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IMMUNOBIOLOGY
From the Dendritic Cell Laboratory, Mater Medical
Research Institute, Brisbane, Australia; and the University of
Queensland Department of Surgery, Mater Misericordiae Hospital,
Brisbane, Australia.
Studies on purified blood dendritic cells (DCs) are hampered by
poor viability in tissue culture. We, therefore, attempted to study
some of the interactions/relationships between DCs and other blood
cells by culturing unseparated peripheral blood mononuclear cell (PBMC)
preparations in vitro. Flow cytometric techniques were used to
undertake a phenotypic and functional analysis of DCs within the
cultured PBMC population. We discovered that both the
CD11c+ and CD11c Dendritic cells (DCs) are a unique leukocyte
population, which control the primary immune response.1
They are extremely potent antigen-presenting cells (APCs),
distinguished by their exceptional ability to prime naive T cells. They
lack the expression of CD3, CD14, CD16, and CD19 molecules but
characteristically express high levels of major histocompatibility and
costimulatory antigens. Two subsets of blood DCs have been described
according to the differential expression of CD11c and CD123
antigens and peanut agglutinin binding.2-4 They
appear to have distinctive characteristics and functions, including
differential regulation by cytokines such as granulocyte
colony-stimulating factor and Flt3 ligand.2 The classical
CD11c+ myeloid DCs traffic into tissues and mucosal
surfaces to act as immune sentinel cells and, after activation by
pathogens or appropriate inflammatory stimuli, migrate by lymphatics to
secondary lymphoid organs, where they initiate immune responses. The
CD11c Although there is general agreement that blood DCs are derived from
hematopoietic stem cells, the concept that the different DC subsets may
represent the progeny of different lineages remains controversial. The
CD11c+ myeloid blood DCs, express the CD13 and CD33 myeloid
differentiation antigens and include precursors for both epithelial and
deep tissue (eg, dermal) DCs. They depend on granulocyte-macrophage
colony-stimulating factor (GM-CSF) for survival in vitro2,6
but apparently not in vivo, as DC populations are unchanged in
GM-CSF-deleted mice.7 In contrast, the
CD11c Blood DCs are commonly purified for functional studies, and similar
preparations are also used in some clinical DC immunotherapy protocols.11 We showed previously that activated blood DCs
were superior to freshly isolated DCs in processing, presenting, and stimulating antigen-specific T-lymphocyte responses.12
However, as described, the ex vivo survival of isolated blood DCs, both CD11c+ and CD123hi, is highly dependent on the
support of cytokines, without which in vitro activation and culture
would result in substantial loss of numbers. We reasoned that it might
be preferable to culture peripheral blood mononuclear cells (PBMCs) as
a whole, before isolating the blood DCs for functional and/or
immunotherapeutic protocols. We found that culturing DCs as part of the
whole PBMC preparation not only provided the required DC activation but
also afforded greater survival and, thus, recovery of functional blood DCs.
Monoclonal antibodies and reagents
FITC-conjugated sheep antimouse immunoglobulin (SAM) was purchased from
Amrad Biotech (Victoria, Australia); PE.Cy5-conjugated streptavidin
from DAKO (Carpinteria, CA); mouse serum, 7-amino-actinomycin-D (7-AAD), propidium iodide (PI), and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St Louis, MO). FITC-Dextran (F-Dx, Mr = 42 000) and Lucifer yellow (LY) from Sigma-Aldrich
were obtained as lyophilized powder and freshly reconstituted in medium
before use. Tetanus toxoid (TT) was obtained from Commonwealth Serum Laboratories (Melbourne, Australia). TT was labeled with FITC (Sigma-Aldrich) in 0.5 M bicarbonate buffer (pH 9.5), dialyzed in
phosphate-buffered saline (PBS) for 48 hours, and the FITC-to-TT molar
ratio was determined to be 9.6:1.
Recombinant human GM-CSF (rhGM-CSF) was obtained from
Sandoz-Pharma (Sydney, Australia), rh interleukin-3 (IL-3) from
Gibco Life Technologies (Melbourne, Australia), rhIL-4 (Sigma-Aldrich), and rh tumor necrosis factor- Complete media included RPMI 1640 supplemented with 10% fetal calf
serum (FCS), penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM), and nonessential amino acids (all purchased from
Gibco Life Technologies) was used throughout the study, except where
indicated. For antigen uptake experiments, the media also contained 25 mM HEPES (Gibco Life Technologies). For some parallel culture
experiments, X-VIVO 10 media (Biowhittaker, Walkersville, MD) was used.
