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Plasmacytoid Dendritic Cells of Different Origins Have Distinct Characteristics and Function: Studies of Lymphoid Progenitors versus Myeloid Progenitors

Guo-Xiang Yang^1,*, Zhe-Xiong Lian^1,*, Kentaro Kikuchi^*, Yuki Moritoki^*, Aftab A. Ansari, Yong-Jun Liu, Susumu Ikehara and M. Eric Gershwin^2,*

^* Division of Rheumatology/Allergy and Clinical Immunology, University of California, Davis, CA 95616; Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322; Department of Immunology, M. D. Anderson Cancer Center, Houston, TX 77030; First Department of Pathology, Kansai Medical University, Moriguchi, Osaka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Plasmacytoid dendritic cells (pDCs) play a central role in host innate and adaptive immunity and are thought to be of lymphoid origin. However, in IL-7R^–/– mice, which are deficient in T and B lymphocytes, pDCs are still found in lymphoid organs, which suggests that there is a lymphoid-independent pathway for the development of pDCs. Previous work has demonstrated that pDCs originate from both lymphoid and myeloid progenitors (MPs). However, it is not clear whether the function of pDCs is different relative to their origin. In an effort to compare the characteristics and functions between pDCs generated from different progenitors, we performed adoptive transfer studies using highly enriched populations of common lymphoid progenitors (CLPs) and MPs from the bone marrow of control mice and examined their potential and developmental kinetics for the generation of pDCs. Interestingly, although CLPs were polarized to generate pDCs, MPs were polarized to generate conventional dendritic cells and the kinetics of pDC generation from MPs was reached earlier than from CLPs. Furthermore, CLPs have the potential to generate more pDCs on a per cell basis. Moreover, MP-derived pDCs produce relatively higher levels of IFN- than CLP-derived pDCs following CpG stimulation. These data indicate that MPs are multipotential and have the capacity to develop into not only myeloid cells, but also pDCs, which have distinct characteristics and function compared to that of lymphoid origin and, therefore, imply a more important role for MP-derived pDCs in conditions where the function of lymphoid progenitors is impaired or compromised.

	Introduction

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There have been a number of studies that have described the phenotype, localization, and function of human and mouse plasmacytoid dendritic cells (pDCs)³ (1). However, their origin still remains controversial. In early studies, human pDCs were described both as plasmacytoid monocytes and plasmacytoid T cells due to markers shared with both of these cell types (2). Previously, the thesis that pDCs are of lymphoid origin was supported by findings that genes originally found to be expressed only in developing T and B cells, such as pre-TCR-, 5, and Spi-B, are also expressed in pDCs, but not in myeloid cells (3, 4). The inhibition of Id2/Id3 expression prevents the development of pDCs, but not that of myeloid dendritic cells (DCs), which also suggests a lymphoid origin for pDCs (5). However, a myeloid origin for pDCs was proposed based on the finding that IL-3R^high pDCs were derived from CD34⁺ M-CSFR⁺ progenitors (6). Mice deficient in IFN consensus sequence binding protein, which is a critical transcription factor for monocyte development, display loss of pDCs (7). The findings that pDCs develop normally in both Notch^–/– and Tcf^–/– mice indicate that pDCs are not linked to the T cell, NK cell, and even B cell lineage (8). Therefore, pDCs may represent a composite group of both myeloid and lymphoid early-committed cells characterized by their capacity to differentiate into DCs (9). Indeed, using mouse transplantation models, recent reports have demonstrated that pDCs can originate from both lymphoid progenitors and myeloid progenitors (MPs) (10, 11, 12). However, whether a difference in the phenotypes or functions of pDCs exists based on their lineage origin remains unclear.

We report, herein, that both lymphoid and MPs have the potential to generate pDCs by intrasplenic (IS) injection. Furthermore, although common lymphoid progenitors (CLPs) are more likely to generate a greater number of pDCs on a per cell basis than that of MPs, our data demonstrate that MPs are the likely source of the majority of pDCs due to the higher abundance in the bone marrow (BM). Moreover, the greater production of select cytokines by MP-derived pDCs indicates that these cells have an important role in innate immunity.

