* Department of Dermatology, Albert-Ludwigs-Universität, Freiburg, Germany; and
Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan
Correspondence: Dr. R. W. Denfeld, Department of Dermatology, Albert-Ludwigs-Universität, Hauptstrasse 7, 79104 Freiburg, Germany. E-mail: denfeld{at}haut.ukl.uni-freiburg.de
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and to a lesser degree of IL-4 and
IL-10, suggesting a preferential down-regulation of Th1 responses by
UVB-DC. FACS analysis of UVB-DC revealed no changes in surface
expression of MHC, costimulatory, and adhesion molecules. To test
tolerance induction, allo- or antigen-specific T cells isolated from
cocultures with unirradiated DC and UVB-DC were restimulated with
unirradiated DC or IL-2. It is interesting that UVB-DC induced
antigen-specific tolerance in the Th1 clone AE7. In contrast, UVB-DC
induced a partial inhibition of allogeneic T-cell proliferation but no
tolerance with similar unresponsiveness to restimulation with IL-2 and
unirradiated DC irrespective of their haplotype. Similar observations
were made when naïve, TCR-transgenic T cells from DO11.10 mice
were used. In conclusion, UVB-DC are impaired in their APC function and
tolerize the primed antigen-specific Th1 clone AE7 but not naive allo-
or OVA-specific T cells.
Key Words: T lymphocyte • anergy • tolerance • costimulatory molecules
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In contrast to LC, human and murine dendritic cells (DC) can be generated in vitro in large numbers when cultured with the appropriate cytokines [11 , 12 ]. Therefore, we became interested in whether low-dose UVBR affects murine bone marrow-derived DC, propagated in granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL)-4, in a similar fashion to LC, which may be of relevance for immunotherapeutic-tolerance induction, for example in autoimmunity, allergy, and transplantation.
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ß [13
], were obtained from
the animal facility of the Max-Planck-Institut für Immunbiologie
in Freiburg, Germany.
Media and reagents
Complete RPMI 1640 (c-RPMI) was supplemented with 10% (v/v)
heat-inactivated fetal calf serum (FCS) and 1% (v/v)
penicillin-streptomycin (100x; all Gibco, Eggenstein, Germany).
Recombinant murine IL-4 and GM-CSF were purchased from Promocell
(Heidelberg, Germany); IL-2, from R&D Systems (Wiesbaden, Germany); the
OVA323–339 peptide, from Calbiochem-Novabiochem
(Schwalbach, Germany); and tritiated thymidine
([3H]-TdR), from Amersham (Freiburg, Germany).
Monoclonal antibodies (mAbs)
mAbs with specificity for the following murine antigens were
used: anti-I-Ab [AF6-120.1, mouse immunoglobulin
(mIgG)2a], anti-I-Ad [2G9, rat IgG
(rIgG)2a], anti-I-Ak (10–3.6,
mIgG2a), anti-CD3 [145-2C11, hamster IgG (haIgG)],
anti-CD4 (RM4-5, rIgG2a, and GK1.5, rIgG2b),
anti-CD8 (53–6.7, rIgG2a), anti-CD11b (M1/70,
rIgG2b), anti-CD11c (HL3, haIgG), anti-CD16/CD32 (2.4G2,
rIgG2b), anti-CD28 (37.51, haIgG), anti-CD40 (3/23,
rIgG2a), anti-CD45R/B220 (RA3-6B2, rIgG2a),
anti-CD54 (3E2, haIgG), anti-CD80 (1G10, rIgG2a), anti-CD86
(GL1, rIgG2a), anti-Ly-6G/Gr-1 (RB6-8C5,
rIgG2c), anti-Mac-3 (M3/84, rIgG1), and control
mIgG, rIgG, and haIgG mAb (all Pharmingen, Hamburg, Germany).
Cell culture
DC were generated from bone marrow cell suspensions cultured in
medium containing FCS, GM-CSF, and IL-4 using a modification of the
procedure described previously [14
, 15
].
For the experiments, DC were used on day 6 following initiation for
culture.
