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IMMUNOBIOLOGY
From the Laboratoire d'Immunologie Cellulaire et
Tissulaire URA CNRS 625, Service d'Urologie, Hôpital de La
Pitié-Salpêtrière, Paris; and Service
d'Hématologie biologique, Hôpital Avicenne, Bobigny,
France.
Although the mouse spleen dendritic cell (DC) is perhaps the most
intensively studied DC type, little has been published concerning its
human equivalent. In this report, rare event flow cytometry and in situ
immunofluorescence were used to study the surface phenotype and
distribution of HLA-DR+
CD3 The initiation of a specific immune response
implies the acquisition and presentation of antigen to naive T
lymphocytes by dendritic cells (DCs).1-3 Like many other
biologic functions, the timeliness and efficacy of this initiation are
regulated by compartmentalization.4 The first level of
compartmentalization is the existence of 2 distinct maturation stages
in DCs: immature DCs are specialized in the uptake of antigens by
macropinocytosis and endocytosis by Fc receptors5,6 and
the macrophage mannose receptor,5,7,8 but they do not have
a high capacity for T-cell costimulation. Conversely, mature or
activated DCs express extremely high levels of major histocompatibility
complex (MHC) and costimulatory molecules such as CD809
and CD86,10,11 and they stimulate T cells far more
effectively than any other cell type12,13 but internalize
antigens poorly.14,15 The second level of
compartmentalization is anatomic: naive T lymphocytes can probably only
be activated in the T-cell areas of lymphoid organs and almost never in
the periphery.3,4,16 Therefore, the initiation of a
specific immune response requires a redistribution of antigen-loaded,
maturing DC toward the T-cell areas of secondary lymphoid
organs.17-20 Indeed, immature DCs are found as sentinels
at nonlymphoid sites, in the interstitia of solid organs, and in the
epithelia as Langerhans cells, whereas mature DCs are mostly found in
the T-cell areas of lymphoid organs.21 However, much has
to be done to understand precisely the micro-anatomy of the immune
response in the lymphoid organs, especially in humans.
In the mouse, the integrin Human spleen DCs have been less extensively studied than either mouse
spleen DCs or human DCs from other lymphoid organs. T-cell zone DCs
were reported to express CD11c, MHC II,29 and CD83.30 In addition, germinal center DCs were observed in
the B-cell zones.31 However, no human equivalent of the
mouse marginating DC population has previously been described.
In the present work, we explore the phenotype of human spleen DCs by
flow cytometry and in situ immunofluorescence to determine how many
subpopulations of human spleen DCs can be defined on the basis of
surface phenotype and distribution. In particular, we describe
the human equivalent of the mouse marginating DC population. Additionally, evidence is presented indicating that spleen DCs in a
subset of organ donors had been activated in vivo, with micro-anatomic distribution changes, perhaps in the process of antigen presentation and specific immune response induction.
Human tissues
Preparation of mononuclear cell suspensions
Flow cytometry Cells were incubated for 30 minutes at 4°C in RPMI containing 10% fetal calf serum and human immunoglobulins (Biotransfusion, Les Ulis, France) at 100 µg/mL and then were incubated with isotype controls or unconjugated antibodies listed in Table 2, which were revealed with polyclonal goat antimouse immunoglobulin G (IgG) conjugated to fluorescein isothiocyanate (FITC) at 7 µg/mL (Caltag, San Francisco, CA). Residual antimouse antibodies were blocked by incubation with 100 µg/mL mouse IgG for 15 minutes at 4°C, and the cells were incubated with the following cocktail of 4 phycoerythrin (PE)-conjugated antibodies: anti-CD3 (S4.1 at 4 µg/mL; Caltag), anti-CD14 (My4 at 5 µg/mL; Coulter, Margency, France), anti-CD16 (3G8 at 2 µg/mL; Caltag), anti-CD19 (B4 at 5 µg/mL; Coulter), and anti-HLA-DR (HL38 at 4µg/mL; Caltag) conjugated to PE-Cy5. When directly FITC-coupled antibodies were used, they were added to cells simultaneously with the cocktail of PE- and PE-Cy5-conjugated antibodies immediately after the blocking step. Cells were washed once between incubation steps in 3 mL phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), then centrifuged at 400g for 6 minutes at 4°C. All incubations were carried out in 100 µL PBS containing 1% BSA for 30 minutes on ice. After a final wash, cells were fixed in PBS containing 1% paraformaldehyde, then analyzed using a FACScan (Becton Dickinson, Le Pont de Claix, France).