Cell preparation and culture
Blood DCs were obtained from PBMCs by a 2-step purification method as described previously with minor modifications.13 Briefly, T cells, B cells, monocytes, and natural killer cells were depleted by using immunomagnetic cell separation (Biomag beads; Polysciences, Warrington, PA; and Variomacs; Miltenyi Biotech, Gladbach, Germany) with antibodies specific for CD3, CD19, CD14, CD11b, and CD16. To remove any remaining lineage-positive cells after the depletion procedure, this cell preparation was labeled with FITC-SAM, blocked with mouse serum, stained with PE-conjugated antibodies specific for CD7, CD20, CD34, CD56, and CD64, and then purified by using flow cytometric sorting (FACS Vantage; Becton Dickinson) for cells that were negative for FITC and PE signals. Monocyte-derived DCs (Mo-DCs) were generated by using the adherence
method as described previously.14 Briefly, PBMCs were plated in 80-cm2 Nunclon tissue culture flasks (Nunc,
Roskilde, Denmark) and incubated for 2 hours. Nonadherent cells were
removed, and the remaining cells were cultured in complete media with
GM-CSF (200 U/mL) and IL-4 (50 U/mL) for 5 days to produce immature
Mo-DCs,15 which were CD1a+
CD14 Flow cytometric analysis To analyze DCs in fresh and cultured PBMCs, cells were stained with PE-conjugated lineage-specific mAbs (CD3, CD14, CD16, CD19) and CD34, and PE.Cy5-conjugated HLA-DR mAb (Figures 1A, 2A, 6B). CD34 was added to the lineage mixture to exclude circulating hematopoietic stem cells.16 The expression of CMRF-44 and -56 was analyzed in the FITC channel. For other 3-color immunofluorescence staining of PBMCs, the combination of FITC fluorochrome for lineage markers, PE.Cy5 for HLA-DR, and PE for CD40, CD80, CD86, CD83, and CD123 and CD11c subset molecules, was used (Figure 1B,D). For each analysis, 3 × 105 to 106 events were collected within the mononuclear gate. To further define the DC subsets in PBMCs, we used 4-color flow cytometric analysis by using the APC channel for CD11c (Figures 3D and 4). Sorted Lin cells
were gated for HLA-DR staining (PE.Cy5) and then analyzed for their
expression of CD11c (FITC) and CD123 (PE). Analysis was performed on a
FACS Calibur flow cytometer (Becton Dickinson) by using CellQuest
software (Becton Dickinson). Data were analyzed by using either
CellQuest 3.1 or FCS Express software (De Novo Software, ON,
Canada).
Viable cells were gated based on forward and side scatter
characteristics (R1, Figure 2A). Within the R1 gate, more than 99.5% of the cellular events in PBMCs and sorted DCs, fresh and cultured (to
3 days), were negative for 7-AAD or PI when labeled (data not shown).
TruCOUNT analysis of absolute cell counts TruCOUNT tubes (Becton Dickinson) were used to determine the absolute counts of DCs in PBMC cultures. Each tube contained a lyophilized pellet that dissolves, releasing a known number of fluorescent beads. The tubes were used according to manufacturer's recommendations with minor modifications.17 PBMCs were seeded and cultured in 96-well flat-bottom plates at 107 cells/mL (200 µL/well) and were harvested for staining at the various time points. The antibody mixture (CD3, CD14, CD16, CD19, CD34)-PE and HLA-DR.PE.Cy5 was prepared for the experiment at a 1:30 dilution first, to ensure a consistent concentration of antibodies for each analysis. Then, 30 µL antibody mix was added to the TruCOUNT tube, followed by 20 µL of cells from the wells. The tube was vortexed gently and incubated in the dark at room temperature for 15 minutes. Finally, 350 µL PBS was added, making a total volume of 400 µL before FACS analysis. A minimum of 500 Lin HLA-DR+ DC
events (R3, Figure 2A), and/or 20 000 to 35 000 beads (R2, Figure 2A)
were acquired for each analysis. Each sample was analyzed in
triplicate. The absolute number of DCs in each sample was calculated as
the average of the triplicate tubes, each being determined by comparing
the cellular events (R3) with bead events (R2) and expressed as DC
counts/104 beads.
