MHC class II-KO mice are resistant to the immunosuppressive effects of UV light.

ARTICLE

UVB radiation is immunosuppressive in both humans and laboratory animals and a major cause of skin cancer [1]. The model of choice for the study of UVB-induced immune suppression is the contact hypersensitivity reaction (CHS), which is a delayed-type hypersensitivity mediated by hapten-specific T cells. In this model, UVB suppress cellular immune responses to antigens applied both at the site of irradiation (local suppression), or at distant, nonirradiated sites (systemic suppression). Although it has been known for years that both systemic [2] and local suppression of CHS [3-5] can be transferred by T cells, the phenotype and mechanism of action of the suppressor T cell subset remain unknown. Several hypotheses have been proposed: 1) UVB block the ability of skin APC to induce priming of antigen-specific effector cells either by killing the APC [6] or by abrogating expression of co-stimulatory signals [7-9]; 2) UVB selectively induce activation of suppressor cells which have been described as CD8⁺ T cells [10] or CD4⁺ T cells (Th2 or Tr1-like) [11-13], by converting dendritic cells (DC) from immunogenic to tolerogenic APCs. UVB-induced suppressor cells may also kill DC or effector T cells of CHS through Fas ligand-mediated apoptosis [5, 14]; 3) UVB exposed skin cells release suppressive cytokines which may inhibit either the APC function of DC or the effector T cell activation or both [15-17].

CHS is a T cell-mediated cutaneous inflammatory reaction occurring after epicutaneous exposure to haptens in sensitized individuals [18, 19]. In humans, it frequently manifests as an inflammatory dermatosis referred to as contact dermatitis. Haptens are low molecular weight chemicals which covalentely bind to discrete amino acid residues on self or exogenous proteins [20]. Hapten-modified proteins could then be processed by APCs into antigenic peptides, which are transported at the cell surface in association with MHC class I and class II molecules. The sensitization phase of CHS, also referred to as the afferent phase, occurs after the first contact of the skin with the hapten. epidermal DC, i.e. Langerhans cells (LC), play a crucial role in the sensitization phase. They capture the hapten in the skin and migrate to draining lymph nodes where they mature into functional APC endowed with the capacity to prime naive T cells. LCs can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8⁺ and CD4⁺ T cell subsets. Recent studies conducted in MHC class I- and class II-deficient mice have shown that: 1) CHS to DNFB was mediated by CD8⁺ T cells whereas CD4⁺ T cells are down-regulatory cells of CHS [21, 22]; 2) priming of CD8⁺ T cells occurs without the need of CD4⁺ T cell help [21, 22]; 3) DC present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8⁺ effector and regulatory T cell subsets, concurrently and independently [22]. The elicitation phase of CHS, also known as the efferent phase, develops a few hours after subsequent contact with the hapten and is mediated by activation of hapten-specific CD8⁺ T cells in the skin. We have recently shown that cytotoxicity is mandatory for CD8⁺ T cell-mediated CHS, since mice deficient in perforine and in Fas-L are unable to develop a hapten-specific skin inflammation [23].

In the present study we took advantage of the model of CHS to DNFB to get better insight into the mechanisms of UVB-induced immune suppression. The existence of a clear-cut functional dichotomy between CD8⁺ and CD4⁺ T cells allowed us to study the effect of UVB on these two T cell subsets and to address several questions: 1) Are UVB able to block antigen-presenting functions of skin DC in vivo? 2) Is UVB-induced immunosuppression the consequence of inhibition of CD8⁺ effector T cells or rather the consequence of activation of CD4⁺ down-regulatory T cells? 3) Do UVB suppress the CHS response by interfering with the sensitization phase or with the elicitation phase of CHS?

Our data show that UVB have differential effects on DC-induced activation of CD8⁺ and CD4⁺ T cells and that CD4⁺ T cells mediate the immunosuppressive effects of UVB through inhibition of expansion of hapten-specific CD8⁺ effector T cells in the lymphoid organs.

