Reciprocal Control of T Helper Cell and Dendritic Cell Differentiation

Abstract

It is not known whether subsets of dendritic cells provide different cytokine microenvironments that determine the differentiation
of either type-1 T helper (TH1) or TH2 cells. Human monocyte (pDC1)–derived dendritic cells (DC1) were found to induce TH1 differentiation, whereas dendritic cells (DC2) derived from CD4+CD3–CD11c–plasmacytoid cells (pDC2) induced TH2 differentiation by use of a mechanism unaffected by interleukin-4 (IL-4) or IL-12. The TH2 cytokine IL-4 enhanced DC1 maturation and killed pDC2, an effect potentiated by IL-10 but blocked by CD40 ligand and interferon-γ.
Thus, a negative feedback loop from the mature T helper cells may selectively inhibit prolonged TH1 or TH2 responses by regulating survival of the appropriate dendritic cell subset.

Full text

oratories (Bar Harbor, ME). All explants were cultured
on 8-mm polycarbonate filters in Transwell plates
(Costar) at the interface of medium (15% fetal bo-
vine serum in Dulbecco’s modified Eagle’s medium,
L-glutamine, penicillin, and streptomycin). For ecto-
derm separation, tissues were incubated with 6%
pancreatin (Gibco-BRL) for 30 min on ice, washed in
10% serum for 20 min, and peeled (13). NIH 3T3 cell
lines were aggregated by culture in hanging drops
(20). Explant cultures shown are representative of at
least four independent experiments. Rat NT-3 and
human g-actin cDNAs were used as probes for the
Northern blot, which was quantified with a multiim-
ager (Fuji).
7. I. Farin˜as, K. R. Jones, C. Backus, X. Y. Wang, L. F.
Reichardt, Nature 369, 658 (1994).
8. A. Patapoutian and L. F. Reichardt, unpublished
observations.
9. D. J. Epstein, M. Vekemans, P. Gros, Cell 67, 767
(1991).
10. T. Franz, Anat. Embryol. 187, 371 (1993).
11. L. C. Schecterson and M. Bothwell, Neuron 9, 449
(1992).
12. G. A. Wilkinson, I. Farin˜as, C. Backus, C. K. Yoshida,
L. F. Reichardt, J. Neurosci. 16, 7661 (1996).
13. A. Neubu¨ser, H. Peters, R. Balling, G. R. Martin, Cell
90, 247 (1997).
14. R. L. Johnson and C. J. Tabin, ibid., p. 979.
15. B. A. Parr, M. J. Shea, G. Vassileva, A. P. McMahon,
Development 119, 247 (1993).
16. A. Kispert, S. Vainio, A. P. McMahon, ibid. 125, 4225
(1998).
17. K. M. Cadigan and R. Nusse, Genes Dev. 11, 3286
(1997).
18. C. van Genderen et al.,ibid. 8, 2691 (1994).
19. I. Farin˜as, G. A. Wilkinson, C. Backus, L. F. Reichardt, A.
Patapoutian. Neuron 21, 325 (1998).
20. T. Serafini et al.,Cell 78, 409 (1994).
21. We thank N. Hong, U. Mu¨ller, A. Neubu¨ser, and
members of Reichardt lab for valuable comments and
A. Saulys for technical help. This work was supported
by research grants from the U.S. Public Health Service
(National Institutes of Health grant MH48200) and
the Howard Hughes Medical Institute. A.P. is a fellow
of the Damon Runyon–Walter Winchell Cancer Re-
search Foundation and L.F.R. is an investigator of the
Howard Hughes Medical Institute.