Depletion assays Single-cell lineages or populations in PBMCs were labeled by using the PE-conjugated mAb: CD14, CD16, CD19, or CD3, respectively. Each cell population was depleted by FACS sorting and cultured in parallel with unseparated PBMCs, as the positive control for each experiment. The cultured cells were labeled and analyzed by FACS to assess the percentage of DCs present at predetermined time points of the culture period.Antigen uptake assays PBMCs were seeded in 6-well plates at 107 cells/mL for culture, harvested each day, and resuspended in complete medium for incubation with the antigens. F-Dx (1 mg/mL), LY (1 mg/mL), or F-TT (0.5 mg/mL) was added and incubated with the cells either at 4°C (control) or 37°C for 60 minutes. Cells were washed 4 times in cold PBS, then stained with the antibody mixture (CD3, CD14, CD16, CD19, CD34)-PE and HLA-DR.PE.Cy5, and analyzed immediately by FACS. The level of antigen uptake by DCs was assessed on the FITC channel after gating for the Lin HLA-DR+ cells and was calculated
as the difference in mean fluorescence intensity ( MFI) between the
test (37°C) and control (4°C) tubes for each sample.
Allogeneic mixed leukocyte reaction Sorted DCs (10 to 20 000 cells per well) were incubated with allogeneic T lymphocytes (105 cells) for 5 days in 96-well U-bottom plates. Sixteen hours before harvesting the cells, 18.5 kBq of 3H-thymidine was added to each well. 3H-thymidine uptake was counted in a liquid -scintillation counter (MicroBeta Trilux Scintillation Counter;
Wallac, Turku, Finland).
Statistical analysis Paired statistical analysis was performed by using the Student 2-tailed t test.
Blood DCs survive in cultured PBMCs without exogenous cytokines Blood DCs were defined within PBMCs by 2-color flow cytometric analysis as HLA-DR+ cells that were lineage (CD3, CD14, CD16, CD19, and CD34) negative. Sorted blood DCs survived poorly in vitro when isolated from the PBMC environment, even when cultured with the cytokines GM-CSF and IL-3.2,8 However, we found that when kept in contact with the other PBMCs, the DCs survived for at least 3 days, in vitro, without the addition of exogenous cytokines (Figure 1A). The relative percentage of Lin HLA-DR+ DCs in PBMCs was the same at the
end of a 3-day culture as at its initiation (n = 10). The
Lin HLA-DR+ DCs in cultured PBMCs appeared to
separate into discrete HLA-DRhi and HLA-DRlo
populations compared with the more homogeneous profile obtained when
examined immediately ex vivo (Figure 1A). An apparent increment in the
relative percentage of Lin HLA-DR+ DCs was
also noted on day 1 (more in next section). When parallel experiments
(n = 3) were performed by using X-VIVO 10 (a serum-free medium), we
observed the same phenomenon. There was no statistically significant
difference between the 2 culture systems (days 1-3, P > .7).
We analyzed these cultured PBMC DCs for their expression of the
costimulatory molecules (CD40, CD80, and CD86) and the activation markers (CMRF-44, CMRF-56, and CD83). DCs within the PBMC cultures spontaneously and progressively up-regulated these molecules in culture
(Figure 1B,C). Both DC subsets, defined by the CD11c and CD123
molecules, were maintained in PBMCs throughout the culture period
(Figure 1D), although the CD11c+ DC population up-regulated
its expression of the CD123 antigen (Figure 3B,D). The
Lin
TruCOUNT analysis quantifies rise of absolute DC counts in cultured PBMCs The definite but variable increase in the percentage of DCs in PBMCs noted after overnight (16-24 hours) culture (n = 10) was investigated further. To assess whether the increase in number reflected an increase in absolute DCs or differential survival with respect to the other PBMC populations in culture, TruCOUNT beads (Figure 2A) were used to obtain absolute DC counts in 8 further experiments. The TruCOUNT analysis confirmed a significant rise in absolute counts of Lin HLA-DR+ events after
the overnight culture period (P < .001, n = 8; Figure 2B left) and showed a close correlation between the changes in the
percentage of DC number (in PBMCs) and absolute cell counts (not
shown). The HLA-DRlo DC population increased by
235% ± 77% (SEM) compared with 150% ± 45% (SEM) in the
HLA-DRhi population (Figure 2B left).
