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,
,§,¶
*
The Center for Blood Research,
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Departments of
Pediatrics,
§
Neurology, and
¶
Pathology, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods to investigate DC biology have been hindered by poor yields.
These methods principally involved DC isolation by negative selection
of specific cell-surface Ags present on other leukocytes but absent
from DC (13, 14, 15, 16). In addition, earlier methods
incorporated some form of density-gradient separation (e.g.,
Metrizamide, Percoll) and short-term tissue culture. The DC yields of
these methods were extremely low, representing 
0.1% of PBMC
(1, 13).
With reports that Mo are precursors of both DC and macrophages (M
)
(17, 18, 19), the problem of low yield has been overcome. Most
contemporary studies rely on Mo-derived DC (MDDC) as the primary source
of DC (19, 20), while others generate DC from
CD34-expressing bone marrow progenitors (2). MDDC are
generated by 7 days of culture of Mo in recombinant human (rh) GM-CSF
and rhIL-4 as described by Sallusto and Lanzavecchia (17).
This method gives rise to immature DC expressing high levels of HLA-DR
and CD86 and low to no CD14 or CD83. Maturation of this immature DC
population to efficient APCs is induced by additional culture
with IL-1ß, LPS, TNF-
, PHA, or calcium ionophore (19, 21). Although this method yields more DC than previous methods,
the necessity for prolonged culture in the presence of cytokines raises
concerns that MDDC may not be phenotypically or functionally similar to
their counterparts in vivo (22, 23).
Our study stemmed from the observation that SRBC-enriched T cell
cultures contain large numbers of contaminating cells with DC
morphology and immunofluorescence staining characteristics
(24). Because SRBC bind to CD2, and 5–50% of circulating
Mo are CD2+ (25), we explored the
possibility that CD2 is a marker for DC in blood. This costimulatory
molecule is a 40- to 60-kDa glycoprotein member of the Ig superfamily
(26). CD2 is principally expressed by T and NK cells
(26, 27), but low expression has also been reported on
subsets of thymic B cells, DC, Mo, and M (1, 25, 28).
CD2 has three regions defined as T111,
T112, and T113
(25). mAbs that bind to the T111
region inhibit binding of its physiological ligand, CD58 (LFA-3), on
SRBC, while mAbs binding to the T112 and
T113 regions do not interfere with binding.
Ligation of CD2 with mAb pairs activates T and NK cells through the
same pathway as the natural ligand CD58 (26, 27).
Our results show that CD2 is present on approximately one-third of
CD14high PBMC and that this CD2-expressing
population is phenotypically, morphologically, and functionally
different from the CD2-
CD14high population. Furthermore, our studies
suggest that CD2+ CD14high
and CD2- CD14high PBMC
represent DC and precursor M (pM), respectively, in the
circulation. Thus, we provide evidence that DC exist at a much higher
frequency in the blood than previously considered and that they can be
isolated from the PBMC without prolonged culture and without the
addition of cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from buffy coats from healthy volunteers (Transfusion Therapy, Children’s Hospital, Boston, MA) by separation on Ficoll (Pharmacia, Piscataway, NJ) gradients, washed twice, and resuspended at 5 x 106 cells/ml. This PBMC-enriched population was layered over a 14.5% w/v discontinuous Metrizamide (Sigma, St. Louis, MO) gradient (13) and centrifuged (Sorvall RT 6000, DuPont, Wilmington, DE) at 650 x g for 10 min to separate the PBMC into low (Mo) and high (T, B, and NK cells) density fractions.