	Materials and Methods

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Animals

C57BL/6-Thy1.2 (B6 Ly5.2) mice, congenic C57BL/6-Ly5.1-Pep3b (B6 Ly5.1) mice, and C56BL/6-IL-7R knockout (IL-7R^–/–) mice were obtained from The Jackson Laboratory. The F₁ mice of B6 Ly5.2 and B6 Ly5.1 were bred at the University of California at Davis animal facility. All mice were maintained under specific pathogen-free conditions and used at 8–12 wk of age for these studies.

Antibodies

FITC-conjugated Sca-1 (E13-161.7); PE-conjugated CD11c (HL3), CD19 (1D3), CD43 (Ly-48, S7), and CD127 (IL-7R, A7R34); biotin-conjugated CD8 (53-6.7), CD11c (HL3), CD40 (3/23), CD80 (B7.1, 16-10A1), CD86 (B7.2, GL1), CD123 (IL-3R, 5B11), CD127 (IL-7R, A7R34), Gr-1 (Ly-6G, RB6-8C5), CD11b (Mac-1, M1/70), and TER119 (TER119); allophycocyanin-CD19 (1D3); and purified CD4 (GK1.5) and CD11c (HL3) Abs were purchased from BD Pharmingen. FITC-conjugated CD4 (GK1.5), CD11c (HL3), Ly5.2 (CD45.2, 104); PE-conjugated IgM (II/41); biotin-conjugated NK1.1 (PK136), class II (M5/114.15.2), Ly5.1 (CD45.1, A20); PE-Cy5.5-conjugated Ly5.1 (CD45.1, A20), Ly5.2 (CD45.2, 104); allophycocyanin-conjugated c-kit (CD117, 2B8); purified CD3 (17A2), CD8 (53-6.7), CD19 (MB19-1), B220 (CD45R, RA3-6B2), CD11b (Mac-1, M1/70), Gr-1 (Ly6-G, RB6-8C5), TER119 (TER119), and CD16/32 (FcIII/IIR, 93). Abs were purchased from eBioscience. Allophycocyanin-conjugated B220 (CD45R, RA3-6B2), biotin-conjugated TCR (H57-597), PE-Cy5.5, and Tri-color-conjugated streptavidin were purchased from Caltag Laboratories. All isotype controls were obtained from BD Pharmingen.

Transplantation of progenitor cells

CLPs and MPs were purified by procedures described elsewhere (13, 14), with some modifications. Briefly, whole BM cells were collected from the femurs and tibiae of B6 Ly5.2 mice. The low-density (LD) cells (p = 1.077) were washed and incubated with a predetermined optimum dilution of rat mAbs against lineage-specific markers CD3, CD4, CD8, CD11c, CD19, B220, Mac-1, Gr-1, and TER119, followed by sheep anti-rat IgG-conjugated magnetic beads (Dynabeads; Dynal Biotech) to deplete the Ab-bound cells. The lineage-negative (Lin^–) cells were stained with FITC-anti-Sca-1, PE-anti-IL-7R, allophycocyanin-anti-c-kit, and biotin-anti-NK1.1 Abs, followed by Tri-color-streptavidin and subjected to flow cytometric-assisted purification. The CLPs were sorted as NK1.1^–IL-7R⁺Sca-1^lowc-kit^low cells; MPs were sorted as NK1.1^–IL-7R^–Sca-1^–c-kit^high cells by a 10-parameter MoFlo cell sorter (DakoCytomation). The purity of sorted cells was usually >97% and both CLPs and MPs were Thy1.2 negative, as determined by reanalyzing an aliquot of the collected cells (data not shown).

For transplantation of progenitor cells, female B6 Ly5.1 recipient mice were irradiated with 5 Gy 7–8 h before transfer. The sorted CLPs (3–5 x 10⁴) and MPs (5–10 x 10⁴) from B6 Ly5.2 mice were injected IS into the recipient mice (15). For cotransfer of progenitors, CLPs isolated from B6 Ly5.2 mice and MPs isolated from B6 Ly5.1 mice were cotransplanted in the spleen of 5 Gy-irradiated F₁ of B6 Ly5.1 and B6 Ly5.2 mice (Ly5.1⁺/Ly5.2⁺). Animals were maintained in a pathogen-free environment and aqueous antibiotics were added to the drinking water after transfer.