7 October 1998; accepted 21 January 1999
Reciprocal Control of T Helper
Cell and Dendritic Cell
Differentiation
Marie-Clotilde Rissoan,
1
* Vassili Soumelis,
1
*
Norimitsu Kadowaki,
2
* Geraldine Grouard,
1
Francine Briere,
1
Rene´ de Waal Malefyt,
2
Yong-Jun Liu
1,2
†
It is not known whether subsets of dendritic cells provide different cytokine
microenvironments that determine the differentiation of either type-1 T helper
(T
H
1) or T
H
2 cells. Human monocyte (pDC1)– derived dendritic cells (DC1) were
found to induce T
H
1 differentiation, whereas dendritic cells (DC2) derived from
CD4
1
CD3
–
CD11c
–
plasmacytoid cells (pDC2) induced T
H
2 differentiation by
use of a mechanism unaffected by interleukin-4 (IL-4) or IL-12. The T
H
2 cytokine
IL-4 enhanced DC1 maturation and killed pDC2, an effect potentiated by IL-10
but blocked by CD40 ligand and interferon-g. Thus, a negative feedback loop
from the mature T helper cells may selectively inhibit prolonged T
H
1orT
H
2
responses by regulating survival of the appropriate dendritic cell subset.
The cytokine microenvironment plays a key
role in T helper cell differentiation toward the
T
H
1orT
H
2 cell type during immune respons-
es (1–6). IL-12 induces T
H
1 differentiation,
whereas IL-4 drives T
H
2 differentiation. Be-
cause T helper cell differentiation requires
the presence of different cytokines at an ini-
tial stage of the T cell– dendritic cell (DC)
interaction (1–7), we investigated whether
distinct DC lineages or subsets may produce
different cytokines and directly induce T
H
1
or T
H
2 differentiation.
Humans have two distinct types of DC pre-
cursors. Peripheral blood monocytes (designat-
ed pDC1) give rise to immature myeloid DCs
after culturing with granulocyte-macrophage
colony-stimulating factor (GM-CSF) and IL-4
(8–10) or after transmigration through endothe-
lial cells and phagocytosis (11). These imma-
ture cells become mature myeloid DCs (desig-
nated DC1) after stimulation with CD40 ligand
(CD40L) or endotoxin (12, 13). The
CD4
1
CD3
–
CD11c
–
plasmacytoid cells (desig-
nated pDC2) from blood or tonsils give rise to
a distinct type of immature DC after culture
with IL-3 (14–16 ). These cells differentiate into
mature DCs (designated DC2) after CD40L
stimulation (17). However, unlike pDC1 and
DC1, pDC2 and DC2 display features of the
lymphoid lineage: (i) pDC2 and DC2 express
few myeloid antigens, such as CD11b, CD11c,
CD13, and CD33 (14); (ii) pDC2 cells do not
differentiate into macrophages, following cul-
ture with GM-CSF and M-CSF; (iii) pDC2 and
DC2 have little capacity to phagocytose or
macropinocytose antigens at all stages of their
maturation (14) (iv) like the putative mouse
lymphoid DCs (18), pDC2 cells depend on
IL-3, but not GM-CSF for their survival and
maturation (14) [this can be explained by high
GM-CSF receptor and low IL-3 receptor ex-
pression in pDC1 cells and low GM-CSF re-
ceptor and high IL-3 receptor expression in
pDC2 cells (Fig. 1)]; and (v) pDC2 cells have
high levels of pre–T cell receptor a-chain ex-
pression (19).
Both myeloid DC1 and “lymphoid” DC2
induce strong proliferation of allogeneic naı¨ve
CD4
1
T cells (14). First, we examined the
profile of cytokine production from DC1 and
DC2 after CD40L activation. DC1 produced
large amounts of IL-12 within 24 hours after
CD40L activation (Fig. 2) as reported (12, 13),
whereas DC2 did not (Fig. 2). In addition,
unlike CD40L-activated DC1, CD40L-activat-
ed DC2 produced little IL-1a, IL-1b, IL-6, and
IL-10, but produced comparable amounts of
chemokine IL-8 (Fig. 2) (20). Neither CD40L-
activated DC1 nor DC2 produced detectable
amounts of IL-4 and IL-13. Quantitative poly-
merase chain reaction (PCR) analyses showed
that CD40L activation up-regulated the expres-
sion of mRNA for IL-12p40, IL-1a, and IL-1b
in DC1, but not in DC2 (Table 1) (21). Neither
DC1 nor DC2 transcribed detectable amounts
of IL-4 mRNA, either before or after CD40L
activation (Table 1).