The CD2+ Mo were isolated from the low-density cells by first removing contaminating T, B, and NK cells with anti-CD3, anti-CD19, and anti-CD56 immunomagnetic beads (Miltenyi Biotech, Heidelberg, Germany). The remaining cell population was >95% CD14high by flow cytometry. These cells were incubated with a 1:100 dilution of mouse mAb (in ascitic fluid) to human CD2 (1Old2-4C1 (anti-T112); Dana-Farber Cancer Institute, Boston, MA) (26) for 30 min at 4°C, washed, and incubated with goat anti-mouse IgG magnetic beads (Miltenyi Biotech). Following incubation, the preparation was passed through a magnetic column according to the manufacturer’s instructions. The magnetic column retained the CD2+ cells, which were >96% pure, while the CD2- cells were >95% pure by flow cytometry with anti-CD2 and anti-CD14. A blocking buffer containing 10% v/v heat-inactivated pooled human serum (PHS) (Nabi, Boca Raton, FL) and human IgG (50 mg/ml; Immuno AG, Vienna, Austria) in HBSS without magnesium and calcium (Cellgro; Fisher Scientific, Pittsburgh, PA) was used to prevent nonspecific mAb binding during each stage of isolation or flow cytometric analysis. For morphologic and functional studies of freshly isolated, noncytokine-incubated CD2+ and CD2- Mo, we used culture medium (CM) containing RPMI 1640 (Cellgro) supplemented with 10% heat-inactivated PHS, 20 µg/ml gentamicin, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD).
Generation of MDDC
MDDC were generated as described by Sallusto and Lanzavecchia (17). In brief, Mo were obtained as described above. This enriched population, >95% CD14high, was subsequently cultured for 7 days in the presence of rhGM-CSF and rhIL-4 at 500 U/ml and 250 U/ml, respectively. Every 2 days, 50% of the CM was removed and replaced with fresh rhGM-CSF/IL-4 medium.
Isolation and activation of naive CD4 T cells
Naive CD4 T cells were isolated from the high-density PBMC fraction of healthy HIV-1-seronegative donors by negative selection, according to the manufacturer’s instructions, with immunomagnetic beads (Miltenyi Biotech) specific for CD8, CD14, CD19, CD56, and CD45RO Ags. The enriched population was >95% pure for CD4/CD45RA-expressing T cells as determined by FACS analysis.
Alloreactive MLR and Ag-specific T cell proliferation assays were performed with naive CD4 T cells obtained as described above and resuspended in CM at 106 cells/ml. Autologous T cells (105/well) were cultured with freshly isolated CD2+ or CD2- Mo (5 x 103) in 96-well U-bottom plates (Falcon; Fisher Scientific) in the absence or presence of 10 µg/ml recombinant HIV-1 gp120 (HIV-1IIIB, catalog no. 1061; Immunodiagnostics, Bedford, MA). Allogeneic T cells (1 x 105) were cultured with freshly isolated CD2+ Mo, CD2- Mo, and total Mo (1 to 3 x 103) or GM-CSF and IL-4 precultured CD2+ Mo, CD2- Mo, or MDDC (5 x 102 to 2 x 104) in 96-well U-bottom tissue culture plates (Falcon; Fisher Scientific). Following incubation at 37°C in humidified 5% CO2 for 6 days, alloreactive and autologous cocultures were then pulsed with 1 µCi (37kBq) [3H]thymidine (NEN-DuPont, Boston, MA) for an additional 18 h and harvested on a UniFilter-96 (Packard Instrument, Meriden, CT). The DNA-associated radioactivity was measured by scintillation counting (TopCount microplate scintillation counter; Packard Instruments, Meriden, CT).
CD2+ and CD2- Mo morphology
The morphology of the freshly isolated or matured CD2+ and CD2- Mo was assessed by scanning electron or phase contrast light microscopy. Freshly isolated cells were suspended in 10% PHS in PBS at a concentration of 2 x 106 cells per ml, and 20 µl (4 x 104 cells) were added to polylysine-coated cover slips and incubated at 37°C in 7% CO2 for 30 min before fixation in 1.25% glutaraldehyde in 150 mM sodium cacodylate buffer at pH 7.2. The samples were postfixed with 1% OsO4, dehydrated, embedded in Epon, and analyzed by the Core Electron Microscopy Facility at Dana-Farber Cancer Institute with a Phillips EM 300 electron microscope. Phase contrast photomicrographs were taken of CD2+ and CD2- Mo plated (2 x 106 cells/ml) in 6-well tissue culture plates and cultured for 36–48 h.
