Ultraviolet B Suppresses Immunity by Inhibiting Effector and Memory T Cells
2008; Elsevier BV; Volume: 172; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2008.070517
ISSN1525-2191
AutoresSabita Rana, Scott N. Byrne, Linda Joanne MacDonald, Carling Yan-Yan Chan, Gary M. Halliday,
Tópico(s)Immunotherapy and Immune Responses
ResumoContact hypersensitivity is a T-cell-mediated response to a hapten. Exposing C57BL/6 mice to UV B radiation systemically suppresses both primary and secondary contact hypersensitivity responses. The effects of UVB on in vivo T-cell responses during UVB-induced immunosuppression are unknown. We show here that UVB exposure, before contact sensitization, inhibits the expansion of effector CD4+ and CD8+ T cells in skin-draining lymph nodes and reduces the number of CD4+ and IFN-γ+ CD8+ T cells infiltrating challenged ear skin. In the absence of UVB, at 10 weeks after initial hapten exposure, the ear skin of sensitized mice was infiltrated by dermal effector memory CD8+ T cells at the site of challenge. However, if mice were previously exposed to UVB, this cell population was absent, suggesting an impaired development of peripheral memory T cells. This finding occurred in the absence of UVB-induced regulatory CD4+ T cells and did not involve prostaglandin E2, suggesting that the importance of these two factors in mediating or initiating UVB-induced immunosuppression is dependent on UVB dose. Together these data indicate that in vivo T-cell responses are prone to immunoregulation by UVB, including a novel effect on both the activated T-cell pool size and the development of memory T cells in peripheral compartments. Contact hypersensitivity is a T-cell-mediated response to a hapten. Exposing C57BL/6 mice to UV B radiation systemically suppresses both primary and secondary contact hypersensitivity responses. The effects of UVB on in vivo T-cell responses during UVB-induced immunosuppression are unknown. We show here that UVB exposure, before contact sensitization, inhibits the expansion of effector CD4+ and CD8+ T cells in skin-draining lymph nodes and reduces the number of CD4+ and IFN-γ+ CD8+ T cells infiltrating challenged ear skin. In the absence of UVB, at 10 weeks after initial hapten exposure, the ear skin of sensitized mice was infiltrated by dermal effector memory CD8+ T cells at the site of challenge. However, if mice were previously exposed to UVB, this cell population was absent, suggesting an impaired development of peripheral memory T cells. This finding occurred in the absence of UVB-induced regulatory CD4+ T cells and did not involve prostaglandin E2, suggesting that the importance of these two factors in mediating or initiating UVB-induced immunosuppression is dependent on UVB dose. Together these data indicate that in vivo T-cell responses are prone to immunoregulation by UVB, including a novel effect on both the activated T-cell pool size and the development of memory T cells in peripheral compartments. UVB radiation (290 to 320 nm) represents ∼5% of the total UV radiation present in sunlight. Exposure to UVB triggers a multitude of molecular and cellular changes in skin, the most deleterious consequence of which is skin cancer. In addition to causing DNA damage in the skin, UVB modulates the immune system in distant lymphoid compartments resulting in the suppression of anti-tumor immunity.1Halliday GM Rana S Waveband and dose dependency of sunlight-induced immunomodulation and cellular changes.Photochem Photobiol. 2008; 84: 35-46Crossref PubMed Scopus (52) Google Scholar An important feature of UVB exposure is the suppression of both primary and memory recall immune responses resulting in antigen-specific tolerance.2Poon TSC Barnetson RSC Halliday GM Sunlight-induced immunosuppression in humans is initially because of UVB, then UVA, followed by interactive effects.J Invest Dermatol. 