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Mechanisms Underlying the Suppression of Established Immune Responses by Ultraviolet Radiation

2002; Elsevier BV; Volume: 119; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2002.01845.x

1523-1747

Dat X. Nghiem, Nasser Kazimi, David L. Mitchell, Arie A. Vink, Honnavara N. Ananthaswamy, Margaret L. Kripke, Stephen E. Ullrich,

Spaceflight effects on biology

The ultraviolet radiation present in sunlight is immune suppressive. Recently we showed that solar-simulated ultraviolet radiation (ultraviolet A + B; 295–400 nm), applied after immunization, suppressed immunologic memory and the elicitation of delayed-type hypersensitivity to the common opportunistic pathogen, Candida albicans. Further, we found that wavelengths in the ultraviolet A region of the solar spectrum (320–400 nm), devoid of ultraviolet B, were equally effective in activating immune suppression as ultraviolet A + B radiation. Here we report on the mechanisms involved. Maximal immune suppression was found when mice were exposed to solar-simulated ultraviolet radiation 7–9 d post immunization. No immune suppression was found in ultraviolet-irradiated mice injected with monoclonal anti-interleukin-10 antibody, or mice exposed to solar-simulated ultraviolet radiation and injected with recombinant interleukin-12. Suppressor lymphocytes were found in the spleens of mice exposed to ultraviolet A + B radiation. In addition, antigen-specific suppressor T cells (CD3+, CD4+, DX5+) were found in the spleens of mice exposed to ultraviolet A radiation. Applying liposomes containing bacteriophage T4N5 to the skin of mice exposed to solar-simulated ultraviolet A + B radiation, or mice exposed to ultraviolet A radiation, blocked immune suppression, demonstrating an essential role for ultraviolet-induced DNA damage in the suppression of established immune reactions. These findings indicate that overlapping immune suppressive mechanisms are activated by ultraviolet A and ultraviolet A + B radiation. Moreover, our findings demonstrate that ultraviolet radiation activates similar immunologic pathways to suppress the induction of, or the elicitation of, the immune response. The ultraviolet radiation present in sunlight is immune suppressive. Recently we showed that solar-simulated ultraviolet radiation (ultraviolet A + B; 295–400 nm), applied after immunization, suppressed immunologic memory and the elicitation of delayed-type hypersensitivity to the common opportunistic pathogen, Candida albicans. Further, we found that wavelengths in the ultraviolet A region of the solar spectrum (320–400 nm), devoid of ultraviolet B, were equally effective in activating immune suppression as ultraviolet A + B radiation. Here we report on the mechanisms involved. Maximal immune suppression was found when mice were exposed to solar-simulated ultraviolet radiation 7–9 d post immunization. No immune suppression was found in ultraviolet-irradiated mice injected with monoclonal anti-interleukin-10 antibody, or mice exposed to solar-simulated ultraviolet radiation and injected with recombinant interleukin-12. Suppressor lymphocytes were found in the spleens of mice exposed to ultraviolet A + B radiation. In addition, antigen-specific suppressor T cells (CD3+, CD4+, DX5+) were found in the spleens of mice exposed to ultraviolet A radiation. Applying liposomes containing bacteriophage T4N5 to the skin of mice exposed to solar-simulated ultraviolet A + B radiation, or mice exposed to ultraviolet A radiation, blocked immune suppression, demonstrating an essential role for ultraviolet-induced DNA damage in the suppression of established immune reactions. These findings indicate that overlapping immune suppressive mechanisms are activated by ultraviolet A and ultraviolet A + B radiation. Moreover, our findings demonstrate that ultraviolet radiation activates similar immunologic pathways to suppress the induction of, or the elicitation of, the immune response. cyclobutane pyrimidine dimer(s) keyhole