At several intervals after transplantation, recipient mice were sacrificed and spleen cells were collected. For detection of donor-derived pDC and DC populations, the LD cells were stained with FITC-anti-Ly5.2, PE-anti-CD11c, allophycocyanin-anti-B220, and PE-Cy5.5-anti-Ly5.1 Abs. Phenotypic characteristics of progenitor-derived pDCs were accomplished by incubation of the LD cells with FITC-anti-Ly5.2 PE-anti-CD11c, allophycocyanin-anti-B220, and biotin-anti-CD4, CD8, Mac-1, CD19, CD40, CD80, CD86, MHC class II, CD45RA, or IL-7R Abs, respectively, followed by PE-Cy5.5-streptavidin staining. The stained populations of cells were analyzed with a dual-laser FACSCalibur using CellQuest software (BD Biosciences).

Isolation and culture of splenic pDC

For sorting of pDCs from recipients transplanted with progenitor cells, spleen cells were collected from recipient mice that were transplanted with CLPs and/or MPs. After depletion of CD3, CD19, Mac-1, and TER119 Ab-bound cells by magnetic beads (Dynabeads; Dynal Biotech), the LD cells were stained with FITC-anti-Ly5.2, PE-anti-CD11c, allophycocyanin-anti-B220, and biotin-anti-Ly5.1, followed by Tri-color-streptavidin staining. After washing, progenitor-derived pDC subsets were sorted by a 10-parameter MoFlo cell sorter (DakoCytomation).

For sorting pDCs from control B6 or IL-7R^–/– mice, spleen cells were collected from 8- to 12-wk-old mice. The LD cells were incubated with a mixture of rat mAbs against CD3, CD19, Mac-1, Gr-1, and TER 119, followed by the addition of sheep anti-rat IgG-conjugated magnetic beads (Dynabeads; Dynal Biotech) to deplete the Ab-bound cells. The leftover DC-enriched cells were stained with FITC-anti-B220, PE-anti-CD11c, biotin-anti-NK1.1, and biotin-TCR, followed by Tri-color-conjugated streptavidin staining. After washing, cells were sorted based on the expression of NK1.1^–TCR^–CD11c⁺B220⁺. The purity of sorted cells was determined by reanalyzing an aliquot of the collected cells and was usually >97%.

All cell cultures were performed in RPMI 1640 culture medium (Invitrogen Life Technologies) supplemented with 10% FCS, 100 µg/ml streptomycin, and 100 U/ml penicillin (Invitrogen Life Technologies). Aliquots of 1–2 x 10⁴ sorted pDCs were cultured in 200 µl of RPMI 1640 medium in round-bottom 96-well plates with or without CpG-2216 (2 µM, ggG GGA CGA TCG TCg ggg gG; purchased from Invitrogen Life Technologies) or CpG-2006 (2 µM, tcg tcg ttt tgt cgt ttt gtc gtt; purchased from TriLink BioTechnologies); small letters indicate phosphorothioate linkage. After 48 h in culture, supernatants were collected and analyzed by ELISA for the following cytokines: IFN- (PBL Biomedical Laboratories), IL-6, IL-12p40, and TNF- (R&D Systems), with known standards.

T cell stimulation

Spleen cells collected from BALB/c (H-2K^d) mice were overlaid onto Histopaque-1.077 (Sigma-Aldrich) and centrifuged for 20 min at 750 x g. LD cells were collected from the interface and, after washing with PBS, incubated with a mixture of rat mAbs consisting of anti-CD8, anti-Mac-1/Gr-1, anti-CD11c, anti-CD19, anti-B220, and anti-TER119. Cells binding the mAbs were depleted using anti-rat Ig magnetic beads (Dynabeads; Dynal Biotech). The CD4⁺ T cells were then purified in the remaining cells by positive selection with CD4⁺ microbeads and MiniMACS separation columns (Miltenyi Biotec).