Flow cytometry
The following directly conjugated mAbs were used:
anti-HLA-A, B, C (B9.12.1; Immunotech, Westbrook, ME);
anti-HLA-DR (B8.12.2; Immunotech); anti-HLA-DQ (Leu-10; Becton
Dickinson, San Jose, CA); anti-CD2 (SFCI3Pt2H9
(T111); Coulter, Miami, FL); Leu 5b (Becton
Dickinson); and 39C1.5 (Immunotech); anti-CD13 (Leu-M7; Becton
Dickinson); anti-CD14 (MY4; Coulter and TUK4; Caltag, Burlingame,
CA); anti-CD33 (Leu-M9; Becton Dickinson); anti-CD19 (J4.119;
Immunotech); anti-CD56 (84H10; Immunotech); anti-Thy-1.2
(5a-8; Immunotech); and anti-TCRß (BMA031; Immunotech). To
increase the staining intensity of CD2 of Mo in some analyses,
biotinylated anti-CD2 (T111; Coulter) and
strepavidin PE (Becton Dickinson) was used. Matched isotype controls
IgG1-FITC (Immunotech) or IgG1-biotin (Immunotech), and IgG2a-PE
(Immunotech) were used for appropriate studies, and limits of
negativity for each histogram reflect the quadrant boundary for the
isotype controls. The samples were washed, fixed in 1%
paraformaldehyde (Sigma), and analyzed on FACScan (Becton
Dickinson).
RT-PCR
Total RNA was isolated using ULTRASPEC (Biotecx Laboratories,
Houston, TX), and cDNA was synthesized by reverse transcription from
mRNA using oligo dT12–18 base primers and
Moloney murine leukemia virus reverse transcriptase (Life
Technologies). PCR reactions were performed in a 24-µl reaction
volume containing 2.4 µl of 10x PCR reaction buffer, 0.75 U
Ampli-Taq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT), 1
µl dNTP (Life Technologies), and 0.5 µl each of 5' and 3'
amplification primers. cDNA from PBMC, T cells, and EBV-transformed
B cells contained 5 ng of reverse transcriptase product;
that from CD2+ Mo contained 25 ng reverse
transcriptase product. The primers used were: CD2, 5'GCATCT
GGCCGATGATCAGGA-3' and 5'-GAGGCTGGTGCTGAACACGGT-3' (Genosys
Biotechologies, Woodlands, TX); TCR
, 5'-CAAGATGAAGT GGAAGGCGC-3' and
5'-AATCCCCTGGGTGTTAGCGA-3' (a gift from Dr. J. Lieberman); CD14,
5'-AGCACTTCCAGAGCCTGT-3' and 5'-CACGACACGTTGCGTAGGC-3' (Genosys
Biotechologies); GAPDH, 5'-GAAGGTGAAGGTCGGAGTC-3' and
5'-CAAAGTTGTCATGGAT GACC-3' (Genosys Biotechologies). All primers were
run for 30 cycles at 95°C for 1 min, 56°C for 1 min, and 72°C for
1 min.
Statistical analyses
Data are presented as mean value ± SEM. Paired and unpaired Student’s t tests were used for analysis of statistical significance. Values of p < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PBMC were first studied by two-color immunofluorescence
analysis for the expression of CD2, CD14, and TCRß Ags (Fig. 1
). Sixty percent of the PBMC expressed
the TCR, and smaller percentages expressed CD14 or neither of the Ags
(Fig. 1A). The population expressing the TCR represents the
T cells; the population expressing CD14 represents Mo; the double
negative population represents B cells and NK cells collectively. When
the PBMC were stained with anti-CD2 and anti-CD14 mAbs, CD2
expression on Mo appeared as a continuum. Nevertheless, four
populations were evident (Fig. 1B):
CD2+ CD14-,
CD2- CD14high,
CD2+ CD14high, and
CD2- CD14-. The
CD2+ CD14high population
constituted approximately one-third of the total
CD14high PBMC, and the level of CD2 expression
was one-half log lower in intensity than on T cells.
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,
A–C). Although there was some variation in the mean
fluorescence intensity from sample to sample of CD13, the percentages
of CD2+ cells among the Mo expressing CD13, CD14,
or CD33 were the same at 33%, 30%, and 32%, respectively. Two-color
analysis of MHC Ags (Fig. 2, D–F) showed that >99% of the
Mo expressed MHC class I Ags (with some variation in mean fluorescence
intensity). In contrast, class II Ags were found on a lower percentage.
