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CD123^bright Plasmacytoid Predendritic Cells: Progenitors Undergoing Cell Fate Conversion?

Discovery Research, Immunex Corp., Seattle, WA 98101

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD123^bright plasmacytoid cells (PC) and CD1c⁺ peripheral blood myeloid dendritic cells (DC) are two human DC precursors that can be expanded in vivo by Fms-like tyrosine kinase 3 ligand (FL). It has been proposed that PC and myeloid CD1c⁺ DC may represent two distinct lineages of DC. However, the phylogenetic affiliation of PC and its relationship with myeloid DC remain controversial. Here we show that CD123^brightHLA-DR⁺ PC from FL-treated healthy volunteers can be divided into mutually exclusive subsets that harbor either lymphoid or myeloid features. Lymphoid-like PC represent the majority of PC and include pT-, CD3-, and CD7-expressing cells. They exhibit TCR- gene loci in germline configuration and show low allostimulatory capacity, but produce type I IFN upon virus infection and can be differentiated in vitro into potent APC. Myeloid-like PC represent a minor fraction of the total PC population. They exhibit a striking PC/myeloid DC intermediate phenotype (CD5⁺CD11c^lowCD45RA^lowCD45RO^-CD101⁺), produce proinflammatory cytokines, and do not require in vitro maturation to act as potent APCs. We propose that, rather than forming a lineage, PC might represent a population of lymphoid cells undergoing an in vivo cell fate conversion from a lymphoid to a myeloid cell type.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human blood dendritic cell (DC)² precursors are commonly divided into two distinct subsets. The so-called myeloid immature DC express CD11c, CD45RO, and CD1c. They are phenotypically and phylogenetically related to CD14⁺ monocytes and can produce IL-12 in response to bacterial extracts and/or cytokines (1, 2, 3). The plasmacytoid DC precursors (PC) express high levels of IL-3R -chain (CD123) (4, 5, 6) and differentiate in the presence of IL-3 into a distinct DC population that lacks most of the typical myeloid markers (7) as well as the ability to produce IL-12 (6). Both human blood DC precursor populations can be efficiently expanded in vivo by Flt3 ligand (FL) (8, 9), a cytokine produced by stromal cells (10) and T lymphocytes (11). Upon herpes or influenza virus infection, PC, but not the myeloid DC precursors, produce high amounts of type I IFNs (9, 12, 13, 14, 15, 16) that act in an autocrine manner to promote PC differentiation into efficient APC (17).

It has been proposed that PC may represent a distinct lineage (14, 18) of DC whose in vivo function might be to link the innate with the adaptive immune responses. However, despite this ever-growing body of data, the phylogenetic affiliation of PC remains controversial, because contradictory evidence supports either a myeloid or a lymphoid origin (19).

Arguments in favor of a myeloid origin for PC are based on phenotypic, functional, and clinical studies. For example, PC co

express CD31, CD36, and CD68, three markers commonly associated with cells from the myelo-monocytic lineage (20). In addition, CD123^bright PC are generated in vitro from M-CSF receptor⁺CD34⁺ bone marrow-committed myeloid progenitors (4). Finally, patients suffering from plasmacytoid T cell leukemia, a rare malignancy characterized by the accumulation of PC-like cells, later develop myelo-monocytic leukemia (21, 22, 23, 24, 25), thus suggesting that PC can differentiate into myeloid cells.

Proponents of a lymphoid origin for PC have provided supportive observations related to both the phenotype and the in vitro derivation of PC. Although PC express several myeloid markers, they do not exhibit all of the classical phenotypic and functional features of myeloid cells. In particular, they fail to express CD11b or CD13 and do not readily differentiate into macrophages in the presence of M-CSF (26, 27). Furthermore, some lymphoid-related molecules, such as CD7 (15, 28, 29, 30, 31, 32) and the pre-TCR -chain (pT) (29, 33), are expressed in PC. Finally, ectopic expression of Id3 (helix-loop-helix motif-containing transcription inhibitor) in CD34⁺ stem cells blocks the development of T and B lymphocytes and reduces the proportion of PC-like cells generated in in vitro cultures, suggesting that most PC are related to T and B lymphocytes (31).

