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Keywords:

  • Human/rodent chimera;
  • Stem cells;
  • Dendritic cells;
  • Inflammation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dendritic cells (DCs) are derived from CD34+ progenitors and play a central role in the development of immune responses and in tolerance. Their therapeutic potential underscores the need for in vivo models that accurately recapitulate human DC development and function to provide a better understanding of DC biology in health and disease. Using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human CD34+ cells as a model of human hematopoiesis, we examined DC ontogeny. Progenitors of both myeloid (m) and plasmacytoid (p) DCs were identified in the bone marrow of mice up to 24 weeks after transplant, indicating ongoing and sustained production of DCs after initial engraftment. To determine whether human DCs derived from transplanted stem cells were functional, their response to acute inflammation using lipopolysaccharide (LPS) was examined. Eighteen hours after LPS administration, a dramatic increase in the plasma levels of the human inflammatory cytokines interleukin (IL)-8, IL-10, tumor necrosis factor-α, and IL-12p70 was observed. Only mDCs and not pDCs responded in vivo to LPS by upregulating CD86 and CD83. In vivo activation of human mDCs resulted in a substantial increase in the ability of mDCs to induce the proliferation of naive human T cells. Taken together, these data indicate that human CD34+ cells seem to have differentiated appropriately within the NOD/SCID microenvironment into DCs that are developmentally, phenotypically, and functionally similar to the DC subsets found in humans.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Human dendritic cells (DCs) are a rare and phenotypically diverse group of bone marrow–derived antigen-presenting cells (APCs) found in tissues throughout the body. DCs have generated significant interest because of their role as adjuvants in vaccines, their role as initiators of graft-versus-host disease and allograft rejection, and their use in immunotherapy for the treatment of cancer and autoimmune disease [1]. Because of their immunotherapeutic potential, the need to analyze human DC development, activation, and function in vivo has become apparent.

Much of what we know about the differentiation of human hematopoietic stem/progenitor cells into the major subsets of DCs (myeloid [m] and plasmacytoid [p]) has been derived from in vitro experiments [28]. Several different groups have defined the culture conditions that result in the generation of the different types of mDCs, and, recently, the pathway of differentiation of human CD34+ cells into pDCs was described [5]. The extent to which these proposed developmental pathways reflect authentic DC ontogeny in humans is still unknown. Because it is not feasible to address these issues in humans, we used an in vivo model, the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse transplanted with human CD34+ hematopoietic progenitor cells that was originally developed to study human hematopoietic stem cell function [9]. NOD/SCID mice have several immunological defects, including lack of B and T lymphocytes, no circulating immunoglobulins, reduced natural killer cell activity, decreased hemolytic complement, and functionally impaired macrophages that contribute to their high levels of engraftment after transplantation with human CD34+ progenitor cells [9,10].

We recently showed that human cells expressing human leukocyte antigen DR (HLA-DR) and exhibiting DC morphology could be detected in tissue sections of skin, lung, liver, and spleen from NOD/SCID mice transplanted with human CD34+ cells [11]. In the bone marrow, we were able to determine the presence of HLA-DR+ lineage–negative, CD11cbright, and HLA-DR lineage–negative CD123+ cells, consistent with their phenotype as human myeloid and plasmacytoid DCs. Moreover, systemic production of human interferon (IFN)-α was induced after the mice were injected with influenza virus, indicating that pDCs were functional [11]. Previously, Nobuyoshi et al. [12] reported human DCs present in the bone marrow of transplant ended/SCID mice with a CD34CD4+HLA-DR+ phenotype. Unfortunately, these analysis did not explore the nature of the different DC precursor/progenitor populations, and the criteria used to identify human DCs did not include analysis of CD11c or CD123 expression or analysis of expression of blood dendritic cell antigens (BDCAs) [13,14]. In this study, we address several novel aspects of human DC development in NOD/SCID mice transplanted with human CD34+ cells and demonstrate that there is a direct correlation between each of the DC progenitor and precursor populations normally found in humans and those found in transplanted mice. We identified the same progenitor/precursor populations of both mDCs and pDCs that have been previously used to generate human DCs in vitro [2, 4, 5]. We show that human mDCs and pDCs are present in the circulation of transplanted mice as well as in the spleen, suggesting that human DC precursors are able to migrate within the mouse. Furthermore, we show to what extent human DCs developed in the NOD/SCID mouse model recapitulate the phenotypic characteristics that define different DC populations in humans. Finally, we demonstrate innate immune recognition by human cells developed in transplanted NOD/SCID mice using a model of acute inflammation (injection of lipopolysaccharide [LPS]). Our results show that in response to LPS, human mDCs are preferentially activated and induced to mature in vivo, resulting in enhanced DC function.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mice, CD34+ Cell Isolation, and Transplantation

NOD/SCID mice were housed and bred in a specific pathogen-free facility at the University of Texas Southwestern Medical Center according to Institutional Animal Care and Research Committee–approved protocols. Six- to eight-week-old NOD/SCID mice were sublethally irradiated with 325 cGys from a cesium source. Twenty-four hours later, mice were transplanted intravenously with purified cord blood CD34+ cells isolated using magnetic beads (Miltenyi Biotech, Auburn, CA), essentially as previously reported [15]. Transplanted CD34+ cells (2 to 9 × 105) were greater than 90% pure, as determined by flow cytometry. Engraftment with human cells was assessed 6–8 weeks after transplant by monitoring the percentage of cells expressing human CD45 in whole peripheral blood. Transplanted mice were harvested at 12–26 weeks of age.