limpet hemocyanin UV radiation between 290 and 320 nm UV radiation between 320 and 400 nm UV radiation between 340 and 400 nm UVA radiation between 320 and 340 nm The adverse effects of ultraviolet (UV) radiation on human health and well-being are well known (Ullrich et al., 2000Ullrich S.E. The effects of ultraviolet radiation on the immune response.in: Kydonieus A.F. Wille J.J. Biochemical Modulation of Skin Reactions: Transdermals, Topicals, Cosmetics. CRC Press, Boca Raton, FL2000: 281-300Google Scholar). Besides being the primary cause of human nonmelanoma skin cancer (Urbach, 1997Urbach F. Ultraviolet radiation and skin cancer of humans.J Photochem Photobiol B Biol. 1997; 40: 3-7Crossref PubMed Scopus (115) Google Scholar), the UV radiation present in sunlight is immune suppressive (Kripke, 1974Kripke M.L. Antigenicity of murine skin tumors induced by UV light.J Natl Cancer Inst. 1974; 53: 1333-1336PubMed Google Scholar;Kripke and Fisher, 1976Kripke M.L. Fisher M.S. Immunologic parameters of ultraviolet carcinogenesis.J Natl Cancer Inst. 1976; 57: 211-215Crossref PubMed Scopus (275) Google Scholar). The immune suppressive effects of UV radiation contribute to skin cancer development by depressing cell-mediated immune reactions that normally serve to destroy the developing skin tumors. Epidemiologic studies with immune suppressed renal transplant patients (Penn, 1984Penn I. Depressed immunity and skin cancer.Immunol Today. 1984; 5: 291-293Abstract Full Text PDF PubMed Scopus (11) Google Scholar), experiments with laboratory mice (Fisher and Kripke, 1982Fisher M.S. Kripke M.L. Suppressor T lymphocytes control the development of primary skin cancers in UV-irradiated mice.Science. 1982; 216: 1133-1134Crossref PubMed Scopus (332) Google Scholar), and immunologic studies with skin cancer patients (Yoshikawa et al., 1990Yoshikawa T. Rae V. Bruins-Slot W. vand-den-Berg J.W. Taylor J.R. Streilein J.W. Susceptibility to effects of UVB radiation on induction of contact hypersensitivity as a risk factor for skin cancer in humans.J Invest Dermatol. 1990; 95: 530-536Abstract Full Text PDF PubMed Google Scholar) support the hypothesis that the immune suppression induced by UV exposure is a major risk factor for skin cancer induction. In addition to suppressing tumor rejection, UV radiation interferes with a wide variety of immune reactions including contact hypersensitivity to chemical allergens applied to the skin (Noonan et al., 1981Noonan F.P. Kripke M.L. Pedersen G.M. Greene M.I. Suppression of contact hypersensitivity by UV radiation is associated with defective antigen presentation.Immunology. 1981; 43: 527-533PubMed Google Scholar;Cooper et al., 1992Cooper K.D. Oberhelman L. Hamilton T.A. et al.UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans: relationship to dose, CD1a–DR+ epidermal macrophage induction, and Langerhans cell depletion.Proc Natl Acad Sci USA. 1992; 89: 8497-8501Crossref PubMed Scopus (347) Google Scholar) and delayed-type hypersensitivity (DTH) to bacterial (Jeevan and Kripke, 1989Jeevan A. Kripke M.L. Effect of a single exposure to ultraviolet radiation on Mycobacterium bovis bacillus Calmette-Guerin infection in mice.J Immunol. 1989; 143: 2837-2843PubMed Google Scholar) and viral (Howie et al., 1986Howie S.E.M. Norval M. Maingay J. Exposure to low dose UVB light suppresses delayed type hypersensitivity to herpes simplex virus in mice.J Invest Dermatol. 1986; 86: 125-128Abstract Full Text PDF PubMed Scopus (102) Google Scholar) antigens. In the majority of studies documenting UV-induced suppression of the immune response to microbial and viral agents, the UV was administered to naive animals prior to immunization (i.e., suppressing the induction of immunity). Of equal concern, however, is the ability of UV exposure to suppress established immune responses. Perhaps the most important medical advance of the past century was the reduction, and in some cases the eradication, of microbial and viral infections through the widespread use of childhood vaccinations. Because UV radiation can suppress the elicitation of certain immune responses (Denkins et al., 1989Denkins Y. Fidler I.J. Kripke M.L. Exposure of mice to UVB radiation suppresses delayed hypersensitivity to Candida albicans.Photochem Photobiol. 