Herein we show that CD123^bright PC from the blood of normal as well as FL-treated healthy volunteers can be further subdivided into different phenotypic and functional subsets exhibiting either myeloid or lymphoid characteristics. We propose that PC are a population of lymphoid cells undergoing an in vivo cell fate conversion from a lymphoid to a myeloid cell type.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients

Healthy volunteers received FL at either 10 or 25 µg/kg/day for 10 consecutive days in conformity with two institutional review board-approved protocols (Baylor Institute, IND no. 7805; Duke University, IND no. 8209). The volunteers had normal blood counts and chemistries and were >18 yr of age. Before entering the study, each volunteer signed an informed written consent that fulfills institutional review board guidelines. FL-treated volunteers underwent apheresis at baseline and after 10 days of FL injection. Mononuclear cells were then selected by a standard Ficoll/Hypaque gradient method or by using a continuous flow cell processor (COBE 2991, Gambro BCT; Lakewood, CO). Purified PBMC were resuspended at 10⁷–10⁸ cells/ml in medium containing 44% RPMI 1640, 44% FBS, and 12% DMSO and then cryopreserved.

Reagents

Human (hu) CSF-1 was purchased from R&D Systems (Minneapolis, MN) and used at 100 ng/ml. Human IL-3, huGM-CSF and trimeric leucine zipper huCD40 ligand (huCD40L) were produced at Immunex (Seattle, WA) and used at 50, 100, and 1 µg/ml, respectively.

Antibodies

Abs used in this study are listed in Table I.

Enrichment for CD123^bright cells was conducted according to a modification of the procedure described by Olweus et al. (4). Briefly, PBMC were labeled with allophycocyanin-conjugated anti-CD123 Ab (BD PharMingen, San Diego, CA) and subsequently incubated with anti-mouse IgG1-coated magnetic microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s specifications. Alternatively, R-PE-conjugated anti-CD123 Ab was used in combination with anti-PE-coated magnetic microbeads (Miltenyi Biotec). CD123^bright cells were then enriched on a high gradient magnetic field. The purity of the CD123^bright cell-enriched fraction was assessed by fluorescence analysis. After 30-min incubation in PBS containing 100 µg/ml purified mouse IgG1 Ab, enriched CD123^bright cells were labeled with anti-CD56 and anti-CD7 Abs or isotype-matched control Abs. Enriched CD123^bright subsets were then sorted into subsets using a double laser-equipped FACSVantage flow cytometer (BD Biosciences, Franklin Lakes, NJ) after adjustment of proper fluorescence detection compensations. Subcellular particles and dead cells were excluded from acquisition data by gating on forward and side angle light scatter parameters. The BDCA-2 cell isolation kit (Miltenyi Biotec) was used as an alternative preenrichment step in one of the four experiments shown in Fig. 8B. CD1b/c⁺ DC and CD14⁺ monocytes were labeled after PC magnetic depletion and were directly purified by flow cytometry. Resulting purity was >97%. For four-color phenotyping experiments, a FITC/PE/CyChrome (PerCP)/allophycocyanin fluorochrome combination was used, and data (at least 60,000 events/sample or at least 2,000 events/cell subset of interest) were collected on a FACSCalibur flow cytometer (BD Biosciences) and subsequently processed using the CellQuest program (BD Biosciences).

View larger version (16K): [in this window] [in a new window]   FIGURE 8. A, Production of IL-1 and IL-6 by in vitro matured PC subsets. PC subsets were purified from the blood of FL-treated healthy volunteers and cultured for 5 days in the presence of huCD40L (1 µg/ml), huIL-3 (50 ng/ml), and huGM-CSF (100 ng/ml). Culture supernatants were then harvested and assayed for the presence of cytokines using LabMAP technology (Luminex). B, IFN- production by freshly isolated PC. Blood PC were purified from FL-treated individuals as indicated in Materials and Methods and then infected with HSV-1. After 24 h in culture, supernatants were harvested, and the concentration of IFN- was determined by ELISA. Horizontal lines indicate the average IFN- production for four independent experiments. The p values were determined by paired t test.

