By
From * Schering-Plough, Laboratory for Immunological Research, 69571 Dardilly, France; and Institute of Anatomy, Division of Cell Biology, University Irchel-Zurich, 8057 Zurich, Switzerland
A subset of CD4+CD11cCD3
blood cells was recently shown to develop into dendritic cells
when cultured with monocyte conditioned medium. Here, we demonstrate that CD4+
CD11c
CD3
cells, isolated from tonsils, correspond to the so-called plasmacytoid T cells, an
obscure cell type that has long been observed by pathologists within secondary lymphoid tissues. They express CD45RA, but not markers specific for known lymphoid- or myeloid-derived
cell types. They undergo rapid apoptosis in culture, unless rescued by IL-3. Further addition of
CD40-ligand results in their differentiation into dendritic cells that express low levels of myeloid antigens CD13 and CD33.
Band T lymphocytes share many similarities, including
their developmental programmes, the structures of
their antigen receptors, and the requirement for costimulatory molecules for activation. B lymphocytes will finally
differentiate into plasma cells that secrete antibody, the key
effector molecule of humoral immune responses. Interestingly, pathologists have long observed the presence of
plasma cells that express the T cell marker CD4 and lack
intracellular immunoglobulins within the T cell zones of human lymph nodes (1). These plasma cells were suggested to represent terminally differentiated T plasma cells
of unknown secretory functions. Paradoxically, these so
called plasmacytoid T cells were found not to express other
T cell markers such as CD3 and TCRs (4, 5). They expressed MHC class II antigens, HLADR and HLADQ, and
the invariant chain CD74 (6, 7).
The CD4+ plasmacytoid cells were also found in human
spleen and thymus from patients with myasthenia gravis
(5). These cells were found in increased number in lymph
nodes taken from patients suffering from lymphomas, leukemias (8), patients with breast cancer metastases (4), and
lymphadenitis (5). In addition, cases of plasmacytoid T cell
lymphomas were reported that paradoxically evolved towards myelomonocytic leukemia (9). These data suggest that the CD4+ plasmacytoid cells represent a neglected but
important cell type of the immune system. However, the
nature and the fate of these plasmacytoid cells has remained
unknown.
Here, we report the isolation of plasmacytoid cells from
human tonsils. The phenotypical, anatomical and functional characterization indicates that these cells correspond
to the CD4+CD11c Immunohistological Localization of CD4+CD11c Purification of CD4+CD11c Purification of Naive T Cells from Adult Blood.
CD4+ CD45RA+
T cells were purified from blood mononuclear cells by immunomagnetic bead depletion using a cocktail of mAbs IOM2 (CD14), ION16 (CD16), ION2 (HLA DR) (Immunotech),
NKH1 (CD56), OKT8 (CD8) (Ortho), 4G7 (CD19), UCHL1
(CD45RO), and mAb 89 (CD40). After three rounds of bead depletion, purity of CD4+ T cells was routinely FACS® Analysis of CD4+CD11c Proliferation Assays and DC Generation in Culture.
CD4+ CD11c T Cell Proliferation Assay.
DC (10 to 5,000) were cultured
with 5 × 104 allogeneic blood CD4+CD45RA+ T cells in
round-bottomed 96-well culture plates in RPMI 1640 containing
10% human AB+ serum. The DC used were generated by culture
of (a) CD4+CD11c Electron Microscopic Analysis of CD4+CD11c In previous studies, plasmacytoid T cells in situ
were shown to express CD4 but to lack lineage markers
specific for B cells, T cells, NK cells, monocytes, Langerhans cells, and granulocytes (1). They expressed CD11a
but not CD11b or CD11c (5). To determine if human tonsils contained CD4+ plasmacytoid T cells, double staining
with anti-CD3 (blue) plus anti-CD11c (blue) together
with anti-CD4 (red) was performed on tonsil sections. Accordingly, whereas CD4+CD3+ helper T cells, CD4low
CD11c+ interdigitating cells and CD4+CD11c+ dendritic
cells within germinal centers (GCDC) (14) should be double stained purple, CD4+CD11c
According to the in situ phenotype of plasmacytoid T
cells, a multistep isolation method was developed including: (a) centrifugation >50% Percoll; (b) magnetic bead depletion of B cells (CD19+CD20+), T cells (CD3+), monocytes (CD14+) and NK cells (CD56+); (c) finally sorting for
CD4+CD11c
Next, the surface phenotype of isolated
plasmacytoid cells was analysed by flow cytometry (Fig. 4).