HLA-DR was expressed on 73% of the Mo, while HLA-DQ was expressed on
<3%. However, no significant difference was seen in the percentage
positive or level of HLA-DR or HLA-DQ expression on
CD2+ and CD2- Mo.
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have greater autofluorescence than other leukocytes
and our results did not show a clear separation of the
CD2+ and CD2- Mo among
CD14high cells, we cultured the PBMC in CM
without added cytokines for 48 h before isolating the Mo. When the
expression of HLA-DR was compared with that of CD2, the staining
pattern was similar to that of the freshly isolated Mo with no point of
clear separation (Fig. 3B).
However, with respect to CD14 and CD2 expression, there was evidence of
two distinct populations; CD2-
CD14high and CD2+
CD14- (Fig. 3C). These findings
indicate that CD2+ Mo lose CD14 expression after
2 days of culture and CD2- Mo do not and confirm
that the limits set from the isotype controls were appropriate. The
loss of CD14 by CD2+ but not
CD2- Mo was also seen in the separated,
incubated populations (data not shown).
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To further characterize CD2 on Mo, we compared
T111, Leu 5b, and 39C1.5 CD2 epitopes on T cells
with their presence on Mo. We used the TUK4 mAb to CD14 to identify Mo.
Each of the three CD2 epitopes was present on 79% of the T cells (Fig. 4, A–C). In contrast,
T111, Leu 5b, and 39C1.5 epitopes were present on
35% (mean = 32.5% ± 0.35%, n = 4), 24%
(mean = 20% ± 3.5%, n = 2), and 18% (mean
= 13.5% ± 3%, n = 2) of the Mo, respectively (Fig. 4, D–F). To exclude the possibility that CD2 detection on
Mo was due to nonspecific binding of the anti-CD2 by Mo,
competitive-inhibition analysis was performed. We used the
T111 and T112 mAbs, which
bind to the same percentage of Mo (not shown), but recognize different
epitopes on CD2 (24), and an irrelevant Ab of the same
IgG2a isotope, anti-Thy-1, to confirm binding specificity. The
CD2+ Mo were selected with
anti-T112 and stained with
anti-T111-PE (Fig. 4G) or
preincubated with unlabeled anti-T111 (Fig. 4H) or anti-Thy-1 (Fig. 4I) and then stained
with anti-T111-PE. Loss of immunofluorescent
staining occurred on T112-selected cells
preincubated with unlabeled anti-T111, but
not on T112-selected cells preincubated with
unlabeled anti-Thy-1. Thus, CD2 positivity represents specific
binding to CD2 on a subpopulation of Mo.
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). There was detectable CD2 mRNA in
PBMC, T cells, and CD2+ Mo. In contrast, TCR mRNA
was detectable only in the PBMC and T cell samples and not in the
CD2+ Mo sample. GAPDH primers were used as an
internal standard and showed that the same quantity of mRNA was present
in all samples. Thus, the CD2 detected on CD2+ Mo
was not due to T cell contamination.
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The preceding experiments demonstrated several distinct epitopes
of CD2 on CD2+ Mo. Consequently, an isolation
protocol was designed based on the unique properties of CD2 (see
Materials and Methods). This protocol rapidly (4–5 h)
produced >95% pure populations of CD2+ and
CD2- Mo without culturing, in a state similar to
that in circulating blood. The CD2+ Mo obtained
by this protocol were 25–36% (mean = 31% ± 1.64%,
n = 6) of the purified Mo population, which correlates
closely with the percentage of CD2+ Mo seen by
immunofluorescence in the unseparated Mo (Fig. 2
). Thus, the apparent
continuum of CD2 expression on Mo could be resolved into distinct (but
usually overlapping) CD2+ and
CD2- subsets. Two-color analysis of the freshly
isolated CD2+ and CD2-
populations showed similar levels of CD14 (Fig. 6, B and F) and
HLA-DR (Fig. 6, C and G), but no detectable
levels of TCR or CD3 (Fig. 6, D and H), CD19, or
CD56 (not shown). As in the unseparated Mo population (Fig. 2), the
CD2+ and CD2- populations
expressed similar levels of CD13, CD33, and HLA-DQ (not shown).