2005; 125: 840-846Crossref PubMed Scopus (72) Google Scholar This study examined the cellular mechanisms of systemic UVB-induced immunosuppression, which is a phenomenon that can be observed when one skin site is exposed to UVB but antigen is applied at a distal, unirradiated site. Exposure to UVB induces the release of numerous soluble mediators that alter immunity by acting on various cell types in both skin and draining lymph nodes (DLNs). Some of these include interleukin (IL-4), IL-10, prostaglandin-E2 (PGE2), platelet-activating factor, histamine, and cis-urocanic acid (cis-UCA).3Ullrich SE Mechanisms underlying UV-induced immune suppression.Mutat Res. 2005; 571: 185-205Crossref PubMed Scopus (283) Google Scholar At the cellular level, UVB induces the generation of various regulatory cells,4Schwarz A Maeda A Wild MK Kernebeck K Gross N Aragane Y Beissert S Vestweber D Schwarz T Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity.J Immunol. 2004; 172: 1036-1043PubMed Google Scholar, 5Byrne SN Halliday GM B cells activated in lymph nodes in response to ultraviolet irradiation or by interleukin-10 inhibit dendritic cell induction of immunity.J Invest Dermatol. 2005; 124: 570-578Crossref PubMed Scopus (93) Google Scholar, 6Moodycliffe AM Nghiem D Clydesdale G Ullrich SE Immune suppression and skin cancer development: regulation by NKT cells.Nat Immunol. 2000; 1: 521-525Crossref PubMed Scopus (283) Google Scholar which are central to the concept of transferable UVB-induced tolerance. UVB can inhibit contact hypersensitivity (CHS) reactions to nonproteineous contact haptens in a systemic and antigen-specific manner. CHS is a cutaneous T-cell-mediated reaction whereby hapten-specific CD4+ and CD8+ T cells are generated in skin DLNs after epicutaneous hapten application. On induction of the efferent phase of CHS, which occurs at a separate site from sensitization, hapten-specific T cells exit lymphoid organs and migrate into the skin site of challenge. The inflammatory response that ensues is primarily thought to involve the cytotoxic killing of hapten-conjugated keratinocytes by infiltrating hapten-specific effector CD8+ T cells.7Akiba H Kehren J Ducluzeau M-T Krasteva M Horand F Kaiserlian D Kaneko F Nicolas J-F Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis.J Immunol. 2002; 168: 3079-3087PubMed Google Scholar How UVB modulates the cellular mechanisms of CHS to reduce the response induced by challenge is not yet known. In particular, it is unknown if UVB has any affect on the magnitude of hapten-specific T-cell responses, which mediate CHS reactions. Activation of naïve T cells on cognate antigenic stimulation by antigen-presenting cells (APCs) causes changes to adhesion and cytokine receptor cell surface molecules. Stimulated naïve T cells up-regulate the adhesion molecule receptor, CD44 to high levels (CD44hi) from an intermediate to low level of expression (CD44int/lo)8Shimizu Y Van Seventer GA Siraganian R Wahl L Shaw S Dual role of the CD44 molecule in T cell adhesion and activation.J Immunol. 1989; 143: 2457-2463PubMed Google Scholar and down-regulate the lymph node homing receptor, L-selectin or CD62L.9Hamann A Jablonski-Westrich D Scholz KU Duijvestijn A Butcher EC Thiele HG Regulation of lymphocyte homing. I. Alterations in homing receptor expression and organ-specific high endothelial venule binding of lymphocytes upon activation.J Immunol. 1988; 140: 737-743PubMed Google Scholar Activated T cells, therefore, have a CD44hiCD62L− phenotype allowing them to leave lymphoid organs and traverse into peripheral tissues. The life span of differentiated cytotoxic or cytokine-secreting effector T cells from activated T cells is partly regulated by the IL-7 receptor (CD127). Naïve T cells express high levels of CD127, whereas effector T cells down-regulate expression of CD127. Therefore, effector T cells are CD44hiCD62L−CD127− and naive T cells are CD44int/loCD62L+CD127+. Alternatively, if a cell is fated to become a long-lived memory T cell, expression of CD127 is maintained.10Kaech SM Tan JT Wherry EJ Konieczny BT Surh CD Ahmed R Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells.Nat Immunol. 2003; 4: 1191-1198Crossref PubMed Scopus (1422) Google Scholar Memory T cells can be further subdivided into populations of central and effector memory. Central memory T cells predominantly circulate through lymphoid organs using CD62L and so are CD44hiCD62L+CD127+. In contrast, effector memory T cells migrate into and monitor peripheral tissues because they lack CD62L and so are CD44hiCD62L−CD127+.11Sallusto F Lenig D Forster R Lipp M Lanzavecchia A Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature. 