We next examined the nature of primary
allogeneic T cell responses induced by DC1 or
DC2. Naı¨ve CD4
1
CD45RA
1
T cells isolated
from human peripheral blood or umbilical cord
blood were cocultured for 7 days with CD40L-
activated DC1, CD40L-activated DC2, or anti-
bodies to CD3 and CD28 (22). The cultured
cells were counted and restimulated with anti-
CD3 and anti-CD28 for either 4 hours for sin-
gle-cell cytokine analyses by flow cytometry
(Fig. 3B) or 24 hours for cytokine secretion
analyses by enzyme-linked immunosorbent as-
say (ELISA) (Fig. 3A). T cells originally cul-
1
Schering-Plough, Laboratory for Immunological Re-
search, 27 chemin des Peupliers, Boite Postale 11,
69571, Dardilly, France.
2
DNAX Research Institute of
Molecular and Cellular Biology, 901 California Ave-
nue, Palo Alto, CA 94304–1104, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-
mail: yliu@dnax.org
Fig. 1. Expression of GM-CSF receptor achain
(GM-CSF Ra) and IL-3 receptor achain (IL-3
Ra) on pDC1 and pDC2 (open curve, isotype
control; shaded curve, specific staining).
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www.sciencemag.org SCIENCE VOL 283 19 FEBRUARY 1999 1183

tured with DC1 secreted large amounts of in-
terferon-g(IFN-g) (34 to 37 ng/ml, from three
independent experiments), but little IL-4, IL-5,
and IL-10. T cells originally cultured with DC2
secreted little IFN-g(2 to 4 ng/ml), but large
amounts of IL-4 (230 to 1500 pg/ml), IL-5 (300
to 900 pg/ml), and IL-10 (4 to 10 ng/ml). T cells
originally cultured with anti-CD3 and anti-
CD28 secreted mainly IL-2 (Fig. 3A). These
polarized cytokine production profiles were
confirmed by single-cell cytokine analysis us-
ing in situ immunocytology (23) and by immu-
nofluorescence flow cytometry (Fig. 3B) (24).
Thus, myeloid DC1 and “lymphoid” DC2, re-
spectively, induce T
H
1 versus T
H
2 differentia-
tion in vitro.
Because DC2 cells do not produce detect-
able amounts of IL-4, as determined by both
mRNA (to a sensitivity of 10
–12
gram) and
protein analysis, it suggests that the DC2-in-
duced T
H
2 differentiation is IL-4 –independent.
T
H
2 development was not blocked by adding
either polyclonal antibody to IL-4 BDA11 (15
mg/ml) or monoclonal antibody (mAb) to IL-4
MP4-25D2 (5 mg/ml) at the beginning of DC–T
cell coculture (22). These two antibodies to
IL-4 completely inhibited the IL-4 – dependent
proliferation of phytohemagglutinin-stimulated
human T cells or CD40- and IL-4 – dependent
human B cell proliferation and immunoglobulin
E synthesis. Although addition of antibody to
IL-4 increased the number of IFN-g–producing
cells, it did not block the generation of IL-4 –
producing cells (Fig. 3B). The DC2-induced
T
H
2 differentiation was not a default mecha-
nism due to an inability of DC2s to produce
IL-12, because polyclonal activation with anti-
bodies to CD3 and CD28 in the absence of
Fig. 2. Cytokine and chemokine secretion by DC 1 after 24 hours of activation with CD40L (open
circles) and DC2 after CD40L activation for 24 hours (solid squares) or 6 days (solid circles). Each
symbol represents an independent experiment.
Fig. 3. DC1 and DC2 in-
duce T
H
1 versus T
H
2 cyto-
kine production, respec-
tively. (A) Quantitation of
T
H
1 and T
H
2 cytokines
by ELISA. Human CD4
1
CD45RO
2
naı¨ve T cells
were cultured for 6 days
with allogeneic CD40L-
activated DC1 or DC2,
or anti-CD3 plus anti-
CD28aCD3/aCD28). Cells
were counted and restim-
ulated with anti-CD3 and
anti-CD28 for 24 hours.