T cells were freshly isolated from lymphnodes. Bulk lymphnode cells contained 80–90% CD3+ T cells and 10–20% CD45R+ B cells and responded only poorly to phytohemagglutinin (PHA; 2.5 µ/ml; Sigma, München, Germany). For most experiments, bulk lymphnode cells were enriched for CD4+ T cells using an immunomagnetic separation method with M-450-labeled anti-CD4 mAb, followed by corresponding detachment mAb according to the manufacturer’s instructions (Dynal, Hamburg, Germany). The resulting population was >95% CD3+ CD4+ as determined by FACS analysis.
The pigeon cytochrome c (PCC)-specific, I-Ek-restricted Th1 clone AE7 [16 ] was kindly provided by Dr. M. Modolell from the Max-Planck-Institut für Immunbiologie. The clone was grown in c-RPMI and maintained in resting and restimulation periods, according to a described protocol [16 ].
Cytokine-enzyme-linked immunosorbent assay (ELISA)
Cytokine ELISA specific for murine interferon (IFN)-, IL-2,
IL-4, and IL-10 was performed according to the manufacturer’s
recommendations (R&D Systems). The absorbance was determined at dual
wave lengths of 450 and 630 nm (MR5000; Dynatech, Hamburg, Germany).
Flow cytometry
For triple-color FACS analysis [10
], DC were
stained in phosphate-buffered saline (PBS; Gibco) supplemented with 2%
(v/v) fetal calf serum (FCS) at 4°C with anti-CD16/32 mAb to block
unspecific binding of mAb to Fc receptors, then fluorescein
isothiocyanate (FITC)-conjugated primary mAb, followed by the
phycoerythrin (PE)-conjugated, anti-CD11c mAb or the PE-conjugated,
isotype-matched, control mAb (all Pharmingen). 7-Aminoactinomycin D
(7-AAD; 2.5 µg/ml) or propidium iodide (PI; 1 µg/ml; both Sigma)
was added to exclude nonviable cells. DC suspensions were analyzed
using a FACScan equipped with CellQuest software (Becton Dickinson,
Heidelberg, Germany).
UVBR
Following resuspension of DC in PBS, low-dose UVBR was performed
as described [10
] with four unfiltered FS20 fluorescent
tubes (broad-band spectrum, 250–400 nm; peak at 313 nm; Westinghouse
Corp., Pittsburg, PA) placed 46 cm above the target. UVBR was
administered as a single dose. Unirradiated DC suspensions served as
controls.
Proliferation assay
Allo- and antigen-specific T-cell proliferation assays were
performed according to published procedures [10
].
Briefly, naïve, allogeneic T cells; naïve,
TCR-transgenic T cells; or primed T cells from the Th1 clone AE7
(1x105) were cocultured with DC (1x104) in
c-RPMI in a MLR setting (96-well plates) for 120 h.
Cocultures were pulsed with [3H]-TdR (1 µCi/well) for
the final 20 h, then harvested with a Canberra Packard Filter Mate
(Canberra Packard, Frankfurt, Germany), followed by measurement of
[3H]-TdR incorporation using a Top-Count (Canberra
Packard).
Induction of tolerance
To test for tolerance induction in a first stimulation,
naïve, allogeneic T cells; naïve, TCR-transgenic T
cells; or T cells from the Th1 clone AE7 (1x105) were
cocultured with unirradiated or UVB-DC (1x104) in c-RPMI
in a MLR setting (96-well plates) for 24 h. After coculture,
clusters were disaggregated using PBS supplemented with 10% (w/v)
bovine serum albumin and 5 mM ethylenediaminetetraacetate (EDTA; both
Sigma). Then T cells were harvested by density-gradient centrifugation
(Histopaque 1.077, Sigma) or magnetic-bead depletion of I-A+ cells
using Dynal beads and rested in medium containing 2 U/ml IL-2 for
24 h up to 120 h. Subsequently, rested T cells
(1x105) were restimulated with unirradiated DC of the same
haplotype as used in the first stimulation or unirradiated, third-party
DC (1x104). To determine the proliferation capacity of all
rested T cells, 100 U/ml IL-2 was added to 1 x 105 T
cells. After 48 h of culture, T-cell proliferation was assessed by
[3H]-TdR uptake as described above.
Statistical analysis
Statistical analysis was conducted using t-test or
analysis of variance (ANOVA).