For 3-color analysis of unseparated spleen mononuclear cells (SMCs),
10 000 ungated events were acquired in addition to 2000 to 5000 gated
CD3 In situ immunofluorescence Small blocks of spleen tissue (approximately 5 mm × 5 mm × 5 mm) were snap-frozen in liquid nitrogen then stored at 80°C until 5-µm thin sections were prepared using a Leica
CM1500 cryotome (Rueil Malmaison, France). Sections were dried
overnight, fixed for 10 minutes in acetone, and used directly for
labeling or stored at 20°C. After rehydration for 2 minutes in
PBS, nonspecific sites were blocked by incubation in PBS containing
0.5% BSA and 0.5% gelatin for 30 minutes. Sections were then
incubated for 60 minutes with primary antibodies listed in Table
3, then washed 3 times in PBS containing
0.5% BSA. Sections were incubated for 30 minutes with biotinylated
goat-antimouse IgG1 at 4 µg/mL (Caltag) and either FITC-conjugated
goat-antimouse IgG2a or FITC-conjugated goat-antimouse IgG2b at 10 µg/mL (Caltag). After 3 further washes, biotinylated antibodies were
revealed by a 15-minute incubation with 5 µg/mL avidin-Texas Red
(Vector Laboratories, Burlingame, CA). Sections were washed again, and
nuclei were counterstained by a 15-second incubation in 20 µg/mL
Hoechst 33258 in PBS. Slides were mounted in 90% glycerol/10% PBS
containing 25 mg/mL Dabco (Sigma).
Cytokine measurement Spleen mononuclear cells (1 × 106/mL) were cultured for 24 hours in RPMI 1640 complete medium containing 10% human AB serum in 24-well plates, with or without 100 ng/mL LPS34 (Escherichia coli serotype 026:B6; Sigma, St Louis, MO). IL-12 was measured in the supernatants using an enzyme-linked immunosorbent assay kit that detects both heterodimeric (p70) and homodimeric (p40-p40) forms (Biosource Europe, Fleurus, Belgium).
Quantitation and phenotype of spleen dendritic cells by flow cytometry The surface phenotype of spleen DCs was examined by rare event flow cytometry, as previously described.35 Briefly, SMCs were stained for 3-color immunofluorescence with a cocktail of PE-conjugated mAbs (anti-CD3, -CD14, -CD56, and -CD19), PE-Cy5 conjugated anti-HLA-DR, and different mAbs directly or indirectly coupled to FITC. Data on DCs were collected by gated acquisition of cocktail HLA-DR+ events (Figure
1A), and large DCs within the acquired
population were analyzed by scatter gating, as previously described
(Figure 1B).35 Thus defined, DCs represented
0.7% ± 0.5% of SMCs (range, less than 0.1%-1.8%; n = 19; Table
1), whereas macrophages and B lymphocytes made up
9.2% ± 6.9% (range, 1%-22%; n = 19) and 52% ± 16% (range,
16%-85%; n = 19) of SMCs, respectively.
Figure 1C shows the results of a typical experiment. Gated DCs were
uniformly HLA-DQ+ CD11a+, CD11b Distribution of spleen dendritic cells Because a difference in CD11c expression intensity between DC and macrophages was noted by flow cytometry (Figure 1), spleen sections were stained for CD11c to investigate the possibility that, as in the mouse, this molecule could be used as a relatively specific marker for spleen DCs in situ.41 Strongly CD11c+ cells were observed in 3 distinct regions at the periphery of the white
pulp, in the T-cell zones, and in the B-cell zones (Figure 2A-F). Double labeling showed that these
CD11c+ cells were CD14 (Figure 2B),
CD3 (Figure 2D), CD20 (Figure 2F),
CD11b, and HLA-DR+ (not shown) and,
therefore, that they had the same phenotype as the
cocktail HLA-DR+ DCs that had been analyzed
by flow cytometry.
