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A Common Pathway for Dendritic Cell and Early B Cell Development

Department of Pathology and Laboratory Medicine and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4⁺ T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In adult mice B cells develop ultimately from hemopoietic stem cells (HSCs)³ in the bone marrow (BM). Although it is generally thought that the earliest B-lineage-committed precursors derive from semirestricted progenitors characterized by their ability to give rise to two or more lymphoid lineages, the precise identities of these pivotal developmental branch points and the signals promoting their differentiation toward the B cell vs alternative lymphoid lineages are not well characterized.

The signals and cellular compartments underlying dendritic cell (DC) development are also unclear (reviewed by Banchereau et al. (1)). Although controversial, several studies suggest that functionally unique subsets of DCs arise from distinct pools of lineage-restricted progenitors. For instance, putative lymphoid DCs, defined by a CD8⁺CD11b^- surface phenotype, are selectively reduced following removal or inhibition of certain lymphoid-specific transcription factors (2, 3). This contrasts with mice lacking RelB or PU.1, which exhibit specific defects in myeloid development and selective reductions in CD8^-CD11b⁺ DCs (4, 5). Furthermore, early thymocyte precursors lacking detectable myeloid potential, including a B/T/NK precursor population and a downstream progenitor pool lacking B and NK cell potential, each give rise to CD8⁺ DCs (6, 7, 8, 9, 10, 11). However, recent studies indicate that different DC surface phenotypes are not determined by ontogeny. For instance, Martin et al. report that thymic CD4^low progenitors give rise to both CD8^- and CD8⁺ DCs (12), and Traver et al. recently demonstrated that a myeloid-restricted progenitor population gives rise to CD8⁺ DCs (13). These observations suggest that DC expression of CD11b and CD8 may be determined by signals unique to particular microenvironments.

In this report we take advantage of a culture system previously described to support DC differentiation from early thymocytes (9) to assess DC precursor potential among a wide spectrum of B-lineage progenitor populations representing varying degrees of B-lineage commitment. These include pluripotent HSCs, common lymphoid progenitors (CLPs) and pre-pro- and pro-B cells previously shown to give rise to B cells, but not T or NK cells. First, we confirm and extend previous reports by Kondo et al. (14, 15) by showing that CLPs can be defined among lineage marker (Lin)^- BM cells via coexpression of IL-7R and the C1q receptor (C1qR/AA4). Second, we show that both CLPs and a subset of pre-pro-B cells, defined previously by their expression of sterile transcripts derived from the IgH locus and a CD4⁺B220⁺CD19^- surface phenotype, rapidly differentiate into DCs in culture. Furthermore, among B-lineage precursor populations, the capacity to give rise to DCs was clearly restricted to CD4⁺B220⁺ pre-pro-B cells, as CD4^- pre-pro-B cells as well as more mature B-lineage cells failed to differentiate into DCs. Together, these findings reveal a previously unrecognized common developmental pathway for early B and DC ontogeny and demonstrate that loss of DC differentiative potential in the final step in the onset of B-lineage commitment.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and cell preparation

Six- to 10-wk-old C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and B6.Ly5.1 (referred to herein as B6.Ly5^SJL) mice were purchased from the National Cancer Institute animal facility (Frederick, MD). BM cells were flushed from tibias and femurs of 8- to 10-wk-old C57BL/6 mice with FACS buffer (PBS containing 3% FCS 1 mM EDTA and 0.05% sodium azide). Following lysis of RBCs with 0.165 M NH₄Cl₂, cells were washed, then stained with the appropriate Abs before sorting.

CD4⁺ T cells were prepared by perfusing spleens from C57BL/6 and BALB/c mice with cold PBS containing 3% FCS. Following RBC lysis, cell suspensions were incubated with ascites containing anti-B220 Abs (RA3-6B2) and purified CD8, CD11b, and CD14 (PharMingen, La Jolla, CA) before washing and depletion with sheep anti-rat Ig-coated magnetic microspheres (Dynal, Lake Success, NY) according to the manufacturer’s instructions.