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 View larger version (20K): [in a new window] |
Figure 1. UVBR dose-dependently inhibits the allo-stimulatory capacity of DC.
Primary MLR were performed as detailed in Materials and Methods using
unirradiated or UVB-DC (H-2b) as stimulators and
allogeneic, naïve lymphnode cells (H-2d) as
effectors. UVBR of DC was carried out prior to tissue culture with a
single dose of 50–200 J/m2 UVB. Proliferation was
determined by [3H]-TdR incorporation from triplicate
measurements (cpm±SD). One of five separate experiments
with similar results is shown.
|
 View larger version (19K): [in a new window] |
Figure 2. UVB-DC suppress proliferation of naïve, antigen-specific T
cells. OVA-specific T-cell responses were assessed as described in
Materials and Methods using unirradiated or UVB-DC (H-2d)
as stimulators; purified, naïve, OVA-specific CD4+
TCR-transgenic T cells from DO11.10 mice as effectors; and OVA peptide
(2.5 nM). UVBR of DC was carried out prior to tissue culture with a
single dose of 100 or 200 J/m2 UVB. Proliferation was
determined by [3H]-TdR incorporation from
triplicate measurements (cpm±SD). One of nine
separate experiments with similar results is shown.
|
 View larger version (20K): [in a new window] |
Figure 3. UVB-DC inhibit the proliferative response of primed, antigen-specific
Th1 cells. As detailed in Materials and Methods, PCC-specific T-cell
responses were analyzed using unirradiated or UVB-DC (H-2k)
as stimulators, primed T cells from the PCC-specific Th1 clone AE7 as
effectors, and PCC (2 µg/ml). UVBR of DC was carried out prior to
tissue culture with a single dose of 100 or 200 J/m2 UVB.
Proliferation was determined by [3H]-TdR incorporation
from triplicate measurements (cpm±SD). One of four
separate experiments with similar results is shown.
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 View larger version (20K): [in a new window] |
Figure 4. UVB-DC render naive T cells partially unresponsive to alloantigen.
Tolerance induction was tested as described in Materials and Methods.
Briefly, in a primary stimulation (1. stimulation), naïve,
allogeneic T cells (H-2d) were cocultured with unirradiated
or UVB-DC (H-2b) for 24 h. Then, T cells were
harvested by density-gradient centrifugation and rested in medium
containing 2 U/ml IL-2 for 24 h. Subsequently, a restimulation (2.
stimulation) was carried out for 72 h using IL-2 (100 U/ml),
unirradiated H-2b DC, or unirradiated, third-party,
H-2k DC. Proliferation was determined by
[3H]-TdR incorporation from triplicate measurements
(cpm±SD; *, statistically significant at
P<0.05). One of three separate experiments with similar
results is shown.
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 View larger version (21K): [in a new window] |
Figure 5. Naïve, antigen-specific T cells are not tolerized by UVB-DC. As
detailed in Materials and Methods in 1. stimulation, naïve,
OVA-specific T cells were cocultured with unirradiated or UVB-DC plus
the OVA peptide for 24 h. Then, T cells were isolated by
magnetic-bead depletion of I-Ad+ cells and rested in medium
containing 2 U/ml IL-2 for 24 h (unpublished results) and 120 h. Restimulation was carried out for 72 h using IL-2 (100 U/ml) or
unirradiated DC with OVA peptide. Proliferation was determined by
[3H]-TdR incorporation from triplicate measurements
(cpm±SD; *, statistically significant at
P<0.001). One of five separate experiments with similar
results is shown.
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 View larger version (18K): [in a new window] |
Figure 6. UVB-DC induce antigen-specific clonal anergy in the Th1 clone AE7. In
1. stimulation, primed, PCC-specific T cells were cocultured with
unirradiated or UVB-DC plus PCC (2 µg/ml) for 24 h. Then, T
cells were isolated by magnetic-bead depletion of I-Ak+
cells and rested in medium containing 2 U/ml IL-2 for 24 h.
