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(Journal of Leukocyte Biology. 2001;69:548-554.)
© 2001 by Society for Leukocyte Biology

UVB-irradiated dendritic cells are impaired in their APC function and tolerize primed Th1 cells but not naive CD4+ T cells

Ralf W. Denfeld*, Hisamichi Hara*,{dagger}, Jens P. Tesmann*, Stefan Martin* and Jan C. Simon*


* Department of Dermatology, Albert-Ludwigs-Universität, Freiburg, Germany; and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown that low-dose UVB radiation converts Langerhans cells (LC) from immunogenic to tolerogenic APC. Therefore, we questioned whether low-dose UVB irradiation of bone marrow-derived dendritic cells (DC) alters their APC function, thereby inducing tolerance in T cells. To address this issue, cocultures of DC; and naïve, allogeneic T cells; naïve, OVA-specific TCR-transgenic T cells from DO11.10 mice; or primed, antigen-specific T cells using the Th1 clone AE7 were analyzed. First, we found low-dose UVB-irradiated DC (UVB-DC) to dose-dependently (50–200 J/m2) inhibit T-cell proliferation of naive and primed T cells. In addition, supernatants harvested from cocultures of UVB-DC and naive T cells showed markedly reduced levels of IL-2 and IFN-{gamma} 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UVB radiation (UVBR) interferes with the process of antigen presentation in vivo in that UVBR induces antigen-specific suppression of T-cell-mediated responses [1 , 2 ]. For example, it is well-established that exposure of the principal antigen-presenting cell (APC) within epidermis, the Langerhans cell (LC), to UVBR in vivo suppresses the development of contact hypersensitivity and results in the development of long-lasting, antigen-specific unresponsiveness that persists long after the effects of UVBR on the animal have disappeared [1 2 3 4 5 6 ]. In addition, low-dose UVBR alters LC APC function and promotes the induction of antigen-specific clonal anergy in CD4+ Th1 cells via perturbation of membrane-bound costimulatory signals in vitro [1 , 2 , 6 7 8 ]. Previously, we have shown that low-dose UVBR prevents the functional expression of CD80 and CD86 on human and murine LC in vitro, thereby inhibiting their capacity to induce allo- and antigen-specific T-cell responses [9 , 10 ].

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Female C57BL/6 (H-2b), BALB/c (H-2d), C3H/HeN (H-2k), and DO11.10 (H-2d) mice (6–16 weeks old), transgenic for the ovalbumin (OVA)323–339-specific and I-Ad-restricted T-cell receptor (TCR)-{alpha}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)-{gamma}, 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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Low-dose UVBR dose-dependently abrogates the stimulatory capacity of murine DC
In earlier studies, we demonstrated low-dose UVBR to distort the capacity of human and murine LC to induce allo- and antigen-specific T-cell proliferation [7 8 9 10 ]. Here, we asked whether UVBR modulates the APC function of murine bone marrow-derived DC. To address this issue, primary, one-way MLR were performed using unirradiated or UVB-irradiated C57BL/6 DC cultured in GM-CSF plus IL-4 as stimulator cells and BALB/c lymphnode cells as effector cells. UVBR of DC was carried out prior to coculture with a single dose of 50 J/m2, 100 J/m2, or 200 J/m2. As shown in Figure 1 , unirradiated DC induced strong allogeneic T-cell proliferation, whereas low-dose UVBR of DC dose-dependently inhibited T-cell proliferation. When purified CD4+ lymphnode T cells were used as effectors, comparable results were obtained (unpublished results). It is important that we could exclude significant effects of UVBR on DC viability as determined by PI staining on CD11c+ DC alone and from cocultures with T cells (unpublished results). Especially during the first 48 h of coculture, the number of unirradiated and UVB-irradiated, viable, PI-negative DC decreased similarly (unpublished results).



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  		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.

 
To analyze antigen-specific, T-cell proliferation, naïve, OVA-specific TCR-transgenic T cells from DO11.10 mice were used as effectors. Again, we found unirradiated DC to induce strong proliferation of naive T cells, which was inhibited when DC were UVB-irradiated (Fig. 2 ). Using primed, antigen-specific T cells from the PCC-specific Th1 clone AE7, similar observations were made (Fig. 3 ). Taken together, low-dose UVBR dose-dependently abrogates the stimulatory capacity of murine DC resulting in an inhibition of the proliferative response of naive and primed antigen-specific T cells.



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  		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.

 


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  		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.

 
UVB-DC induce antigen-specific clonal anergy in primed Th1 cells but not in naïve T cells
In previous in vitro studies, we have shown that UVBR converts LC from immunogenic to tolerogenic APC using a keyhole limpet hemocyanin (KLH)-specific Th1 clone [8 ]. Therefore, the same T-cell populations as described above (Figs. 1 2 3) were used to determine whether a primary stimulation with UVB-DC induces antigen-specific tolerance in naive versus primed T cells in a recall experiment (restimulation). To test tolerance induction, we first cocultured naive H-2d T cells with unirradiated or UVB-irradiated, allogeneic H-2b DC for 24 h to prime T cells to alloantigen. Then, T cells were isolated and rested in medium containing 2 U/ml IL-2. Subsequently, a restimulation of T cells was carried out using unirradiated H-2b DC, unirradiated third-party H-2k DC, or 100 U/ml IL-2 in the absence of APC. After coculture with unirradiated DC, T-cell proliferation was similar upon restimulation with unirradiated H-2b DC, unirradiated third-party H-2k DC, and IL-2 (Fig. 4 ). It is interesting that coculture with UVB-DC induced a partial inhibition of T-cell proliferation upon restimulation (Fig. 4) . This effect was also observed, although less pronounced, when T cells were left in coculture with irradiated DC for only 4 h in the first stimulation (unpublished results). However, T cells showed similar unresponsiveness upon restimulation with IL-2 or unirradiated DC irrespective of their haplotype, indicating a failure of tolerance induction.