CD11c+ cells at the periphery of the white pulp surrounded both T-cell and B-cell zones, effectively separating them from the red pulp (Figure 2A-D). Macrophages expressing CD14 and CD11b were not present in this zone. Indeed white pulp CD11c+ cells and the first line of red pulp CD14+ macrophages seemed to form concentric rings around the white pulp (Figure 2A-B). These marginal zone CD11c+ cells had a distribution similar to that of marginating DCs in the mouse, though more consistently surrounding the white pulp.22,26,27 Large CD11c+ cells with clear dendritic morphology, corresponding to the previously described germinal center DCs,31 were scattered throughout the B-cell zones (Figure 2E-F). In some sections, they appeared to stain weakly positive for CD14 (not shown). CD11c+ DCs were distributed evenly throughout the T-cell zones (Figure 2D). Although they were not rigorously counted, they seemed more densely distributed than B-cell zone DCs. Among them, a minority of mature or activated CD86+ DCs were found (Figure 2H), a subpopulation of which expressed CD83 (also see Figure 5). No staining for CD83 or CD86 was observed in the B-cell zones (not shown). Therefore, in 5 of 8 human spleens analyzed in situ, 3 populations of CD11c+ DCs were clearly identified: marginal zone DC, T-cell zone DC, and B-cell zone DC, with a minority of activated cells in the T-cell zone. In vivo activation of dendritic cells in a subset of organ donors By flow cytometry, spleen DCs from most donors were found to be CD8083, and CD86 expression did not seem
significant, implying that spleen DCs were not fully mature or
activated, as previously observed.35 However, because both
CD83+ and CD86+ DC were observed in situ,
fluorescence histograms were analyzed in more detail to quantify these
DC subpopulations (Figure 3A). The
proportion of DCs expressing high levels of CD86 varied from 4.0% to
81% (median, 12%) but was greater than 50% in only 3 of 18 donors
analyzed (donors 93, 106, and 176; Table 1). The frequency histogram of
these percentages (Figure 3B) was suggestive of a bimodal distribution,
though the low sample number does not allow this to be affirmed with
certainty. CD83+ DCs made up 3.4% to 58% of spleen DCs
(median, 5.8%; n = 9). As with CD86, high proportions of spleen DC
from donors 93, 106, and 176 expressed CD83. Indeed, the expression of
CD83 and CD86 by DCs was significantly correlated (Figure 3C;
P < .005; n = 9), implying that these molecules are
up-regulated in a coordinated fashion by DCs in vivo, as previously
observed in vitro.35,42
In donor 93, who had 75% CD86+ spleen DCs, the
distribution and the surface phenotype of spleen DCs was strikingly
different from those seen in most of the other donors. Extremely large
numbers of CD11c+ DCs were concentrated in the T-cell
zones, and the layer of CD11c+ DCs encircling the white
pulp seemed to be absent, though CD11c+ DC were present in
the B-cell zones (Figure 4E-F; Figure
5). Activated CD83+ and
CD86+ DCs were located exclusively in the DC-laden T-cell
zones. As in other donors, CD83+ DC were a subpopulation of
the CD86+ DCs (Figure 5C-D). In donors 106 and 176, the
distribution of spleen DCs appeared more normal, with strongly
CD11c+ cells clearly tracing the edge of the white pulp,
but 1 to 2 T-cell zones per section did contain heavy concentrations of
CD11c+ DCs (Figure 4C-D), many of them expressing
CD83+ and CD86+.