Abs, cell sorting, and analytical flow cytometry

For cell sorting experiments BM suspensions were stained with optimal dilutions of directly conjugated fluorescent Abs for 30 min on ice, then washed twice in FACS buffer. For HSCs, cells were stained with fluorescein (FL) anti-Sca-1/Ly6 A/E (E13-161.7), PE-labeled lineage markers (B220 (RA3-6B2), CD11b/Mac-1 (M1/70), Gr-1 (8C5), Ter-119, and CD3 (2C11), and allophycocyanin anti-CD117/c-kit (2B8). For CLPs, cells were stained with FL-anti-Sca-1, PE-anti-Lin, PE-Cy5-anti-IL-7R (A7R34) (16), and allophycocyanin-anti-C1qR/AA4.1 (17, 18). For pre-pro-B cell subsets, cells were stained with FL-anti-B220, PE-anti-AA4.1, biotin (BI)-anti-CD24/HSA (30F1), and allophycocyanin-anti-CD4 (GK1.5). For pro- and pre-B cells, cells were stained with FL-anti-CD43 (S7), PE-anti-CD19 (1D3), and BI-anti-sIgM (R6-60). Biotinylated Abs were revealed with streptavidin-coupled PE-Cy5, and all reagents were purchased from PharMingen except for B220, A7R34, AA4.1, and GK1.5, which were purified and conjugated by standard methods in our laboratory.

Each cell population was purified on a 10-parameter MoFlo cell sorter (Cytomation, Fort Collins, CO) equipped with Summit software and three lasers, including an I-90C argon laser tuned to 488 nm and an I-70C Spectrum argon/krypton laser (both obtained from Coherent, Santa Clara, CA) tuned to 647 nm for excitation of allophycocyanin and its derivatives.

The following Abs were purchased from PharMingen and used for analysis of adoptive transfers and culture assays described below: FL-anti-I-A^b (AF6-120.1), anti-Ly5^B6 (104), and anti-Ly5^SJL (A20); PE-anti-CD11c (HL3), CD19, or CD3 (2C11); BI-anti-CD11b; and allophycocyanin-anti-CD8 (53-6.7), CD11b, or TCR (H57). After staining, propidium iodide (final concentration, 1 µg/ml) was added for dead cell exclusion, and cells were analyzed on a dual laser FACSCalibur (BD Biosciences, San Jose, CA) equipped with CellQuest software. All flow cytometric data were analyzed by uploading files into FlowJo (Tree Star, San Carlos, CA).

Adoptive transfers

For each recipient, 3000 sorted CLPs or HSCs from C57BL/6 mice were mixed with 10⁵ unfractionated BM cells from B6.Ly5^SJL mice, then transferred i.v. into B6.Ly5^SJL recipients given 900 rad 20 h previously. Twenty to 30 days later, recipient peripheral blood was stained with FL-anti-Ly5^B6, PE-anti-CD19, BI-anti-CD11b (revealed with PE-Cy5 streptavidin), and allophycocyanin-anti-CD3, and CLP or HSC-derived cells were identified by gating on Ly5^B6+ cells.

Stromal cell cultures

Single HSCs or CLPs were sorted directly onto 96-well flat-bottom plates containing pre-established monolayers of S17 stromal cells and 200 µl complete medium (Opti-MEM with 5% FCS (Irvine, CA, Santa Ana, CA), 10 mM glutamine, 10 mM HEPES, 0.5 mg/ml gentamicin, and 5 x 10^-5 2-ME) supplemented with 100 ng/ml rIL-7 (R&D Systems, Minneapolis, MN). Single-cell sorting was accomplished using single-sort mode with a one-drop sort envelop and the CyCLONE automated cell cloner (Cytomation). Fourteen days later wells containing cell growth were counted, and cells were stained for flow cytometry.

Myeloid lymphoid progenitor (MLP) assay

A variation of the MLP assay originally described by Kawamoto et al. (19) was used to simultaneously assess B cell, T cell, and myeloid activities from individual HSCs or CLPs. For these experiments single-sorted cells were microinjected into day 15 fetal thymi given 2700 rad 20 h previously, and individual inoculated thymi were placed onto polycarbonate filters (Millipore, Bedford, MA) and floated in 2 ml complete medium containing 3 µg/ml IL-3, 10 ng/ml IL-7, and 10 ng/ml stem cell factor (SCF; R&D Systems). After 7 days medium was replaced with complete medium containing IL-3 and IL-7, but not SCF. Seven days later single-cell suspensions were prepared from individual thymi and stained for flow cytometric analysis.