All cultures were performed in RPMI 1640 medium enriched with 2 mM L-glutamine (JRH Biosciences, Lenexa, KS), 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT), 5000 U/ml penicillin G (Calbiochem, La Jolla, CA), 5000 U/ml streptomycin sulfate (Mediatech, Herndon, VA), and 200 mM L-glutamine (JRH Biosciences). Morphological examination of cultured cells was performed after staining with DIFF/quick (Dade-Behring, Newark, DE). To assay the allostimulatory capacity of freshly isolated or in vitro-activated PC cells, increasing numbers of irradiated (504 rad) APC were added to a fix number of purified CD3⁺ allogeneic peripheral blood T cells and cultured for a period of 5 days. Tritiated thymidine (NEN, Boston, MA) was then added at 0.5 µCi/well (20.0 Ci/mmol) for the last 16 h of culture. For proinflammatory cytokine production, cultures were seeded with 2.5 x 10⁴ cells/ml and allowed to proceed for 48 h. The presence of IL-1 and IL-6 in culture supernatants was then assessed by immunoassay using LabMAP technology (Luminex, Austin, TX). For IFN- production, cells were infected with human HSV-1 (American Type Culture Collection, Manassas, VA; catalog no. VR-260; multiplicity of infection, 10). Cells were then seeded in round-bottom 96-well plates at 20,000 cells/well in a final volume of 200 µl. Supernatants were collected at 24 h, and the presence of IFN- was assayed using the huIFN- ELISA kit (BioSource International, Camarillo, CA).

TCR -chain locus analysis

Rearrangement of the TCR -chain locus was assessed according to Ktorza et al. (34). Briefly, genomic DNA from 2000 purified PC was PCR-amplified using a set of primers complementary to a genomic DNA sequence upstream of D1 (TBF1, 5'-TGGGAGGGGCTGTTTTTGTA-3'; sense primer) and downstream of J1 minigenes (TBR1, 5'-TCCAGGTAAGAAGGGGTGAC-3'; antisense primer). PCR products were blotted and hybridized with the TBR3 probe (5'-CTGACCTCCGTTCTTACACT-3'). CD3⁺ T cells and CD14⁺ monocytes were also purified and included as positive and negative controls, respectively.

CD3 chains and pT expression

Expression of CD3-, -, and -chains as well as pT was assessed by PCR analysis. The primer sequences and the PCR conditions used are summarized in Table II.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
FL preferentially expands two discrete subsets of CD123^brightHLA-DR⁺ cells

Subcutaneous injections of FL transiently enhance by 10-fold the number of circulating CD123^bright PC in the blood of healthy human volunteers (8, 9). PC generated in vivo in response to FL were shown to exhibit a similar phenotype to those normally found in nontreated donors (9). In particular, they express surface CD4 and HLA-DR, but no CD3, CD11b, or CD13 (9). However, most phenotypic studies conducted on PC used a lineage depletion step in which cells expressing typical NK, T cell, B cell, or monocyte markers are electronically or physically excluded from the analysis gate (7, 9, 15, 26, 27, 35). Noticeably, the actual composition of lineage mixtures varies between studies, which makes cross-study comparison difficult. Because PC can be unambiguously identified by virtue of coexpression of HLA-DR and high CD123 expression (4), we tested whether all the common lineage markers used in lineage depletion mixtures were actually absent from the surface of CD123^brightHLA-DR⁺ cells. Thus, CD123^bright cells were first enriched by positive selection with magnetic beads, then labeled with anti-HLA-DR and one Ab from the lineage mixture. Consistent with previous studies, we could not detect CD3, CD11b, CD15, CD16, CD19, CD20, CD34, or CD94 on the surface of PC (not shown). In contrast, some CD7⁺ cells were clearly present within CD123^brightHLA-DR⁺ PBMC. Surprisingly, a minor population of CD56 (B159)⁺ cells was detected within the CD123^brightHLA-DR⁺ PBMC from healthy donors. CD56⁺ PC only represented 1% (range, 0.4–2.2%; n = 6) of the total PC pool from normal donors (Fig. 1). Four-color labeling experiments showed that CD7⁺ and CD56⁺ PC are distinct PC subsets that are preferentially expanded by FL treatment. Typically, after FL treatment, 4–15% (n = 7) of PC express CD7, and 4–12% (n = 7) express CD56. CD7⁺CD56⁺ PC were only sporadically detected in FL-treated healthy volunteers, representing <0.3% of the total PC population.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. FL preferentially expands two discrete subsets of PC. Human healthy volunteers received a daily injection of 10 µg/kg FL for 10 days. PBMC were collected by apheresis before the first and after the last injection of FL, and cryopreserved PC from pre-FL and post-FL samples were enriched through MACS as indicated in Materials and Methods. Enriched PC were then labeled with Abs against HLA-DR-PerCP, CD7-FITC, and CD56-biotin plus streptomycin-PE. Results are representative of six different experiments.