Amounts of IFN-g, IL-4,
IL-5, IL-10, and IL-2 within
culture supernatants were
collected after 24 hours
and measured by ELISA.
Results represent one of the three independent
experiments. (B) Two-color analysis of IL-4 and
IFN-gexpression by flow cytometry. (Upper pan-
els) DC1–T cell cocultures with control goat im-
munoglobin G antibodies and goat antibody to
IL-12. (Middle panels) DC2–T cell coculture with
control antibody and goat antibody to IL-4. (Low-
er panels) DC2–T cell coculture with anti–IL-12
and IL-12. Some 10
4
cells were analyzed, and the
percentages of each T cell population are indicat-
ed in the plots. Figure 4 represents the results
from one of the four independent experiments
performed.
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IL-12 did not induce T
H
2 differentiation (Fig.
3A). To support this conclusion, we performed
two experiments. First, neutralizing antibody to
IL-12 (AB-219-NA, 25 mg/ml) was added at
the beginning of the DC–T cell cocultures to
see whether anti–IL-12 could induce IL-4 –pro-
ducing T cells in DC1–T cell culture or increase
the number of IL-4 –producing cells in the
DC2–T cell cocultures. Although addition of
antibody to IL-12 decreased the percentage of
IFN-g–producing cells in both DC1–T cell and
DC2–T cell cultures (Fig. 3B), it did not induce
IL-4 –producing cells nor did it significantly
increase IL-4 –producing cell number in
DC1–T cell or DC2–T cell cultures (Fig. 3B).
Second, IL-12 (5 ng/ml) was added at the be-
ginning of DC2–T cell cocultures, to see if it
could block the generation of IL-4 –producing
T cells. Although the addition of IL-12 in-
creased the percentages of IFN-g–producing T
cells, it did not inhibit the number of IL-4 –
producing cells. However, IL-12 induced the
IL-4 –producing cells to produce IFN-g(Fig.
3B). Thus, DC2 may produce one or more
positive T
H
2 differentiation factors distinct
from IL-4, and its activity can neither be
blocked by IL-12 nor enhanced by anti–IL-12.
A common feature of T cell cytokine-medi-
ated T helper cell differentiation is the positive
autocrine effect. IL-2 promotes the IL-2–pro-
ducing T
H
0 cells, IL-4 promotes the IL-4 –
producing T
H
2 cells, IFN-gpromotes the IFN-
g–producing T
H
1 cells, IL-10 promotes the IL-
10 –producing regulatory CD4
1
T cells (25),
and TGF-bpromotes the TGF-b–producing
T
H
3 cells (26 ). Because negative feedback reg-
ulation represents a general mechanism used by
living organisms to maintain homeostasis of
physiological processes, the immune system
may need a negative feedback mechanism to
control the balance between T
H
1 and T
H
2 re-
sponses in order to prevent T
H
-mediated auto-
immune inflammatory responses or T
H
2-medi-
ated allergic responses. The studies on the reg-
ulation of DC1 and DC2 maturation allowed us
to identify a potential negative feedback loop in
which IL-4 and IFN-gmay negatively regulate
T
H
1 and T
H
2 development, respectively, by
interfering with the survival and maturation of
pDC1 and pDC2.
We observed that IL-4, IL-10, and CD40L
inhibited the IL-3– dependent proliferation of
pDC2 (Fig. 4A, a) (27). In contrast to CD40L,
which enhanced the survival and maturation of
pDC2, IL-4 and IL-10 decreased pDC2 num-
bers during a 6-day culture period in the pres-
ence IL-3 in a concentration-dependent fashion
(Fig. 4A, b and c) (27). IL-4 and IL-10 have
an additive effect in killing pDC2 (Fig. 4B).
The ability of IL-4 and IL-10 to kill pDC2 by
apoptosis was confirmed by direct culture
morphology, Giemsa staining of cytospin
preparations, and double-staining with an-
nexin–fluorescein isothiocyanate (FITC) and
propidiumiodide.