Additionally, there were no detectable differences in the expression of
CD1a, CD11a/c, CD50, CD54, CD58, CD80, or CD86 (Table I). The CD83 Ag, which is expressed by
cultured DC (21, 29, 30, 31), was not expressed on freshly
isolated CD2+ or CD2- Mo.
After 2 days of culture, CD83 became detectable on
CD2+ Mo, but this expression varied from donor to
donor.
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Previous studies have shown that after 36–48 h of culture, Mo are
large, round or oval cells with an eccentric nucleus, while DC are
large, irregularly shaped cells with dendritic-like projections
(1). These morphologic features also distinguish the two
populations from other leukocytes. We examined the correlation of these
morphologic features with the presence or absence of CD2. Under phase
contrast microscopy, the freshly isolated CD2+
and CD2- Mo were indistinguishable (not shown).
However, when examined by scanning electron microscopy, a distinct
difference in cell-surface topography was seen. The
CD2+ population had large prominent ruffles (Fig. 7, A and C), while,
in contrast, the CD2- population had small
knob-like projections (Fig. 7, B and D). When
cultured for 36–48 h and viewed under phase contrast microscopy, the
CD2+ cells were irregular in shape with prominent
dendritic processes (Fig. 7E), while the
CD2- CD14high cells were
rounded or oval with dense centers (Fig. 7F). These
morphologic characteristics have been previously ascribed to DC and Mo,
respectively, and suggest that the CD2+ Mo
represent DC and the CD2- Mo represent those
cells that later give rise to M.
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To test the capacity of CD2+ Mo to stimulate
allogeneic and primary Ag-specific T cell responses, we investigated
their ability to activate naive CD4 T cells in MLR and Ag-specific
proliferation assays. First, we compared the ability of freshly
isolated CD2+, CD2-, and
total Mo to induce an alloreactive MLR (Fig. 8A). The freshly isolated
CD2+ population induced 2- to 3-fold greater
stimulation (p < 0.02) than the total or
CD2- population at various stimulator/responder
ratios. Because of the recent introduction of MDDC as a source of DC,
we compared the allogeneic T cell stimulation by GM-CSF- and
IL-4-cultured CD2+ and
CD2- Mo to that of MDDC (Fig. 8B).
GM-CSF- and IL-4-cultured CD2+ Mo, at low
concentrations, were significantly more efficient than MDDC
(p < 0.02) in naive T cell activation.
However, at APC concentrations of 20%, the efficiency of naive T cell
activation by MDDC and CD2- Mo decreased, while
that of the CD2+ Mo population increased to
levels that were 10- and 40-fold greater (p <
0.0005) than that of CD2- Mo and MDDC,
respectively. This suggests that fresh or cytokine-incubated
CD2+ Mo are much more potent stimulators of naive
CD4 T cells than MDDC, particularly at higher concentrations, and
further supports the hypothesis that the CD2+ Mo
are DC.
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C).
Both CD2+ and CD2- Mo
induced naive CD4 T cell activation with stimulatory indices of 41 and
6, respectively. However, the induction of naive CD4 T cell activation
by CD2+ Mo was significantly different from
control values (p < 0.02), while the induction
by CD2- Mo was not (p >
0.05). These findings further corroborate our hypothesis that
CD2+ Mo are DC. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and
2–4% of the total PBMC. Furthermore, we showed that CD2 is expressed
on both freshly isolated and cultured DC and thus can be used as a
means of identification and positive selection. The apparent continuum
of CD2 expression on fresh Mo was shown to represent overlapping
populations of distinct CD2+ and
CD2- Mo because 1) they could be separated by
anti-CD2 immunomagnetic beads, 2) they differed with respect to
surface morphology on scanning electron microscopy and in the
development only by incubated CD2+ Mo of 10–20
cell diameter dendrites visible on phase microscopy, 3) the loss of
CD14 expression by CD2+ Mo (but not
CD2- Mo) on incubation for 2 days, 4) the 2- to
3-fold greater allogeneic T cell stimulation by
CD2+ Mo than by CD2- Mo or
MDDC, and 5) the significantly greater ability of fresh or
GM-CSF/IL-4-incubated CD2+ Mo to induce
proliferation of naive T cells in the presence of foreign Ag compared
with CD2- Mo or MDDC. In contrast to CD2 expression, myeloid Ags (CD13, CD14, and CD33), MHC, or costimulatory and adhesion molecules were expressed at similar levels on freshly isolated DC and Mo. Therefore, these cell-surface Ags were not useful in distinguishing the two populations in the circulation. Collectively, these results suggest that what is thought to be a subset of circulating CD2+ Mo consists, in fact, of DC and provide support for the concept that DC and Mo exist in the circulation as distinct populations.