1999; 401: 708-712Crossref PubMed Scopus (4581) Google Scholar The effects of UVB on effector and memory T-cell development are unknown. Using a CHS model we investigated the responses of T cells in vivo with and without exposure to UVB before contact sensitization. We examined whether suberythemal low-level UVB exposure, typically acquired during normal daily activities, could modulate T-cell immunity. By monitoring T-cell activation, proliferation, and infiltration during primary CHS and in long-term resting mice, the influence of sensitization and UVB on the development of effector and memory CD4+ and CD8+ T cells was examined. We show that sensitization induces T-cell activation and proliferation in skin DLNs, and that CHS elicitation caused the infiltration of these cells into skin. UVB inhibited the primary T-cell response in skin DLNs during sensitization and decreased T-cell accumulation in challenged skin. Unirradiated mice 10 weeks after sensitization developed dermal effector memory CD8+ T cells at the challenged skin site; however, this was lost in mice that were exposed to UVB. DLN cells from UVB-irradiated mice could not transfer suppression and treatment with a cyclooxygenase (COX) inhibitor to prevent PGE2 production did not reverse the reduction of CD4+ and CD8+ T-cell expansion caused by UVB. These results indicate a novel affect of UVB, in which it can profoundly inhibit in vivo the magnitude of the effector T-cell response and modulate peripheral memory T-cell development. This occurred independently of UVB-induced regulatory T cells and the PGE2 pathway, probably because the UVB dose was not high enough to activate these pathways. C57BL/6J female mice were used at 8 weeks of age (Animal Resource Centre, Perth, Australia). FVB transgenic GFP (T-GFP) mice on a CD4 promoter were obtained from Ulrich H. von Andrian (The Center for Blood Research, Harvard Medical School, Boston, MA)12Manjunath N Shankar P Stockton B Dubey PD Lieberman J von Andrian UH A transgenic mouse model to analyze CD8+ effector T cell differentiation in vivo.Proc Natl Acad Sci USA. 1999; 96: 13932-13937Crossref PubMed Scopus (81) Google Scholar and were backcrossed to C57BL/6J mice. T-GFP mice were used at 8 weeks of age. All experiments were conducted under the approval of the University of Sydney Animal Ethics Committee. A 1000 W xenon arc lamp solar simulator (Oriel, Stanford, CT) filtered with two 200- to 400-nm dichroic mirrors and a 310-nm narrowband interference filter (CVL Laser, Albuquerque, NM) was used to produce the UVB spectra that had a peak irradiance of 3.69 × 10−2 mW/cm2 at 311-nm wavelength, and a halfband width of ∼11 nm. UVA (more than 320 nm) and UVC (less than 290 nm) contaminated the spectra by ∼16% and 0.31%, respectively. Spectral output and intensity was measured with an OL-754 spectroradiometer (Optronics Laboratories, Orlando, FL) and a broadband radiometer (International Light Technologies, Inc., Peabody, MA) calibrated against the source was used continuously to monitor fluctuations in output. Timing of UVB delivery was accurately maintained using an automated timing device. Dorsums were shaved with animal clippers (Oster, McMinnville, TN) and an electric razor (Remington, Braeside, Australia) 24 hours before irradiation. Mice were restrained during irradiation within black Perspex boxes with a quartz lid. Ears and heads were protected from UVB radiation with black Perspex. Mouse dorsums were exposed to 90 mJ/cm2 of UVB daily for 3 consecutive days, which is ∼0.3 of a minimal erythema dose. To examine the effect of COX inhibition, mice were treated with 40 μl of 0.06% (w/v) indomethacin (Sigma, St. Louis, MO) in acetone on their dorsum immediately after irradiation. UVB-irradiated but unsensitized mice were included in every experiment to control against any nonspecific effects caused by irradiation. Three days after the last UVB irradiation, mouse abdomens were shaved and sensitized with a 50-μl epicutaneous application of 2% (w/v) oxazolone (Ox, 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma) in acetone. Ears were challenged topically 7 days later with 10 μl of 2% (w/v) Ox in acetone per ear. Increases in ear thickness were calculated based on previous and 24 hours after Ox challenge measurements using micrometer calipers (Interapid, Rolle, Switzerland). To determine the CHS, increases in ear thickness of unsensitized but ear-challenged irritant controls were subtracted from sensitized mice. For secondary CHS responses, mice were resensitized with Ox on their abdomens 8 weeks after the initial sensitization and were then rechallenged on the ears 1 week after resensitization. Equivalent Ox concentrations were used as during primary CHS.13Byrne SN Spinks N Halliday GM Ultraviolet A irradiation of C57BL/6 mice suppresses systemic contact hypersensitivity or enhances secondary immunity depending on dose.J Invest Dermatol. 