Although CD7⁺ PC have been previously described, the presence of CD56⁺ PC was surprising, because previous studies failed to detect CD56-expressing cells within the PC population (4, 26). Interestingly, among three anti-CD56 mAbs tested (Mem188, T199, B159), B159 was the only Ab that distinctly labeled a subset of PC in both normal and FL-treated donors (not shown). Further studies are warranted to establish whether B159 identifies a specific CD56 (neural cell adhesion molecule) epitope or a cross-reactive molecule on PC. To confirm that these PC subsets are indeed related cell types, CD7⁺, CD56⁺, and CD7^-CD56^- (double-negative (DN)) subsets were labeled with a large panel of Abs, and their phenotypes were compared. All three subsets expressed CD4, CD36, CD44, CD68, CXCR3, CCR5, LIR5(IL-T3), and HLA-DR, yet they did not express several markers, i.e., activation (CD10, CD23, CD25, CD26, CD45R0, CD69, CD152), lymphocytic related (CD21, CD27, CD28), myelo-monocytic related (CD11b, CDw17, CD33, CDw65, CD89), or NK-related (CD57, CD94, CD122, CD161, CD244) markers (Fig. 2 and not shown). In addition, those three PC subsets are clearly distinct from CD123^bright blood basophils that express FcRI, CCR3, CD11b, CD45R0, and CDw125, but no HLA-DR or CXCR3 (data not shown). Therefore, all three PC subsets share many phenotypic characteristics suggesting that they are closely related cells. However, some minor phenotypic differences were also detected (Fig. 2). In particular, CD56⁺ PC differ from the other PC subsets in that they express significantly lower levels of CD162 (PSGL1) and CD45RA, but higher levels of CD2, CD5, CD101, and CD49e. BDCA2 and BDCA4, two PC-specific Ags (32), had a bimodal distribution on CD56⁺ PC. A sizeable fraction of CD56⁺ PC was CD45RA^-, and very few cells expressed detectable levels of CD45RO. Finally, CD11c was detectable on some CD56⁺ PC, although at reduced levels compared with monocytes from the same donors (not shown). Globally, CD7⁺ and DN subsets appeared more closely related, with CD7 being the only discriminatory marker.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Comparative phenotypic analysis of PC subsets and CD1b/c+ DC from FL-treated individuals. PBMC from FL-treated healthy volunteers were collected by leukapheresis and labeled as described in Materials and Methods. Vertical bars on histograms indicate the maximal labeling intensity obtained with isotype-matched control Abs. Results are representative of at least three independent experiments.

A fraction of PC express the pre-TCR -chain and CD3, but not CD3 or CD3

An unusual population of surface CD4⁺CD3^-CD14^- peripheral blood cells that could efficiently differentiate into mature CD3⁺ T lymphocytes in fetal thymic organ cultures was recently described (33). A fraction of these circulating pre-T cells expressed pT mRNA as well as mRNA for the -, -, and -chains of the CD3 complex (33). Because CD4⁺CD3^-CD14^- pre-T cells display many phenotypic similarities with PC, it has been proposed, although not clearly demonstrated, that these two cell types may be identical (6). To address this issue we examined the levels of pT, CD3-, -, and -chain mRNA expression in CD123^bright PC subsets. Fig. 3 shows the PCR amplification products of pT as well as CD3, , and obtained from 1–50 highly purified cells of each PC subset. Among the three CD3 chains tested, CD3 was the only gene for which a product could be detected in PC. CD3 was consistently detectable in both CD7⁺ and DN PC populations down to the five cells per well level, whereas it remained undetectable in CD56⁺ PC even at the highest cell concentrations tested (50 cells/well). As a control, under the same experimental conditions, all CD3 chains could be detected in mature T lymphocytes from the same donor down to the single-cell level. We were able to detect pT expression in as few as five sorted DNPC (Fig. 3). By contrast, pT mRNA could only be detected in CD7⁺ PC at the 50-cell level. Assuming that the level of pT/CD3 expression per cell is equivalent in each PC subset, this result would indicate that most pT- and CD3-expressing PC are included within the DN fraction. CD3-, - and -chains are part of the T cell Ag receptor complex and, as such, are coexpressed on T lymphocytes. However, since NK cells can express CD3 in the absence of the other CD3 chains (36), we tested the ability of CD7⁺, CD56⁺, and DN PC to differentiate in vitro into mature NK cells. To this end, PC subsets were cultured in the presence of IL-2, IL-7, and stem cell factor or in the presence of IL-7, IL-15, and FL as previously described (37). Under these culture conditions, none of the PC cultured could survive >6 days in culture, and no NK cell development was noted (not shown).