For mixed lymphoid reaction assays, 1 x 10⁵ CD4⁺ T cell were cocultured in triplicate with freshly isolated pDCs derived from different progenitor cells in the presence or absence of CpG-2006 (2 µM) in 200 µl of RPMI 1640 culture medium for 5 days in round-bottom 96-well plates. Supernatants were collected and IFN- was measured by ELISA. [³H]Thymidine (1 µCi/well) was added during the last 18 h of culture, and the uptake of [³H]TdR was quantitated in a liquid scintillation counter (PerkinElmer). The mean cpm of triplicate cultures was calculated.

Statistical analyses

Differences in the amount of cytokine production among pDC subsets were analyzed using the unpaired Student t test (StatView); values of p < 0.05 were considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Existence of pDC in IL-7R^–/– mice

IL-7R^–/– mice are severely deficient in both T and B lymphocytes (16, 17, 18). To investigate the distribution of pDCs and conventional DCs in IL-7R^–/– mice, we collected LD cells from the spleen, BM, and liver of these mice and analyzed them by flow cytometry. Fig. 1A shows that the population of CD11c⁺B220⁺ pDCs and CD11c^highB220^– DCs were detected in all organs examined. Both CD4⁺ and CD4^– pDC subpopulations (19, 20, 21), as well as CD8⁺ and CD8^– DC subpopulations, were detected in these tissues as well (data not shown). However, although the percentages of DC subsets were higher in the knockout mice, the cell number is significantly lower than that of the control B6 mice (pDCs 1.5 x 10⁵ vs 5 x 10⁵; DCs 5.3 x10⁵ vs 10 x 10⁵, both p < 0.05; Fig. 1B). These data indicate that although all known DC subsets were detected in IL-7R^–/– mice, the loss of IL-7R signaling affects the development of DCs.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Distribution of pDCs in IL-7R–/– mice. A, Spleen, BM, and liver mononuclear cells from IL-7R–/– and control B6 mice were stained with CD11c-PE and B220-allophycocyanin, and biotin-labeled CD3, CD19, NK1.1, CD11b, and TER119, followed by Tri-color-streptavidin. Tri-color-negative/CD11c-positive cells were analyzed for subsets of DC. B, Frequency and total cell number of pDCs and conventional DCs in spleens of in IL-7R–/– mice and control B6 mice. C, IFN- production. pDCs (2 x 104) isolated from spleen of IL-7R–/– or control B6 mice were cultured with CpG-2216 for 48 h; concentrations of IFN- were measured by ELISA. Data represent the mean ± SD and are representative of three independent experiments (*, pDC from IL-7R–/– mice vs pDC from B6 mice; p < 0.05).

IS injection of progenitors and analysis of donor cell phenotype

The Lin^–IL-7R^–Sca-1^–c-kit⁺ MPs cell population includes common MPs (CMP; CD34⁺CD16/32^low), granulocyte-macrophage progenitors (CD34⁺CD16/32^high), and megakaryocyte-erythrocyte progenitors (CD34^–CD16/32^low) (14). Granulocyte-macrophage progenitors demonstrate very poor DC precursor activity and megakaryocyte-erythrocyte progenitor do not give rise to detectable numbers of pDCs (10, 11). Therefore, in this study, we injected the Lin^–IL-7R^–Sca-1^–c-kit^high population as MPs. To compare differences between i.v. injection and direct IS injection, CLPs and MPs were purified from B6 Ly5.2 BM cells, as shown in Fig. 2A, and then transferred i.v. or IS into 5 Gy-irradiated B6 Ly5.1 mice. Significantly higher levels of donor-derived cells were detected in the recipient spleen injected by IS compared with i.v. (Fig. 2B). Therefore, IS injection was subsequently used to examine the developmental kinetics of pDCs for all of the remaining experiments in this study.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Comparison of IS and i.v. injection. A, Sorting gate and reanalysis of CLPs and MPs. B, Note the increase in donor cells by IS injection. Freshly sorted CLPs and MPs from B6 Ly5.2 mice were either IS or IV injected into B6 Ly5.1 congenic mice. One week after transfer, recipient spleen cells were collected and stained with FITC-Ly5.2, PE-CD11c, PE-Cy5.5-Ly5.1, and allophycocyanin-B220. The percentages of donor-type Ly5.2+Ly5.1– cells are illustrated.