Although CD4+CD11c
The nature of these
plasmacytoid cells was further analyzed by cell culture with
different cytokines. Strikingly, these cells undergo rapid
spontaneous apoptosis in culture, resulting in 40% of cells
displaying apoptotic figures at 4 h (Fig. 5, B and D) and
>90% at 16 h (Fig. 5 F). Among a wide range of tested cytokines (IL-1
As described above, CD40-ligand did
not affect the survival of plasmacytoid cells and consistently
blocked the [3H]thymidine incorporation induced by IL-3.
Most strikingly, CD40-ligand induced the development of
long dendrites on these plasmacytoid cells in the presence
of IL-3. Fig. 7 A shows that after 3 and 6 d of culture,
many clusters of cells with long dendrites appeared. This
culture morphology was similar to that of dendritic cells generated from CD34+ progenitor cells cultured with GM-
CSF+TNF
The phenotype of plasmacytoid cells cultured for 6 d
with IL-3 or with IL-3+CD40-ligand (Fig. 8) was subsequently determined by flow cytometry. With IL-3, the adhesion molecule CD58 and the costimulatory molecules
B7.1/CD80 and B7.2/CD86 were induced, and the adhesion molecule CD54, the costimulatory molecule CD40 and MHC class II DR were upregulated. CD40-ligand further upregulated the expression of the above molecules.
Antigens specifically expressed by myeloid cells (CD13,
CD33, CD11b, CD14, CD35), B cells (CD21), Langerhans
cells (CD1a), NK cells (CD16), or activated cells (CD25 and CD45RO) were not induced under these two culture
conditions. CD11c, a marker highly expressed by different
subsets of DC appeared to be induced on a fraction of cells.
To test
the functional properties of DCs generated from plasmacytoid cells cultured with IL-3 or IL-3+CD40-ligand, their
antigen processing and presentation capacities were analyzed. In contrast to DC generated from monocytes with
GM-CSF+IL-4, DC derived from plasmacytoid cells after
6 d of cultures either with IL-3 alone or with IL-3 plus
CD40-ligand did not uptake FITC-dextran as analyzed by
FACS® (Fig. 9). In addition, they displayed a poor ability
to phagocytose FITC-latex beads as analyzed by confocal
microscopy and electron microscopy (not shown). Moreover, fresh isolated CD4+ plasmacytoid cells also failed to
uptake FITC-dextran during a 40-min culture with FITC-
dextran (not shown). However, DC derived from plasmacytoid cells stimulated allogeneic CD4+CD45RA+ naive T
cells and were at least as potent as monocyte-derived DC
or CD34+ progenitor cell-derived DC (Fig. 10). The cells
with pseudopods generated from plasmacytoid cells after 6 d
of culture with IL-3 also significantly stimulated the proliferation of CD4+CD45RA+ naive T cells (Fig. 10).
The experiments described in this article demonstrate
that the plasmacytoid T cells, which have long been observed in the T cell areas of peripheral lymphoid tissues,
correspond to CD4+CD11c DC represent an heterogeneous population of hematopoietic-derived cells that display potent ability to prime naive T cells and to stimulate memory T cells (22). The past
few years have witnessed remarkable advances in the isolation of DC precursors and in the definition of culture conditions allowing the generation of DCs (23, 24). DCs have
been generated from early hematopoietic progenitors that
can be isolated from human and mouse bone marrow (25-
28), thymus (29), or human blood/cord blood (12, 13, 33, 34). Hematopoietic progenitors isolated from bone marrow were shown to have the potential of becoming dendritic cells as well as granulocytes, monocytes, and macrophages (27, 34). This together with the fact that
monocytes have the potential to differentiate into both
macrophages and dendritic cells (11, 12, 37) suggests a myeloid DC developmental pathway. A critical cytokine for
myeloid DC development was shown to be GM-CSF.
Other factors such as TNF The capacity of CD40-ligand to promote the differentiation of CD4+CD11c Tonsillar CD4+CD11c The increased accumulation of CD4+CD11c blood dendritic cell (DC)1 presursors,
that either undergo rapid apoptosis or differentiate into DC
upon culture with IL-3 and CD40-ligand.
CD3
Cells.