2002; 119: 858-864Crossref PubMed Scopus (60) Google Scholar In hapten challenge control experiments, Ox-sensitized mice were challenged with 20 μl of 1% (w/v) 2,4,6-trinitrochlorobenzene (TNCB; Tokyo Kasei, Toyo, Japan) in acetone. The inguinal lymph nodes (ILNs) were disassociated through 70-μm cell strainers (BD Falcon, Bedford, MA) into RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 2% fetal calf serum (Invitrogen). Viable cells were enumerated by trypan blue exclusion on a Vi-CELL counter (Beckman Coulter, Hialeah, FL). Cells (2 × 107 total) from the ILNs and brachial lymph nodes of sensitized UVB-irradiated and unirradiated mice were adoptively transferred intravenously into naïve mice. Twenty-four hours after transfer, mice were sensitized with Ox and 7 days later, challenged on their ears with Ox. The CHS response was measured 24 hours after challenge. Untransferred sensitized and unsensitized control mice were included. Ears were removed from mice, split into dorsal and ventral sides and were incubated in 20 mmol/L ethylenediaminetetraacetic acid (Sigma) in Tris-buffered saline (pH 7.3) for 2 hours at 37°C. Epidermal and dermal layers were first separated before they were finely chopped together into small pieces. Minced skin pieces were then incubated in 2 ml of RPMI 1640 containing 1 mg/ml of collagenase IV (Sigma), 0.02 mg/ml of DNase I (Sigma), and 5% fetal calf serum for 1.5 hours at room temperature with constant agitation. Digestion was stopped by the addition of 200 μl of 0.1 mol/L ethylenediaminetetraacetic acid for 5 minutes. Digested skin pieces were then mashed through a 100-μm steel strainer, washed, and numerated. A skin sample containing spiked lymphoid cells was used as a positive gating control for flow cytometry. Monoclonal antibodies used for flow cytometry included rat anti-mouse CD3 (145-2C11; fluorescein isothiocyanate, APC), CD4 (RM4-5; PerCP, PE-Cy7), CD8α (53-6.7; PerCP, PE-Cy7), CD16/CD32 (2.4G2; purified), CD25 (PC61; APC, APC-Cy7), CD45 (30-F11; PerCP), CD62L (MEL-14; biotin), CD152 (UC10-4F10-11; PE), and interferon (IFN)-γ (XMG1.2; PE). These were purchased from BD Pharmingen (San Diego, CA). Rat anti-mouse CD62L (MEL-14; APC-Cy7), CD127 (A7R34; APC, APC-Cy7, biotin), and FoxP3 (FJK-16s; PE) were purchased from eBiosciences (San Diego, CA) and rat anti-mouse CD44 (IM7.8.1; PE) from Caltag (Burlingame, CA). Antibodies for immunohistochemistry included purified goat anti-rat/mouse IFN-γ (AF-585-NA; R&D Systems, Minneapolis, MN), rat anti-mouse CD4-biotin (H129.19; BD Pharmingen), rat anti-mouse CD8α-biotin (53-6.7; BD Pharmingen), and donkey anti-goat F(ab′)2 (biotin) (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were initially blocked with anti-CD16/CD32 (anti-FcRγIII/II receptor; clone 2.4G2) antibody, before staining with T-cell activation surface antibodies. When required, secondary streptavidin-APC-Cy7 (eBiosciences) was applied. All incubations were performed at 4°C for 30 minutes in fluorescence-activated cell sorting buffer (phosphate-buffered saline, 5% fetal calf serum, and 0.1 mol/L ethylenediaminetetraacetic acid, pH 7.2). Except after anti-CD16/CD32 antibody block, cells were washed three times between incubations. FoxP3 staining was performed following the manufacturer's instructions. Stained cells were analyzed on a six-color FACSAria (BD Immunocytometry Systems, San Jose, CA) and a minimum of 200,000 events were acquired for every sample. Data analysis was performed using FlowJo software v. 6.4 (Tree Star Inc., Ashland, OR). From the time of sensitization, mice were given fresh 0.8 mg/ml of BrdU (BD Pharmingen) in their drinking