Because IL-4 and IL-10 are T
H
2 cyto-
kines, we investigated whether CD40L and
IFN-gcould block the negative effect of IL-4
and IL-10 on the survival of pDC2 main-
tained by IL-3. CD40L blocked the killing
effects of IL-4 or IL-10 on pDC2 during a
6-day culture period with IL-3 (Fig. 5A).
CD40L partially rescued pDC2 in the pres-
ence of both IL-4 and IL-10. IFN-galso
blocked the negative effect of IL-4 or IL-10
Fig. 4. (A) IL-4 and IL-10 inhibit the
IL-3–dependent proliferation and sur-
vival of pDC2 in a dose-dependent
fashion. (a) The IL-3–dependent
3
H-
thymidine incorporation by pDC2 at
day 3 of culture is suppressed by IL-4,
IL-10, and CD40L. In contrast to
CD40L, which enhances the survival of
pDC2, IL-4 and IL-10 (b and c) de-
crease in a dose-dependent fashion
the numbers of viable cells after 6
days of culture. Cell viability was de-
termined by trypan blue exclusion.
Results are expressed as means 6SD
of culture triplicates. One representa-
tive of eight independent experiments
is shown. (B) IL-4 and IL-10 have ad-
ditive effects in killing pDC2 over a 6-day time course. Kinetics of cell survival at days 1, 3, and 6 of
culture with IL-3 alone, IL-3 1IL-4, IL-3 1IL-10, and IL-3 1IL-4 1IL-10. The initial input of cells was
45,000 or 25,000 per well. Each symbol represents one independent experiment.
Fig. 5. CD40L and
IFN-grescue pDC2
from cell death in-
duced by IL-4 and IL-
10. (A) CD40L rescues
a large proportion of
DC2 precursors after 6
days of culture with
combinations of IL-3,
IL-4, and IL-10. (B)
IFN-grescues a large
fraction of DC2 pre-
cursors after 6 days of
culture with IL-3 1
IL-4 and IL-3 1IL-10.
It did not rescue DC2
precursors when cultured with IL-3 1IL-4 1IL-10. Results are representative of three independent
experiments.
Table 1. Quantitation of cytokine mRNA by PCR
(expressed as femtograms mRNA per 50 ng cDNA)
(21). Results before (–) and after (1) CD40L acti-
vation are shown after experiment number.
Expt.
number
(CD40L)
IL-12p40 IL-1aIL-1bIL-4
DC1
1 (–) 38 34 41 0
1(1) 12,835 573 2,175 0
2 (–) 7 45 60 2
2(1) 12,460 346 4,165 0
3 (–) 5 44 45 0
3(1) 40,194 441 763 0
DC2
1 (–) 0 12 20 0
1(1) 605 9 16 0
2 (–) 0 37 102 0
2(1) 714 21 50 0
3 (–) 0 7 14 0
3(1) 0 12 45 0
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www.sciencemag.org SCIENCE VOL 283 19 FEBRUARY 1999 1185

on pDC2 (Fig. 5B). However, IFN-gdid not
rescue the cells when IL-4 and IL-10 were
both added to the culture. Cells rescued by
either CD40L or IFN-gexpressed high levels
of major histocompatibility complex (MHC
class II DR) and costimulatory molecules
(B7.1/CD80 and B7.2/CD86), and stimulated
the proliferation of allogeneic CD4
1
T cells.
Our study suggests that a negative feed-
back loop may exist in regulating the balance
between T
H
1 versus T
H
2 responses. IL-4, a
key T
H
2 cytokine, kills the pDC2, a profes-
sional antigen-presenting cell subset that in-
duces T
H
2 differentiation. The ability of
CD40L (a potent DC maturation factor) to
prevent IL-4 –induced killing suggests that
IL-4 cannot kill mature DC2 during their
cognate interaction with T cells in established
responses. This may allow the rapid and ef-
ficient development of T
H
2 responses needed
for the host defense. However, overproduc-
tion of IL-4 may inhibit the development of
pDC2. By contrast, IL-4 promotes DC1 mat-
uration together with GM-CSF (12, 13).