Reagents

RPMI was purchased from Gibco Invitrogen Corporation (Carlsbad, CA) and was supplemented with 10% fetal calf serum (Gibco Invitrogen Corporation). LPS Escherichia coli serotype O111:B4 was purchased from Sigma Chemical Co. (St. Louis) and was resuspended in endotoxin-free 0.9% sodium chloride solution (B. Braun Medical Inc., Irvine, CA).

Antibodies

To identify human DCs in samples from NOD/SCID mice reconstituted with human CD34+cells, we first evaluated each of our antibodies for possible cross-reactivity with the endogenous mouse cells. None of the antibodies tested showed any significant cross-reactivity with mouse antigens (data not shown). The phenotype of human DCs in the bone marrow, peripheral blood, and spleen of NOD/SCID mice was determined by multiparameter flow cytometry using the following monoclonal antibodies (mAbs): anti-CD3-fluorescein isothiocyanate (FITC) (immunoglobulin G1 [IgG1], clone SK7), anti-CD11c-APC (IgG2b, clone S-HCL-3), anti-CD19-Per-CP (IgG1, clone SJ25C1), anti-CD33-APC (IgG1, clone WM53), anti-CD45-APC (IgG1, clone 2D1), anti-CD123-PE (IgG1, clone 9F5), anti-HLA-DR-Per-CP (IgG2a, clone L243), and anti-lineage cocktail-FITC (IgG1, anti-CD3, clone SK7; anti-CD14, clone MØP9; anti-CD16, clone 3G8; anti-CD19, clone SJ25C1; anti-CD20, clone L27) from Becton, Dickinson (San Jose, CA). Anti-CD11c-PE (IgG1, clone B-ly6), anti-CD19-PE (IgG1, clone SK7), anti-CD19-PE (IgG1, clone HIB19), anti-CD33-APC (IgG1, WM53), anti-CD34-APC (IgG1,clone 581), anti-CD45RA-PE-Cy5 (IgG2b, clone HI100), anti-CD83-APC (IgG1, Hb15e), and anti-CD86-APC (IgG1, FUN-1) were purchased from BD Pharmingen (San Diego). APC-conjugated anti-human BDCA-1 (IgG2a, clone AD5-8E7), BDCA-2 (IgG1, clone AC144), BDCA-3 (IgG1, clone AD5-14H12), and BDCA-4 (IgG1, clone AD5-17F6) were purchased from Miltenyi Biotec. Anti-CD19-APC (IgG2a, clone 1D3) from BD Pharmingen and anti-NK1.1-PE (IgG2a, clone PK136) and anti-CD3-Cy5 conjugated (IgG1, clone 500A2) from Caltag (Burlington, CA) were used to confirm the absence of murine lymphocytes in samples from transplanted mice.

Human Cytokine Analysis

Human cytokines present in the plasma of NOD/SCID mice were quantified using the Cytometric Bead Array (CBA) kit for inflammatory cytokines (BD Pharmingen). Data were acquired on a FACSCalibur instrument (BD Biosciences, San Jose, CA) and analyzed using the BD CBA data analysis software. Human IFN-α levels were determined by enzyme-linked immunosorbent assay according to the manufacturer's instructions (PBL Biomedical Laboratories, New Brunswick, NJ). No cross-reactivity between mouse cytokines produced in response to LPS and the human cytokine reagents used was detected (not shown).

Flow Cytometry

To determine the percentage of cells expressing human HLA-DR and to determine the presence of human DCs, whole-blood staining was performed using FACS Lysing Solution (BD Biosciences) according to the manufacturer's protocol. For bone marrow and spleen samples, single-cell suspensions were prepared as previously reported [16]. To reduce nonspecific binding of mAb, cells were incubated with mouse IgGs (10 μg/ml, Sigma Chemical Co.) on ice for 20 minutes before the addition of human-specific mAb or isotype control mAb. When indicated, stains were performed in combination with FITC-conjugated anti-mouse CD45toexcludemousecellsfromtheanalysis.HumanDCs were identified using the Becton, Dickinson DC kit consisting of a FITC-conjugated lineage cocktail (comprised of anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20, and anti-CD56), Per-CP–conjugated anti-HLA-DR, APC-conjugated anti-CD11c, and PE-conjugated anti-CD123. Nonspecific binding of mAb was assessed by the use of isotype-matched control mAb. Flow cytometry data were acquired on a FACS Calibur instrument (BD Biosciences) and analyzed using the Cellquest Pro (BD Biosciences) or Flowjo (Treestar, Inc., Ashland, OR) software packages.

Isolation of Human mDCs for Mixed Lymphocyte Reactions

Plasma and tissue from control mice injected with endotoxin-free saline or injected with 10 μg LPS in saline were harvested 18 hours after injection. Before isolation of human mDCs from bone marrow, mouse cells were depleted using positive selection magnetic separation with murine anti-CD45 mAb (StemCell Technologies, Vancouver, Canada). Myeloid DCs from control (saline injected) and LPS-treated mice were obtained using the CD1c (BDCA-1) Dendritic Cell Isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. Graded doses of irradiated (3,000 rads) DCs were cocultured for 6 days with 5 × 104 allogeneic CD4+CD45RA+ or CD4+CD45RO+ T cells isolated from peripheral blood mononuclear cells using T-cell enrichment columns from R&D Systems (Minneapolis). Mixed lymphocyte reactions (MLRs) were pulsed with 3H-thymidine (1 μCi/well, New England Nuclear Co., Boston) for the last 18 hours of culture and then collected onto glass fiber filters (Wallac, Finland) with a Tomtec Cell Harvester (Orange, CT). Incorporation of radioactivity was determined using a 1205 Betaplate scintillation counter (Wallac, Finland).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Transplantation and Engraftment of NOD/SCID Mice with Human CD34+ Cells