DC precursor cultures

Progenitor cells were sorted directly into complete medium containing 2 ng/ml IL-1, 400 ng/ml IL-3, 10 ng/ml IL-7, 10 ng/ml SCF, 100 ng/ml Flt3L, and 1 ng/ml TNF- (R&D Systems) as previously described (20). After sorting, 1000 cells/well in 200 µl were added to V-bottom 96-well plates and incubated for 4–6 days before harvesting and determination of viable cell numbers and expression of indicated cell surface molecules by flow cytometry.

MLR

DC precursor cultures were initiated with 5000 cells/well and extended to 9 days to generate sufficient numbers of DCs. Fifty percent of the medium was exchanged with fresh cytokine-containing medium every 3 days. On day 9 viability was determined by trypan blue exclusion and graded doses of DCs mixed in quadruplicate at a final volume of 200 µl with 100,000 purified CD4⁺ splenic T cells in 96-well round-bottom plates containing complete medium. Six days later cultures were pulsed with 1 µCi [³H]thymidine for 18 h before scintillation counting.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Resolution of early B-lineage progenitor populations

Fig. 1 illustrates our strategy for the resolution of several early B-lineage precursor populations in adult mouse BM. These include a Lin^- IL-7R⁺ C1qR/AA4⁺ Sca-1^low population representing 0.04% of total BM, which we will show below to correspond to CLPs (21) (Fig. 1A), and two subsets of early B-lineage cells (pre-pro-B cells, fractions (Fr.) A₁ and A₂). Significantly, each pre-pro-B cell subset represents 0.1% of total BM (Fig. 1B) and is characterized by the expression of sterile IgH locus transcripts but lack of the pan-B cell marker CD19 (15), and was shown previously to give rise to pro-B cells, but not T or NK cells or cells of the myeloid or erythroid lineage (14).

View larger version (82K): [in this window] [in a new window]   FIGURE 1. Resolution of CLPs and pre-pro-B cells in adult mouse BM. A, Unfractionated BM cells from an 8-wk C57BL/6 mouse were stained with FL-anti-Sca-1, PE-anti-lineage markers (see Materials and Methods), PE-Cy5-anti-IL-7R, and allophycocyanin-AA4.1. B. BM cells from the same mouse were stained with FL-anti-B220, PE-AA4.1, BI-anti-CD24/HSA, and allophycocyanin-anti-CD4. For A and B, data were generated by analyzing 200,000 events and are representative of at least five separate experiments.

Identification of CLPs in adult BM via coexpression of IL-7R and AA4

Several lines of evidence suggest that the Lin^-IL-7R⁺C1qR/AA4⁺ Sca-1^low population illustrated in Fig. 1A corresponds to the BM CLP population described by Kondo et al. (21). First, in competitive adoptive transfer experiments Lin^- IL-7R⁺ C1qR/AA4⁺ Sca-1^low cells gave rise to donor-derived B and T cells, but not CD11b⁺ myeloid lineage cells (Fig. 2). This finding contrasted with recipients of Lin^-c-kit⁺Sca-1⁺HSCs, in which CD11b⁺ cells were readily detected. Second, single Lin^-IL-7R⁺ AA4⁺Sca-1^low cells gave rise to B cells, but not macrophages, when cultured on S17 stromal cells, and this also contrasted with cultures seeded with HSCs that typically contained both CD19⁺ CD11b^- B cells and CD19^-CD11b⁺ macrophages (Table I). Third, because S17 cultures do not support T cell development, we injected single HSCs vs CLPs into the MLP assay previously shown to support B cell, T cell, and myeloid development from HSCs (19). As shown in Fig. 3, donor-derived (Ly5^SJL+) CD11b⁺ myeloid as well as TCR⁺ T cells and CD19⁺ B cells were detected when fetal thymi were seeded with single HSCs. In contrast, seven of seven thymi seeded with BM CLPs (Lin^-IL-7R⁺ AA4⁺Sca-1^low cells) contained detectable numbers of donor-derived TCR⁺ T cells and CD19⁺ B cells, but not CD11b⁺ myeloid cells. Finally, Lin^-IL-7R⁺AA4⁺Sca-1^low cells also expressed a c-kit^low CD24/HSA^intermediate Thy-1.2^- surface phenotype as previously described for BM CLPs (data not shown) (21). Together, these data confirm the existence of a CLP population in adult mouse BM and support the possibility that particular combinations of environmental stimuli can drive CLPs and perhaps early B-lineage cells to adopt different cell fates.