To exclude the possibility that DCs or DC precursor proliferation
during the culture period was responsible for the increase, fresh PBMCs
were irradiated (3000 Gy), then cultured, and analyzed in parallel with
their nonirradiated controls, again using TruCOUNT beads. Irradiation
of the starting PBMC preparation did not affect the rise of absolute DC
counts (n = 3): Similar increases in absolute Lin Isolated DCs survive poorly in culture as determined by TruCOUNT analysis By using the TruCOUNT assay, we confirmed previous data,2,8 indicating that isolated DCs survive poorly, even when cultured with GM-CSF and IL-3. Sorted Lin cells
increased in size and granularity with culture as indicated by changes
in the forward and side scatter profiles (Figure 3A). Three-color FACS
analysis with the fluorochrome combination of HLA-DR-PE.Cy5,
CD11c.FITC, and CD123.PE was used. The (HLA-DR+)
CD11c+ DC subset can be easily distinguished from the
(HLA-DR+) CD11c CD123hi
population immediately ex vivo, but after overnight culture, some of
the CD11c+ cells, which were CD123lo,
up-regulated the intensity of CD123 expression (Figure 3B), whereas
CD11c CD123hi cells died rapidly. In freshly
isolated DCs, less than 1% was double-positive (ie, CD11c+
CD123hi) but this double positivity rose to 33% after
overnight incubation. The isolated CD11c+ DC subset
survived better (74% to 78% of starting cells on days 1 to 3),
whereas the isolated CD11c CD123hi DC subset
numbers fell sharply to 34% of the starting population after overnight
culture and to 2% and 1% of the original cells on days 2 and 3, respectively (Figure 3B,C).
Because the CD11c+ DC subset up-regulated CD123 antigen
expression (Figure 3B), we used 4-color flow cytometry (with the
fluorochrome combination of Lin.FITC, HLA-DR-PE.Cy5, CD11c.APC, and
CD123.PE) to define the 2 DC subsets in cultured PBMCs accurately. The
CD11c
DC numbers increase rapidly in cultured PBMC Because the rise in Lin HLA-DR+ DCs
occurred mainly within the first 24 hours, we proceeded to evaluate
this phenomenon more closely. The increase was rapid and occurred
within the first 4 hours of incubation, with the peak and plateau
attained after 8 to 12 hours of incubation (Figure
5). The DC number returned to baseline
level after 48 hours of culture (Figure 5). Taken together with the
cell irradiation experiments above, this finding suggested that the
increase in cell numbers was due to the contribution of either a
population of Lin+ cells (down-regulating their markers) or
a population of Lin HLA-DR cells,
up-regulating HLA-DR expression to enter the Lin
HLA-DR+ DC pool on culture in vitro.
PBMC CD14+ and CD16+ cells contribute to
the Lin
HLA-DR+ DCs, we performed a series of depletion
experiments. Single-cell populations were removed from PBMCs by using
appropriate CD markers (CD19, 14, 16, and 3), and each depleted PBMC
population was then cultured in parallel with the control-starting
whole PBMC preparation. The Lin HLA-DR+ gate
was used to follow changes in cell numbers and cell dot plot profiles.
In contrast to the characteristic rise in DC numbers in whole PBMC
cultures (Figure 6), the
Lin HLA-DR+ DC number remained remarkably
constant in the PBMC cultures that were depleted of CD14+
monocytes (Figure 6). In the CD16+ cell-depleted cultures,
the Lin HLA-DR+ DC number only rose after the
4-hour time point, and this rise was considerably attenuated (Figure
6). When CD19+ B cells were depleted, the effect was
minimal (Figure 6). Similarly, the removal of CD3+ T cells
from the culture system did not affect the rise in DC number (n = 2,
not shown). This finding clearly demonstrated that the rapid and
spontaneous in vitro emergence of "new" Lin
HLA-DR+ cells required the presence of CD14+
and/or CD16+ PBMCs in the culture.
The new Lin Activated DCs increase dextran but decrease TT uptake capacity Next, we tested the antigen uptake capacity of the DCs within PBMCs. Previous reports have shown that TNF-
differentiated/activated Mo-DCs and that cultured Langerhans cells
down-regulate their antigen uptake capacity and their increased
allostimulatory activity.14 In direct contrast to this,
the culture and activation of DCs in PBMC preparations increased their
uptake capacity of F-Dx (Figure 7A,
left). The uptake of the soluble agent LY did not change greatly during
culture (Figure 7A, middle). The greatest uptake of F-TT occurred with
fresh DCs and, thereafter, decreased progressively with culture and
activation (Figure 7A, right). In each test system, the
HLA-DRhi population (Figure 7A, hatched bars) appeared to
have better antigen uptake capacity than the HLA-DRlo
population (Figure 7A, open bars).