Materials and methods

Mice

Mice with a mutation in the beta2-microglobulin (I-) or Abeta gene (II-) have been created by gene targeting techniques [24, 25]. Heterozygous (I- and II-) knockout mice were backcrossed for more than 8 generations to C57BL/6 mice and used to generate the offsprings of the present study. They were obtained from C. Benoist and D. Mathis (IBMG, INSERM U. 184, Stasbourg, France) and bred in specific-pathogen-free conditions in Iffa Credo/Transgenic alliance, L'Arbresle, France. C57BL/6 (H-2^b) mice were obtained from Iffa-Credo, L'Arbresle, France. Mice were used between 2 and 4 months of age.

Assay for CHS: the mouse ear swelling test

The procedure of the mouse ear swelling test has been described in detail elsewhere [26]. DNFB (2,4-dinitro-fluorobenzene, Sigma, St. Louis, MO) was diluted in acetone: olive oil (4:1) immediately before use. For sensitization, irradiated and nonirradiated mice were painted once (day -5) on the shaved dorsal skin with 25 muL of 0.5% DNFB. Five days later (day 0), mice were challenged by the application of 5 µl 0.15% DNFB on each side of the right ear, while the left ear received the vehicle alone. Ear thickness was assessed before and at various intervals after challenge using a spring-loaded micrometer (J15, Blet SA, Lyon, France). Ear swelling was calculated by substracting the initial value from the values recorded on the corresponding day, and further substracting any swelling recorded for the vehicle-painted ear from the swelling recorded for the hapten-challenged ear.

UV radiation

UV radiation was provided by a bank of 14 F8T5 UV lamps (Sankio Denki, Japan), emitting a broad spectrum of UV radiation between 280 and 360 nm, with a peak at 314 nm. Approximately 67% of the energy emitted is within the UVB range. It produced an irradiance of 19 W/m² in the UVB range and 7 W/m² in the UVA range at a distance of 20 cm from the source, as determined by an IL-1700 radiometer with a SED 240 detector (International Light, Newburyport, MA). Groups of 5 mice were anesthetized with sodium pentobarbital injected i.p. and subsequently irradiated on shaved dorsal skin on 4 consecutive days (day-9 to day-6), 24 hrs prior to sensitization (Fig. 1A). The ears of the mice were protected from UV radiation by covering them with opaque tape. Control mice were treated in an identical manner, but were not exposed to UV radiation.

In some experimental groups, mice were killed at different time intervals after DNFB challenge, for histological and RT-PCR analysis.

Antibody depletion of CD4⁺ and CD8⁺ T cells in vivo

The rat anti-mouse CD4 mAb GK 1.5 was obtained from the ATCC (Rockville, MD) and the rat anti-mouse CD8 mAb H35.17.2 was kindly provided by G. Milon (Institut Pasteur, Paris, France). Hybridomas were grown as ascites in pristane-primed nude mice and used to treat mice. Mice were given i.p. injections of 200 muL diluted ascites on days - 1, 0, + 1 and + 4 of skin sensitization. The amount of antibodies required for > 95 depletion of directly detectable CD4⁺ molecules on spleen, lymph node and blood lymphocytes was determined in preliminary experiments. Cell depletion was assessed in each mouse by staining for CD4 molecules on PBMC from venous blood drawn from the tail vein.

In vitro secondary T cell proliferation

Spleen cells from DNFB-sensitized C57BL/6 mice were collected 5 days after sensitization. T lymphocytes were purified through negative selection using anti-Ig columns (Biotex, Edmonton, Alberta, Canada) as described else-where [22]. The resulting cell suspensions contained > 90% CD3⁺ viable cells. CD8⁺ T cells were isolated from the spleen T cells by elimination of CD4⁺ T cells using columns coated with goat anti-mouse and goat anti-rat IgG and a rat anti-mouse CD4⁺ mAb (YTS191.1) (Biotex). FACS analysis of cells eluted from the column showed < 0.5% of contaminating CD4⁺ T cells. In vivo DNFB-primed unfractionned or CD8⁺T cells (2.5 x 10⁵/well) obtained on day 5 after DNFB sensitization were co-cultured for 3 days at 37° C in 96-well plates with 10⁶ mitomycin C-treated syngenic spleen cells from naive nonirradiated mice, that were either DNBS-derivatized as described [22] or left untreated. Briefly, 10⁷ cells were incubated for 30 min with 25 µg/mL of mitomycin C (Sigma, St. Louis, MO), washed and haptenated by 30 min incubation at 37° C with 4 mM DNBS, ph 8.0, in serum-free RPMI medium. The proliferative responses were assessed on day 3 of culture by ³H thymidine incorporation (1 muCi/well) for the last 6 hours of culture. The results are expressed as proliferation indexes: (cpm in cultured T cells + DNBS-treated spleen cells)/(cpm in cultured T cells + untreated spleen cells).

