Development and Activation of Human Dendritic Cells In Vivo in a Xenograft Model of Human Hematopoiesis
http://www.100md.com
《干细胞学杂志》
Department of Internal Medicine, Division of Infectious Diseases, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Key Words. Human/rodent chimera ? Stem cells ? Dendritic cells ? Inflammation
Correspondence: J. Victor Garcia, Ph.D., Division of Infectious Diseases Y9.206, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9113, USA. Telephone: 214-648-9970; Fax: 214-648-0231; e-mail: victor.garcia@utsouthwestern.edu
ABSTRACT
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 . 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 and plasmacytoid ) has been derived from in vitro experiments . 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 . 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 . 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 .
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 . 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)- was induced after the mice were injected with influenza virus, indicating that pDCs were functional . Previously, Nobuyoshi et al. reported human DCs present in the bone marrow of transplant ended/SCID mice with a CD34–CD4+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) . 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 . 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 ). 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
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 x 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 , 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 1. Frequency of human cells and human dendritic cell subsets in nonobese diabetic/severe combined immunodeficiency mice transplanted with human CD34+ cells
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 . 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+, CD33–CD34+, and CD33–CD34– 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 CD33–CD34+ early progenitor population (region c) or the CD33–CD34– population that corresponds to human CD19+ B cells (region d and data not shown) . 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 .
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 CD33–CD34+ early progenitor cells; and (d) includes CD33–CD34– 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.
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
To identify the pDC progenitors present in the bone marrow of reconstituted mice, the phenotyping criteria previously established by Blom et al. 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), CD34–CD45RA+ pre-pDC (region D), and CD34–CD45RA– 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 and normal human cord blood (Table 3).
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. and revealed the presence of CD34++CD45RA– early progenitor cells (A), CD34++CD45RA+ progenitor cells (B), CD34+CD45RA+ pro-DC2 (C), CD34–CD45RA+ pre-DC2 (D), and non-pDC CD34–CD45RA– 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 . 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.
Table 3. Progenitors of human pDCs in the bone marrow of NOD/SCID mice transplanted with human CD34+ cells and in human cord blood
In addition to their lack of lineage markers, human pDCs are characterized by coexpression of HLA-DR, CD123, and CD4 . 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 . 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 Lin–HLA-DR+CD123+ pDC and Lin–HLA-DR+CD11c+ mDCs in the bone marrow of NOD/SCID mice transplanted with human CD34+ cells . 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 and the frequency of human CD45+ cells in the lymph nodes is negligible , 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.
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 Lin–HLA-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 Lin–HLA-DR+ is indicated in the upper left corner of the dot plot. Lin–HLA-DR+ cells in both tissues were then examined for CD123 or CD11c expression. The percentage of each DC subset identified within the Lin–HLA-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.
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 . 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 . The differential expression of BDCA markers has been used to facilitate the isolation of human DC subsets from peripheral blood . 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 . 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.
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.
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 .
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 . In response to intravenously administered LPS, humans produce several proinflammatory cytokines such as interleukin (IL)-1?, tumor necrosis factor (TNF)-, IL-6, and IL-8 . 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-1?, TNF-, IL-6, IL-8, IL-10, IL-12p70, and IFN- 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- (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-1?, IL-6, and IFN- 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.
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.
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 . Specifically, LPS has been shown to induce maturation of human monocyte-derived DCs in vitro . 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.
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.
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 . 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.
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 -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.
DISCUSSION
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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Key Words. Human/rodent chimera ? Stem cells ? Dendritic cells ? Inflammation
Correspondence: J. Victor Garcia, Ph.D., Division of Infectious Diseases Y9.206, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9113, USA. Telephone: 214-648-9970; Fax: 214-648-0231; e-mail: victor.garcia@utsouthwestern.edu
ABSTRACT
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 . 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 and plasmacytoid ) has been derived from in vitro experiments . 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 . 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 . 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 .
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 . 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)- was induced after the mice were injected with influenza virus, indicating that pDCs were functional . Previously, Nobuyoshi et al. reported human DCs present in the bone marrow of transplant ended/SCID mice with a CD34–CD4+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) . 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 . 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 ). 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
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 x 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 , 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 1. Frequency of human cells and human dendritic cell subsets in nonobese diabetic/severe combined immunodeficiency mice transplanted with human CD34+ cells
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 . 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+, CD33–CD34+, and CD33–CD34– 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 CD33–CD34+ early progenitor population (region c) or the CD33–CD34– population that corresponds to human CD19+ B cells (region d and data not shown) . 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 .
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 CD33–CD34+ early progenitor cells; and (d) includes CD33–CD34– 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.
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
To identify the pDC progenitors present in the bone marrow of reconstituted mice, the phenotyping criteria previously established by Blom et al. 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), CD34–CD45RA+ pre-pDC (region D), and CD34–CD45RA– 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 and normal human cord blood (Table 3).
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. and revealed the presence of CD34++CD45RA– early progenitor cells (A), CD34++CD45RA+ progenitor cells (B), CD34+CD45RA+ pro-DC2 (C), CD34–CD45RA+ pre-DC2 (D), and non-pDC CD34–CD45RA– 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 . 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.
Table 3. Progenitors of human pDCs in the bone marrow of NOD/SCID mice transplanted with human CD34+ cells and in human cord blood
In addition to their lack of lineage markers, human pDCs are characterized by coexpression of HLA-DR, CD123, and CD4 . 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 . 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 Lin–HLA-DR+CD123+ pDC and Lin–HLA-DR+CD11c+ mDCs in the bone marrow of NOD/SCID mice transplanted with human CD34+ cells . 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 and the frequency of human CD45+ cells in the lymph nodes is negligible , 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.
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 Lin–HLA-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 Lin–HLA-DR+ is indicated in the upper left corner of the dot plot. Lin–HLA-DR+ cells in both tissues were then examined for CD123 or CD11c expression. The percentage of each DC subset identified within the Lin–HLA-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.
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 . 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 . The differential expression of BDCA markers has been used to facilitate the isolation of human DC subsets from peripheral blood . 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 . 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.
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.
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 .
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 . In response to intravenously administered LPS, humans produce several proinflammatory cytokines such as interleukin (IL)-1?, tumor necrosis factor (TNF)-, IL-6, and IL-8 . 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-1?, TNF-, IL-6, IL-8, IL-10, IL-12p70, and IFN- 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- (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-1?, IL-6, and IFN- 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.
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.
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 . Specifically, LPS has been shown to induce maturation of human monocyte-derived DCs in vitro . 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.
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.
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 . 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.
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 -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.
DISCUSSION
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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