For this study, NOD/SCID mice were transplanted with human CD34+ cells (2 to 9 × 105 per mouse) enriched by immunomagnetic isolation. Multilineage engraftment and initial levels of reconstitution with human cells were assessed using whole-blood flow cytometry at 6–8 weeks after transplant (not shown). Bone marrow and spleen cells were examined for expression of human panleukocyte marker CD45 12 weeks after transplant (n = 6) (Table 1). Human CD45+ cells were present in the bone marrow and spleen of all transplanted animals, with an average of 31% (± 9) and 10% (± 5), respectively (n = 6). Human mDCs and pDCs present in these mice were identified by their lack of expression of lineage markers (CD3, CD14, CD16, CD19, CD20, and CD56), by the expression of HLA-DR [13, 17, 18], and by the expression of CD11c+ for mDCs and CD123+ for pDCs. Plasmacytoid DCs and mDCs were present in the bone marrow of all mice examined regardless of their overall levels of reconstitution (Table 1).

Table Table 1.. Frequency of human cells and human dendritic cell subsets in nonobese diabetic/severe combined immunodeficiency mice transplanted with human CD34+ cells
  • a

    aPercentage of total human bone marrow or spleen cells.

  • b

    bPercentage of HLA-DR+Lin cells in bone marrow or spleen.

  • c

    Abbreviations: HLA-DR, human leukocyte antigen DR; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell.

  Bone marrowSpleen
MouseWeeks after transplant% hCD45a% pDCb% mDCb% hCD45a% pDCb% mDCb
        
1124112912156
21229551333
31238551442
41236751622
5122297414
6121963320
Mean (± standard deviation)31 (9)7 (3)6 (2)10 (5)5 (5)3 (2)

Characterization of Human DC Progenitor Cells in the Bone Marrow of NOD/SCID Mice Transplanted with Human CD34+ Cells

To examine the development of human DCs in reconstituted NOD/SCID mice, we took advantage of the fact that human mDCs are generated from two different sources, CD34+ hematopoietic progenitor cells and CD14+ monocytes [2, 4, 8, 19]. To identify the progenitors of myeloid DCs, bone marrow cells from reconstituted NOD/SCID mice 12 or 14 weeks after transplant (n = 5 and 2, respectively) were stained for expression of HLA-DR, CD34, and the myeloid lineage antigens CD33 and CD14. We then examined expression of these markers in HLA-DR+ cell populations (Fig. 1, panel A). Four distinct populations of cells could be discerned by expression of CD34 and CD33: CD33++CD34, CD33++CD34+, CD33CD34+, and CD33CD34 in regions athrough d, respectively (Fig. 1, panel B). To identify mature monocytes in each of these populations, coexpression of CD33 and CD14 was examined (Fig. 1, C–F). Cells expressing CD14 were present in CD33++CD34 cells in region a and in the CD33+CD34+ myeloid progenitor cells in region b. No CD14+ monocytes were present in the CD33CD34+ early progenitor population (region c) or the CD33CD34 population that corresponds to human CD19+ B cells (region d and data not shown) [16]. The results obtained from seven different mice are summarized in Table 2. For comparison, the same populations of myeloid precursor cells that can be found in normal human cord blood is also shown in Table 2. These results demonstrate the presence in these mice of the same mDC precursors that are normally isolated from human peripheral blood, cord blood, and fetal bone marrow [2, 4, 13, 20, 21].

Table Table 2.. Progenitors of human myeloid dendritic cells in the bone marrow of NOD/SCID mice transplanted with human CD34+ cells and in human cord blood
  1. a

    Bone marrow from transplanted and reconstituted NOD/SCID mice (n = 7; 5 harvested 12 weeks after transplant and 2 harvested 14 weeks after transplant) and human cord blood mononuclear cells (n = 5) were stained with anti-human HLA-DR, anti-human CD33, anti-human CD34, and anti-human CD14. HLA-DR+ cells were examined for the presence of myeloid progenitors according to their expression of CD34 and CD33 as described in the legend of Figure 1.

  2. b

    Abbreviations: HLA-DR, human leukocyte antigen DR; NOD/SCID, nonobese diabetic/severe combined immunodeficiency.

ProgenitorRegionPhenotypeMEAN (standard deviation)Range
NOD/SCID bone marrow
    MyeloidaCD33++CD346 (2)3.8–10
    Early myeloidbCD33++CD34+2 (0.8)1.4–3.5
    Early progenitorcCD33CD34+3.6 (2.7)1.8–9
    NonmyeloiddCD33CD3468 (11)52–78
Human cord blood
    MyeloidaCD33++CD3420.7 (16.1)9.1–43.0
    Early myeloidbCD33++CD34+2.8 (1.4)0.7–3.3
    Early progenitordCD33CD34+1.2 (0.9)0.5–2.4
    NonmyeloideCD33CD3440.1 (22.6)12.1–59.9
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Figure Figure 1.. Analysis of the human myeloid dendritic cell progenitor/precursor populations present in the bone marrow of nonobese diabetic/severe combined immunodeficiency mice transplanted with human CD34+ cells. HLA-DR+ cells (A) were examined for expression of CD34 and the myeloid lineage marker CD33 (B). Four populations of cells were defined according to their expression of these markers and were labeled as follows: (a) includes CD33++CD34 myeloid cells; (b) includes CD33++CD34+ myeloid progenitor cells; (c) includes CD33CD34+ early progenitor cells; and (d) includes CD33CD34 nonmyeloid cells. The cells within each region were additionally examined for coexpression of CD33 and CD14 to identify mature monocytes (C–F). Numbers in the upper right corner of the dot plots represent the percentage of mature CD33+CD14+ monocytes present in each region. Data shown are from one representative mouse of the seven mice shown in Table 2. Abbreviation: HLA-DR, human leukocyte antigen DR.