IFN-gamma ELISA

Murine IFN-gamma was measured by ELISA in secondary T cell proliferation culture supernatants using commercially available kits HyCult biotechnology (Liden, The Netherlands). Assays were performed according to the manufacturer's instructions.

IFN-gamma ELISPOT assay

Inguinal and axillary lymph nodes were harvested 5 days after DNFB sensitization. Cell suspensions were restimulated in vitro by overnight culture in complete RPMI medium supplemented with 10% FCS and containing a final concentration of 0.4 muM DNBS. Control cultures included cells cultured overnight in medium supplemented with 0.2 mM of the irrelevant hapten TNBS, or in medium alone. The number of IFN-gamma producing cells was determined using an Elispot assay. Briefly, 96 well nitrocellulose plates (MAHA 45 Millipore) were coated overnight at 4° C with anti-IFN-gamma antibody (R46A2) and blocked with PBS/2% BSA for 2 hrs at 37° C. The plates were washed 3 times with PBS/Tween 0.1% before use. The cell suspensions were washed, and incubated at different concentrations in duplicate wells for 4hrs at 37° C, 5% CO₂. Plates were washed 3 times with PBS/0.1% Tween and incubated with a biotinylated anti-IFN-gamma antibody (AN18). IFN-gamma spot forming cells (SFC) were developed using streptavidine-alkaline phosphatase (Boehringer Mannheim), incubated for two hrs and extensively washed before adding the substrat (5-Bromo-4-Chloro-3-Indolyl-Phosphate, Sigma). The number of IFN-gamma SFC present in each well was counted using a microscope and the results expressed as IFN-gamma SFC/106 cells.

RNA extraction and RT-PCR analysis of CD8 and IFN-gamma mRNA

At different intervals after challenge, ear samples were collected from irradiated and nonirradiated mice and frozen in liquid nitrogen. The detection of RNA was conducted as described in detail elsewhere [23, 27]. Briefly, total RNA was extracted using a RNAXEL kit (Eurobio, Les Ullis, France). After DNase I treatment, 1 mug of total mRNA was reverse transcribed using poly dT15 primers and Superscript II RT (Gibco, BRL) (90 min 37° C). The amount of RNA to be used for detection was normalized using the housekeeping gene HPRT (hypoxanthine phosphoribosyltransferase) as reference. The cDNA obtained was amplified using different sets of primers, for HPRT (5'primer: 5'GTA ATG ATC AGT CAA CGG GGG AC 3'-3'primer: 5'CCA GCA AGC TTG CAA CCT TAA CCA 3'), for CD4 (5'primer: 5'AGC AAC TCT AAG GTC TCT AAC C 3'-3'primer: 5'AGA GTC AGA GTC AGG TTG CC 3'), for CD8 (5'primer: 5'AGG ATG CTC TTG GCT CTT CC 3'-3'primer: 5'TCA CAG GCG AAG TCC AAT CC 3') and for IFN-gamma (5'primer: 5'GCT CTG AGA CAA TGA ACG CT 3'-3'primer: 5'AAA GAG ATA ATC TGG CTC TGC3'). The amplifications were carried out with 29 cycles for HPRT, 31 cycles for CD4 and 33 cycles for IFN-gamma and CD8 (1 min at 94° C, 1 min 30 s at 60° C, 2 min at 72° C). The PCR products were analysed on 1.5% agarose gel.

Statistical analysis

Panels consisted of 5 mice each and all experiments were performed at least twice. Data were examined for normality and equal variance. Statistical significance of differences between mean values of experimental groups was evaluated using the two-tailed Student's t test (p < 0.05).

