⁻Foxp3^hi and CD45RA⁻Foxp3^lo T cells. Whereas CD45RA⁻Foxp3^hi T cells were increased only after treatment with GM-CSF alone, CD45RA⁻Foxp3^lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3⁺ T cells. The correlation analysis demonstrated that the development of these Foxp3⁺ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.</p

image_2_Introduction of Human Flt3-L and GM-CSF into Humanized Mice Enhances the Reconstitution and Maturation of Myeloid Dendritic Cells and the Development of Foxp3+CD4+ T Cells.PDF

<p>Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3^null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14⁻CD1c⁺ conventional DCs (cDCs) and CD14⁻CD141⁺ cDCs, in addition to CD14⁺ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123⁺ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c⁺ myeloid cells, CD141⁺ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3⁺ cells within splenic CD4⁺ T cells were significantly increased in the presence of GM-CSF. Foxp3⁺ T cells could be subdivided into two subpopulations, CD45RA⁻Foxp3^hi and CD45RA⁻Foxp3^lo T cells. Whereas CD45RA⁻Foxp3^hi T cells were increased only after treatment with GM-CSF alone, CD45RA⁻Foxp3^lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3⁺ T cells. The correlation analysis demonstrated that the development of these Foxp3⁺ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.</p

Influence of irradiation on the survival and growth of hNOJ mice.

<p>Newborn NOJ mice (1−2 days after birth) were irradiated (1 Gy) or not before transplantation with CD34⁺CD133⁺ HSCs isolated from human cord blood. (A) Survival curves for hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 16 and <i>n</i> = 28, respectively). Significant differences (^***<i>P</i><0.001) were determined by the log-rank test. (B) Changes in the body weight of hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 7 and <i>n</i> = 10, respectively). Data are expressed as the mean ± SD. Significant differences (^***<i>P</i><0.001) were determined by the unpaired <i>t</i> test.</p