It has been previously found that all DC subsets including pDCs are generated not only from lymphoid, but also from MPs (10, 11). To examine the developmental kinetics, purified CLPs and MPs from B6 Ly5.2 mice (Fig. 2A) were IS injected into sublethally irradiated 5 Gy congenic B6 Ly5.1 mice. DC production in the spleen by these progenitors was analyzed by flow cytometry after transplantation. As shown in Fig. 3A, CD11c⁺B220⁺ pDC and CD11c^highB220^– conventional DC populations appeared at 3 days and nearly disappeared 4 wk following transplantation with either CLPs or MPs, indicating that the pDC and DC populations by both CLPs and MPs were transient and lack self-renewal capacities. An interesting fact is that as early as 3 days after CLPs transfer, donor-derived CD11c⁺B220⁺ pDCs had reached 3% of the total donated population, whereas CD11c^highB220^– DCs were nearly undetectable at that time (Fig. 3A). Moreover, the percent of pDCs present in CLP-derived cells were always higher than that of DCs at any time point of detection. In contrast, MP-derived pDCs and DCs appeared at the same time as early as 3 days, but subsequently the pDC population was always smaller than that of DC (Fig. 3A). To address the kinetics of DC development from different progenitors, the total numbers of CLP- or MP-derived pDCs and DCs in the spleen were calculated and are shown in Table I. pDCs and DCs produced by MPs reached a peak at 7 days, whereas the two populations produced by CLPs reached a peak at 10 days after transfer. At the peak level, the total numbers of pDCs produced by CLPs were very much higher than those from MPs, but DC numbers were not significantly different between CLP-derived and MP-derived cells (Table I). Therefore, although MPs generate pDCs and DCs more rapidly, CLPs have a greater potential to quantitatively generate more pDCs on a per cell basis. In addition, the ratios of pDC:DC after transplantation were always >1 when they were generated by CLPs and <1 when they were MP derived (Fig. 3B). At peak levels of pDC generation, the average ratio of pDC:DC is 1.8:1 for CLP-derived cells and 0.7: 1 for MP-derived cells. These results indicate that CLPs are polarized to generate pDCs, whereas MPs are polarized to generate DCs. Additionally, using this transfer model, we found that MPs and CLPs can both generate the CD4⁺ and CD4^– pDC subsets and both CD8⁺ and CD8^– DC subsets (data not shown). Expression of CD4 and CD8 indicates that this is a reflection of the developmental stage of DC subsets.

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Production of splenic DC by different progenitors. Freshly sorted CLPs and MPs from B6 Ly5.2 mice were IS injected into B6 Ly5.1 congenic mice. At serial time points after transfer, recipient spleen cells were collected and stained with FITC-Ly5.2, PE-CD11c, PE-Cy5.5-Ly5.1, and allophycocyanin-B220. Donor-type Ly5.2+Ly5.1– cells were analyzed for CD11c+B220+ pDC and CD11chighB220– DC subpopulations. A, Examples of the phenotypes of pDCs and DCs at various time points after the transfer of CLPs or MPs. B, Cell numbers of pDCs and DCs generated per 105 CLPs or MPs are illustrated as ratios of pDCs:DCs. The data present here are the mean ± SD of three to four such experiments, and each time point included six to eight mice.

To compare the difference (if any) in the relative levels of expression of cell surface molecules between pDCs derived from CLPs and MPs, the phenotype of CD11c⁺B220⁺ pDCs was analyzed following transplantation. Both CLP- and MP-derived murine CD11c⁺B220⁺ pDC populations have the characteristic phenotype of pDCs. Thus, they are negative or weakly positive for the activation markers CD40, CD80, and CD86 and express MHC class II and CD45RA (data not shown). MP-derived pDCs do not express Mac-1, a cell surface molecule, normally restricted to myeloid lineage cells, including putative myeloid DCs, and express low levels of IL-7R, which is in line with CLP-derived pDCs (data not shown). We did not find significant differences between phenotypes of CLP- and MP-derived pDCs and normal splenic pDCs.