View larger version (52K): [in this window] [in a new window]   FIGURE 2. Generation of B and T cells, but not myeloid lineage cells, following adoptive transfer of CLPs. Three thousand sorted BM HSCs (Lin-IL-7R-Sca-1+CD117/c-kit+) or CLPs (Lin-IL-7R+AA4+Sca-1low) from C57BL/6 (Ly5B6) mice were mixed with 105 unfractionated BM cells from B6.Ly5SJL congenics before transfer to lethally irradiated B6.Ly5SJL recipients. Data show analyses of PBL 21 days post-transfer, and are representative of five mice per group at 17–40 days post-transfer. CLP- and HSC-derived cells are identified as Ly5B6+.

View this table: [in this window] [in a new window]   Table I. Lin-IL-7R+AA4+Sca-1low progenitors yield pro-B cells but not macrophages in stromal cultures1

View larger version (23K): [in this window] [in a new window]   FIGURE 3. CLPs give rise to clonal populations of B and T cells. Single HSCs vs CLPs derived from B6.Ly5SJL mice were injected into irradiated C57BL/6 day 15 fetal thymi that were then placed in fetal thymic organ cultures supplemented with the cytokines described in Materials and Methods. A, Representative flow cytometric analysis of individual thymi inoculated with single HSCs or CLPs. B, The percentages of HSC- and CLP-derived (Ly5SJL+) cells giving rise to B, T, and myeloid lineage cells in individual thymi are shown.

We assessed the degree to which several lymphoid precursor populations differentiated into DCs in culture by exposing 1000 sorted cells from each population to a cytokine combination previously shown to promote lymphoid DC development from thymic progenitors (9). The progenitor populations tested include HSCs, CLPs, CD4⁺B220⁺ (Fr. A₁), and CD4^-B220⁺ (Fr. A₂) pre-pro-B cells and pro-B cells (Frs. B/C) defined by a CD19⁺CD43⁺sIgM^- surface phenotype and readily detectable heavy chain rearrangements (20, 22). Significantly, pre-pro-B cells and pro-B cells each lack detectable T-lineage progenitor activity when derived from wild-type mice (14) and thus serve as a test of whether DC progenitor activity is maintained after loss of T-lineage potential during BM lymphopoiesis. We chose this in vitro DC progenitor assay over in vivo adoptive transfer experiments for several reasons. First, these cultures allow a direct and immediate assessment of DC differentiative potential and thus avoid complications arising from the potential maturation of multipotent progenitors into more immediate DC precursors. Second, unlike adoptive transfer of CLPs, we have found that detection of donor cells derived from Fr. A₁ requires the transfer of at least 20,000 cells/recipient, making it difficult to control for small numbers of contaminating HSCs. Therefore, we controlled for potential contamination by directly comparing parallel short term cultures seeded with as few as 1000 cells derived from a wide array of progenitor populations. Finally, with this system we can readily assess differences in proliferation, surface phenotype, and function that result from exposure of different progenitors to the combination of cytokines used.