We then generated Mo-DCs and confirmed reports of our
own15,18 and others14 that their F-Dx uptake
capacity was dramatically reduced when they become
differentiated/activated (Figure 7B, left). However, the cultured
sorted Lin DCs isolated from cultured PBMCs are efficient stimulators in the mixed leukocyte reaction Finally, to test their costimulatory function, DCs were sorted from PBMCs after 3 days of in vitro culture and tested for their allostimulatory capacity. DCs cultured with PBMCs were as efficient as freshly isolated DCs in stimulating the allogeneic mixed leukocyte reaction (MLR) in 3 separate experiments (data not shown).
This study produced the entirely novel finding that DCs survive in
cultured PBMCs for up to 3 days without the addition of exogenous
cytokines. This data contrasted notably with what appeared to be the
mandatory addition of cytokines to maintain even modest isolated blood
DC survival.2,8 Of particular importance, we were able to
identify the CD123hi CD11c We also established a second phenomenon, namely an increase in the
absolute numbers of Lin The differentiation pathway of DCs from CD34+ hematopoietic
progenitors has been studied in vitro.28 CD34+
cells obtained from peripheral blood, bone marrow, or cord blood cultured with GM-CSF and TNF- Circulating blood DCs do not up-regulate expression of costimulatory (CD40, CD80, CD86) or activation (CMRF-44, CMRF-56, CD83) markers in the face of stress and associated cytokine changes,33 yet they invariably up-regulate these molecules, when isolated from the blood. The presence of other PBMCs, although enhancing DC survival in vitro, did not prevent the up-regulation of DC differentiation/activation markers in the whole PBMC cultures. This finding led us to speculate that the vascular endothelium, which prevents activation of the clotting and coagulation cascades, may also be important in maintaining the quiescent state of the circulating DCs during surgical and physical stress.33 It is interesting to speculate that the rapid increase in circulating DC counts noted in those studies may, in part, be due to the recruitment of the PBMC progenitors documented here. In contrast to what has been described for Mo-DCs, we have established that blood DCs increase their dextran uptake capacity with extended in vitro culture and activation. This finding confirms preliminary observations in this regard18 and our previous functional antigen presentation data.12 The macrophage mannose receptor (MMR), which mediates dextran uptake in Mo-DCs,14 is not constitutively expressed on blood DCs ex vivo or after activation,18 (K. A. MacDonald et al, unpublished data, December 2001). Therefore, although the dextran uptake may still be receptor dependent, this mechanism is unlikely to be mediated by the MMR. Further studies are now under way to determine if DEC 205 (CD205), another C-type lectin receptor constitutively expressed on blood DCs, may be responsible for this function. There is intense interest in using DCs and exploiting their distinctive
immune function for cancer immunotherapy. Although they can be isolated
from tissues like tonsils and skin, the most accessible source of DCs
is in the blood. Most clinical immunotherapy trials for various cancers
have used DCs derived in vitro from monocytes
(Mo-DCs).11,34 The use of monocytes as the source of DCs,
rather than directly harvested blood DCs, is facilitated by the fact
that monocytes make up 10% to 15%, whereas blood DCs constitute less
than 1% PBMCs. Mo-DCs are prepared in vitro from blood monocytes under
the control of cytokines (generally, GM-CSF and IL-4) over a period of
5 to 14 days and, once produced, require a further defined stimulus to
activate/mature them.14 DCs generated from
CD34+ hematopoietic stem cells in vitro have also been used
in some immunotherapeutic protocols.11,34 We champion the
use of DCs isolated directly from blood. This latter approach offers
clear theoretical advantages in that these blood DCs are in their
natural and defined state of differentiation, free from the influence of exogenous cytokines, and presumably capable of responding to and
stimulating immune responses in a more physiologic
manner.35 DCs that are directly harvested from blood
spontaneously acquire an activated phenotype after a brief period
(hours) of in vitro culture.33 PBMCs can be cultured as a
whole to induce an appropriate amount of differentiation/activation
before isolating the blood DCs, perhaps using mAbs CMRF-44 and
-5613,36 selection, and magnetic bead separation
(J.A.L. et al, unpublished data, January 2002). Blood DCs are,
in our hands, more efficient than Mo-DCs in inducing primary
proliferative and interferon- We have described an entirely new assay for studying the physiology of blood DCs. This system maintained blood DC survival in culture, provided the requisite DC activation (without the need for exogenous cytokines or stimuli), and permitted effective antigen loading into DCs. Furthermore, these findings may represent a more physiologic option for antigen-loading DCs in cultured PBMCs before their isolation for immunotherapeutic protocols.