View larger version (85K): [in this window] [in a new window]   FIGURE 3. Expression of pT and CD3 chains in PC subsets. Total RNA was generated from lysates of automated cell deposition unit-sorted cells using Qiagen’s RN-easy kit. Each RNA preparation was then split into six equal fractions and used in one-step RT-PCR with intron/exon-spanning probes. -Actin was used as a positive control and could be detected down to a single-cell level in every one of the populations tested (not shown). One-fifth of RT-PCR reactions were run on 1.6% agarose gel containing ethidium bromide and photographed.

Cells from all PC subsets exhibit a TCR--chain locus in germline configuration

Rearrangement of the DJ junction within the TCR--chain DNA locus is believed to represent the earliest irreversible molecular event in the T cell lineage differentiation pathway and is often used to assess commitment of early T cell precursors. Bruno et al. (33) reported that pT⁺ PBMC exhibit a partially recombined TCR--chain DNA locus. Since pT-expressing cells are found within the CD7⁺ and DN CD123^bright PC subsets, we investigated whether these PC also displayed a partially recombined TCR- locus. To this end, a PCR-based method was used to amplify the DJ1 gene region of genomic DNA from each PC subset. Mature CD3⁺ T lymphocytes were purified from the same donor and used as a positive control. Although our PCR amplification protocol was capable of detecting TCR- gene rearrangement down to the 100-T cell level, we were unable to detect any DJ rearrangement for up to 20,000 PC (Fig. 4). Thus, if present among PC, TCR--rearranged, committed pre-T cells represent a minority (<0.5%) of each PC subpopulation.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. PC subsets show TCR- DNA loci in germline configuration. Genomic DNA was extracted from 2 x 104 highly purified PC. DNA contained within the D1 and J1 minigenes of the TCR- DNA locus was then amplified by PCR. The resulting cDNA was subjected to electrophoresis, blotted, and probed as indicated in Materials and Methods. The presence or the absence of D1-J1 recombination events within PC subsets DNA was then determined by comparing the size of the cDNA products with that of CD14+ monocytes (2 x 104 cells, negative control) and CD3+ T lymphocytes (100 cells, positive control).

CD56⁺ PC fail to express lymphoid-related markers such as pT and CD3 chains, although they do express CD5 and low, but significant, levels of CD11c (Fig. 2). Because others have shown that CD56 (32), CD5 (38), and CD11c (39) are coexpressed in CD1c⁺ myeloid DC, we examined whether CD56/CD5⁺ PC and CD1c⁺ DC may be phenotypically related. To this end, PBMC from FL-treated healthy volunteers were labeled with Abs directed against CD123, CD5, and CD1b/c. The phenotypes of CD123^bright PC and CD1b/c⁺ DC were compared on a CD123/CD5 scatter plot (Fig. 5). Although CD1b/c⁺ DC and PC appear as distinct populations, cells with an intermediate PC/DC phenotype were detected (Fig. 5). These intermediate cells expressed variable levels of surface CD5 and were more readily identifiable within a typical horseshoe-like profile (Fig. 5). These data suggest that CD56⁺/CD5⁺ PC might be part of a continuum that links PC to CD1b/c⁺ DC.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. CD56/CD5+ PC and CD5+CD1b/c+ DC from FL-treated individuals are part of a phenotypic continuum. PBMC from FL-treated healthy volunteers were labeled with anti-CD1b/cFITC, anti-CD123PE, and anti-CD5-biotin and streptomycin-allophycocyanin. Viable PC and DC were first selected by gating on forward/side scatter parameters (R1, left panel). The phenotype of CD123bright PC was then compared with that of CD1b/c+ DC (grey dots) on a CD123/CD5 scatter plot (right panel).