Results

MHC class II-deficient mice are resistant to UVB-induced immunosuppression

We studied the dose-response of UVB-induced suppression of the CHS reaction in C57BL/6 (I⁺II⁺) and in class II-deficient (I⁺II^-) mice (Fig. 1). Daily UVB doses equal to or higher than 600 J/m² induced dose-dependent inhibition of the inflammatory reaction in I⁺II⁺ mice. The suppression was significant at 800 J/m² and at 1,200 J/m² the ear-swelling response was equal to the negative controls (naive challenged mice) (Fig. 1B). We have therefore used 1,200 J/m² as a suppressive daily UVB dose in our further experiments.

I⁺II^- mice developed an enhanced and prolonged DNFB-specific CHS reaction, confirming previous studies which demonstrated the down-regulatory role of CD4⁺ T cells [21] (Fig. 2A). UV irradiation doses which were suppressive in I⁺II⁺ mice failed to inhibit the CHS reaction in I⁺II^- mice. 2,000 J/m² was the lowest daily irradiation dose at which a mild reduction of CHS was observed. Irradiation at higher doses induced erosive lesions at irradiation sites but failed to completely abolish the hapten-specific ear-swelling. As shown on Figure 2B, ear sections obtained from I⁺II⁺ mice irradiated at 1,200 J/m² displayed none of the characteristics of an inflammatory reaction and thus resemble naive nonirradiated controls. Nonirradiated as well as irradiated I⁺II^- mice exhibited an enhanced CHS reaction as compared to nonirradiated I⁺II⁺ mice, namely exagerated dermal edema, vascular enlargement and mononuclear cell infiltration.

CD4⁺ T cells mediate UVB-induced suppression of CHS to DNFB

The lack of inhibition of the inflammatory reaction in irradiated I⁺II^- mice (which lack class II-restricted CD4⁺ T cells) suggested that MHC class II-restricted CD4⁺ T cells could mediate UVB-induced suppression. To test this hypothesis we performed CD4⁺ and CD8⁺ T cell depletion experiments on irradiated and nonirradiated I⁺II⁺ C57BL/6 mice (Fig. 3). As reported earlier [2], CD4⁺ T cell-depleted nonirradiated I⁺II⁺ mice developed an enhanced and prolonged inflammatory reaction, while CD8⁺ T cell depletion completely abolished hapten-specific ear swelling. In contrast, irradiated I⁺II⁺ mice did not develop a CHS reaction and depletion of CD8⁺ T cells did not affect this lack of response. Surprisingly, UV-irradiated mice, depleted of CD4⁺ T cells exhibited a normal CHS response with a magnitude and kinetics similar to that of nonirradiated mice. Taken together these data confirm that the lack of inhibition of the inflammatory reaction in UV-irradiated class II-deficient mice is due to the deficiency in CD4⁺ T cells and that CD4⁺ T cells mediate UVB-induced immune suppression in contact sensitivity to DNFB.

UVB irradiation is associated with a lack of infiltration of IFN-gamma-producing CD8⁺ effector T cells in the challenged skin

Two possibilities could explain the suppression of CHS reaction by UVB-induced CD4⁺ T cells: a) they exert their action at the periphery, at hapten-challenged sites, by inhibiting effector mechanisms initiated by effector T cells of CHS that have already migrated to the skin; b) they impair the priming and expansion of effector populations in the lymphoid organs.

To discriminate between these two hypotheses we studied the expression of CD4, CD8 and IFN-gamma mRNA in the skin of irradiated and nonirradiated mice after sensitization and challenge (Fig. 4). CD4 mRNA is normally expressed at low levels in the skin of naive mice and probably reflects the infiltration of recirculating skin homing CD4⁺ T cells. In sensitized challenged mice it is down-regulated, declining to its lowest values at 24 hrs post-challenge. It is up-regulated thereafter, returning to pre-irradiation values at 48 hrs. PCR analysis in this study confirmed these previous findings as to mRNA expression in the skin of nonirradiated mice. CD4 mRNA in the skin of irradiated mice did not show any significant changes in magnitude and kinetics in comparison to nonirradiated mice. Thus, UVB-induced immunosuppression is not due to increased infiltration of the skin by down-regulatory CD4⁺ T cells.