Because the phenotype and distribution of DCs in these 3 atypical
donors suggested in vivo activation, the clinical records of the organ
donors were examined to identify possible clinical correlates of spleen
DC activation (Table 1). The duration between trauma and death might be
a factor: 2 of 5 patients with 28% or more CD86high DCs
were in intensive care for 10 and 4 days, respectively. However, 2 other patients with normal percentages of CD86high DC had
hospital stays of 5 and 4 days, respectively. The median duration
between trauma and death was 1 day (n = 16; Table 1). Particular
attention was paid to bacterial infection given that LPS and endotoxins
are known to result in DC activation in the mouse17,18,43
and in the rat.44 One striking correlation was that donor
176 had a localized, nosocomial infection (lung abscess) with E
coli and Staphylococcus aureus. There was no other recorded infection (all were organ donors and, therefore, extensively tested). It seems that the 2 other donors with more than 50% activated DCs had experienced more extensive trauma than the others: donor 106 had a myocardial trauma that might have resulted in the release of
tumor necrosis factor (TNF)- IL-12 secretion To determine whether these phenotypically activated DCs had a function related to their potential bacterial stimulation and to T-cell activation, the spleen cells from the donor with the most activated DCs (donor 176) were compared with those of a donor with a few activated DCs (donor 172) for the secretion of IL-12. The spontaneous secretion of IL-12 p40 after 24-hour culture was 1554 ± 967 pg/mL for donor 176 compared to 433 ± 0 for donor 172, indicating a functional activation of dendritic cells but perhaps also of other cell types.
Human spleen dendritic cell labeling by flow cytometry and in situ Although mouse spleen DCs have been intensively studied for the past 20 years, relatively little has been published concerning their human counterpart.29,33,35,46 In particular, the phenotype and distribution of human spleen DCs has not yet been described in great detail. In the present work, the surface phenotype of fresh human spleen DCs was studied by flow cytometry, and their distribution was studied by in situ immunofluorescence. Fresh human spleen DCs were defined as large, HLA-DR+ cells negative for markers of other cell lineages. No single DC-specific cell surface marker was found for these cells. For example, as in earlier immunocytochemical studies,29,46 CD1a+ DCs were not found in the spleen. In the mouse, CD11c expression is restricted to DCs, allowing the CD11c promoter to be used to direct specific expression of genes into DCs.41 In contrast, in human spleen and blood, DCs are not the only cells to express CD11c. Nevertheless, we found that DCs expressed CD11c at a higher level than any other cell type, including macrophages, which provided a discriminative tool for in situ immunofluorescence studies. In situ, a clear-cut exclusion between strong CD11c labeling and CD14 (My4) expression was found (except in some B-cell zone DCs), whereas immunocytochemical studies using other monoclonal antibodies had found DC-like cells to express CD14 at various levels.29Overall, there was a close agreement between the flow cytometry and in
situ results concerning the phenotype of CD11c+ DCs. By
both methods, only a minority of CD86+ or CD83+
DCs were detected in most donors, and if many CD83+ and
CD86+ DCs were found in situ (see below), these DCs were
also detected by flow cytometry. Hence, both methods analyzed the same
cells, and in situ immunofluorescence did not reveal the existence of any DC subtypes that escaped analysis by flow cytometry, probably because all DCs had been efficiently released from tissue by
DNAse/collagenase digestion of spleen.32 This combination
of flow cytometric and in situ immunofluorescence data allowed us to
divide spleen DC into 4 subpopulations: CD11c CD11c Marginal zone DCs. First, CD11c+ DCs were situated at the edge of the white pulp. These cells were negative for CD83 and CD86 and, hence, were not fully differentiated for optimal stimulation of T cells. They made up most of CD11c+ DC in most donors. In the human spleen, CD11c+ cells with this distribution have been previously termed marginal zone macrophages29; however, their lack of expression of CD11b and CD14 indicates that they are in fact the human equivalent of the mouse marginating DC population.21,26,27 In the mouse, marginating DCs appear to play a sentinel role, as they retain the capacity to capture and process antigen efficiently.49 The phenotype and localization of their human equivalent, which we shall also refer to as marginal zone DCs, suggest that they fulfill a similar function, sampling blood-borne antigens as they pass through the marginal zone. By flow cytometry, human spleen DCs were found to be positive for the Fc receptors CD32 and CD64. These molecules could be involved in antigen capture by spleen DCs. However, the absence of CD11b, CD21, and CD35 implies that complement receptors are not used for antigen uptake by spleen DCs. In addition, the distribution of CD11c+ cells in donor 93 (discussed below) implies a direct relation in vivo between the CD11c+ DCs at the periphery of the white pulp and those in the T-cell zones, analogous to the relation between mouse spleen marginating DCs and peri-arteriolar DCs.18,26 On the other hand, the distribution of CD11c+ marginal zone DCs in human spleen differed somewhat from that of mouse marginating DCs, which are clustered at the edge of the white pulp, usually adjacent to a T-cell zone.21,26,27 In human spleen, we did not observe such DC clusters. Rather, CD11c+ marginal zone DCs formed a ring surrounding both the T-cell zones and the B-cell zones, in a distribution reminiscent of mouse marginal zone metallophilic macrophages.50 Hence, human CD11c+ marginal zone DCs, while phenotypically equivalent to mouse marginating DCs, seem to have a slightly different anatomic localization.T-cell zone DCs.