We thank Dr Slavica Vuckovic for constructive discussion of the data, L. Brown, G. Chojnowski, C. Schmidt, and M. Rist for assistance in cell sorting and 4-color flow cytometry. We thank all our volunteers and the Australian Red Cross Blood Service, Brisbane, for blood supplies.
Submitted August 23, 2001; accepted December 3, 2001.
Supported by a Mater Medical Research Institute grant. C.S.K.H. was supported by the Royal Australasian College of Surgeons' Raelene Boyle and the Paul Mackay Bolton Cancer Research Scholarships.
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: Derek N. J. Hart, Mater Medical Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, QLD 4101, Australia; e-mail: dhart{at}mmri.mater.org.au.
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© 2002 by The American Society of Hematology.
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  V. R. Cicinnati, J. Kang, G. C. Sotiropoulos, P. Hilgard, A. Frilling, C. E. Broelsch, G. Gerken, and S. Beckebaum Altered chemotactic response of myeloid and plasmacytoid dendritic cells from patients with chronic hepatitis C: role of alpha interferon J. Gen. Virol., May 1, 2008; 89(5): 1243 - 1253. [Abstract] [Full Text] [PDF]
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 I. G. Rodrigue-Gervais, L. Jouan, G. Beaule, D. Sauve, J. Bruneau, B. Willems, R.-P. Sekaly, and D. Lamarre Poly(I:C) and Lipopolysaccharide Innate Sensing Functions of Circulating Human Myeloid Dendritic Cells Are Affected In Vivo in Hepatitis C Virus-Infected Patients J. Virol., June 1, 2007; 81(11): 5537 - 5546. [Abstract] [Full Text] [PDF]
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 S. Vuckovic, D. Khalil, N. Angel, F. Jahnsen, I. Hamilton, A. Boyce, B. Hock, and D. N. J. Hart The CMRF58 antibody recognizes a subset of CD123hi dendritic cells in allergen-challenged mucosa J. Leukoc. Biol., March 1, 2005; 77(3): 344 - 351. [Abstract] [Full Text] [PDF]
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 S. Beckebaum, X. Zhang, X. Chen, Z. Yu, A. Frilling, G. Dworacki, H. Grosse-Wilde, C. E. Broelsch, G. Gerken, and V. R. Cicinnati Increased Levels of Interleukin-10 in Serum from Patients with Hepatocellular Carcinoma Correlate with Profound Numerical Deficiencies and Immature Phenotype of Circulating Dendritic Cell Subsets Clin. Cancer Res., November 1, 2004; 10(21): 7260 - 7269. [Abstract] [Full Text] [PDF]
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 F. F. Fagnoni, B. Oliviero, G. Giorgiani, P. De Stefano, A. Deho, C. Zibera, N. Gibelli, R. Maccario, G. Da Prada, M. Zecca, et al. Reconstitution dynamics of plasmacytoid and myeloid dendritic cell precursors after allogeneic myeloablative hematopoietic stem cell transplantation Blood, July 1, 2004; 104(1): 281 - 289. [Abstract] [Full Text] [PDF]
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 M. Dauer, B. Obermaier, J. Herten, C. Haerle, K. Pohl, S. Rothenfusser, M. Schnurr, S. Endres, and A. Eigler Mature Dendritic Cells Derived from Human Monocytes Within 48 Hours: A Novel Strategy for Dendritic Cell Differentiation from Blood Precursors J. Immunol., April 15, 2003; 170(8): 4069 - 4076. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, continuous line), CD14-depleted (
),
CD16-depleted (
), and CD19-depleted (
) was evaluated over a
24-hour period (3 experiments shown). Analyses at each time point were
performed in triplicates. Error bars show SEM (A). Dot plots
demonstrating the Lin
4°C), and expressed as relative fold 
responses (Y. Osugi et al, unpublished
data, January 2002).