Although all DC subsets in mice can be identified via surface coexpression of MHC class II and CD11c (23), we did not find detectable class II surface expression in any of the progenitor populations examined (data not shown). However, as shown in Fig. 4, among HSCs and the lymphoid-restricted progenitor populations tested, both CLPs and CD4⁺B220⁺ (Fr. A₁) pre-pro-B cells gave rise to class II⁺CD11c⁺ cells after 4 days in culture. Significantly, these cells also exhibited a high forward and side light scatter profile typical of DCs (data not shown), and cultures seeded with CLPs and CD4⁺B220⁺ (Fr. A₁) pre-pro-B cells contained 60–70% and nearly 100% class II⁺CD11c⁺ cells, respectively (Fig. 4). These data clearly contrasted with those for cultures seeded with equivalent numbers of CD4^-B220⁺ pre-pro-B cells (Fr. A₂) and CD19⁺CD43⁺ pro-B cells (Frs. B/C), in which significant numbers of DCs were not detected (Figs. 4 and 5). There were also significant differences in numbers of viable cells and DCs recovered from cultures seeded with Fr. A₁ vs CLPs, with 4-fold increases in numbers of CLP-derived DCs over input cell numbers compared with no more than 2-fold increases in cultures seeded with Fr. A₁ pre-pro-B cells (Fig. 5). Although CD19⁺CD43⁺ pro-B cells readily expanded in response to these culture conditions on day 4, and we were unable to detect class II⁺ CD11c⁺ cells in these cultures. Furthermore, most of these cells remained CD19⁺ and also failed to up-regulate expression of surface molecules associated with DC function such as CD80 and CD86 (Fig. 5 and data not shown). Thus, among these early B-lineage precursor populations, we found that only CLPs and CD4⁺ B220⁺ pre-pro-B cells contained significant numbers of cells susceptible to cytokine-induced DC differentiation.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. CLPs and CD4+ pre-pro-B cells (Fr. A1) selectively give rise to DCs in culture. One thousand sorted cells from the indicated progenitor populations from C57BL/6 (H-2b) adults were cultured in quadruplicate wells with IL-1, IL-3, IL-7, Flt-3L, SCF, and TNF- as described in Materials and Methods. On day 4 of culture the resulting cells were stained with Abs to the indicated surface molecules and analyzed by flow cytometry. A large forward side scatter gate was drawn to include both lymphocytes and DCs, and dead cells were excluded with propidium iodide. Data are representative of four separate experiments.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. CLPs and early B-lineage cells exhibit unique degrees of proliferation in response to cytokines inducing DC differentiation. Numbers of viable and class II+ CD11c+ cells in quadruplicate cultures containing the cytokines described in Fig. 4 and seeded with the indicated population were determined by flow cytometry. The percentages of cells that were viable (PI-) or expressed a DC surface phenotype were determined by multiplying total events derived from each tube by the percentages of cells that were PI- and PI- class II+CD11c+, respectively. Data are representative of four separate experiments.

To examine DC function, we tested whether graded doses of CLP- and Fr. A₁-derived DCs could stimulate the proliferation of resting allogeneic T cells and tested for up-regulation of CD80 and CD86. Because Fr. A₁ yields limiting numbers of DCs in these cultures (Fig. 5), only three doses of Fr A₁-derived DCs were used in these experiments compared with six doses of CLP-derived DCs. As shown in Fig. 6, C57BL/6 (H-2^b) CLP-derived (Fig. 6A) and Fr. A₁-derived (Fig. 6B) class II⁺ CD11c⁺ cells readily stimulated the proliferation of CD4⁺ T cells purified from BALB/c (H-2^d), but not C57BL/6 splenocytes. Second, class II⁺ cells induced from both CLPs and Fr. A₁ also expressed the CD28 costimulatory ligands CD80 and CD86 (Fig. 7). Together, these data demonstrate that exposure of CLPs and CD4⁺B220⁺ (Fr. A₁) pre-pro-B cells to cytokines known to induce DC differentiation resulted in the generation of functional DCs.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. MLR initiated with CLP- and Fr. A1-derived DCs. A, Graded doses of CLP-derived DCs sorted from C57BL/6 mice were mixed in quadruplicate with 100,000 purified splenic CD4+ T cells from BALB/c (•) or C57BL/6 () mice and proliferation assessed by pulsing cultures with [3H]thymidine for 18 h beginning on day 6. B, Graded doses of Fr. A1-derived DCs sorted from C57BL/6 mice used to stimulate BALB/c (•) or C57BL/6 () CD4+ T cells.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Cell surface phenotypes of CLP and CD4+ pre-pro-B (Fr. A1)-derived DCs. Cells from cultures seeded with the indicated progenitor population were stained for expression of class II and the indicated surface molecule. Unfilled curves represent staining of cells with an isotype-matched control Ab. All plots are gated on class II+ cells. Data are representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our data demonstrate a previously unrecognized link between B and DC development and allow three additional novel conclusions regarding early B and DC ontogeny. First, both CLPs and CD4⁺ B220⁺ pre-pro-B cells (Fr. A₁), the latter of which lacks detectable T and NK cell precursor activity (14), gave rise to DCs in culture. Thus, we conclude that the loss of DC precursor potential is the final step in B-lineage commitment. Second, among lymphoid progenitors, the capacity to give rise to DCs is not limited to a single population, but, in fact, appears to be restricted to early B-lymphoid progenitors that remain susceptible to signals that promote adoption of alternative cell fates. Third, although lymphoid progenitors were previously thought to yield only CD8⁺ CD11b^- DCs with a marginal ability to stimulate alloreactive T cells, we found that CLP and CD4⁺B220⁺ pre-pro-B cell-derived DCs each exhibited a CD8^lowCD11b⁺ surface phenotype and readily stimulated the proliferation of allogeneic CD4⁺ T cells. Thus, our data further support the idea that DC phenotype and function are dictated, in part or in whole, by unique environmental stimuli rather that ontogeny (13).