Second, numerous CD11c+ DCs were present in the T-cell
zones. Some of these DCs were CD83+ and CD86+,
and, hence, the T-cell zones appeared to be the site of spleen DC
maturation. Labeling in situ showed that in all donors,
CD83+ DCs were a subset of CD86+ DCs, and the
strong positive correlation between the percentage of CD83+
and CD86+ DCs showed that
CD83+CD86+ mature DCs formed a relatively
stable proportion (64% ± 20%; n = 9) of all CD86+
DCs. Hence, spleen DCs in the T-cell zones seem to be progressing along
a differentiation pathway in which immature or sentinel CD83 B-cell zone DCs. Finally, the B-cell zones also contained CD11c+ DCs, which by microscopy appeared larger than the CD11c+ cells in the T-cell zones and in some sections stained weakly positive for CD14. These 2 observations, together with the previously reported expression of CD11b by tonsil germinal center DCs,31 imply that B-cell zone DC differ in some respects from marginal zone and T-cell zone DCs and may be closer to macrophages. In addition, they were never observed to express CD83, CD80, or CD86 by immunofluorescence microscopy and were, therefore, not fully differentiated for T-cell stimulation, even in the 3 donors who had very high proportions of activated DCs. This indicates clearly that in vivo activation of these cells requires different signals from those that activate marginating and T-cell zone DCs. DC activation in a subset of donors. Very high proportions of CD83+ CD86+ CD11c+ DCs, suggesting in vivo DC activation, were observed in a subset (3 of 18; 17%) of organ donors. Spleens of the same 3 donors (of 8 spleens examined by in situ immunofluorescence) also showed signs of DC activation in situ. Donor 93 in particular showed a striking alteration in the distribution of spleen DCs, with few marginal zone DCs and large numbers of CD83+ CD86+ DCs concentrated in the T-cell zones. Similar changes in DC distribution were observed only in a few T-cell zones in donors 106 and 176. The high proportion of DCs (1.8% of SMC) in donor 93 may account for the fact that changes in DC distribution were more clearly visible in this donor. However, there was no relation between DC number and DC phenotype or distribution; donor 108, in whom DCs made up 1.7% of SMC, showed a normal distribution of DCs. Moreover, a high spontaneous secretion of IL-12 was found in the spleen cells from donor 176, who had the most activated DC phenotype and a localized bacterial infection. This interleukin may have been secreted by other cell types, but the data are compatible with a secretion by activated DC.3 The up-regulation of CD83 and CD86 on human DC in vitro occurs in response to TNF- , IL-1, and microbial products such as LPS.5,51 In the mouse, the same stimuli provoke in vivo
activation of DCs and their migration to the T-cell regions of lymphoid
organs.43,52 In particular, relocalization of mouse spleen
DCs from the marginal zone to the T-cell zones, with simultaneous
up-regulation of CD86, is caused by the injection of LPS or of
T gondii-derived antigens.18,19 The
similarity of the observed phenotype and distribution of DCs in donors
93, 106, and 176 to those in mice led us to search for a similar cause
of DC activation. This was distinctly possible for donor 176, who was
the only subject with an ongoing bacterial infection. The possible
cause of DC activation in donors 93 and 106 was less clear and was
perhaps related to more extensive trauma than in the other donors,
which may have increased the serum levels of TNF-, such as after
myocardial infarction or cardiac surgery.45,53,54 However,
we were unable to verify the TNF- hypothesis because of the lack of
serum samples. In addition, the kinetics of DC activation and
redistribution after microbial challenge in mice is transient, with a
maximum after 6 hours,17-19,55 and other organ donors may
have displayed similar changes transiently.