In general, PC are defined as pre-DC because they typically do not exhibit high allostimulatory capacity or DC morphology unless activated or matured in vitro (7, 39). Because APC function has been assayed with bulk populations of PC, it is possible that a putative minor fraction of cells with high allostimulatory capacity would not be detected under these conditions. We therefore purified PC subsets from FL-treated healthy volunteers and assessed their capacity to activate allogeneic T lymphocytes in a mixed leukocyte culture. CD14⁺ monocytes and CD1b/c⁺ DC were purified from the same donor and included as negative and positive controls, respectively. PC subsets exhibited differential allostimulatory capacities; freshly isolated CD7⁺ PC and CD14⁺ monocytes were poor stimulators of allogeneic T lymphocytes, whereas CD56⁺ PC and CD1b/c⁺ were equivalently potent in inducing alloreactive T cell proliferation (Fig. 6A). The DN fraction of PC exhibited an intermediate potency. Therefore, the CD56⁺ fraction and, to a lesser extent, the DN fraction of PC contain potent APC. In contrast, freshly isolated CD7⁺ PC are not efficient APC. Interestingly, no significant differences in cell surface expression of MHC class II and costimulatory molecules (CD80, CD86) could be observed on PC subsets (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 6. PC subsets exhibit differential allostimulatory capacities. Freshly isolated (A) or CD40L- plus IL-3-stimulated (B) PC subsets were irradiated and cultured in the presence of allogeneic T lymphocytes as described in Materials and Methods. Proliferation of T lymphocytes was then assessed by [3H]thymidine incorporation 4 days after the initiation of the culture. Freshly isolated CD14+ monocytes and CD1b/c+ DC were used as negative and positive controls, respectively. Error bars show the range of experimental duplicates. Results are representative of three independent experiments.

PC undergo spontaneous apoptosis when cultured in medium. However, stimulation with IL-3 and CD40L can partially counteract this spontaneous cell death (7). Surviving cells exhibit a DC phenotype and are potent allostimulatory cells. We tested the ability of each PC subset to survive and differentiate in vitro into DC. CD7⁺, CD56⁺, and DN populations of PC were purified and cultured for 5 days in the presence of rIL-3 and CD40L. Cells were then counted, and their allostimulatory capacity was assessed in a mixed leukocyte reaction with highly purified allogeneic CD3⁺ peripheral blood T cells (Fig. 6B). CD40L plus IL-3 stimulation appeared equally potent in maintaining survival of the PC subsets (not shown). Surviving cells displayed a DC morphology (Fig. 7), although CD40L- plus IL-3-stimulated CD56⁺ PC appeared larger, with a more abundant and vacuolated cytoplasm, but with shorter processes than the other PC subsets. After CD40L plus IL-3 culture, all three subsets were capable of inducing proliferation of alloreactive T cells, with CD56⁺ PC being more potent than the other subsets. Adjunction of GM-CSF to the CD40L plus IL-3 maturation culture did not significantly alter the allostimulatory potentials of PC subsets (not shown).

View larger version (179K): [in this window] [in a new window]   FIGURE 7. Morphology of PC subsets. PC subsets were purified from FL-treated healthy volunteers. Cells were then cultured for 5 days in the presence of huCD40L (1 µg/ml) and huIL-3 (50 ng/ml). The pictures illustrate the morphology of each PC subset before and after culture.

CD56⁺ PC and CD1b/c⁺ produce IL-6 and IL-1 after in vitro maturation

IL-1 and IL-6 exert a synergistic effect on the activation of T lymphocytes in an allogeneic mixed leukocyte cultures by increasing T lymphocyte IL-2 production and responsiveness (40, 41). Because PC subsets show differences in their allostimulatory capacity, we tested their ability to produce IL-1 and IL-6 after in vitro maturation. PC, CD14⁺ monocytes, and CD1b/c⁺ cells were purified from the blood of FL-treated healthy volunteers and cultured for 48 h in the presence of IL-3, CD40L, and GM-CSF. The presence of IL-1 and IL-6 in culture supernatants was then assessed by immunoassay using LabMAP technology (Luminex, Austin, TX; Fig. 8A). Both IL-1 and IL-6 were detectable in the supernatants of cultured CD56⁺ PC at levels comparable to those observed for CD1b/c⁺ DC. In contrast, matured CD7⁺ or DN PC only produced subnanomolar concentrations of these cytokines. Consistent with previous observations (42), cultured CD14⁺ monocytes produced large amounts of both cytokines. Thus, after in vitro maturation, PC subsets display differences in their ability to produce proinflammatory cytokines such as IL-1 or IL-6; CD56⁺ PC and myeloid CD1b/c⁺ DC produce comparable amounts of these cytokines.