1989; 49: 615-619Crossref PubMed Scopus (73) Google Scholar;Magee et al., 1989Magee M.J. Kripke M.L. Ullrich S.E. Suppression of the elicitation of the immune response to alloantigen by ultraviolet radiation.Transplantation. 1989; 47: 1008-1013Crossref PubMed Scopus (20) Google Scholar;Damian et al., 1997Damian D.L. Halliday G.M. Barnetson R.S.C. Broad-spectrum sunscreens provide greater protection against ultraviolet-radiation-induced suppression of contact hypersensitivity to a recall antigen in humans.J Invest Dermatol. 1997; 109: 146-151Crossref PubMed Scopus (122) Google Scholar;Moyal et al., 1997Moyal D. Courbière C. Le Corre Y. de Lacharrière O. Hourseau C. Immunosuppression induced by chronic solar-simulated irradiation in humans and its prevention by sunscreens.Eur J Dermatol. 1997; 7: 223-225Google Scholar), sunlight exposure may compromise the ability of prior vaccination to control infectious disease. Recently we reported that exposing mice to solar-simulated UV radiation suppressed immunologic memory and the elicitation of DTH in vivo (Nghiem et al., 2001Nghiem D.X. Kazimi N. Clydesdale G. Ananthaswamy H.N. Kripke M.L. Ullrich S.E. Ultraviolet A radiation suppresses an established immune response: implications for sunscreen design.J Invest Dermatol. 2001; 117: 1193-1199Crossref PubMed Scopus (103) Google Scholar). We found that UVA radiation, essentially devoid of UVB, was equally effective at suppressing the elicitation of DTH as was solar-simulated UV radiation. In addition, we found that sunscreens that absorbed only UVB radiation were ineffective at protecting against immune suppression and immune protection was observed only with sunscreens that absorbed both UVB and UVA radiation.Moyal and Fourtanier, 2001Moyal D.D. Fourtanier A.M. Broad-spectrum sunscreens provide better protection from the suppression of the elicitation phase of delayed-type hypersensitivity response in humans.J Invest Dermatol. 2001; 117: 1186-1192Crossref PubMed Google Scholar came to a similar conclusion in a study using human volunteers and natural sunlight; UVA protection was required for maximal protection against UV-induced suppression of established immune reactions. Unfortunately little is known concerning the underlying immunologic mechanism(s) of UV-induced suppression of an established immune response. The focus of the experiments presented here is to understand the immunologic mechanisms involved. Dr. Stanley Wolf, Genetics Institute (Cambridge, MA), provided us with the recombinant interleukin-12 (IL-12). The hybridoma secreting anti-IL-10 (JES-2A5.11) was kindly provided by Dr. Anne O'Garra, DNAX Research Institute, Palo Alto, CA. The hybridoma cells were grown in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% newborn bovine serum (HyClone Laboratories, Logan, UT). Supernatants were collected, the IgG fraction was enriched by 33% ammonium sulfate precipitation, and the IgG was further purified by passage over protein A/G columns (Pierce Immunochemicals, Rockford, IL). Protein concentration was determined by use of the bicinchoninic acid protein determination kit (Pierce Immunochemicals). Control rat IgG was purchased from Sigma (St. Louis, MO). Liposomes containing the bacteriophage DNA excision repair enzyme T4N5 were kindly provided by Dr. Dan Yarosh, AGI-Dermatics, Freeport, NY. They were prepared and used as described previously (Kripke et al., 1992Kripke M.L. Cox P.A. Alas L.G. Yarosh D.B. Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.Proc Natl Acad Sci USA. 1992; 89: 7516-7520Crossref PubMed Scopus (463) Google Scholar). Keyhole limpet hemocyanin (KLH) was purchased from Pierce Immunochemicals. Specific-pathogen-free female C3H/HeNCr (MTV) mice were obtained from the National Cancer Institute Frederick Cancer Research Facility Animal Production Area (Frederick, MD). The animals were maintained in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International, in accordance with current regulations and standards of the National Institutes of Health. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee. Within each experiment all the mice were age matched. The mice were 8–10 wk old at the start of each experiment. A 1000 W xenon UV solar simulator equipped with a Schott WG-320 atmospheric attenuation filter (1 mm thick), a visible/infrared bandpass blocking filter (Schott UG-11; 1 mm thick), and a dichroic mirror to further reduce visible and infrared energy (Oriel, Stratford, CT) was used to provide solar-simulated UV radiation (UVA + UVB). Replacing the WG-320 filter with a 3 mm thick WG-335 filter resulted in a UVA source deficient in UVB. The WG-320 and WG-335 filters were purchased from Oriel. The intensity and spectral output of the WG-320 and WG-335 equipped solar simulator were measured with an Optronics model OL 754 scanning spectrophotometer interfaced to an Acer model 330 notebook computer (Optronics Laboratories, Orlando, FL). The spectral output of both light sources has been published (Nghiem et al., 2001Nghiem D.X. Kazimi N. Clydesdale G. Ananthaswamy H.N. Kripke M.L. Ullrich S.E. Ultraviolet A radiation suppresses an established immune response: implications for sunscreen design.J Invest Dermatol. 2001; 117: 1193-1199Crossref PubMed Scopus (103) Google Scholar). During irradiation of the shaved dorsal skin, the mice were held individually in a specially constructed Plexiglas container with a quartz glass top, to prevent cage mates from climbing on top of each other and interfering with the UV dose applied. Spectrophotometer readings were taken through the quartz glass top. During the irradiation period (15–90 min in duration) the mice were conscious and had full range of movement. Female C3H/HeN mice were immunized by subcutaneous injection of 107 formalin-fixed Candida albicans into each flank. Nine days later the immunized mice were shaved and exposed to solar-simulated UV radiation as described previously (Ananthaswamy et al., 1999Ananthaswamy H.N. Ullrich S.E. Mascotto R.E. et al.Inhibition of solar simulator-induced p53 mutations and protection against skin cancer development in mice by sunscreens.J Invest Dermatol. 1999; 112: 763-768Crossref PubMed Scopus (43) Google Scholar). The next day each hind footpad was measured with an engineer's micrometer (Mitutoyo, Tokyo, Japan) and then challenged by intrafootpad injection of 50 µl of Candida antigen (Alerchek, Portland, ME). Eighteen to 24 h later the thickness of each foot was re-measured and the mean footpad thickness for each mouse was calculated (left foot + right foot ÷ 2). Generally, there were five mice per group; the mean footpad thickness for the group ± the standard deviation of the mean was calculated. The background footpad swelling (negative control) was determined in a group of mice that were not immunized but were challenged. The specific footpad swelling response was calculated by subtracting the background response observed in the negative controls from the mean footpad swelling found in mice that were immunized and challenged. Each experiment was repeated at least three times. Statistical differences between the controls and experimental groups were determined by use of the two-tailed Student's t test, with a probability of less than 0.05 considered significant (Prism Statistical Software, GraphPad, San Diego, CA). Percentage immune suppression was determined using the following formula: % immune suppression = 1 - (specific footpad swelling of the UV-irradiated mice ÷ specific footpad swelling of the positive control) × 100. Splenic CD4+ T cells were purified by negative selection using antibody cocktails and magnetic microbeads (Stem Cell Technologies, Vancouver, Canada) as described previously (Moodycliffe et al., 2000Moodycliffe A.M. Nghiem D. Clydesdale G. Ullrich S.E. Immune suppression and skin cancer development: regulation by NKT cells.Nature Immunol. 