Alternatively, UVB irradiation was associated with an impaired infiltration of CD8⁺ T cells at challenged site. CD8 and IFN-gamma mRNA were found neither in the skin of naive mice, nor of naive challenged mice. Up-regulation of both CD8 and IFN-gamma mRNA occurred as early as 6 hrs after challenge in nonirradiated mice, with a gradual increase in the intensity thereafter (Fig. 4). Interestingly, sensitized irradiated mice showed only a mild increase in CD8⁺ and IFN-gamma mRNA which appeared later than in nonirradiated mice, at 24 hrs (Fig. 4).

Thus, the lack of CHS reaction observed in UVB-irradiated mice is associated with a lack of infiltration of CD8⁺ T cells in situ.

Quantitative and qualitative alterations of DNFB-specific CD8⁺ T cells in the lymphoid organs of UV-irradiated mice

The lack of infiltration of IFN-gamma-producing CD8⁺ effector cells in the challenged site of irradiated mice could be secondary either to their inability to migrate from blood to skin or to an impaired priming and expansion of specific CD8⁺ T cells in the lymphoid organs. To test for the latter hypothesis, lymphoid cells from irradiated and nonirradiated animals were recovered 5 days post-sensitization and tested for specific T cell proliferation and IFN-gamma production.

Both total T cells and CD8⁺ T cells obtained from nonirradiated mice responded vigorously in a secondary T cell proliferation assay to DNBS-treated but not to TNBS-treated syngeneic splenocytes (Fig. 5A). Conversely, hapten-specific responses were significantly diminished when total T cells and CD8⁺ T cells were obtained from irradiated mice (Fig. 5A). CD4⁺ T cells recovered from lymphoid organs of both irradiated and nonirradiated animals yielded only low proliferative responses.

We next studied IFN-gamma production by T cell subsets recovered from lymph nodes of irradiated and nonirradiated mice (Fig. 5B). Total T cells, CD4⁺ and CD8⁺ T cells were restimulated in vivo as previously described and 24 hrs later IFN-gamma release in culture supernatants was analysed using an ELISA assay. As shown on Figure 5B, IFN-gamma is produced exclusively by the CD8⁺ T cell subset and its secretion is greatly diminished in cultures of total T cells and CD8⁺ T cells recovered from lymphoid organs of irradiated animals.

We next used an ELISPOT assay to determine the frequency of DNFB-specific, IFN-gamma-producing lymph node cells in irradiated and nonirradiated animals (Fig. 5C). Previous studies using lymph node cells from CD8⁺ T cell-depleted mice showed that IFN-gamma producing cells were entirely contained in the CD8⁺ T cell subset [23]. UVB irradiation resulted in a major reduction in the frequency of spot forming lymph node cells. An average number of only 10 IFN-gamma-SPF/10⁶ LN cells was detected in irradiated mice, compared to 35/106 in nonirradiated animals (Fig. 5C).

Taken together, these results indicate that UVB irradiation is responsible for an impairment in the induction of hapten-specific CD8⁺ T cells in the lymphoid organs.

CD4⁺ T cells impair the induction of hapten-specific CD8⁺ T cells in the lymphoid organs of irradiated animals

Since UVB-induced CD4⁺ T cells do not inhibit the CHS reaction by infiltrating the challenged site and since CD8⁺ hapten-specific activation is impaired, we examined the hypothesis that the immune suppression could be generated in the lymphoid organs, through CD4⁺ T cell-induced inhibition of priming and expansion of effector CD8⁺ T cell population of CHS.

We determined the effect of CD4⁺ and CD8⁺ T cell depletion on the frequency of IFN-gamma-producing T cells in the lymphoid organs of irradiated I⁺II⁺ C57BL/6 mice (Fig. 6). As expected, depletion of CD8⁺ T cells totally abrogated the expansion of hapten-specific T cells. Interestingly, depletion of CD4⁺ T cells induced a dramatic increase in the number of IFN-gamma-producing SFC, compared to that obtained in irradiated C57BL/6 mice (180 and 38 SFC/10⁶ LNC respectively). More importantly, the number of IFN-gamma SFC was identical in irradiated and nonirradiated mice, i.e. UVB irradiation did not modify the number of hapten-specific CD8⁺ T cell precursors in CD4⁺ T cell-depleted mice.