water daily. Single-cell suspensions of ILNs and skin were prepared as described above. Labeling for T-cell surface markers and intracellular BrdU was performed using a fluorescein isothiocyanate BrdU Flow kit (BD PharMingen) by following the manufacturer's instructions. Flow cytometry acquisition and analysis was as described above. Mouse ears were snap-frozen in liquid nitrogen in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA). Cryostat sections, 7 μm thick, were cut onto SuperFrost Plus slides (Menzel-Glasser, Braunschweig, Germany), air-dried, fixed in 4% paraformaldehyde (Amresco, Solon, OH), and blocked for endogenous biotin activity using a biotin blocking kit (DAKO, Glostrup, Denmark). Nonspecific antibody labeling was prevented by incubating sections in TNB blocking buffer (Perkin Elmer Life Sciences, Wellesley, MA) supplemented with 5% normal rabbit serum (Hunter Antisera, Jesmond, Australia) for 30 minutes. This blocking buffer was also used to dilute all labeling reagents. Sections were first labeled for T cells with biotinylated anti-CD4 or anti-CD8 antibodies for 1 hour. All subsequent incubations were then performed in the dark. Secondary streptavidin-Alexa Fluor 488 (Molecular Probes, Eugene, OR) was applied for 30 minutes before sections were again blocked to limit biotin and nonspecific antibody labeling. Sections were incubated with anti-IFN-γ antibody for 1 hour, which was followed by biotinylated donkey anti-goat and streptavidin-Alexa Fluor 594 (Molecular Probes) for 30 minutes each. Staining controls included antibody isotype and omission of primary T-cell or IFN-γ antibodies. Between all incubations, except after the nonspecific antibody labeling block, sections were washed three times with Tris-buffered saline and 0.05% (v/v) Tween 20 (Amersco). Sections were counterstained and coverslipped in SlowFade Gold antifade reagent with 4,6-diamidino-2-2phenylindole (DAPI, Molecular Probes). A BX51 fluorescent microscope with a DP70 camera attachment (Olympus, Tokyo, Japan) was used to visualize and photograph the sections. CHS responses were evaluated by one-way analysis of variance analyses with Tukey post hoc tests. To determine the effect of UVB on Ox-induced T-cell activation, proliferation, and accumulation into skin, background unsensitized (irritant control) groups were subtracted from sensitized groups to enable the direct comparison of sensitization in unirradiated and UVB-irradiated groups. Unpaired Student's t-tests were applied to compare the parameters of T-cell activation between groups of unsensitized and sensitized, or groups of sensitized unirradiated and UVB-irradiated mice. SPSS v.11 software (SPSS Inc., Chicago, IL) was used to determine significance, where P < 0.05 was considered to be significant. UVB-irradiated mice had a significantly reduced CHS response (19.7 mm−2 ± 2.0, n = 18) compared to unirradiated control mice (28.4 mm−2 ± 1.0, P = 0.002). T-cell reactivity to Ox after sensitization was investigated within the ILNs because this drains both abdominal and dorsal skin with CD44hiCD62L−CD127− effector T cells being numerated 9 days after sensitization. Application of a contact hapten significantly increased the total number of effector CD4+ and CD8+ T cells by more than threefold compared to the number of effector T cells present in the ILNs of unsensitized mice (data not shown). To investigate whether UVB modulates this expansion of ILN effector T cells, mice were irradiated with UVB before sensitization. In contrast to the increases observed in control unirradiated sensitized mice, exposure to UVB significantly reduced the expansion of effector CD4+ and CD8+ T cells by threefold (P = 0.0030) and twofold (P = 0.011), respectively (Figure 1A). A survey of other nondraining lymphoid organs (mesenteric and auricular lymph nodes, spleen), as well as blood and liver in unirradiated and UVB-irradiated sensitized mice showed that these responses to Ox are limited to the skin-draining ILNs (data not shown). This suggests that UVB inhibits the primary activation and expansion of effector CD4+ and CD8+ T cells to contact sensitization locally in the ILNs. To confirm that the T-cell expansion was attributable to antigen-induced proliferation rather than nonspecific lymph