2000; 1: 521-525Crossref PubMed Scopus (284) Google Scholar). They were then stained with rat antimouse pan natural killer cell monoclonal antibody (DX5, IgM, PharMingen, San Diego, CA) followed by mouse antirat IgM (IgG). After staining, the T cells were mixed with antimouse IgG-coated magnetic beads, at a cell to bead ratio of 4:1 (Dynal, Great Neck, NY), and the mixture was enriched for DX5+ cells by passing over a magnetic column. Relative purity of each population was determined by flow cytometry using monoclonal antibodies specific for T cell receptor αβ, CD3, CD4, and CD8. Two methods were used to document the formation of CPD by UVA, immunohistochemistry and radioimmunoassay. The shaved dorsal skin of adult C3H/HeN was exposed to 80 kJ per m2 of UV radiation supplied by the WG-320- or WG-335-filtered xenon solar simulator. Twenty-four hours after UV exposure the mice were killed and epidermal DNA was extracted according to the procedures described byAnanthaswamy et al., 1999Ananthaswamy H.N. Ullrich S.E. Mascotto R.E. et al.Inhibition of solar simulator-induced p53 mutations and protection against skin cancer development in mice by sunscreens.J Invest Dermatol. 1999; 112: 763-768Crossref PubMed Scopus (43) Google Scholar. The numbers of CPD present in the epidermal DNA of control nonirradiated animals, or found in the DNA of mice exposed to UVA + UVB or UVA only, were determined by use of a radioimmunoassay, as described previously (Mitchell and Henderson, 1999Mitchell D.L. Quantification of DNA photoproducts in mammalian cell DNA using radioimmunoassay.in: Henderson D.S. Methods in Molecular Biology, DNA Repair Protocols. The Humana Press, Totowa, NJ1999: 165-175Google Scholar). There were three mice per group. DNA was isolated from each animal and samples from each mouse were run in triplicate. The data are expressed as the mean number of CPD per 1 million bases of DNA ± the standard deviation of the mean. Statistical differences between the numbers of CPD found in the control DNA and the DNA isolated from the UV-irradiated mice were determined by use of Student's t test. UVA-induced CPD were also measured by immunohistochemical analysis. Mice were exposed to 80 kJ per m2 solar-simulated UVA + UVB radiation or 80 kJ per m2 of UVA radiation. Skin sections from the UV-irradiated mice and sections from normal nonirradiated control mice were harvested 24 h after irradiation. They were embedded in Tissue-Tek OCT medium (Miles Laboratories, Elkhart, IN) and snap frozen in liquid nitrogen, and 5 µm thick sections were cut with a cryostat. A mouse monoclonal antibody (H3; IgG1λ) and goat antimouse IgG fluorescein-labeled secondary antibodies were used for CPD immunostaining of the skin sections. The antibody was developed against cyclobutane thymine dimers in single-stranded DNA (Roza et al., 1988Roza L. Van der Wulp K.J.M. MacFarlane S.J. Lohman P.H.M. Baan R.A. Detection of cyclobutane thymine dimers in DNA of human cells with monoclonal antibodies raised against a thymine dimer containing tetranucleotide.Photochem Photobiol. 1988; 48: 627-633Crossref PubMed Scopus (132) Google Scholar) and has high affinity for 5′-T-containing dimers (Fekete et al., 1998Fekete A. Vink A.A. Gáspár S. Bérces A. Módos K. Ronotó G.Y. Roza L. Assessment of the effects of various UV sources on inactivation and photoproduct induction in page T7 dosimenter.Photochem Photobiol. 1998; 68: 527-531Crossref PubMed Scopus (48) Google Scholar). CPD were detected by fluorescent microscopy. From our previous experiments we know that exposure to solar-simulated UV radiation, given 9 d post immunization, suppresses the elicitation of DTH to the fungal antigen C. albicans. Exposing mice to 80 kJ per m2 of solar-simulated UV radiation yielded 50% immune suppression (Nghiem et al., 2001Nghiem D.X. Kazimi N. Clydesdale G. Ananthaswamy H.N. Kripke M.L. Ullrich S.E. Ultraviolet A radiation suppresses an established immune response: implications for sunscreen design.J Invest Dermatol. 