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To identify the pDC progenitors present in the bone marrow of reconstituted mice, the phenotyping criteria previously established by Blom et al. [5] were used. According to these criteria, pDC progenitors and precursors can be distinguished by their differences in CD45RA and CD34 expression. Therefore, lineage-negative cells (lacking expression of CD3, CD14, CD16, CD19, CD20, and CD56) were stained with human anti-CD34 and human anti-CD45RA. In the bone marrow of transplanted NOD/SCID mice, we identified the following five distinct populations of cells (A through E), as shown in Figure 2, left panel: CD34++CD45RA early progenitor cells (region A), CD34++CD45RA+ late progenitor cells (region B), CD34dimCD45RA+ pro-pDC (region C), CD34CD45RA+ pre-pDC (region D), and CD34CD45RA non-pDC (region E). A summary of the data obtained from five different mice harvested 12 weeks after transplant is shown in Table 3. These populations are in accordance with those observed in normal human hematopoietic tissues [5] and normal human cord blood (Table 3).

Table Table 3.. Progenitors of human pDCs in the bone marrow of NOD/SCID mice transplanted with human CD34+ cells and in human cord blood
  1. a

    Bone marrow from transplanted and reconstituted NOD/SCID mice (n = 5 harvested 12 weeks after transplant) and human cord blood mononuclear cells (n = 8) were stained with lineage cocktail, anti-human CD34, and anti-human CD45RA. Lineage-negative cells were examined for the presence of pDC and pDC progenitors according to their expression of CD34 and CD45RA, as described in legend of Figure 2.

  2. b

    Abbreviations: NOD/SCID, nonobese diabetic/severe combined immunodeficiency; pDC, plasmacytoid dendritic cell.

ProgenitorRegionPhenotypeMean (standard deviation)Range
NOD/SCID bone marrow
    Early progenitoraCD342+CD45RA0.7 (0.2)0.63–1.02
    Late progenitorbCD342+CD45RA+4.4 (1.5)2.2–5.5
    Pro-DC2cCD34+CD45RA+28.8 (14.9)7.1–40.9
    Pre-DC2dCD34CD45RA+14.7 (5.6)7.1–19.1
    Non-pDCeCD34CD45RA23.7 (10.8)13.4–38.6
Human cord blood
    Early progenitoraCD342+CD45RA12.2 (9.4)5.0–34.0
    Late progenitorbCD342+CD45RA+2.0 (1.4)0.4–4.0
    Pro-DC2cCD34+CD45RA+3.0 (5.1)0.5–15.0
    Pre-DC2dCD34CD45RA+10.3 (4.5)3.0–15.6
    Non-pDCeCD34CD45RA46.3 (5.9)40.8–53.9
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Figure Figure 2.. Analysis of the progenitor/precursors of human plasmacytoid dendritic cells in the bone marrow of nonobese diabetic/severe combined immunodeficiency mice transplanted with human CD34+ cells. Expression of CD34 and CD45RA by lineage-negative human-derived bone marrow cells was analyzed according to Blom et al. [5] and revealed the presence of CD34++CD45RA early progenitor cells (A), CD34++CD45RA+ progenitor cells (B), CD34+CD45RA+ pro-DC2 (C), CD34CD45RA+ pre-DC2 (D), and non-pDC CD34CD45RA cells (E). To confirm the identity of each of the populations described above as pDC precursors, each subset of cells was further examined for expression of HLA-DR, CD123, and CD4. Mouse cells stained with anti-mouse CD45 were excluded from the analysis. Numbers in the upper right corner of histograms represent the mean fluorescence intensity of cells expressing a specific antigen. Histograms shaded in black represent staining with isotype control mAb; white histograms represent staining with the test mAb. Data shown are from one of three independent experiments. Note that cells coexpressing human CD123 and CD4 (i.e., pDC) are only present in regions B, C, and D, as described for human adult blood, bone marrow, cord blood, and fetal liver [5]. Data shown are from one representative mouse of the five mice in Table 3, all harvested 12 weeks after transplant. Abbreviations: HLA-DR, human leukocyte antigen DR; mAb, monoclonal antibody; pDC, plasmacytoid dendritic cell.

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In addition to their lack of lineage markers, human pDCs are characterized by coexpression of HLA-DR, CD123, and CD4 [5]. Despite the fact that cells in regions A through E in Figure 2 expressed HLA-DR, only cells in regions B, C, and D coexpressed CD123 and CD4 (Fig. 2, right). These pDC phenotypes are consistent with those previously described in human adult peripheral blood, cord blood, fetal bone marrow, fetal liver, and fetal thymus [5]. Together, these data demonstrate that human CD34+ cells differentiate in vivo in NOD/SCID mice into progenitors of both the lymphoid and myeloid DC lineages. The presence of these progenitor populations persists up to 26 weeks after transplant (the longest time evaluated), demonstrating that the bone marrow of NOD/SCID mice provides a microenvironment capable of sustaining long-term DC development and that the developmental pathway of human pDC in transplanted NOD/SCID mice reflects that previously described for human hematopoietic tissues.