PC subsets produce different levels of type I IFN after viral infection

It is well established that PC can produce type I IFN in response to viral infection (14, 15). However, it is still unknown whether all or only a subset of PC can produce this IFN. We purified each PC subset (CD7⁺, DN, CD56⁺) and cultured them in the presence of HSV-1 for 24 h. Culture supernatants were then collected, and the presence of IFN- was assayed by ELISA. The results (Fig. 8B) show that CD7⁺ and DN PC produce comparable amounts of IFN- in response to HSV-1 infection. Although IFN- could also be clearly detected in culture supernatants from the HSV-1-infected CD56⁺ PC subset, production levels were significantly lower than in the two other PC subsets. Thus, it appears that all three PC subsets can produce type I IFN in response to virus infection, with CD56⁺ PC being less potent IFN-producing cells than the two other PC subsets.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is generally held that leukocyte differentiation can follow two mutually exclusive developmental paths that will give rise to myeloid and lymphoid cells, respectively. Indeed, in vivo reconstitution experiments have demonstrated the existence of two distinct progenitors that, upon transfer into a lethally irradiated host, will give rise to either myeloid or lymphoid lineages. Since each progenitor generates a clearly distinct population of cells, it is possible to represent the lymphoid and myeloid differentiation processes in the form of a bifurcated tree that links common myeloid or lymphoid progenitors to their respective progeny.

DC, however, represent a noticeable exception to this rule, since these cells can differentiate from both lymphoid (43) and myeloid (44) progenitors. Attempts to reconcile this paradox have led to the proposition that there might be two DC lineages, one myeloid and one lymphoid (reviewed in Ref. 45). However, DC differentiation might not be the exclusive privilege of common myeloid and lymphoid progenitors, because after adoptive transfer T cell and granulocyte-macrophage progenitors are also capable of differentiating into DC (46). Taken together, these results suggest that there might not be one (or even two) DC lineages. Instead, DC might be the product of a lateral differentiation process, or conversion, that might affect different lymphoid as well as myelo-granulocytic early committed cells. In this context cytokines could play a major role by instructing early precursor cells to deviate from their original lineage commitment and undergo a cell fate conversion (46, 47). Thus, it is conceivable that disturbances in the homeostatic cytokine network, by introduction of exogenous cytokines or by an ongoing immune response, might result in the conversion of early committed cells into DC.

PC are a rare population of human leukocytes present in the blood and lymphoid organs that can differentiate in vitro into DC (7, 39). By many phenotypic criteria, PC appear as a homogeneous population. Yet we show that PC can still be divided into discrete subpopulations that exhibit either lymphoid or myeloid features.

We found that lymphoid-like PC are confined to two distinct subsets: CD7⁺ and CD7^-CD56^- (DN) PC. DN PC and CD7⁺ PC include cells expressing both pT and CD3 mRNA. In contrast, they do not express CD3 or CD3 mRNA. T lymphocytes and NK cells are believed to derive from a CD56^- common progenitor expressing CD7 (48) as well as CD3-, -, and -chains (36, 49). Cells that commit to T cell differentiation will express pT (50), maintain expression of CD7 and CD3 chains, and initiate the rearrangement of the TCR--chain locus (48). By contrast, progenitor cells that commit to the NK pathway would maintain expression of CD7 and down-regulate the expression of CD3, then CD3, and finally CD3 (49, 51). Unfortunately, pT expression has not been studied in the context of NK cell differentiation. Thus, the CD7⁺ PC phenotype (germline-TCR-, CD7⁺CD3⁺,^-,^-) would be consistent with that of an NK cell precursor. Yet, in accordance with previous studies (29), we were unable to differentiate in vitro CD7⁺ or DN PC into NK cells (see Ref. 37 for method). It is possible that if indeed CD7⁺ PC derive from NK cell precursors, they might lose their ability to differentiate into NK cells during their transition to a PC stage, but might retain some phenotypic features (expression of CD7 and CD3) of their initial lineage commitment. The DN PC phenotype (germline-TCR-, pT⁺, CD7^-;CD3⁺,^-,^-) is more ambiguous and could relate to either T cell or NK cell progenitors. As for recently described human thymic DC precursors (29), phenotypic characteristics of CD7⁺ and DN PC are reminiscent of NK cell and/or T cell progenitors, suggesting that these cells are of lymphoid origin. Our results do not exclude the possibility that earlier multipotent progenitors or other lineage-committed cells might contribute to the formation of PC. The idea that other committed progenitors might contribute to the formation of PC is supported by the fact that -like 14.2, a gene specifically expressed in pro-B cells (52), can also be detected within the PC compartment (53).