Thus, MHC class II-restricted CD4⁺ T cells are the targets of UVB-induced immunosuppression, since in their absence there is a full development of hapten-specific IFN-gamma producing CD8⁺ T cells and the expression of a normal CHS reaction to DNFB.

Discussion

In the present study, we addressed the question of the mechanisms involved in immune suppression induced by UVB radiation using the classical model of CHS, which is a hapten-specific inflammatory skin DTH response mediated by hapten-specific MHC class I-restricted CD8⁺ T cells and down-regulated by MHC class II-restricted CD4⁺ T cells. The immunosuppressive effect of UVB in this system is attested by the dose-dependant inhibition of the CHS response. We show that CD4⁺ T cells mediate UVB-induced immune suppression through inhibition of expansion of the CD8⁺ effector T cell pool. More importantly, UVB have differential effects on CD4⁺ and CD8⁺ T cell subsets. Using mice deficient in MHC class II molecules, we demonstrate that UVB do not affect directly the MHC class I-restricted CD8⁺ T cell activation pathway. Conversely, UVB induce suppressor CD4⁺ T cells which are able to prevent expansion of CD8⁺ effector T cells.

A major finding in this study is that UVB have differential effects on CD4⁺ and CD8⁺ T cell subsets. Although the final effect of UVB is the inhibition of hapten-specific CD8⁺ effector T cell expansion, UVB do not directly impair the priming and expansion of the CD8⁺ T cell subset. Indeed, MHC class II-deficient mice and CD4⁺ T cell-depleted mice are resistant to the immune suppression induced by UVB and develop a normal antigen-specific skin inflammation. In other words, in the absence of CD4⁺ T cells, UVB radiation is unable to suppress CD8⁺ T cell-mediated CHS reaction, even if UV doses are increased up to skin toxicity. Thus, CD4⁺ T cells are the targets of UVB radiation whereas CD8⁺ T cells are unaffected by UVB. These data confirm and extend earlier studies suggesting that CD4⁺ T cells mediate UVB-induced immune suppression [2, 3, 12, 13].

That UVB do not alter the development of antigen-specific CD8⁺ T cells in CD4⁺ T cell-deficient mice, has important implications for the understanding of the immunosuppressive effect of UVB. Indeed, in CD4⁺ T cell-deficient mice, the kinetics and magnitude of the CHS reaction is similar to that of non-irradiated mice. Priming of CD8⁺ effector T cells in the lymph nodes is unaffected by UVB, as shown by a normal number of hapten-specific IFN-gamma-producing CD8⁺ T cells in irradiated mice. Thus, in contradiction to several data obtained using in vitro irradiated DC [7-9, 28], our results show that UVB do not impair the functional properties of APCs which are necessary for priming CD8⁺ effector T cells of CHS, in the lymphoid organs. Therefore, we have to assume that UVB do not affect the migration of epidermal DC from the irradiated skin to the draining lymph nodes and do not alter their functional maturation into potent APCs able to present antigen to MHC class I-resctricted CD8⁺ T cells. Similarly, in the absence of CD4⁺ T cells, CD8⁺ T cell functions are unaffected by UVB. Infiltration of the challenged site by CD8⁺ T cells is normal in these irradiated mice (data not shown) and constitution of the inflammatory cell infiltrate occurs with identical kinetics as in non irradiated mice. We conclude from these data that the MHC class I-restricted pathway for antigen-specific CD8⁺ T cell activation is not sensitive to UVB.