DC activation in organ donors may have consequences for the fate of
transplanted organs. Interstitial DCs in transplanted organs are good
candidates for allostimulatory passenger leukocytes, postulated as
initiators of graft rejection.4,56 Indeed, artificially increasing the numbers of phenotypically mature donor DCs in
transplanted liver by donor pretreatment with Flt-3 ligand resulted in
acute rejection after transplantation in mice.57 In
conclusion, the data presented here indicate that the human spleen can
be the site of activation and of microanatomic redistribution of DCs on
bacterial stimulation or extensive trauma, pointing to events potentially occurring at the initiation of the immune response.
We thank Prof T. F. Tedder (Duke Medical University, Durham, NC) for generously providing the HB15a monoclonal; Dr C. Sylla for providing spleen samples; M. S. Laye for technical help; Drs G. Milon, J. G. Guillet, and E. Souil for kind advice; and Drs M. Moser and C. Müller for critical review of the manuscript.
Submitted May 25, 2000; accepted January 15, 2001.
Supported by grants from the Agence Nationale de Recherches sur le SIDA and the Fondation pour la Recherche Médicale (A.H. and D.M.), SIDAction (Ensemble Contre le SIDA) (A.S., C.T.), and the CAPES (F.G).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Anne Hosmalin, Unité INSERM 445, 27 rue du Faubourg St Jacques, 75014 Paris, France; e-mail: hosmalin{at}cochin.inserm.fr.
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C. Gurer, T. Strowig, F. Brilot, M. Pack, C. Trumpfheller, F. Arrey, C. G. Park, R. M. Steinman, and C. Munz Targeting the nuclear antigen 1 of Epstein-Barr virus to the human endocytic receptor DEC-205 stimulates protective T-cell responses Blood, August 15, 2008; 112(4): 1231 - 1239. [Abstract] [Full Text] [PDF]
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C. Nobile, M. Lind, F. Miro, K. Chemin, M. Tourret, G. Occhipinti, S. Dogniaux, S. Amigorena, and C. Hivroz Cognate CD4+ T-cell-dendritic cell interactions induce migration of immature dendritic cells through dissolution of their podosomes Blood, April 1, 2008; 111(7): 3579 - 3590. [Abstract] [Full Text] [PDF]
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C. Lecureuil, B. Combadiere, E. Mazoyer, O. Bonduelle, A. Samri, B. Autran, P. Debre, and C. Combadiere Trapping and apoptosis of novel subsets of memory T lymphocytes expressing CCR6 in the spleen of HIV-infected patients Blood, May 1, 2007; 109(9): 3649 - 3657. [Abstract] [Full Text] [PDF]
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M. Lindstedt, K. Lundberg, and C. A. K. Borrebaeck Gene Family Clustering Identifies Functionally Associated Subsets of Human In Vivo Blood and Tonsillar Dendritic Cells J. Immunol., October 15, 2005; 175(8): 4839 - 4846. [Abstract] [Full Text] [PDF]
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G. Ferlazzo, M. Pack, D. Thomas, C. Paludan, D. Schmid, T. Strowig, G. Bougras, W. A. Muller, L. Moretta, and C. Munz Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs PNAS, November 23, 2004; 101(47): 16606 - 16611. [Abstract] [Full Text] [PDF]
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N. S. Wilson, D. El-Sukkari, and J. A. Villadangos Dendritic cells constitutively present self antigens in their immature state in vivo and regulate antigen presentation by controlling the rates of MHC class II synthesis and endocytosis Blood, March 15, 2004; 103(6): 2187 - 2195. [Abstract] [Full Text] [PDF]
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N. S. Wilson, D. El-Sukkari, G. T. Belz, C. M. Smith, R. J. Steptoe, W. R. Heath, K. Shortman, and J. A. Villadangos Most lymphoid organ dendritic cell types are phenotypically and functionally immature Blood, September 15, 2003; 102(6): 2187 - 2194. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
receptors: half (54% ± 3%;
n = 3) of the gated DCs expressed high levels of Fc