To address functional differences between CLP- and MP-derived pDCs, we sorted splenic CD11c⁺B220⁺ pDC subpopulations from recipient B6 Ly5.1 mice that had been IS injected 1wk previously with CLPs or MPs (Ly5.2) (Fig. 4A). Freshly sorted cells were cultured in vitro with or without CpG-2216 or CpG-2006 for 48 h. As shown in Fig. 4B, following CpG-2216 stimulation, MP-derived pDCs produced higher levels of IFN- than CLP-derived pDCs. These cells also produced higher amounts of IL-12p40 and the proinflammatory cytokines IL-6 and TNF- following stimulation with CpG-2006 (Fig. 4B). Therefore, on a per cell basis, MP-derived pDCs appear to synthesize higher levels of these cytokines than CLP-derived pDCs.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Production of cytokines by pDC. A, Sorting gate for pDCs. Freshly sorted CLPs and MPs from B6 Ly5.2 mice were IS injected into B6 Ly5.1 congenic mice. Seven or 10 days after transplantation, spleen cells of recipients were collected and stained with FITC-Ly5.2, PE-CD11c, PE-Cy5.5-Ly5.1, and allophycocyanin-B220. Donor-derived Ly5.2+Ly5.1–CD11c+B220+ pDCs were sorted as described in Materials and Methods. B and C, Cytokine production by CLP- or MP-derived pDCs sorted 7 days (B) and 10 days (C) after transfer. Aliquots of 1 x 104 pDCs sorted from spleen of recipient were cultured with CpG-2216 or CpG-2006 for 48 h. Concentrations of cytokines in the culture medium were measured by ELISA. Data represent the average values of triplicate samples ± SD and are representative of two independent experiments (MP-derived pDC vs CLP-derived pDC; *, p < 0.05).

Cotransplantation of myeloid and lymphoid progenitors

The differences between pDCs derived from lymphoid and MPs may be based on the fact the pDCs developing from CLP- or MP-transplanted animals were exposed to different milieus in vivo; this probably reflected in the following in vitro analysis of cytokine production. Thus, to exclude environmental influences, CLPs isolated from B6 Ly5.2 mice and MPs isolated from B6 Ly5.1 mice were cotransplanted in 5 Gy-irradiated B6 Ly5.1⁺/Ly5.2⁺ F₁ mice. Both CLPs and MPs generated pDCs in this model (Fig. 5A). Similarly, pDCs produced by MPs reached their peak after 7 days, whereas CLPs reached their peak after 10 days (Fig. 5B).

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Production of splenic pDCs following cotransplantation with different progenitors. Freshly sorted CLPs (from B6 Ly5.2 mice) and MPs (from B6 Ly5.1) were IS injected into B6 Ly5.1+Ly5.2+ congenic mice. At serial time points after the transfer, recipient spleen cells were collected and stained with FITC-Ly5.2, PE-CD11c, PE-Cy5.5-Ly5.1, and allophycocyanin-B220. Donor-type Ly5.2+Ly5.1– or Ly5.2–Ly5.1+ cells were analyzed for CD11c+B220+ pDC and CD11chighB220– DC subpopulations. A, Note the pDCs 10 days after the transfer of CLPs and MPs. B, The time course of pDC generated by CLPs or MPs are illustrated; each time point includes six to eight mice. C, Production of cytokine by different original pDCs. Seven and 10 days after transfer, aliquots of 1 x 104 pDCs sorted from spleen of recipient were cultured with CpG-2216 or CpG-2006 for 48 h. Concentrations of cytokines in the culture medium were measured by ELISA. Data represent the average values of triplicate samples ± SD. (MP-derived pDC vs CLP-derived pDC; *, p < 0.05).