These opposing effects of IL-4 on DC1 ver-
sus DC2 may enhance T
H
1 development, but
inhibit T
H
2 development at a late stage of
immune response. The ability of IFN-gto
protect pDC2 from IL-4 –and IL-10 –induced
apoptosis and promote DC2 differentiation
may represent an indirect mechanism to in-
hibit T
H
1 development at later stages of T
H
1
responses. This represents another example
of antagonism between IL-4 and IFN-g(28).
During the last two decades, studies on the
relationship between lineage and function have
been a main focus of B and T lymphocyte
immunology. Mouse DC cells may also have
different lineages with distinct functions (29–
35). Whereas the CD8a
–
CD11c
1
CD11b
1
my-
eloid DC cells are immunogenic for T cells, the
CD8a
1
CD11c
1
CD11b
–
lymphoid DC cells
may be tolerogenic (31). This concept is sup-
ported by two findings: (i) the lymphoid DC
subset that appears to be localized within the T
cell areas of mouse spleen highly express MHC
class II–self peptide complexes (32); and (ii)
the myeloid subset that appears to be localized
around the marginal zone– bridging channels
migrates into the T cell areas and produces
IL-12 after endotoxin stimulation (33, 34). Our
results here extend the concept regarding the
functional heterogeneity of DC subsets and
suggest two additional mechanisms for T
H
1 and
T
H
2 regulation. DC1 and DC2 stimulate naı¨ve
T helper cells and directly induce their differ-
entiation toward T
H
1orT
H
2. pDC1 and pDC2
provide the potential targets for negative feed-
back regulation by IL-4 and IFN-g. Two im-
portant relationships still need to be established:
(i) the relationship between the mechanisms
regulating immunity/tolerance versus T
H
1/T
H
2
and (ii) the correlation between mouse and
human DC subsets. These studies may ulti-
mately lead to the understanding of the molec-
ular mechanism underlying DC2-induced IL-
4 –independent T
H
2 differentiation and the dis-
tinct functions of DC subsets in normal and
disease states.
References and Notes
1. T. R. Mosmann and R. L. Coffman, Annu. Rev. Immu-
nol. 7, 145 (1989).
2. W. E. Paul and R. A. Seder, Cell 76, 241 (1994).
3. S. Romagnani, Annu. Rev. Immunol. 12, 227 (1994).
4. A. K. Abbas, K. M. Murphy, A. Sher, Nature 383, 787
(1996).
5. S. L. Constant and K. Bottomly, Annu. Rev. Immunol.
15, 297 (1997).
6. A. O’Garra, Immunity 8, 1 (1998).
7. J. Banchereau and R. Steinmann, Nature 392, 245
(1998).
8. F. Sallusto and A. Lanzavecchia, J. Exp. Med. 179,
1109 (1994).
9. N. S. Romani et al.,ibid. 180, 83 (1994).
10. Generation of DC1 from monocytes in vitro. Blood
mononuclear cells were isolated as described (8, 9).
To generate DC1, monocytes were cultured with
GM-CSF (200 ng/ml) and IL-4 (5 ng/ml; Schering-
Plough, Kenilworth, NJ) for 5 days in RPMI-1640
medium supplemented with 10% fetal bovine serum
(FBS), 2 mML-glutamine, and 0.08 mg/ml gentamy-
cin. The resulting immature DC1 were cultured with
CD40L-transfected L cells (one L cell per four DC
cells) for 24 hours.
11. G. J. Randolph, S. Beaulieu, S. Lebecque, R. M. Stein-
man, W. A. Muller, Science 282, 480 (1998).
12. M. D. Cella et al.,J. Exp. Med. 184, 747 (1996).
13. F. Koch et al.,ibid., p. 741.
14. G. Grouard et al.,ibid. 185, 110 (1997).
15. U. O’Doherty et al.,J. Immunol. 82, 487 (1994).
16. J. Olweus et al.,Proc. Natl. Acad. Sci. U.S.A. 94,
12551 (1997).