Double stainings on human tonsil sections were performed using
mouse IgG1 anti-CD3 (Immunotech, Marseille, France) and antiCD11c (Dako, Glostrup, Denmark) together with mouse IgG2a
anti-CD4 (Innotest, Besancon, France). The binding of mouse IgG1
antibodies was revealed by sheep anti-mouse IgG1 (The Binding
Site, Birmingham, UK), followed by mouse anti-alkaline phosphatase-alkaline phosphatase complexes (Dako), the APAAP technique. The binding of mouse IgG2a antibodies was revealed by
sheep anti-mouse IgG2a-biotin (The Binding Site), followed by
ExtrAvidin-peroxidase (Sigma Chem. Co., St. Louis, MO). Alkaline phosphatase activity was developed by the Fast Blue substrate, whereas peroxidase activity was developed by 3-aminoethylcarbazole.
Lin
Cells.
CD4+CD11c
cells
were isolated from human tonsils. In brief, tonsils were cut into
small pieces and digested for 12 min at 37°C with collagenase IV
(1 mg/ml; Sigma) and deoxyribonuclease I (50 KU/ml, Sigma) in
RPMI 1640. The cells, pooled from two rounds of tissue digestion, were centrifuged over 50% Percoll (Pharmacia, Uppsala,
Sweden) for 20 min at 400 g. CD3+ T cells, CD14+ monocytes,
CD19+ and CD20+ B cells, and CD56+ NK cells were depleted
from the resulting low density cells by immunomagnetic beads
(sheep anti-mouse Ig-coated Dynabeads; Dynal, Oslo, Norway).
The resulting cells were stained with mouse anti-CD4-PE-Cy5 (Immunotech), anti-CD11c-PE (Becton Dickinson), and a cocktail of FITC-labeled mAbs anti-CD3 and anti-CD34 (Immunotech), anti-CD20, anti-CD57, anti-CD7, anti-CD14, and antiCD16 (Becton Dickinson), and anti-CD1a (Ortho). Then CD4+
CD11c
Lin
cells were isolated by cell sorting. Reanalysis of the
sorted cells confirmed a purity of 98%.
96%.
Cocktail FITC
-sorted
Cells.
FITC-labeled mAbs for surface phenotyping of sorted (or
cultured) cells were purchased from Immunotech (CD13, CD33,
CD34, CD8, CD11b, CD21, CD18, CD54, CD58, and CD38),
Becton-Dickinson (CD14, CD2, TCR
, MHC class II DR
and DQ, CD16, and CD71), Coulter (TCR-
), Sigma (CD45,
CD44), Dako (CD45RO, CD23, CD11a, and CD11c), PharMingen (CD35), Medarex (CD32). PE-labeled mAbs were purchased from Becton Dickinson (CD80), Immunotech (CD40), and
PharMingen (CD86). Cells, incubated with antibody for 15 min
at 4°C, were analyzed after one wash with a FACScan® flow cytometer. Negative controls were performed with unrelated murine mAbs (Dako). These negative controls are indicated by filled histograms.
Lin
cells were cultured with IL-3 in the presence or absence of
CD40L fibroblasts (10). All cultures were performed in RPMI
1640 supplemented with 10% of FCS, 2 mM L-glutamine and antibiotics. Cells (1.5 × 104) were seeded in 96-well flat-bottom
microtiter plates for the DNA synthesis assay. After 3 d, cells were
pulsed with 1 µCi [3H]thymidine for 8 h before harvesting and
counting. Tests were carried out in triplicate and standard deviations are indicated with bars. For viable cell recovery estimation,
cells were counted by Trypan blue dye exclusion. rhIL-3 or GMCSF (Schering-Plough Research Institute, Kenilworth, NJ) were
used at a saturating concentration of 10 ng/ml (50 U/ml) and 100 ng/ml (200 U/ml), respectively. rhTNF
(Genzyme, Boston,
MA) was used at 2.5 ng/ml (50 U/ml). For phenotypic studies, 3 × 105 to 5 × 105 cells were cultured in 48-well flat-bottom plates (5 CD40L fibroblasts for 1 CD4+CD11c
cocktail FITC
cell).