Our results do not support the concept that DC exist as two
populations, CD2+ CD14-
and CD2- CD14- (14, 16). Rather, they suggest that most peripheral blood DC are
CD2+ CD14high. The
discrepant results are likely to be explained by the use of different
mAbs to detect CD2 (14, 16) and the use of anti-CD14
and anti-CD2 mAbs or SRBC to deplete Mo, T, and NK cells (31, 33). Most studies of CD2 on DC have used
non-T111 mAbs such as Leu 5b (13, 14, 16). However, our results suggest that the Leu 5b and 39C1.5 CD2
epitopes are detectable on only a fraction of DC (Fig. 4, E
and F) that may vary from 10–70% (our unpublished
observations). Furthermore, the failure of some studies to detect CD2
expression on DC presumably has been because of the one-half log lower
expression of CD2 by freshly isolated DC compared with T cells (Fig. 4, A and D) or NK cells (our unpublished
observations). Together, these findings suggest that isolation methods
that enrich for HLA-DRhigh
CD14- DC (1, 13, 14, 16) lose 90%
of DC primarily because of the reagents used to deplete T cells, NK
cells, and Mo. Although Weissman et al. demonstrated enrichment of T
cells and DC with SRBC (24), which bind to CD2
(26), some studies have continued to use
neuraminidase-treated SRBC or anti-CD2 mAbs to deplete T and NK
cells (34, 35).
CD14 expression on DC but not on Mo is lost after 2 days of culture in
either PBMC (Fig. 3) or in isolated populations (data not shown).
Because cultured DC after incubation have characteristic morphology and
are largely CD14-, whereas Mo largely retain
CD14 expression, the concept arose that DC circulate as
CD14- myeloid cells (14). Our
studies suggest that the use of anti-CD14 mAbs to deplete Mo
(13, 14, 16) not only depletes the majority of DC but
leaves a heterogeneous population of CD14- Mo
and DC (Fig. 4D).
The ultimate criteria for distinguishing DC from Mo/M are the DCs
unique morphology after culture, greater capacity to induce an
alloreactive MLR (36), and the induction of Ag-specific
proliferation of naive T cells (8). Morphologically,
freshly purified Mo are indistinguishable from DC by light microscopy
(37, 38). In this report, evidence is presented that
CD2+ Mo (DC) are morphologically distinguishable
from CD2- Mo (pM).
In contrast to previous findings by others (14, 16) that freshly isolated DC require preculture in conditioned medium before a potent T cell response is generated, we observed full Ag-presenting capacity in freshly isolated DC. One possible explanation for the preculturing requirement of HLA-DR-sorted CD14- DC is to allow the recovery of APC function. APC function is inhibited by anti-HLA-DR mAbs, which can block HLA-DR-TCR interaction (39, 40) or inhibit the phagocytic stimuli required for efficient activation (41). Although we enriched by positive selection with anti-CD2 mAbs, our method does not appear to affect DC function.
Although CD2 expression on Mo/DC has been known for many years (13, 25), it was not until recently that emphasis has been placed on CD2-expressing subsets. Takamizawa et al. demonstrated that the CD2+ CD14- and CD2- CD14- DC were functionally distinct populations (16). Both populations could induce allogeneic T cell activation; however, only the CD2+ CD14- DC population had the ability to activate naive T cells. This is consistent with our finding that the CD2+ population is able to initiate an HIV-1 gp120-specific T cell response. In addition to HIV-1 gp120, we have found that DC efficiently present other nominal Ags, such as hepatitis B surface Ag and keyhole limpet hemocyanin, to naive T cells (data not shown).