Identification of Human mDCs and pDCs Present in the Blood and Spleen of Transplanted and Reconstituted NOD/SCID Mice

We have previously demonstrated the presence of human LinHLA-DR+CD123+ pDC and LinHLA-DR+CD11c+ mDCs in the bone marrow of NOD/SCID mice transplanted with human CD34+ cells [11]. We therefore proceeded to characterize the human DCs in blood and spleen 12 weeks after transplant. Cells from these tissues were prepared for flow cytometry using whole blood and mononuclear cell staining protocols, respectively. Representative staining of peripheral blood from a transplanted animal is shown in Figure 3 (top). The left panels show the staining of lineage cocktail versus HLA-DR for each tissue. Lineage-negative and HLA-DR+ cells were then examined for expression of CD123 and CD11c. Human pDCs and mDCs were present in the blood of all transplanted and reconstituted NOD/SCID mice, with a mean frequency of 2.2% (range, 0.3 to 3.7) and 2.4% (range, 0.6 to 11) (n = 6), respectively. Because the lymph nodes are underdeveloped in NOD/SCID mice [9] and the frequency of human CD45+ cells in the lymph nodes is negligible [22], the spleen represents the largest secondary lymphoid tissue available for the study of human DCs in this model. Representative staining of spleen cells from a transplanted mouse is shown in Figure 3 (bottom). Myeloid DCs and pDCs were present in the spleen of almost all transplanted and reconstituted mice, with a mean frequency of 5 ± 5% (range, 1 to 15) and 3 ± 2% (range, 0 to 6), respectively (Table 1). Taken together, the data described above and the data showing the presence of DC progenitors and precursors in the bone marrow of transplanted mice (Figs. 1, 2) demonstrate the presence of human DCs in peripheral blood and in the primary and secondary lymphoid tissues of transplanted mice.

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Figure Figure 3.. Human pDCs and mDCs from the blood and spleen of transplanted and reconstituted nonobese diabetic/severe combined immunodeficiency mice. Whole blood and spleen of transplanted mice were examined for the presence of human DC subsets by flow cytometry. Single-cell suspensions of spleen or whole blood were stained with anti-human lineage cocktail, anti-human HLA-DR, and anti-human CD123 or anti-human CD11c, as indicated in Materials and Methods. Dot plots on the left show staining of lineage cocktail versus HLA-DR to identify LinHLA-DR+ cells present in the tissue. Gates were set for both tissues using the appropriate isotype-matched monoclonal antibody controls. The percentage of total live cells that were LinHLA-DR+ is indicated in the upper left corner of the dot plot. LinHLA-DR+ cells in both tissues were then examined for CD123 or CD11c expression. The percentage of each DC subset identified within the LinHLA-DR+ fraction of each tissue is noted in the upper right corner. The data shown are from one representative mouse of six harvested 12 weeks after transplant. Abbreviations: HLA-DR, human leukocyte antigen DR; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell.

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Expression of BDCA by Human DC Subsets in Transplanted NOD/SCID Mice

Recently, the antigens BDCA-1, BDCA-2, BDCA-3, and BDCA-4 have been used to discriminate between different human DC subsets [14]. For example, in human peripheral blood, BDCA-2 and BDCA-4 are expressed by pDCs, whereas mDCs express BDCA-1. In addition, a subset of mDCs expressed BDCA-3 [14]. The differential expression of BDCA markers has been used to facilitate the isolation of human DC subsets from peripheral blood [23]. To further characterize the human DC populations in transplanted mice, freshly isolated bone marrow and spleen cells were stained for expression of the BDCA markers in combination with the mAbs used above to identify pDCs and mDCs (i.e., anti-lineage cocktail, anti-HLA-DR, and anti-CD123 or anti-CD11c, respectively). In the bone marrow, approximately 70% of the mDCs expressed BDCA-1, whereas only 30% expressed BDCA-3 (n = 2), consistent with their identification as mDCs (Fig. 4A, top). As previously seen in humans, only a few mDCs coexpressed BDCA-2, and virtually none expressed BDCA-4 [14]. By contrast, most bone marrow pDCs (Fig. 4A, bottom) expressed BDCA-2 and BDCA-4 (approximately 90% and 80%, respectively), whereas only a small percentage of pDCs expressed BDCA-3, and virtually none expressed BDCA-1.

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Figure Figure 4.. Expression of BDCA by human DC subsets in the bone marrow and spleen of nonobese diabetic/severe combined immunodeficiency mice reconstituted with human CD34+ cells. Bone marrow and spleen cells were stained with lineage cocktail, anti-HLA-DR, and anti-CD123 or CD11c to identify pDCs and mDCs, respectively, and the expression of BDCA-1, BDCA-2, BDCA-3, or BDCA-4 was determined for both DC subsets. Gates were set using isotype-matched monoclonal antibody controls for each stain. The percentage of each DC subset expressing the different BDCA antigens is noted in the right corner of the dot plots. (A): Expression of BDCA antigens by bone marrow mDC and pDC. (B): Expression of BDCA antigens by splenic mDC and pDC (n = 2). Note that expression of the different BDCA markers by DC subsets is mutually exclusive; mDCs expressed BDCA-1 and BDCA-3, and pDCs expressed BDCA-2 and BDCA-4. The mice used for these experiments were those in Table 3 and were harvested 12 weeks after transplant. Abbreviations: BDCA, blood dendritic cell antigen; HLA-DR, human leukocyte antigen DR; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell.