We also confirm and extend previous reports characterizing CLPs in adult BM (21). In particular, we show that B/T-restricted progenitors can be identified among Lin^- BM cells based on an IL-7R⁺C1qR/AA4⁺Sca-1^low surface phenotype and suggest that these cells correspond to the BM CLP population described by Kondo et al. (21). Supporting this conclusion, each population expressed a Lin^-IL-7R⁺Sca-1^lowCD117/c-kit^low Thy-1^- CD24/HSA^intermediate surface phenotype (Fig. 1 and data not shown) (21), and both populations gave rise to B and T cells, but not myeloid lineage cells, in a competitive adoptive transfer assay (Fig. 2) and in fetal thymic organ culture under conditions previously shown to support multilineage progenitor differentiation (Fig. 3) (19). In addition, single CLPs, as defined here, exhibited highly efficient cloning efficiencies and failed to give rise to detectable numbers of myeloid lineage cells on S17 stromal cells (Table I), yet gave rise to clonal populations of B and T cells in fetal thymic organ culture. Therefore, these cells coupled with each pre-pro-B cell subset constitute pivotal stages of early B cell development and B-lineage commitment.

Our data support the idea that loss of DC differentiative potential is the final step in B-lineage commitment, because CD4⁺ B220⁺ pre-pro-B cells (Fr. A₁) yield DCs (Figs. 4 and 5), but not T or NK cells (14). Interestingly, a potentially analogous relationship has been reported for T-lineage commitment in the adult thymus, because both an early B/T progenitor and a more restricted downstream progenitor lacking B-lineage potential reportedly give rise to DCs (10). Regarding DC precursors among early B-lineage cells, it is noteworthy that cells within Fr. A₁ were previously shown to express transcripts for several B-lineage-restricted genes, including Pax5 (15), a zinc finger transcription factor associated with maintaining and perhaps inducing B cell commitment (24, 25). Although this finding might appear to be at odds with the capacity of Fr. A₁ to give rise to DCs, we would point out that the precise activity of Pax5 in CLPs and early B-lineage precursors remains to be determined.

DC differentiation potential of several B-lineage progenitors clearly correlated with developmental maturity, with the least mature of two subsets of pre-pro-B cells (Fr. A₁) defined by low CD4 expression clearly giving rise to DCs, while later stages did not (Figs. 4 and 5). Indeed, we did not observe measurable numbers of DCs when later B-lineage subpopulations, including CD4^- pre-pro-B cells (Fr. A₂) and CD19⁺CD43⁺ pro-B cells, were introduced into this system. At present it is difficult to reconcile these data with a previous report using this culture system in which low frequencies of DC precursors were detected among CD19⁺ CD43⁺ pro-B cells (20). However, because disparate results might result from differences in sorting gates, cytokine concentrations, and/or duration of cultures, we were particularly careful to use consistent sorting gates, single-use aliquots of each cytokine, and to restrict our phenotypic analyses to 4- to 5-day cultures. In fact, we have been able to detect small numbers of class II^bright CD11c^dull cells by extending pro-B cultures to 8 days (data not shown), suggesting that small numbers of contaminating cells may be responsible for this discrepancy. We would also point out that while early CD4⁺ pre-pro-B cells readily differentiated into DCs, more mature CD4^- pre-pro-B cells, which have been shown to readily differentiate into pro-B cells in culture (14), failed to either proliferate or adopt a DC phenotype (Figs. 4 and 5). Therefore, these data suggest that loss of DC precursor potential accompanies the development of late (CD4^-) pre-pro-B cells and their CD19⁺ progeny and should therefore serve as a baseline for studies exploring the signals that induce and prevent B-lineage progenitors from adopting a DC fate.