Lin
cells for 6 d in the presence of IL-3
with or without CD40L transfected fibroblasts; (b) blood monocytes for 10 d with GM-CSF plus IL-4 (50 U/ml) (11, 12); and
(c) CD34+ cells for 12 d with GM-CSF and TNF
(13). After 5 d
of DC-T cell cocultures, cells were pulsed with 1 µCi [3H]thymidine for 8 h before harvesting and counting. Tests were carried
out in triplicate and standard deviations are indicated with bars.
Lin
Cells.
For electron microscopy, CD4+CD11c
Lin
cells (freshly isolated or cultured cells) were fixed in 2.5% glutaraldehyde plus 0.8% paraformaldehyde in 0.1 M cacodylate buffer and postfixed with an
aqueous solution of 1% OsO4 containing K4Fe(CN)6. For scanning electron microscopy (SEM), cells were allowed to settle on
coverslips precoated with poly-L-lysine, dehydrated in an acetone
series, dried by the critical point method (CO2), and coated with
10 nm gold in sputter device. Observations were made with a
SEM 505 instrument (Philips, Eindhoven, Netherlands). For
transmission electron microscopy (TEM), the cells were dehydrated in an alcohol series and embedded into epon. Ultrathin
sections (~50 nm) were stained with lead citrate and uranyl acetate and studied with a CM 100 instrument (Philips).
Localization and Isolation of Tonsillar Plasmacytoid T
Cells.
CD3
plasmacytoid T cells
should be single stained red (Fig. 1). Indeed, red CD4+
CD11c
CD3
cells were found in the T cell-rich extrafollicular areas (Fig. 1, A, C, D, E) but not within germinal
centers (Fig. 1, A and B), confirming the previous observations in lymph nodes (5). Interestingly, many red CD4+
CD11c
CD3
cells were found to be close to or within
the high endothelial veinules (HEV) (Fig. 1, C, D, E), suggesting that these cells might be blood derived cells that
were crossing the HEV. The CD4+CD11c
CD3
cells
within the T cell rich areas indeed displayed a plasmacytoid morphology (Fig. 1, C, D, E).
Fig. 1.
Anatomical localization of CD4+CD11cCD3
plasmacytoid cells. To distinguish CD4+CD11c
CD3
plasmacytoid cells from
CD4+CD3+ T cells and CD4+CD11c+ germinal center dendritic cells, double staining with anti-CD3 (blue), anti-CD11c (blue), and anti-CD4 (red) on
tonsillar sections was performed. Accordingly, whereas CD4+CD3+ T cells and CD4+CD11c+ germinal center dendritic cells should be double stained
purple, CD4+CD11c
CD3
plasmacytoid cells should be single stained red. (A) shows a germinal center and its adjacent T cell-rich extrafollicular areas containing HEV. Red CD4+CD11c
CD3
plasmacytoid cells can only be found in T cell area but not in germinal center. (B) shows that the same germinal center displayed in A does not contain red CD4+CD11c
CD3
plasmacytoid cells. (C) shows that the same T cell area displayed in A contains
many red CD4+CD11c
CD3
plasmacytoid cells, located within or around HEV. (D) shows a red CD4+ CD11c
CD3
plasmacytoid cell within the
lumen of HEV. (E) shows many red CD4+CD11c
CD3
plasmacytoid cells, one in particular is within the endothelial wall of HEV. Original magnification: (A) ×100; (B, C) ×200; (D, E) ×400.
[View Larger Version of this Image (180K GIF file)]
cells that were negative for CD7, CD57,
CD20, CD3, CD14, CD1a (Langerhans cells), CD34, and
CD16 (Fig. 2). Two CD4+Lin
populations were identified (Fig. 3 A). The CD4+CD11c+ population that represents GCDCs has been described earlier (14). On Giemsa
staining, freshly sorted CD4+CD11c
cells display a plasma
cell-like morphology, characterized by an eccentric nucleus, a blue basophilic cytoplasm and pale Golgi zone (Fig.
3 A). By scanning electron microscopy, these cells display a
smooth round lymphoid morphology and are 8-10 µm in diameter (Fig. 3 B). By transmission EM, the cells display
nuclei with marginal heterochromatin and cytoplasm containing juxtanuclear Golgi apparatus and parallel arrays of
rough endoplasmic reticulum (RER) (Fig. 3, C and D).
However, unlike immunoglobulin containing CD38++
CD20
plasma cells isolated from human tonsils (Fig. 3 F),
these CD4+CD11c
Lin
plasmacytoid cells did not contain intracellular immunoglobulin (Fig. 3 E).