2001; 117: 1193-1199Crossref PubMed Scopus (103) Google Scholar). What was not clear was the timing between immunization and UV exposure required for optimal activation of immune suppression. Therefore, we set up an experiment in which groups of mice were immunized with C. albicans on day 0 and then exposed to 80 kJ per m2 solar-simulated UV radiation on subsequent days. All mice were challenged with antigen on day 10 and DTH was measured 18–24 h after challenge. Data from this experiment are found in Table I. In this particular experiment, maximal immune suppression (74%, p <0.01 versus the positive control) was observed when the mice were exposed to UV 9 d after immunization. Significant immune suppression (p 0.05 considered not significant (NS).Negative control1.8 ± 1.5––Positive control18.5 ± 7.616.7––UV 3 d post immunization11.4 ± 2.79.643NSUV 5 d post immunization12.8 ± 3.91134NSUV 7 d post immunization9.1 ± 1.47.3560.05UV 9 d post immunization6.1 ± 1.84.3740.01a Mice were immunized with C. albicans on day 0, exposed to 80 kJ per m2 of UV radiation 3–9 d post immunization, and challenged with antigen on day 10. DTH was measured 18–24 h post challenge. Negative control refers to mice that were not immunized but were challenged; positive control refers to mice that were immunized and challenged.b mm × 10-2 ± SD, N = 5.c Change in footpad swelling of the positive control or experimental groups minus the background swelling found in the negative control.d % immune suppression = 1- (specific footpad swelling of the experimental groups ÷ specific footpad swelling of the positive control) × 100.e p-values determined by two-tailed Student's t test versus the positive control; p >0.05 considered not significant (NS). Open table in a new tab First we tested the hypothesis that UV-induced IL-10 is involved in suppressing established immune reactions. Mice were immunized on day 0 and exposed to UV on day 9. Four hours following UV exposure, 100 µg of monoclonal anti-IL-10 or 100 µg of rat IgG was injected into the peritoneal cavity. The next day the mice were challenged with antigen and the effect anti-IL-10 had on the suppression of DTH was measured (Figure 1). Two different radiation sources were used in this experiment, the WG-320-filtered solar simulator (UVA + UVB) and the WG-335-filtered solar simulator (UVA). Exposing mice to 80 kJ per m2 of solar-simulated radiation (UVA + UVB) suppressed the elicitation of DTH. Injecting the UV-irradiated mice with rat IgG had no effect on the degree of immune suppression. When the mice were injected with monoclonal anti-IL-10, however, the suppressive effect was lost (p >0.05 versus the positive control). Identical results were observed when the mice were exposed to UVA radiation and injected with monoclonal anti-IL-10. As shown previously, UVA suppresses the elicitation of DTH (Nghiem et al., 2001Nghiem D.X. Kazimi N. Clydesdale G. Ananthaswamy H.N. Kripke M.L. Ullrich S.E. Ultraviolet A radiation suppresses an established immune response: implications for sunscreen design.J Invest Dermatol. 2001; 117: 1193-1199Crossref PubMed Scopus (103) Google Scholar). Treating the UVA-irradiated mice with rat IgG failed to reverse the immune suppression, but when the UVA-irradiated mice were injected with neutralizing anti-IL-10, the DTH response generated was not significantly different from the positive control (p >0.05). These data indicate that UV-induced IL-10 plays a role in suppressing the elicitation of DTH by UVA and by solar-simulated UV radiation. Previously, we demonstrated that injecting UV-irradiated mice with recombinant IL-12 reversed the UV-induced suppression of the induction of immunity (Schmitt et al., 1995Schmitt D.A. Owen-Schaub L. Ullrich S.E. Effect of IL-12 on immune suppression and suppressor cell induction by ultraviolet radiation.J Immunol. 