Restimulation was carried out for 72 h using IL-2 (100 U/ml) or
unirradiated DC with PCC (2 µg/ml). T cells plus PCC without DC and T
cells plus DC without PCC served as controls. Proliferation was
determined by [3H]-TdR incorporation from triplicate
measurements (cpm±SD; *, statistically significant at
P<0.005). One of three separate experiments with similar
results is shown.
|
, whereas type 2 T cells produce IL-4
and IL-10 [17
]. To examine the cytokine profiles of the
responding T-cell populations in our experiments, supernatants from
cocultures with naïve, allogeneic T cells were harvested on day
5 following initiation of cocultures and analyzed for cytokine
production by ELISA. Unirradiated DC induced IL-2 and IFN-
production by responding allogeneic T cells (Fig. 7
). In contrast, supernatants harvested from cocultures with UVB-DC
exhibited dose-dependently reduced levels of the type 1 cytokines IL-2
and IFN- compared with unirradiated DC. Albeit to a small degree,
the low IL-4 secretion by type 2 T cells induced by unirradiated DC was
also reduced in cocultures with UVB-DC, which was significant in some
but not all experiments, whereas IL-10 production by type 2 T cells
remained unaffected (Fig. 7)
.
 View larger version (25K): [in a new window] |
Figure 7. UVB-DC down-regulate preferentially IL-2 and IFN- secretion by
allogeneic T cells. Supernatants from MLR were harvested on day 5 of
coculture and analyzed by cytokine-specific ELISA. Mean values of
triplicate measurements are shown (ng/ml). This experiment represents
one of three separate experiments with similar results.
|
production by OVA-specific type 1 T cells, which was reduced
markedly when UVB-DC were used as stimulators (Fig. 8
). Also, IL-4 production by type 2 T cells was dose-dependently
reduced following coculture with UVB-DC and to a lesser degree IL-10
secretion (Fig. 8)
, underlining our findings with allogeneic T cells.
Taken together, our data suggest a preferential down-regulation of type
1 T-cell polarization by UVB-DC versus type 2 T-cell polarization.
 View larger version (28K): [in a new window] |
Figure 8. IL-2, IFN-, and IL-4 production by OVA-specific T cells is reduced
after contact with UVB-DC. Naïve, OVA-specific T cells were
cocultured with unirradiated or UVB-DC plus the OVA peptide (2.5 nM)
for 3 days. For the last 24 h of coculture, these cells were
transferred to anti-CD3 mAb (10 µg/ml)-coated wells. Subsequently,
supernatants were harvested and analyzed for cytokine production by
ELISA. Mean values of triplicate measurements are shown (ng/ml;
statistically significant at *, P<0.001; **,
P<0.01). This experiment represents one of three separate
experiments with similar results.
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 View larger version (42K): [in a new window] |
Figure 9. Surface expression of MHC class II, costimulatory, and adhesion
molecules on DC is not affected by UVBR. The surface expression of MHC
class II, CD40, CD80, CD86, CD54, and CD11c on DC was determined by
triple-color FACS analysis as described in Materials and Methods. Day 6
DC were irradiated with a single dose of 200 J/m2 UVB
(+UVB) or left unirradiated (no UVB). FACS analysis was performed
48 h later. MHC class II (I-Ab), CD40, CD80, CD86, and
CD54 expression (FL1) was analyzed on 10,000 viable CD11c+ DC. For
CD11c expression, 10,000 viable DC were analyzed with the PE-conjugated
anti-CD11c mAb (FL2). Isotype controls are indicated (Ctrl).