17. Generation of DC2 from the CD4
1
CD11c
–
lineage
–
pDC2 precursors. CD4
1
CD11c
–
lineage
–
plasmacytoid
cells were isolated from human tonsils or peripheral
blood as detailed (13). In two experiments, pDC1 and
pDC2 were isolated from the same blood donor.
These cells were cultured with IL-3 in the presence or
absence of CD40L-transfected L cells for 6 days or
with IL-3 alone for 5 days, followed by culture with
CD40L-transfected L cells for 24 hours.
18. D. Saunderset al.,J. Exp. Med. 184, 2185 (1996).
19. L. Bruno, P. Res, M. Dessing, M. Cella, H. Spits, ibid.
185, 875 (1997).
20. Quantitation of cytokine secretion by ELISA. The
presence of cytokines in culture supernatants of DC1
and DC2 after 24 hours of CD40L activation or in T
cell cultures for 24 hours with anti-CD3 and anti-
CD28 was determined by ELISA. ELISA kits for IL-1a,
IL-1b, IL-5, IL-6, IL-10, IL-12, IL-13, and IFN-gwere
obtained from R&D Systems (Minneapolis, MN); for
IL-2 and IL-4 from Cayman Chemical (Ann Arbor, MI).
21. Quantitation of mRNA expression. RNA isolation was
according to P. Chomcznski and N. Sacchi [Anal. Bio-
chem. 162, 156 (1987)]. The reverse transcription was
performed with SuperScriptII (Gibco-BRL, Rockville,
MD). We analyzed 50 ng of cDNA for the expression of
cytokine genes by the Fluorogenic 59-nuclease PCR
assay [P. M. Holland et al.,Proc. Natl. Acad. Sci. U.S.A.
88, 7276 (1991)], using a Perkin-Elmer ABI Prism 7700
Sequence Detection System (SDS; ABI–Perkin-Elmer,
Foster City, CA). Reactions were incubated for 2 min at
50°C, denatured for 10 min at 95°C and subjected to 40
two-step amplification cycles with annealing/extension
at 60°C for 1 min followed by denaturation at 95°C for
15 s. The following amplicons were used and analyzed
with 6-carboxy-fluorescein–labeled predeveloped Tag-
man assay reagents (Perkin-Elmer, Foster City, CA):
IL-1a, IL-1b, IL-4, and IL-12p40. Cytokine amplicons
spanned at least one intron/exon boundary. An 18S
ribosomal RNA amplicon was analyzed with a labeled
probe (Perkin-Elmer, Foster City, CA) and used as an
internal control for quantitation of the total amount of
cDNA in a multiplex reaction. Seven 10-fold dilutions of
plasmids (10 ng/ml) containing cytokine cDNAs were
used to create a standard curve for quantitation of
cytokine cDNA using the SDS software, then these
values were adjusted for the amount of total cDNA.
Values are expressed as femtogram of cDNA per 50 ng
input total RNA.
22. Purification of naı¨ve T cells and DC–T cell cocultures.
CD4
1
CD45RA T cells were incubated with a cocktail
of mAbs, including IOM2 (CD14); ION16 (CD16);
ION2 (HLA-DR) (Immunotech); NKH1 (CD56); OKT8
(CD8) (Ortho); 4G7 (CD19); UCHL1 (CD45RO); and
mAb 89 (CD40). This was followed by incubation
with anti-mouse immunoglobin-coated magnetic
beads and magnetic depletion. This was repeated two
times to create .96% pure CD4
1
naı¨ve T cells. T
cells were cocultured with allogeneic DC1 or DC2 at
a 2:1, 4 :1, and 8 :1 ratios in Yssel’s medium (Irvine
Scientific, Santa Ana, CA) containing 10% FBS in
24-well culture plates for 6 days with or without: (i)
polyclonal antibody to IL-4 (BDA11, 15 mg/ml; R&D
Systems); (ii) mAb to IL-4 (MP4-25D2; Pharmingen);
(iii) antibody to IL-12 (AB-219-NA, 25 mg/ml; goat
polyclonal immunoglobin G, R&D Systems); and (iv)
IL-12 (5 ng/ml; R&D Systems). T cells were also
cultured with anti-CD3 (5 mg/ml) and anti-CD28 (1
mg/ml; Pharmingen) coated on culture plates for 6
days. After 6 days of priming, T cells were restimu-
lated with anti-CD3 and anti-CD28 for either 4 or 24
hours.