T cell allostimulation by pDCs of different origins

CpG studied herein has the potential to enhance the expression of costimulatory molecules expressed by pDC and stimulate them to differentiate into mature DC (20, 21). To examine whether pDC of different origins have altered capacities to activate T cells, CD4⁺ T cells were cocultured with varying numbers of allogeneic CLP- or MP-generated pDCs. Freshly isolated CLP- or MP-generated pDCs from the spleen of recipients cotransferred with progenitors did not stimulate T cell proliferation when cultured in vitro without CpG-2006 (data not shown). In the presence of CpG-2006, however, both CLP- and MP-generated pDC stimulated allo-T cell proliferation. The stimulating capacity of MP-derived pDCs proved to be stronger than that of CLP-derived pDCs (Fig. 6A). Importantly, we also demonstrated that T cells stimulated by MP-generated pDCs synthesized higher level of IFN- (Fig. 6B). These results indicate that MP-derived pDCs have a greater potential to activate allogeneic T cells than those of lymphoid origin.

View larger version (10K): [in this window] [in a new window]   FIGURE 6. Allogeneic T cell stimulation. Freshly isolated pDCs from spleen of cotransferred mice were cultured with 1 x 105 CD4+ T cells from BALB/c mice in the presence or absence of CpG-2006. After 5 days of culture, cells were pulsed with [3H]thymidine for 16 h before harvesting (A). Cell proliferation was determined by [3H]thymidine incorporation. B, IFN- in the supernatant was measured by ELISA. Data represent the average values of triplicate samples ± SD (MP-derived pDC vs CLP-derived pDC; *, p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

It is of interest whether pDCs derived from lymphoid and MPs have distinct functions. Conventional DCs, regardless of derivation from lymphoid progenitors or MPs, are able to present allogeneic Ags efficiently and are equivalent in IL-12 production (24, 25). However, we found that MP-derived pDCs produce significantly higher levels of IFN- and other cytokines after CpG stimulation (Figs. 4 and 5). These pDCs are also stronger in their stimulation of allo-T cell reactions (Fig. 6). Although CLPs are more efficient in producing pDCs on a per cell basis, it is plausible that due to the large number of MPs in the BM, most peripheral pDCs are of myeloid origin. Therefore, pDCs derived through the myeloid pathway may play important roles in host innate immunity. However, it is still unclear whether pDCs isolated from mouse spleen and liver express B cell genes and are more closely related to lymphoid rather than myeloid cells (20). In fact, pDCs have been demonstrated to share early stages of development with cells of the lymphoid lineage (26), and there is a common pathway for DCs and early B cell development (27). It has been recently suggested that precursor activities of pDCs branch off between the stages of CLP conversion to pro-B cells (10). Interestingly, our transfer data demonstrate that MPs also have the capacity to differentiate into not only pDCs and DCs but also mature B cells (data not shown). B cell precursors were further found to predominantly exist within Flt3⁺ fractions of the CMP, which is a population, enriched in DC progenitors (10). These data suggest that myeloid-derived pDCs also have a developmental linkage with B lineage cells, similar to lymphoid-derived pDCs. If so, it may explain why pDCs have both myeloid and lymphoid origins but show mainly lymphoid characteristics. Indeed, it has been demonstrated that myeloid original pDCs, like B lymphoid cells, also express RAG, pre-TCR-, and D-J IgH rearrangements (11). Therefore, the relationship between gene expression in pDCs and lymphoid cells may not reflect their lineage origin.

The fact that all subsets of pDCs and conventional DCs can be generated by both lymphoid and MPs raises the question as to whether a common DC progenitor lineage exists in the BM of adult mice, and whether the isolation methods used herein caused them to be distributed into lymphoid and MP subsets. In both CMPs and CLPs, the potential of DC in the BM is best defined by the level of expression of Flt3 rather than the established myeloid or lymphoid precursor markers (10). A common progenitor for all DCs, including CD8⁺ and CD8^– DCs, as well as B220⁺ pDCs, has been found in mouse peripheral blood and BM (28, 29). However, the relationship between CLP and CMP to this common DC progenitor in BM and in peripheral blood is unclear. Our data show that lymphoid and MPs have different developmental kinetics and polarizations for pDC and DC development (Fig. 2 and Table I). This indicates that there may not be a single DC progenitor population that is resident in both BM CLPs and MPs, despite the fact that Flt3L can drive DC development along the lymphoid and myeloid developmental pathways from Flt3⁺ progenitors to Flt3⁺ DCs (30). In addition, the fact that MP-derived pDCs produce higher amounts of cytokines in response to in vitro CpG stimulation than that of CLPs also supports the thesis that different DC progenitors exist in CLPs and MPs (Fig. 4). It would be important to compare the response of pDCs to other stimuli, including viral challenges, since these cells play a major role in antiviral defense.