CD56 (B159)⁺ PC exhibiting several phenotypic and functional myeloid characteristics is also identifiable in human PBMC. Although myeloid-like PC are rare in normal donors, this population can represent up to 12% of all PC in FL-treated healthy volunteers. Unlike the other PC subsets, freshly isolated CD56 (B159)⁺ PC produce low levels of IFN- in response to viral infection and appear allocompetent, for they can induce proliferation of allogeneic T lymphocytes as efficiently as blood CD1c⁺ DC. After culture in the presence of IL-3, GM-CSF, and CD40L, CD56⁺ PC and myeloid CD1c⁺ DC are able to produce comparable amounts of IL-1 and IL-6.

Upon in vitro maturation, PC progressively lose their ability to produce IFN- and acquire the ability to produce proinflammatory cytokines (14, 54). If indeed the in vivo fate of PC is to differentiate into allocompetent DC producing proinflammatory cytokines, then our results suggest that CD7⁺ and DN (lymphoid-like) PC subsets will give rise to CD56⁺ (myeloid-like) PC.

Conversion of early committed lymphoid progenitors into myeloid cells has been documented both in vivo (47) and in vitro. In particular, pro-B (55, 56) and pro-T cells (57) seem to have the capacity to divert from their initial lineage commitment to convert into myeloid cells. This conversion could be seen as a rescue mechanism by which lymphocyte progenitors unable to undergo recombination of their Ag receptor DNA loci are diverted toward a myeloid differentiation pathway. It is conceivable that introduction of exogenous FL, by artificially increasing the number of T cell (58) and B cell (59, 60, 61) progenitors, would exceed the selection capacity of primary lymphoid organs, thus favoring cell fate conversion.

Our results raise the intriguing possibility that PC could represent a common intermediate stage for lymphoid cells, possibly of diverse origins, undergoing an in vivo conversion to a myeloid (DC) population. The idea that PC may give rise to myeloid cells is in line with data showing that when differentiating into DC, PC lose pT expression (29) and, in the presence of GM-CSF, acquire CD11c (62). It is also consistent with reports on patients with plasmacytoid-T cell lymphoma (PTL). PTL are PC-related tumors that have been observed mostly in elderly men with generalized lymphadenopathy (21, 22, 23, 24, 25, 63). All patients diagnosed with PTL also developed acute or chronic myelo-monocytic leukemia. It has been debated whether the PC observed in this pathology are part of the neoplastic clone, a separate neoplasm, or a reactive phenomenon (21, 64, 65). Recently, however, it has been shown that PTL and the associated myeloid leukemia obtained from the same patients exhibit a common chromosomal abnormality (66), demonstrating that chronic myelo-monocytic leukemic cells and PTL derive from a common ancestor (63). pT⁺ PC as well as CD11c⁺LIR5(IL-T3)⁺ myeloid DC tumor cells also have been identified in a subset of patients with acute myeloid leukemia (67). In these patients pT⁺ PC and myeloid DC tumor cells are clonally related, because both tumor cells expressed common chromosomal abnormalities. Another hemodermic neoplasm composed of CD56⁺ PC-like cells expressing pT could differentiate upon culture into cells expressing typical myeloid markers (CD11c, CD13, and CD33) (68).

We would like to propose that an oncogenic transformation event affecting cells undergoing a cell fate conversion from a lymphoid pT⁺ stage to a myeloid DC stage might be responsible for this phenotype. Identification of PC subpopulations as cellular intermediates in cell fate conversion offers a unique opportunity to decipher the molecular mechanisms regulating this process.

	Acknowledgments

 
We thank Drs. Jacques Banchereau, Kim Lyerly, and Michael Morse for providing cells from FL-injected healthy volunteers, and Daniel Hirschstein, Steven Braddy, and Julie Hill for flow cytometry expertise. We are grateful to Anne Aumell for expert editorial assistance.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Laurent Galibert, Discovery Research, Immunex Corp., 51 University Street, Seattle, WA 98101-2936. E-mail address: galibertl{at}immunex.com

² Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; DN, double negative; FL, fms-like tyrosine kinase 3 ligand; hu, human; PC, CD123^bright plasmacytoid cell; PTL, plasmacytoid-T cell lymphoma.

Received for publication December 10, 2001. Accepted for publication April 29, 2002.

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