Fig. 2.
Cell sorting of CD4+CD11cCD3
plasmacytoid cells. After
preparation of tonsillar cells that are negative for CD3, CD14, CD19, CD20, and CD56 (detailed in Materials and Methods), cells were stained
with mouse anti-CD4-PE-Cy5 (Immunotech), anti-CD11c-PE (Becton
Dickinson), and a cocktail of FITC-labeled mAbs anti-CD3 and CD34
(Immunotech), anti-CD20, anti-CD57, anti-CD7 and anti-CD16 (Becton Dickinson), and anti-CD1a (Ortho). As shown in the left panel, cells
were first selected by their cocktail FITC
(Lin
). This selected population contains CD4+CD11c+ germinal center dendritic cells (14) and
CD4+ CD11c
plasmacytoid cells, as shown in the right panel.
[View Larger Version of this Image (29K GIF file)]
Fig. 3.
Morphology of freshly isolated CD4+CD11cCD3
plasmacytoid cells. (A) Giemsa staining; (B) SEM; (C) TEM; (D) TEM. (E) negative antiIg
+
light chain staining of CD4+CD11c
Lin
plasmacytoid cells; (F) positive anti-Ig
+
light chain staining of CD38++CD20
Ig-secreting
plasma cells. Original magnification: (A, F) ×1,000; (B) ×3,500; (C) ×2,000; (D) ×8,000.
[View Larger Version of this Image (111K GIF file)]
Lin
Tonsillar
Plasmacytoid Cells.
cells express CD45RA, indicating their hematopoietic origin, they lack all the lineage
markers for B cells (CD19, CD21), T cells (CD2, CD3,
TCR, CD8), natural killer cells (CD56 and CD57), myeloid cells (CD13 and CD33), monocytes (CD14 and
CD11b), granulocytes (CD16 and CD35), and Langerhans
cells (CD1a). However, they express MHC class II antigens
and the adhesion molecules CD11a, CD18, CD44, and
CD54 (but not CD58). They also express low levels of CD40 and CD38. They do not express detectable levels of
B7.1/CD80 and B7.2/CD86. This phenotypic profile fits
well with that of the in situ plasmacytoid T cells characterized by immunohistochemistry (5). In addition, the phenotype of these tonsillar CD4+CD11c
Lin
plasmacytoid
cells is similar to that of the blood CD4+CD11c
Lin
cells,
that differentiate into dendritic cells when cultured with
monocyte-conditioned medium (15).
Fig. 4.
Surface phenotype of isolated CD4+CD11cCD3
plasmacytoid cells analyzed by flow cytometry. Filled histograms represent isotype matched
controls, open histograms represent staining with specific antibodies. Data shown are one representative of six experiments.
[View Larger Version of this Image (35K GIF file)]
, IL-1
, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10,
M-CSF, GM-CSF, SCF) and CD40-ligand, IL-3 alone was
found to completely block the apoptotic cell death of plasmacytoid cells (Fig. 5, A and C). In contrast to cells cultured in medium alone, cells cultured with IL-3 rapidly
formed aggregates within 4 h (Fig. 5 A) and few cells displayed apoptotic figures (Fig. 5 C). During the next 6 d of
culture with IL-3, >90% of cells remained viable (Fig. 6 A)
and displayed: (a) pseudopods after 16 h of culture (Fig. 5 E);
(b) blastic nuclei and mitotic figures after 3 d of culture (Fig. 5 G); and (c) veiled morphology after 6 d of culture
(Fig. 5 H). The CD4+ plasmacytoid cells cultured for 3 d in
the presence of IL-3 incorporated [3H]thymidine (Fig. 6 B), a
finding consistent with the appearance of mitotic figures at
this stage. Two cytokine combinations (GM-CSF+TNF
and GM-CSF+IL-4) that, respectively, induce CD34+ progenitor cells (13) and blood monocytes (11) to become DC
did not have significant survival (not shown) and proliferation (Fig. 6 B) effects on plasmacytoid cells. CD40-ligand,
a key survival and activation signal for several cell types
(16), including B cells (19, 20) and DC (21), did not interfere with the IL-3-dependent survival of plasmacytoid
cells, but inhibited the IL-3-dependent [3H]thymidine uptake (Fig. 6 B).
Fig. 5.