The lower T cell stimulation induced by fresh or GM-CSF/IL-4-incubated
CD2- Mo at higher APC-to-T cell ratios suggests
the presence of an inhibitory activity not present in
CD2+ Mo. This activity would be consistent with
the presence of classic Mo/M and could represent either suppression
or killing of the T cells.
Recent studies by Randolph et al. (42) demonstrated that GM-CSF and IL-4 are not required for DC differentiation from Mo, suggesting that either transmigration across an endothelial barrier, prolonged culture, or adherence to collagen are sufficient to induce the DC phenotype. Once differentiated, the DC described by these authors are phenotypically similar to our cultured DC, expressing HLA-DR, CD83, and CD86, but no or low CD14. Our results are consistent with their findings, and suggest that prolonged culture on tissue culture plastic is sufficient to induce further DC differentiation.
Because endothelial cells do not secrete IL-4 or IL-13 (43), it appears likely that DC differentiation in vivo is different from the proposed GM-CSF and IL-4 or IL-13 models (17, 18). It may be that DC expansion from GM-CSF- and IL-4-cultured Mo does not induce the differentiation of DC from Mo, but instead induces activation of that fraction of DC already present in the starting CD14high population. Our results do not preclude the possibility that DC can arise from Mo, but rather suggest that the CD2+ CD14high Mo subpopulation consists of DC. The ability to isolate pure primary DC in high yield from the peripheral blood without cytokine stimulation has considerable importance for the study of human DC biology and will help to elucidate the potential role of these cells in graft-vs-host disease, in immune-mediated diseases, and for immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chester A. Alper, The Center for Blood Research, 800 Huntington Avenue, Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cells; CM, culture medium; Mo, monocytes; MDDC, Mo-derived DC; M, macrophage; pM, M precursors; rh, recombinant human; PHS, pooled human serum.
Received for publication October 20, 1998. Accepted for publication September 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Res. Immunol. 144:277.[Medline]
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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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K. Crawford, A. Stark, B. Kitchens, K. Sternheim, V. Pantazopoulos, E. Triantafellow, Z. Wang, B. Vasir, C. E. Larsen, D. Gabuzda, et al. CD2 engagement induces dendritic cell activation: implications for immune surveillance and T-cell activation Blood, September 1, 2003; 102(5): 1745 - 1752. [Abstract] [Full Text] [PDF]
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Y. Osugi, S. Vuckovic, and D. N. J. Hart Myeloid blood CD11c+ dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes Blood, September 26, 2002; 100(8): 2858 - 2866. [Abstract] [Full Text] [PDF]
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L. Y. Poluektova, D. H. Munn, Y. Persidsky, and H. E. Gendelman Generation of Cytotoxic T Cells Against Virus-Infected Human Brain Macrophages in a Murine Model of HIV-1 Encephalitis J. Immunol., April 15, 2002; 168(8): 3941 - 3949. [Abstract] [Full Text] [PDF]
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S. Parlato, S. M. Santini, C. Lapenta, T. Di Pucchio, M. Logozzi, M. Spada, A. M. Giammarioli, W. Malorni, S. Fais, and F. Belardelli Expression of CCR-7, MIP-3beta , and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities Blood, November 15, 2001; 98(10): 3022 - 3029. [Abstract] [Full Text] [PDF]
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M. O. Muench and A. Barcena Broad Distribution of Colony-Forming Cells with Erythroid, Myeloid, Dendritic Cell, and NK Cell Potential Among CD34++ Fetal Liver Cells J. Immunol., November 1, 2001; 167(9): 4902 - 4909. [Abstract] [Full Text] [PDF]
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H. Schultz, J. Weiss, S. F. Carroll, and W. L. Gross The endotoxin-binding bactericidal/permeability-increasing protein (BPI): a target antigen of autoantibodies J. Leukoc. Biol., April 1, 2001; 69(4): 505 - 512. [Abstract] [Full Text]
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),
CD2- Mo (
), or total Mo (
, MDDC). Autologous naive
CD4 T cells (105) were cocultured with CD2+
(
) Mo (5 x 103) in the
absence (control) or presence of HIV-1 gp120 (10 µg/ml)
(C). The values in brackets represent the stimulatory
index for each group. Naive T cell proliferation in the absence of APC
was <100 cpm. The error bars represent SEM. Results are representative
of at least three independent experiments.