node shutdown, BrdU incorporation was assessed 9 days after sensitization. A significantly greater proportion of BrdU+ cells were detected within the activated subset of CD44hi CD4+ and CD8+ T cells in sensitized mice (63% and 45%, respectively) compared to unsensitized mice (45%, P = 0.0004 and 32%, P = 0.0054, respectively), indicating that sensitization does induce active T-cell proliferation leading to an increase in effector CD4+ and CD8+ T-cell populations. In contrast to unirradiated mice, a smaller BrdU+ frequency of CD44hi T cells was detected in the ILNs of UVB-irradiated mice (Figure 1B). This was significantly decreased in the CD4+ subset of T cells (P = 0.0396). Similarly, a reduced number of BrdU+CD44hi CD4+ (P = 0.0355) and CD8+ (P = 0.0231) T cells were found in UVB-irradiated mice (Figure 1C), suggesting that UVB hindered the proliferation of T cells after sensitization. To investigate whether regulatory cells were being generated in the DLNs by the irradiation regime used in this study, total DLN cells from sensitized UVB-irradiated or unirradiated mice were adoptively transferred into naïve mice, which were then sensitized and challenged to elicit a CHS reaction. As expected, recipients of cells from unirradiated donors exhibited an increased CHS response compared to untransferred sensitized mice (P = 0.0307), as these transferred cells included Ox-specific effector T cells (Figure 2A). However, no difference was found between recipients of UVB-irradiated or unirradiated cells, indicating no transfer of suppression. UVB-induced regulatory T cells have a CD4+CD25+CTLA-4+(CD152)14Schwarz A Beissert S Grosse-Heitmeyer K Gunzer M Bluestone JA Grabbe S Schwarz T Evidence for functional relevance of CTLA-4 in ultraviolet-radiation-induced tolerance.J Immunol. 2000; 165: 1824-1831PubMed Google Scholar phenotype and are FoxP3+.15Gorman S Tan JWY Yerkovich ST Finlay-Jones JJ Hart PH CD4+ T cells in lymph nodes of UVB-irradiated mice suppress immune responses to new antigens both in vitro and in vivo.J Invest Dermatol. 2007; 127: 915-924Crossref PubMed Scopus (28) Google Scholar Because recent evidence indicates that UVB increases the number and percentage of these cells in DLNs,15Gorman S Tan JWY Yerkovich ST Finlay-Jones JJ Hart PH CD4+ T cells in lymph nodes of UVB-irradiated mice suppress immune responses to new antigens both in vitro and in vivo.J Invest Dermatol. 2007; 127: 915-924Crossref PubMed Scopus (28) Google Scholar regulatory CD4+ T cells were examined in the ILNs. UVB-irradiated and unirradiated mice did not differ in the total number of FoxP3+CD4+ (Figure 2B) and CD4+CD25+CTLA-4+ T cells (Figure 2D). The percentages of these cells were also unchanged (Figure 2, D and E). Together, these data suggest that the systemic UVB regime used in this study does not induce the generation of regulatory CD4+ T cells. Prostaglandin production was inhibited to investigate whether it is a mediator of UVB inhibition of T-cell activation and expansion. Immediately after each UVB irradiation, indomethacin, a COX inhibitor, was applied to the UVB-irradiated skin. Mice were then sensitized and challenged. Indomethacin administration had no effect on the expansion of ILN effector T cells in unirradiated mice, and it did not prevent UVB-induced reduction in effector CD4+ (absence of indomethacin, P = 0.0392, and presence of indomethacin, P = 0.0233) and CD8+ T cells (absence of indomethacin, P = 0.0309, and presence of indomethacin, P = 0.0234) (Figure 3). This suggests that UVB-induced PGE2 production in irradiated skin is not responsible for suppressing local T-cell activation in DLNs. To determine whether UVB influences CD45+ leukocyte and T-cell recruitment into the challenge site, we examined ear skin by flow cytometry 48 hours after challenge because this was determined to be the time of maximal leukocyte infiltrate. Single-cell suspensions of total ear skin were prepared from epidermal and dermal skin layers and then stained with antibodies against CD45, CD44, CD62L, CD4, CD8, and CD3. Representative dot plots of CD4+CD3+ and CD8+CD3+ labeling in ear skin are shown in Figure 4A. Uninflamed ear skin from unsensitized mice contained a small resident population of CD4+ T cells, representing ∼3% of the CD45+ population. Few