CD4+CD11cCD3
plasmacytoid cells undergo rapid apoptosis when cultured in medium, unless rescued by IL-3. (A) Many cell clusters at
4 h of culture with IL-3; (B) Dispersed cells at 4 h of culture in medium alone; (C) Few apoptotic cells within clusters at 4 h of culture with IL-3; (D)
Many apoptotic cells at 4 h of culture in medium alone; (E) Cells display pseudopods at 16 h of culture with IL-3; (F). All cells are dead at 16 h of culture
in medium; (G) Mitotic figures at 3 d of culture with IL-3; (H) cells with pseudopods at 6 d of culture with IL-3. Data shown are one representative of
six experiments. Original magnification: (B) ×200.
[View Larger Version of this Image (119K GIF file)]
Fig. 6.
IL-3 maintains the viability of CD4+CD11cCD3
plasmacytoid cells for up to 6 d. (A) Total numbers of viable cells at day 1, day 3, and day 6 of culture with medium (open circles), IL-3 (open squares), CD40ligand transfected L cells (open triangles), CD40-ligand negative L cells = ORIENT (solid squares) and CD40-ligand transfected L cells plus IL-3 (solid
triangles). (B) IL-3 induces [3H]thymidine incorporation by plasmacytoid cells
at day 3 of culture. Data shown are one representative of four experiments.
[View Larger Version of this Image (23K GIF file)]
(13). Indeed, Giemsa staining of cytospin
preparation of plasmacytoid cells cultured for 6 d with IL-3
and CD40-ligand revealed a typical mature dendritic cell
morphology (Fig. 7 B). These observations were further
confirmed by TEM and SEM (Fig. 7, C and D). The cells
cultured with CD40-ligand and IL-3 developed large dendrites (like spines of hedgehog) (Fig. 7 C) and did not have
a trace of RER (Fig. 7 D).
Fig. 7.
CD4+CD11cCD3
plasmacytoid cells acquire the morphology of mature interdigitating dendritic cells after 6 d of culture with IL-3 and
CD40-ligand. (A) Culture morphology; (B) Giemsa staining; (C) SEM; (D) TEM. Original magnification: (A) ×100; (B) ×400; (C) ×3,000; (D) ×6,000.
[View Larger Version of this Image (94K GIF file)]
Fig. 8.
CD4+CD11cCD3
plasmacytoid cells express high levels of costimulatory molecules, but few myeloid antigens, after 6 d of culture with
IL-3 or IL-3+CD40-ligand. Filled histograms represent isotype matched controls, open histograms represent staining with specific antibodies. Data
shown are one representative of six experiments.
[View Larger Version of this Image (34K GIF file)]
Fig. 9.
Dendritic cells derived from CD4+CD11cCD3
plasmacytoid cells after 6 d of culture with IL-3+CD40-ligand did not uptake
FITC-dextran. Methods are as described by Sallusto et al. (11). Time 0 represents cells that were incubated with FITC-dextran for 30 min at
4°C. After three washes, the FITC-dextran uptake by plasmacytoid cells derived DC cultured with IL-3 (open squares) or with IL-3 plus CD40ligand (solid circles) and monocyte-derived DC (solid triangles) at 37°C were
analyzed with a FACScan® flow cytometer. MFI, mean fluorescence intensity. Data shown are one representative of three experiments.
[View Larger Version of this Image (18K GIF file)]
Fig. 10.
DC derived from CD4+CD11cCD3
plasmacytoid cells
induces strong proliferation of CD4+CD45RA+ naive T cells. Purified
allogeneic blood CD4+CD45RA+ naive T cells were cocultured with
different numbers of DC generated from: CD14+ monocyte-derived
DCs (solid circle), CD34+ progenitor-derived DCs (solid square), plasmacytoid cells after 6 d of culture with IL-3 (open square) and plasmacytoid
cells after 6 d of culture with IL-3 and CD40L (open circle). After 5 d of
DC-T cell cocultures, cells were pulsed with 1 µCi [3H]thymidine for 8 h
before harvesting and counting. Data shown are one representative of six
experiments.
[View Larger Version of this Image (20K GIF file)]
blood DC precursors (15).