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  		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.

 
Then, we used a similar protocol to test tolerance induction in naïve, OVA-specific TCR-transgenic T cells from DO11.10 mice. In the first stimulation, these naive T cells were cocultured with unirradiated or UVB-DC plus peptide for 24 h. Restimulation was carried out using unirradiated DC with different concentrations of OVA peptide or 100 U/ml IL-2. Again, T cells showed markedly reduced OVA-specific proliferation upon restimulation when cocultured with UVB-DC in the first stimulation (Fig. 5 ). However, T cells cocultered with UVB-DC or unirradiated DC did not proliferate in reponse to IL-2, indicating no induction of OVA-specific anergy (Fig. 5) .



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  		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.

 
In contrast, when T cells from the PCC-specific Th1 clone AE7 were tested for tolerance induction, these primed T cells, when cocultured with UVB-DC in the first stimulation, failed to proliferate in reponse to unirradiated DC plus antigen upon restimulation, however they responded vigorously when restimulated with IL-2 (Fig. 6 ). This demonstrates that UVB-DC induce PCC-specific clonal anergy in primed T cells from the Th1 clone AE7.



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  		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.

 
UVB-DC preferentially down-regulate type 1 T-cell responses
Now, we wanted to study whether UVB-DC interfere with T-cell differentiation. Depending on the eliciting stimulus, polarized type 1 T cells secrete IL-2 and IFN-{gamma}, 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-{gamma} 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-{gamma} 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) .



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  		Figure 7. UVB-DC down-regulate preferentially IL-2 and IFN-{gamma} 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.

 
Because supernatants of cocultures with DC and naïve, OVA-specific T cells did not contain significant amounts of the aforementioned cytokines on day 5, a modified protocol to induce cytokine secretion by these T cells was used. Cocultures of naïve, OVA-specific T cells with unirradiated or UVB-DC were transferred to anti-CD3 mAb-coated wells for the last 24 h of coculture. Then, supernatants were harvested and subjected to ELISA for cytokine production. Again, unirradiated DC induced strong IL-2 and IFN-{gamma} 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.



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  		Figure 8. IL-2, IFN-{gamma}, 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.

 
Low-dose UVBR does not affect the surface expression of major histocompatibility complex (MHC) class II, costimulatory, and adhesion molecules on DC
Recently, we found that the reduced stimulatory capacity of human and murine LC to induce allo- and antigen-specific T-cell responses is linked to a decreased surface expression of CD80 and CD86 on LC [9 , 10 ]. In contrast, FACS analysis of bone marrow-derived UVB-DC compared with unirradiated DC revealed no changes in surface expression of MHC class II (I-Ab), the costimulatory molecules CD40, CD80, and CD86, as well as the adhesion molecules CD11c and CD54, whether DC were irradiated with 50 J/m2, 100 J/m2 (unpublished results), or 200 J/m2 UVB (Fig. 9 ). We also considered the possibility that, although no quantitative alterations were detectable by FACS, CD80 and CD86 were functionally perturbed following UVBR, resulting in deficient T-cell costimulation via CD28. However, exogenous triggering of CD28 by addition of a stimulatory mAb for CD28 to cocultures of UVB-DC and T cells did not reverse the functional inhibition of DC-stimulatory capacity in MLR (unpublished results). Thus, murine DC respond differently to low-dose UVBR than LC in that their reduced stimulatory capacity following UVBR is not associated with a perturbation of CD28 costimulation.



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  		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).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated that low-dose UVBR (50–200 J/m2) distorts the capacity of murine bone marrow-derived DC to stimulate naïve, allo- and antigen-specific T cells as well as primed, antigen-specific Th1 cells in a dose-dependent manner. This finding is in line with experiments by us and others using different sources of DC. Specifically, low-dose UVBR (i.e., 200 J/m2) of freshly isolated LC [6 , 9 , 10 , 18 , 19 ] and the murine, epidermal-derived DC line XS52 [20 ] inhibited their stimulation of primary T-cell responses by at least 80%. In contrast, Young et al. [21 ] found that human peripheral blood DC required UVBR in excess of 1000 J/m2 to inhibit allogeneic T-cell responses by more than 60%. Because the stimulatory capacity of matured LC is much less susceptible to UVBR compared with freshly isolated LC [18 ], it seems likely that the DC used by Young et al. [21 ] exhibited a more mature DC phenotype compared with the DC used in our study.

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-{gamma}, 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-{gamma} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Si 397/7-1, 8-1) and the Deutsches Zentrum für Luft- und Raumfahrt (1GC9701/7). The authors thank A. Fehrenbach for excellent technical support. R. W. D., H. H., and J. P. T. contributed equally to this paper.

Received August 20, 2000; revised December 4, 2000; accepted December 5, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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