23. Immunostaining of cytokines on cytospins. Cytospin
slides were fixed in cold paraformaldehyde for 5 min
and washed in Hank’s balanced salt solution. For
IL-12p40 staining, the slides were incubated with
mAb 609 (5 mg/ml; R&D Systems) or an isotype
control mAb for 45 min. The binding of antibody was
revealed by a Vectastain ABC kit ( Vector Laboratory,
Burlingame, CA). DAB chromogen was used to reveal
the peroxidase activity. For IFN-gstaining, slides
were incubated with mAb B27 (J. Abrams, DNAX
Research Institute of Molecular and Cellular Biology)
followed by staining with the Vectastain ABC kit.
24. Flow cytometry analysis of intracellular cytokines. After
6 days of DC–T cell coculture, T cells were reactivated
with anti-CD3 and anti-CD28 for 5 hours. Brefeldin A (1
mg/ml; Sigma) was added into the cultures for 2 hours
before the staining to prevent cytokine secretion. Cells
were washed and fixed with paraformaldehyde and
incubated with the following antibodies (Pharmingen):
anti-IL-4 –phycoerythrin (PE) plus anti-IFN-g–FITC; anti-
IL-10–PE plus anti-IFN-g–FITC; and anti-IL-4–FITC plus
anti-IL-10–PE. Samples were analyzed on a FACScan
(Becton-Dickinson).
25. H. Groux et al.,Nature 389, 737 (1997).
26. R. A. Seder et al.,J. Immunol. 160, 5719 (1998).
27. pDC2 were cultured in complete medium alone (RPMI-
1640 medium supplemented with 10% FBS, 2 mM
L-glutamine, and 40 mg/ml gentamycin) or with differ-
ent cytokines. Cytokines used were IL-3 (10 ng/ml), IL-4
(50 U/ml), IL-10 (100 ng/ml), and IL-13 (50 ng/ml) from
Schering-Plough Research Institute, Kenilworth, NJ, and
IFN-g(25 ng/ml) and IL-2 (20 U/ml) from Genzyme,
Boston, MA. Irradiated CD40L-transfected L cells were
used at a concentration of one L cell per four DC2 cells.
Nontransfected or CD32-transfected L cells were used
as controls. We cultured 3 310
4
cells per well in
triplicate in 96-well plates. After 3 days, cells were
pulsed with 1 mCi of
3
H-thymidine for 12 hours before
harvesting and of counting. Viable and dead cell num-
bers at 1, 3, and 6 days after culture were determined
by trypan blue exclusion assay.
28. U. Boehm, T. Klamp, M. Groot, J. C. Howard, Annu.
Rev. Immunol. 15, 749 (1997).
29. C. Ardavin, L. Wu, C. L. Li, K. Shortman, Nature 362,
761 (1993).
30. L. Wu, C.-H. Li, K. Shortman, J. Exp. Med. 184, 903
(1996).
31. G. Suss and K. Shortman, ibid. 183, 1789 (1996).
32. K. Inaba et al.,ibid. 186, 665 (1997).
33. T. De Smedt et al.,ibid. 184, 1413 (1996).
34. C. R. e Sousa et al.,ibid. 186, 1819 (1997).
35. B. Pulendran et al.,J. Immunol. 159, 2222 (1997).
36. We thank L. Lanier, R. Coffman, A. O’Garra, H. Kanzler,
and K. Moore for critical reading of the manuscript, S.
Ho, P. Larenas, B. Bennett, and I. Durand for technical
help, and J. Katheiser for editorial assistance. DNAX
Research Institute of Molecular and Cellular Biology is
supported by Schering-Plough Corporation.
10 November 1998; accepted 11 January 1999
REPORTS
19 FEBRUARY 1999 VOL 283 SCIENCE www.sciencemag.org1186