These cells display six striking features: (a) they have a plasmacytoid morphology, characterized by well developed
RER and Golgi apparatus, which would normally suggest a
terminally differentiated cell type with an active secretory function; (b) they die very rapidly by apoptosis in simple
culture medium, consistent with the previous report that
nuclear pyknosis and tingible body macrophages were
found within clusters of plasmacytoid T cells in situ (5) and
suggesting a rapid cell turn over rate; (c) they are found either close to the HEV or within the lumen of HEV; this,
together with the identification of CD4+CD11c
blood
DC precursors (15), indicates that the tissue plasmacytoid DC precursors are blood derived; (d) IL-3 represents the
only cytokine tested so far that maintains their survival and
promotes their proliferation; (e) CD40-ligand promotes
their differentiation into mature DC; ( f ) the plasmacytoid
cell derived DC display features similar to a subset of tonsillar interdigitating cells (Björck, P., and Y.-J. Liu, unpublished observation), including low expression of myeloid
antigens CD13 and CD33 and poor ability to uptake soluble antigens or to phagocytose particles.
, IL-4, SCF, and CD40-ligand
were shown to enhance the myeloid DC maturation (23,
24). DC have also been generated from the most immature
thymic T cell precursors or from bone marrow lymphoid
progenitors, which could differentiate into T cells, B cells,
and NK cells (28). These experiments suggest a lymphoid DC developmental pathway. Interestingly, GM-CSF
does not appear to be required for DC generation from
lymphoid precursors (31). In contrast, IL-3 seems to be one
of the key cytokine required for lymphoid DC development (28). A recent experiment by Maraskovsky et al.
(38) showed that Flt3L could potently enhance the development of both myeloid and lymphoid DCs in mice. The
dependence of CD4+CD11c
plasmacytoid cells on IL-3
but not GM-CSF to differentiate into DCs, which express
relatively low levels of CD13 and CD33 myeloid antigens,
suggests that these plasmacytoid cells may represent lymphoid-derived immediate DC precursors.
Lin
plasmacytoid cells into DC is
consistent with the previous reports on the diverse effects
of CD40-ligand on the maturation and function of DCs.
Ligation of CD40 on dendritic cells was shown to trigger production of high levels of IL-12 (39, 40), enhance T cell stimulatory capacity (21, 41) and improve DC survival
(21, 42, 43). A recent experiment showed that ligation of
CD40 induced a subset of human CD34+ hematopoietic
progenitors to proliferate and differentiate into DC (44).
Taken together, these data suggest that T cells play important roles in DC generation and maturation.
plasmacytoid cells share many
similar features with the blood CD4+CD11c
(15) or CD33low
CD14low DC precursors (45). However, blood CD4+CD11c
cells appear to survive better than tonsillar CD4+CD11c
cells in culture medium (15, our unpublished observation),
suggesting that the apoptosis program of these cells may be
further triggered upon their arrival within tissues. Blood
CD4+CD11c
cells were shown earlier to differentiate
into DCs in the presence of undefined factors within
monocyte-conditioned medium (15). In our hands, these
blood CD4+CD11c
cells were found to become DCs in the
presence of IL-3 and CD40-ligand (data not shown), as tonsillar CD4+CD11c
cells. This strongly suggests that the tonsillar CD4+CD11c
plasmacytoid cells are derived from blood
CD4+CD11c
cells.
plasmacytoid cells in the draining lymph nodes of tumor patients
suggests important immunological functions in these pathological conditions. They may be constantly produced in
bone marrow and may migrate through blood to the T cell
areas of the spleen by crossing the marginal zone sinuses or
the T cell areas of lymph nodes or tonsils by crossing the
HEV. There, they would undergo rapid apoptosis, unless rescued by T cell signals (IL-3+CD40-ligand) generated by
on going antigen-driven T cell-dependent immune responses. Thus, the IL-3 and CD40-ligand-dependent DC
development from plasmacytoid precursors may represent a
unique peripheral regulatory pathway that can rapidly recruit large numbers of DC into immune responses.
Address correspondence to Yong-Jun Liu, Schering-Plough, Laboratory for Immunological Research, 27 Chemin des Peupliers, BP 11, 69571 Dardilly, France.
Received for publication 30 December 1996 and in revised form 20 January 1997.
G. Grouard was supported by the Foundation Marcel Merieux, Lyon, France.We thank C. Caux and K. Shortman for helpful discussions and critically reading the manuscript; M. Balzer and M. Erni for technical help; J. Chiller for support; S. Bonnet-Arnaud and M. Vatan for editorial help.
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