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In the spleen, most mDCs express BDCA-1 (73%), and approximately 26% express BDCA-3 (Fig. 4B, top). In addition, in the spleen, virtually all of the pDCs expressed BDCA-2 (>90%), but only 40% expressed BDCA-4 (Fig. 4B, bottom). These results show that expression of BDCA markers by human DC subsets in the bone marrow and spleen of transplanted mice is very similar to that previously described for human DCs in peripheral blood and tonsil [14,24].

LPS Induces the Production of Human Inflammatory Cytokines In Vivo

The data described above indicate that human CD34+ cells transplanted into NOD/SCID mice can give rise to prolonged hematopoietic reconstitution and the production of human DC progenitor and precursor populations. We then sought to determine whether these cells were functional by examining the in vivo induction of an innate immune response, specifically, acute inflammation. LPS is an endotoxin derived from the cell wall of Gram-negative organisms that has been used to model acute inflammation in humans [2527]. In response to intravenously administered LPS, humans produce several proinflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8 [2527]. Therefore, we determined whether a similar cytokine response is obtained in this model. In the steady state, analysis of plasma from control mice reconstituted with human CD34+ cells examined 12 weeks after transplant for the presence of human IL-1β, TNF-α, IL-6, IL-8, IL-10, IL-12p70, and IFN-α failed to detect any of these cytokines (Fig. 5). In sharp contrast, the plasma collected from mice 18 hours after intravenous LPS administration (12 weeks after transplant) had high levels of human TNF-α (mean, 142 ± 74; range, 118 to 175), IL-8 (mean, 177± 43; range, 135 to 221), IL-10 (mean, 1172 ± 692; range, 807 to 1,686), and IL-12p70 (mean, 1,100 ± 296; range, 818 to 1,423) (Fig. 5). IL-1β, IL-6, and IFN-α were also detected in the plasma of LPS-treated mice, albeit at lower levels (32, 21, and 29 pg/ml, respectively; data not shown). These results indicate that transplanted mice are able to produce human inflammatory cytokines and in this way recapitulate this aspect of the acute human response to LPS.

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Figure Figure 5.. Human inflammatory cytokine production in response to LPS administration by NOD/SCID mice reconstituted with human CD34+ cells. Reconstituted mice at 12 weeks after transplant were injected via tail vein with either saline solution (control) or with 10 μg LPS in saline. Eighteen hours after injection, levels of human cytokine present in EDTA anticoagulated plasma was assessed using a human inflammatory cytokine cytometric bead array kit. Results are presented as picograms of cytokine per milliliter. Plasma from a LPS-treated NOD/SCID control mouse (mouse with no human cells) was also tested, and no cross-reactivity between mouse and human cytokines was observed (not shown). Note that different scales were used for the top and bottom panels. Results are shown for six different mice (three control and three LPS-treated), harvested in parallel at the same time. They are representative of one of four independent experiments. Abbreviations: IL, interleukin; LPS, lipopolysaccharide; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; TNF, tumor necrosis factor.

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LPS Induces the Specific Activation and Maturation of Human mDCs In Vivo

Human DC subsets have been shown to respond differentially to activating stimulus in vitro [2830]. Specifically, LPS has been shown to induce maturation of human monocyte-derived DCs in vitro [31]. To determine whether the human CD34+ cell-derived DCs respond adequately to LPS in vivo, we evaluated their response to this stimulus. In addition to the increase of human cytokine levels in the plasma of treated mice shown in Figure 5, administration of LPS resulted in phenotypic changes of DC in vivo. In the bone marrow, up to 91% of the mDCs strongly upregulated expression of CD86 (mean, 64 ± 21%; range, 41 to 91), whereas the proportion of cells expressing CD83 remained low (12% positive) (Fig. 6A). Interestingly, the levels of CD86 and CD83 expression by bone marrow pDCs were minimally affected at 18 hours after LPS administration (Fig. 6A). Myeloid DCs isolated from the spleens of LPS-treated mice (Fig. 6B) expressed high levels of CD86 (mean, 78 ± 22%; range, 40 to 93) and strongly upregulated CD83 (mean, 87 ± 10%; range, 76 to 95), indicating their activation and maturation. By contrast, few splenic pDCs expressed CD86 (mean, 13 ± 7; range, 7 to 30) or CD83 (mean, 15 ± 15; range, 1 to 32) in response to LPS. Therefore, systemic administration of LPS induced the in vivo phenotypic activation and maturation of human mDCs.

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Figure Figure 6.. In vivo activation of human mDCs by LPS. (A): Bone marrow cells from control (saline-injected) and LPS-treated mice (10 μg/mouse) were stained with anti-lineage cocktail, anti-HLA-DR, anti-CD123, or anti-CD11c (to identify human pDCs or mDCs, respectively), and either anti-CD86 or CD83 (to determine the level of activation and maturation, respectively). The numbers in the gates indicate the percentage of gated mDCs or pDCs that express CD86 or CD83. (B): Spleen cell suspensions were stained and gated as described above for bone marrow cells. Note the dramatic upregulation of both CD86 and CD83 by mDCs in the spleen. Gates to determine positive staining for each plot were set using the appropriate isotype-matched control for every monoclonal antibody used and for each tissue. Data shown are from one of three independent experiments. Abbreviations: HLA-DR, human leukocyte antigen DR; LPS, lipopolysaccharide; mDC, myeloid dendritic cell; PBS, phosphate-buffered saline; pDC, plasmacytoid dendritic cell.