Certain previous studies suggest that lymphoid-restricted progenitors yield exclusively CD8⁺ CD11b^- DCs. However, both CLP and Fr. A₁-derived DCs were CD8^low CD11b⁺ (Fig. 7), suggesting that environmental stimuli can dictate or modulate DC phenotype and perhaps function. Supporting this model, two recent studies demonstrate that BM common myeloid progenitors and thymic CLPs each give rise to CD8⁺ and CD8^- DCs (12, 13). This idea is also supported by recent experiments showing induction of CD8 expression in Langerhans cells (26) and CD11b⁺ DCs (27). However, it is noteworthy that a recent report from Kamath et al. failed to find evidence for frequent interconversion of these phenotypes among splenic DCs (28). Together these studies suggest that signals inducing unique DC phenotypes and functions are probably restricted to unique microenvironmental niches associated with initiation and/or maintenance of T cell responses in situ.

The in vitro DC progenitor assay used here also revealed differences in proliferative responses to the cytokines used. We suggest that this difference might be accounted for by previous reports demonstrating that B-lineage commitment correlates with loss in expression of receptors for pro-proliferative cytokines such as IL-7 or SCF (29, 30, 31). In fact, we previously reported that CD4⁺ B220⁺ (Fr. A₁) pre-pro-B cells specifically lack detectable levels of the SCF receptor c-kit and the IL-7R -chain (14), suggesting that down-regulation of these receptors may be a key factor in DC vs B cell differentiation from early B-lineage progenitors. Indeed, compared with Fr. A₂, cells within Fr. A₁ yield low frequencies of pro-B cell precursors (14), suggesting that cells in Fr. A₁ may be more likely to yield DCs rather than pro-B cells.

In summary, we find that both CLPs and a subset of pre-pro-B cells readily differentiate into functional DCs in culture when exposed to the appropriate cytokines. These data provide the first clear evidence for a shared pathway for DC and early B cell ontogeny and support a model in which the onset of B-lineage commitment coincides with the loss of DC differentiative potential. Therefore, these findings should lead to studies to enhance our understanding of DC and B cell ontogeny and may lead to novel approaches for selectively expanding and functionally manipulating lymphoid-derived DCs.

Note added in proof:

A recent paper from (32) demonstrates that CLPs but not CD19⁺CD43⁺ pro-B cells differentiate into DCs.

	Acknowledgments

 
We thank Drs. Gwenn Danet, Katia Schlienger, and Martin Carroll for helpful discussions and reviewing the manuscript, and Drs. Carl June and Mark Greene for giving us the privilege and opportunity to perform these studies.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant RO1AI/CA47833 and intramural American Cancer Society Grant IRG-78-002-23.

² Address correspondence and reprint requests to Dr. David Allman, University of Pennsylvania School of Medicine, Biomedical Research Buildings II and III, 421 Curie Boulevard, Room 553, Philadelphia, PA 19104-6160. E-mail address: dallman{at}mail.med.upenn.edu

³ Abbreviations used in this paper: HSC, hemopoietic stem cell; BI, biotin; BM, bone marrow; CLP, common lymphoid progenitor; DC, dendritic cell; FL, fluorescein; Fr., fraction; Lin, lineage marker; MLP, myeloid lymphoid progenitor; SCF, stem cell factor.

Received for publication March 19, 2001. Accepted for publication June 5, 2001.

	References

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