MHC class II-restricted CD4⁺ T cells are clearly the targets of UVB and mediate the UVB-induced immune suppression since their presence is mandatory for the immunosuppressive effect of UVB. Indeed, I⁺II⁺ C57BL/6 mice, but not MHC class II-KO mice, are sensitive to UVB and show an impaired CHS response and deficient infiltration of the skin by CD8⁺ effector cells. The possibility that CD4⁺ T cells exert their suppressive functions at the periphery, in the skin, during the elicitation phase of CHS was ruled out. Indeed, the pattern and kinetics of infiltration of CD4⁺ T cells at the challenge site was identical in irradiated versus non-irradiated mice, excluding the possibility that massive and early recruitment of CD4⁺ T cells was responsible for impaired CD8⁺ T cell infiltration. Alternatively, our data show that CD4⁺ T cells act at the time of sensitization by blocking the expansion of CD8⁺ T cells in the lymphoid organs. In C57BL/6 mice, UVB irradiation was associated with diminished CD8⁺ T cell proliferation, IFN-gamma production and with decreased induction of hapten-specific, IFN-gamma-producing, CD8⁺ T cells in lymph nodes. The role of CD4⁺ T cells in impeding the development of CD8⁺ T cells was further supported by the observation that depletion in CD4⁺ T cells restores the number of antigen-specific CD8⁺ T cells in the lymphoid organs. Thus, CD4⁺ T cells exert their suppressive effect in lymphoid organs at the time of the sensitization. These data are in keeping with recent studies, which showed that UV-induced CD4⁺ suppressor cells were found in lymphoid organs of irradiated mice [12, 13, 29].

The mechanisms by which UV-induced CD4⁺ T cells suppress CHS is not fully understood although recent data have pointed to a major role of IL-10-producing Th2/Tr1 cells [11-13]. Cloned CD4⁺ T cells [13] and CD4⁺ T cell lines recovered from LN of UV-irradiated mice produce IL-10 and high levels of IL-10 are found in the serum of irradiated mice [13]. Furthermore, IL-10-KO mice are resistant to UVB-induced immune suppression. Interestingly, IL-10 which was initially decribed as an immunosuppressant for CD4⁺ T cells, was recently shown to inhibit the antigen-specific proliferation and cytolytic functions of CD8⁺ T cells [30, 31]. These effects were indirect and were mediated through inhibition of MHC class I antigen expression and co-stimulatory function of APC. Since CD8⁺ T cells are effector cells of CHS through their cytotoxic activity [23], the pathophysiology of UV-induced immune suppression could be as follow: 1) UVB, delivered before the skin sensitization, are able to activate CD4⁺ T cells which produce IL-10; 2) upon skin sensitization, epidermal DC migrate to the lymph nodes for presentation of haptenated peptides to MHC class I-restricted CD8⁺ T cells; 3) activation and expansion of hapten-specific CD8⁺ T cells is inhibited because of the presence in the LN microenvironment of high quantities of IL-10.

UV-induced suppressive CD4⁺ T cells share many phenotypic and functional properties with down-regulatory CD4⁺ T cells of CHS. In CHS, CD4⁺ T cells have been clearly identified as down-regulatory cells, since MHC class II-deficient (I⁺II^-) mice develop an enhanced and prolonged CHS reaction [21, 32]. Both types of cells produce Th2/Tr1 cytokines and are MHC class II-restricted suggesting they are activated by epidermal DC through presentation of peptide/MHC class II complexes [12, 13, 33]. IL-10 is the major suppressive cytokine in both CHS and UVB-induced immune suppression of CHS, since IL-10-KO mice develop an enhanced CHS to DNFB [34] and are resistant to the UVB-induced immune suppression [35]. Therefore, it is tempting to speculate that UVB is able to activate IL-10 producing CD4⁺ T cells which are involved in the physiological down-regulation of the CHS response. In this respect, we have previously shown in the classical model of CHS to DNFB, that injection of haptenated MHC class I-II⁺ DC before sensitization with DNFB was responsible for suppression of the CHS response and was associated with activation of DNFB-specific CD4⁺ T cells in the lymph nodes [22]. Thus, suppression of the CHS response may be due to activation of down-regulatory CD4⁺ T cells before that of effector CD8⁺ T cells. Based on the above data, we postulate that UVB light, which is delivered before the sensitization phase, is able to activate the normal IL-10 producing CD4⁺ T cells responsible for the down-regulation of CHS.

Abbreviations

DC dendritic cell

HPRT hypoxanthine phosphoribosyltransferase

CHS contact hypersensitivity

DNBS 2,4-dinitrobenzenesulfonic acid

DNFB dinitrofluorobenzene

DTH delayed-type hypersensitivity

LC Langerhans cell

SPF spot forming cell

TNBS 2,4,6-trinitrobenzenesulfonic acid

Article accepted on 31/8/01

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