Similarly, we noted that pDC and DC reached their peak level at 2 wk after i.v. transfer (10, 11, 24, 30), whereas IS injection shortened the pDC and DC peak time from both CLPs and MPs (Table I). This suggests that the splenic microenvironment is important for the differentiation of progenitor cells into DCs. However, it should be noted that both lymphoid and MPs are physiologically contained in BM, and BM is likely the ideal developmental environment. Therefore, future work should attempt the injection of progenitor cells directly into the BM of recipients to investigate developmental characteristics.

IL-7 is a stromal-derived, lymphoid-specific cytokine known to regulate lymphocyte development and homeostasis. The loss of IL-7R signaling results in mice severely deficient in both T and B lymphocytes (16, 17); however, pDCs still persist in various tissues of IL-7R^–/– mice (Fig. 1). When cultured in vitro in the presence of a mixture containing IL-7, stem cell factor, and Flt3L, CLPs generated high numbers of B cells but not pDCs (12), suggesting that pDCs develop along the IL-7-independent pathway. In contrast, cell numbers of pDCs in the lymphoid tissue of IL-7R^–/– mice were significantly decreased, which may imply that a lymphocyte-deficient environment influences pDC development (Fig. 1). Although both lymphoid- and myeloid-derived pDCs expressed higher levels of IL-7R by RT-PCR (11), we found IL-7R expression to be low on the cell surface of all of pDCs, regardless of their origin (data not shown), Therefore, IL-7R expression may not, similar to D-J IgH and RAG, reflect pDC derivation (31). In addition, higher IFN- production may not be a feature of IL-7R-deficient pDCs. pDCs exposed to a different environment in IL-7R^–/– mice may influence their function compared with normal mice.

Lymphoid and myeloid cells generally differentiate along independent pathways, where CLPs and CMPs likely represent the earliest committed branch points (13, 14). Therefore, we submit that lymphoid- or myeloid-derived pDCs are characteristically distinct. Although we could not find any apparent phenotypic differences, developmental and functional differences exist between the myeloid- and lymphoid-derived pDCs. Indeed, it has been found that only MPs give rise to liver pDCs using an in vivo reconstitution assay (12). Adding Flt3L alone to in vitro cultures was sufficient to support the development of pDCs from MPs, whereas CLPs required additional survival factors provided either by stromal cells or by introduction of transgenic Bcl-2 (12). In RAG1/GFP knock-in mice, GFP⁺ pDCs, which appear to be derived from early lymphoid progenitor and relate to B-lineage cells, were less potent than GFP^– pDCs in T cell allostimulation and cytokine production (32). These observations are consistent with our observations that pDCs derived from different progenitors have distinct functional characteristics. Further studies are needed to clarify whether pDCs of different origins have different capabilities to interact with immune effector cells and whether they vary in their response to different viral and microbial pathogens. It is also plausible that their cytokine profiles will contribute to their specific roles in autoimmune diseases.

	Acknowledgments

 
We thank Carol Oxford for FACS sorting, Loreli Coleman for mouse irradiation, and Nikki Phipps for manuscript preparation.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ G.-X.Y. and Z.-X.L. contributed equally to this work.

² Address correspondence and reprint requests to Dr. M. Eric Gershwin, Division of Rheumatology, Allergy and Clinical Immunology, Genome and Biomedical Sciences Facility, Suite 6510, University of California, Davis, CA 95616. E-mail: megershwin{at}ucdavis.edu

³ Abbreviations used in this paper: pDC; plasmacytoid dendritic cell; DC, dendritic cell; IS, intrasplenic injection; CLP, common lymphoid progenitor; MP, myeloid progenitor, BM, bone marrow; LD, low density; CMP, common MP.

Received for publication November 24, 2004. Accepted for publication September 15, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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