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Functional Consequences of In Vivo Activation and Maturation of Human mDCs

The LPS-induced phenotypic changes of human mDCs in vivo indicate that endotoxin specifically activates mDCs, leading to their maturation. Based on these results, we examined whether the phenotypic changes associated with LPS administration resulted in a quantifiable functional change as determined by the ability of mDCs to stimulate T-cell proliferation in the MLR. Because most human mDCs present in the bone marrow of transplanted mice expressed BDCA-1 (Fig. 4A, top), we used expression of this marker and a commercially available kit to positively select human mDCs from control and LPS-treated mice (as described in Materials and Methods). Isolated mDCs were irradiated and cocultured with human allogeneic CD4+CD45RA+ naive or CD4+CD45RO+ memory T cells for 6 days, and the results of a representative MLR are shown in Figure 7. No proliferation was observed in control cultures of T cells or DCs alone. Myeloid DCs isolated from control mice were able to stimulate both naive and memory T cells to proliferate in a dose-dependent manner in the absence of any exogenous human cytokines and without being cultured in vitro for any period of time. Coculture of 5,000 mDCs with T cells (1:10 DC:T cell ratio) resulted in modest thymidine incorporation, but when 10,000 mDCs were used (1:5 DC:T cell ratio), T-cell proliferation was four-fold greater. Myeloid DCs isolated from LPS-treated mice and cocultured with allogeneic naive T cells induced a 15-fold stronger MLR reaction at a 1:10 DC:T cell ratio than human mDCs from control mice (Fig. 7). We were not able to perform the MLR at the higher 1:5 DC:T cell ratio, because we observed that fewer mDCs could be recovered from LPS-treated mice. Human mDCs isolated from LPS-treated mice were also able to induce proliferation of memory T cells. However, as previously shown for human mDCs, the T-cell proliferation observed was significantly less than that for the naive T-cell cocultures [32]. These results demonstrate that human mDCs generated in NOD/SCID mice are functional and responsive to LPS in vivo, resulting in DC activation and maturation into potent APCs.

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Figure Figure 7.. Human mDCs isolated from LPS-injected nonobese diabetic/severe combined immunodeficiency bone marrow are potent stimulators of naive T-cell proliferation. Graded doses of BDCA-1+ mDCs isolated from the bone marrow of two control mice (top) or from two LPS-treated mice (bottom) harvested 16 weeks after transplant were cocultured with 50,000 allogeneic human CD4+CD45RA+ naive or CD4+CD45RO+ memory T cells for 6 days. Cells were pulsed with [3H]-thymidine for the last 18 hours of culture before harvesting. Thymidine incorporation into the DNA of proliferating T cells is represented as cpm. Note the differences in the scale of the y-axis for the top and bottom panels. Results are representative of three independent experiments performed with mice reconstituted with CD34+ cells from three different donors and harvested 12, 14, and 20 weeks after transplant. Abbreviations: BDCA, blood dendritic cell antigen; cpm, counts per minute; LPS, lipopolysaccharide; mDC, myeloid dendritic cell.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The in vivo analysis of human DC development from hematopoietic progenitor/stem cells and the in vivo analysis of their role in initiating an immune response has been hindered by the lack of adequate model systems. For the most part, our understanding about the origins of DCs and their pivotal role in linking the innate immune response to the development and regulation of an adaptive immune response has been acquired from elegant in vitro and in vivo studies with murine DCs [3343]. In vitro studies using human cells have shown that human DCs can differentiate from several different progenitor or precursor populations. Both mDCs and pDCs can be obtained in vitro from CD34+ cells that have been stimulated for a period of time with combinations of cytokines such as GM-CSF and TNF-α or Flt3-ligand [2,5]. Myeloid DCs can also be derived in vitro from CD14+ monocytes that have been stimulated for a period of time with GM-CSF and IL-4 or from monocytes that have undergone reverse transmigration through an endothelial cell layer [4,8]. In this manuscript, we report the use of a xeno-graft model, the NOD/SCID mouse transplanted with human CD34+ cells, to examine in vivo human DC development, DC activation, and the response of DCs to inflammation.

In NOD/SCID mice transplanted with human CD34+ progenitor/stem cells, we identified cell populations that are known to give rise to mDCs in vitro, including human CD34+ early progenitors and CD34CD33+CD14+ monocytes. We also identified human progenitor and precursor populations of pDCs by using differential expression of CD34 and CD45RA [5]. The presence of these populations in the bone marrow of transplanted animals indicates that the production of human DCs from CD34+ cells is ongoing and sustained up to 24 weeks after transplant and that the NOD/SCID bone marrow provides a suitable microenvironment for DC ontogeny. The presence of both human mDCs and pDCs in murine hematopoietic tissues suggests that human DCs in transplanted mice are able to migrate to murine tissues. This notion is consistent with our previous study in which we demonstrated that human DCs are also distributed in nonhematopoietic tissues, including the skin, lungs, liver, and pancreas [11]. In addition, similar to human peripheral blood mDCs, both BDCA-1+ and BDCA-3+ mDC subsets were identified in transplanted animals. The presence of both BDCA-1 and BDCA-3 mDC subsets in the bone marrow and spleen of transplanted NOD/SCID mice gives additional credence to the authenticity of human DC ontogeny within the NOD/SCID mouse.