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Furthermore, we assessed whether UVB-DC induce tolerance in naive versus primed T cells, which may be of relevance for immunotherapeutic applications [11 , 12 ]. It is interesting that short-time coculture (4–24 h) of naïve, allo- and OVA-specific T cells with UVB-DC, plus peptide in the latter case, revealed diminished cluster formation (unpublished results), which is crucial for T-cell activation [22 , 23 ]. Consequently, a partial inhibition of allo- and OVA-specific T-cell proliferation upon restimulation with unirradiated DC was detectable. However, the proliferative capacity of restimulated allo- and OVA-specific T cells to IL-2 (in the absence of APC) was also suppressed markedly, indicating a failure of allo- and OVA-specific tolerance induction. In accordance with our results, Young et al. [21 ] also found UVB-irradiated human peripheral blood DC not to tolerize T cells to alloantigen in vitro using a slightly different protocol. By contrast, antigen-specific tolerance, that is clonal anergy, could be induced by UVB-DC when primed T cells from the PCC-specific Th1 clone AE7 were used. Together with an earlier study that shows UVB-irradiated LC to induce T-cell tolerance [8 ], it must be noted that in these studies, antigen-specific Th1-cell clones were used. Therefore, in the experiments with naïve, TCR-transgenic T cells, an OVA-peptide dose of 2.5 nM was used, known to result in the development of a Th1 phenotype (unpublished results) [24 ], suggesting that the Th-cell phenotype, i.e., Th1 versus Th2, does not determine whether T cells can be tolerized by UVB-DC. It needs to be pointed out, that although the primary response of the three different T-cell types used in our study (naïve, allogeneic; naïve, OVA-specific; and primed, PCC-specific T cells) was reduced similarly when stimulated by UVB-DC compared with unirradiated DC, their secondary response upon restimulation with unirradiated DC was different in that primed antigen-specific T cells could be tolerized (clonal anergy) but not naive allo- or antigen-specific T cells. One explanation for these observations could be that the requirement for sufficient priming of naive versus primed T cells is different, because naive allo- and OVA-specific T cells responded only poorly upon restimulation with IL-2, whereas the primed T cells from the Th1 clone AE7 responded efficiently to IL-2. In the future, we are planning to use the antigen-specific, TCR-transgenic DO11.10 mouse model to test tolerance induction by UVB-DC in primed OVA-specific Th1 versus Th2 cells [24 ], derived from naive T cells after a relatively short period of cell culture.
In addition, we observed DC-UVBR to dose-dependently reduce secretion
of the type 1 T-cell cytokines IL-2 and IFN-, whereas secretion of
the type 2 cytokines IL-4 and IL-10 was affected to a lesser extent.
Because it is well-known that proliferating T cells in a MLR with DC
belong mainly to the CD4+ T-cell subset [22
,
23
], this could indicate that UVBR interferes with the
ability of DC to induce type 1 CD4+ T-cell responses that are Th1
responses but not Th2 responses [17
]. In support of this
notion, our experiments revealed that following interaction with UVB-DC
proliferation of CD4+ T cells from the Th1 clone, AE7 was inhibited
markedly in a primary and secondary stimulation, resulting in classical
clonal-anergy induction in the latter case. These findings parallel
experiments with UVB-irradiated LC, which lost their capacity to
stimulate KLH-specific Th1 T cells but retained their ability to
stimulate KLH-specific Th2 T cells [7
]. In a therapeutic
setting, the down-regulation of IFN- secretion by UVB-DC may be
especially beneficial for the treatment of Th1-mediated diseases.
Our previous observations that the reduced allo- and antigen-specific, T-cell stimulatory capacity of UVB-irradiated LC is related to their deficient, costimulatory molecule expression [9 , 10 ] tempted us to speculate that DC might react similarly to UVBR. However, neither CD80 nor CD86 surface expression on DC was affected by UVBR as determined by FACS analysis. Moreover, the addition of a stimulatory antibody for their ligand CD28 could not restore T-cell proliferation, as it was the case with UVB-irradiated LC as stimulators. Another candidate, CD54, whose expression on LC is suppressed significantly by UVBR [18 ], remained unaffected on DC. Likewise, MHC class II, CD40, and CD11c expression was not affected. Also the finding by Schuhmachers et al. [20 ] that UVBR interrupts cytokine-mediated support of the DC cell line XS52 via an inhibition of their GM-CSF receptor and CSF-1-receptor expression was considered and tested. FACS analysis revealed no differences in the surface expression of the GM-CSF and CSF-1 receptors on unirradiated compared with UVB-DC (unpublished results). Taken together, the molecular mechanism(s) by which UVB-DC are impeded in the induction of allo- and antigen-specific T-cell responses remain to be elucidated.
In conclusion, low-dose UVB-DC are impaired in their APC function and tolerize a primed, antigen-specific Th1 clone but not naive, allo- or OVA-specific T cells. These results suggest that UVB-DC have differential effects on primary and secondary T-cell responses, which may be of interest for immunotherapeutic applications.
Received August 20, 2000; revised December 4, 2000; accepted December 5, 2000.
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