to no CD8+ T cells were detected in unsensitized mice. Consistently, a sizeable population of CD3hi, CD4−CD8− cells were found in the ear skin (∼40% of the CD45+ population) that were also γδTCR+, suggesting the presence of dendritic epidermal T cells (data not shown). Sensitization followed by ear challenge induced an influx of CD45+ cells into ear skin, including CD4+ and CD8+ T cells. Both unirradiated and UVB-irradiated sensitized mice showed a similar recruitment frequency of CD4+ and CD8+ T cells (of total CD45+) into ear skin, suggesting that UVB does not affect the proportional recruitment of T cells against other inflammatory cell types. The majority of T cells were CD44hiCD62L− (Figure 4B) and were BrdU+ (Figure 4C), indicating that these cells are activated and proliferating T cells. UVB significantly decreased the total number of recruited CD45+ leukocytes (P = 0.0248), CD4+ T cells (P = 0.0448), and CD8+ T cells (P = 0.0415) by ∼50% compared to unirradiated sensitized mice (Figure 5A). To determine the contribution of nonspecific cells migrating into irritated skin, Ox-sensitized mice were challenged with a different unrelated hapten, TNCB. Neither Ox sensitization nor UVB irradiation significantly altered the CHS response compared to the reaction measured in unsensitized TNCB-challenged mice (7.9 mm−2 ± 0.9). Moreover, the ears of these TNCB-challenged mice contained significantly fewer CD45+ cells, CD4+ T cells, and CD8+ T cells compared to Ox-sensitized and challenged mice. These results confirm that the cellular infiltrate detected in the ear skin of Ox-sensitized and Ox-challenged mice were recruited in a hapten-specific manner. We examined the functional capacity of skin-localized T cells by assessing their in situ IFN-γ expression 48 hours after challenge (Figure 5B). CD4+ cells in sensitized mice were localized to both the epidermal and dermal layers (Figure 5Ba, arrows). In contrast, CD8+ cells were mostly observed in the dermis (Figure 5Bc, arrows). IFN-γ+ cells were detected in the dermis of mice, although, none were co-labeled with CD4+ cells (Figure 5Ba, arrowheads). Rather, a proportion of CD8+ cells expressed IFN-γ (Figure 5Bc, asterisk), indicating that production of this cytokine is limited to CD8+ T cells in ear skin. No IFN-γ reactivity was observed in the ear skin of unsensitized mice (data not shown). UVB-irradiated mice maintained a suppressed secondary memory response to Ox resensitization and rechallenge compared to unirradiated mice (22.9 mm−2 ± 1.9 versus 29.6 mm−2 ± 1.5, P = 0.0102, n = 14). To determine whether UVB inhibited these reactions by affecting the development of memory T cells, UVB-irradiated, sensitized, and challenged mice were rested for 10 weeks after sensitization (memory mice). As mice were not resensitized or rechallenged during this period, T cells with an activated phenotype were defined as memory T cells. A comparable number of central memory T cells (CD44hiCD62L+CD127+) in draining and nondraining lymphoid organs, blood, and liver were detected in sensitized and unsensitized mice, indicating that the lymphoid system was in a resting and homeostatic state (data not shown). The ear skin of memory mice was examined to determine whether previous hapten exposure or UVB had any effect on the resident T-cell population in skin. The number of total CD45+ leukocytes and CD4+ T cells in ear skin was similar between unirradiated and UVB-irradiated mice, suggesting that UVB does not play a role in regulating these cells in memory mice (data not shown). In contrast, the ears of unirradiated memory mice were populated by a significant number of effector memory (CD44hiCD62L−) CD8+ T cells that represented ∼4% of skin CD45+ cells, compared to 0.60% in unsensitized mice (Figure 6A). UVB-irradiated, sensitized mice had a CD8+ T-cell frequency similar to unsensitized mice. Moreover, compared to unirradiated memory mice, the total number of memory CD8+ T cells in the ears of mice previously exposed to UVB was significantly decreased by more than 30-fold (P = 0.0121) (Figure 6B). Hence, UVB inhibited the development of peripheral effector memory CD8+ T cells that would normally localize to the skin of sensitized mice. No
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