Because a lineage cocktail that included anti-human CD14 and anti-human CD16 among its components was used to identify human DCs in the transplanted mice, phenotyping information about other human myeloid cell populations, including human CD14+ and CD14+CD16+ monocytes, was not obtained. Experiments to examine the phenotype and function of human myeloid populations, including monocytes, monocyte subsets, macrophages, and granulocytes in this model, are currently underway. The ability to examine human myeloid cell populations in vivo will provide new insights into the interrelationships between human myeloid cells, their possible regulation, and their unique contributions to innate immune responses.

To further characterize the human cells that have developed from CD34+ cells within transplanted NOD/SCID mice, the in vivo response during acute inflammation using LPS was examined. Administration of LPS resulted in the release into the mouse plasma of human inflammatory cytokines, including IL-1β, TNF-α, IL-6, IL-8, IL-10, and IL-12p70. Furthermore, after administration of LPS, the highly selective activation and maturation of mDCs was documented. IL-1β, TNF-α, and IL-6 are cytokines well known to contribute to maturation of myeloid monocyte-derived DCs in vitro and therefore are the likely contributors to the maturation of human mDCs in vivo [44]. One of the advantages of using NOD/SCID mice for these types of experiments is that they have multiple defects in innate and adaptive immune responses [10], but it should be noted that a contribution by murine cells to the human DC activation cannot be completely ruled out. We compared human mDCs and pDCs in the steady state and during inflammation. Consistent with their activated phenotype, human mDCs from mice treated with LPS were found to be potent stimulators of allogeneic naive human T-cell proliferation in vitro compared with mDCs isolated from control mice. In addition, the capacity of mDCs to stimulate a more robust proliferative response in naive T cells than in memory T cells is consistent with a study in which it was reported that only a small percentage of human memory T cells (less than 5%) can respond in the MLR, whereas a larger percentage of human naive T cells (20%–30%) are responsive in the MLR [32]. These results confirm the functionality of the mDCs generated in vivo in these mice.

Differential Toll-like receptor (TLR) expression on DC subsets correlates with distinct cytokine response profiles after in vitro stimulation with pathogen-associated molecular patterns (PAMPs) [30,45]. For example, mDCs express TLR4, TLR7, and not TLR9, whereas pDCs do not express TLR4 but express TLR7 and TLR9 [46]. The fact that LPS interacts with TLR4 expressed by mDCs and not by pDCs explains why human mDCs that have developed in the transplanted mice respond to LPS by upregulating costimulatory molecules, whereas pDCs do not. Even though human TNF-α was produced in response to LPS stimulation and was present in the plasma of treated mice at 18 hours after LPS administration, we did not observe a change in the phenotype of human pDCs at this time point. However, it should be noted that this is consistent with the fact that upregulation of costimulatory molecules, CD83, and HLA-DR by CD11cneg pDCs occurs after 3 days of stimulation in vitro with TNF-α [47]. It should be noted that pDCs in these mice are not unresponsive, because we have previously shown that they respond to infection with influenza virus [11]. These in vivo responses are therefore consistent with the differential expression of TLR4 by mDCs.

A recent study identified DC precursors in the bone marrow of transplanted NOD/SCID mice as CD34HLA-DR+CD4+ cells that required an in vitro differentiation in GM-CSF and TNF-α for 14 days to become functional APCs, as assessed by their ability to stimulate allogeneic T cells in the MLR [12]. Using a different isolation procedure and different markers to identify human DCs, we found that freshly isolated human mDCs from the bone marrow of transplanted NOD/SCID mice could stimulate allogeneic human CD4+CD45RA+ in a dose-dependent manner, without cytokine stimulation in vitro. Moreover, Traggiai et al. [48] recently documented the presence of human CD11c+ mDCs and CD123+ pDCs in the hematopoietic and lymphoid tissues of Rag2−/− γc−/− mice that were reconstituted after intrahepatic injection with human CD34+ cells, confirming that other murine backgrounds in addition to the NOD/SCID can permit human DC development [48]. Unlike the transplanted NOD/SCID mice used in the studies described here, the Rag2−/–γc−/− mice develop human T cells. The molecular basis for the differential production of T cells in Rag2−/− γc−/− mice versus NOD/SCID mice is unknown at this time.

In conclusion, the presence of human DC progenitors, their distribution, and their functional response upon stimulation indicate that human DCs that have developed from human CD34+ cells seem to have differentiated appropriately within the microenvironment provided by the NOD/SCID mouse. In addition, our results highlight the usefulness of this model to examine the in vivo response to PAMPs by human DCs and to investigate the early events occurring during activation of innate immunity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Shana O'Reilly for expert technical assistance, Angela Mobley of the Dallas Cell Analysis Facility for help with flow cytometry, Alejandra Herrera for help with figures, Nancy Monson for the use of the cell harvester, and Alecia Curry for help with different aspects of this manuscript. We are grateful to James Thomas and Nitin Karandikar at University of Texas Southwestern Medical Center for helpful discussions and Laurie Davis, Iwona Stroynowski, Akira Takashima, and James Thomas for critical review of the manuscript and helpful discussions. This work was supported by grant CA82055 from the National Cancer Institute of the National Institutes of Health (to J.V.G.).

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  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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