1995; 154: 5114-5120PubMed Google Scholar;Schmitt et al., 2000Schmitt D.A. Walterscheid J.P. Ullrich S.E. Reversal of ultraviolet radiation-induced immune suppression by recombinant IL-12: suppression of cytokine production.Immunology. 2000; 101: 90-98Crossref PubMed Scopus (51) Google Scholar). Next we wished to determine if IL-12 would reverse the suppression of the elicitation of DTH by UV radiation. Mice were immunized with C. albicans and then exposed to solar-simulated UVA + UVB radiation. Four hours after UV exposure (80 kJ per m2) recombinant IL-12 or vehicle (phosphate-buffered saline containing 1% fetal bovine serum) was injected into the peritoneal cavity. Significant immune suppression (p <0.001) was observed in mice exposed to UV radiation, or exposed to UV and injected with the vehicle. When the UV-irradiated mice were injected with 1–2 µg of IL-12, doses of IL-12 that totally reversed the UV-induced suppression of the induction of immunity (Schmitt et al., 1995Schmitt D.A. Owen-Schaub L. Ullrich S.E. Effect of IL-12 on immune suppression and suppressor cell induction by ultraviolet radiation.J Immunol. 1995; 154: 5114-5120PubMed Google Scholar), no suppression of the elicitation of DTH was noted. These data (Figure 1,Figure 2) indicate that cytokines are involved in suppressing the elicitation of DTH by solar-simulated UV radiation. The activation of immune regulatory T cells is a prominent feature of the immune suppression induced by UV exposure. We wished to determine whether suppressor T cells can be found in the lymphoid organs of mice first immunized with C. albicans and then exposed to UV radiation. Donor mice were exposed to 80 kJ per m2 of solar-simulated UV (UVA + UVB) radiation 9 d post immunization. In addition, one set of donor mice were injected with 100 µg of isotype-matched control IgG, and another set were injected with 100 µg of anti-IL-10 antibody. As shown above, significant immune suppression was noted in donor mice exposed to UVA + UVB radiation, or exposed to UVA + UVB radiation and injected with control IgG. The spleens of the donor mice were removed, single cell suspensions were prepared, and the cells (108 per mouse) were injected into the tail veins of recipient mice. The recipient mice were then immunized with C. albicans and the DTH reaction was measured 11 d later. The data from this experiment are found in Figure 3(a)). The positive control for this experiment consisted of measuring the immune response in a group of mice that were simply immunized and challenged with antigen. An additional control consisted of injecting 108 spleen cells from mice immunized with antigen but not exposed to UV (NR-SC) into another group of recipient mice. The magnitude of the immune reaction observed in these two groups was identical, indicating that the simple transfer of spleen cells does not adversely affect DTH in the recipients. Significant immune suppression (p <0.001) was observed in mice that received spleen cells from the solar-simulator-irradiated donors, indicating that UV exposure post immunization activates suppressor cells. Injecting UV-irradiated mice with control antibody did not affect the activation of suppressor cells. No immune suppression was noted when spleen cells were isolated from donor mice that were exposed to UV and then injected with anti-IL-10, however. These data indicate that IL-10 plays an essential role in the activation of suppressor cells. Next we asked if UVA radiation could activate suppressor cells. Spleen cells were transferred from three different groups of donor mice. The first set of donors were immunized and challenged but not exposed to UV radiation. The second set of donors were exposed to solar-simulated UV radiation (UVA + UVB), 9 d post immunization. The third set of donor mice were exposed to 80 kJ per m2 of UVA radiation 9 d post immunization. As shown above, irradiating the donor mice with UVA or UVA + UVB significantly suppressed DTH. The spleens of the donor mice were removed, single cell suspensions were prepared, and the cells (108 per mouse) were injected into the tail veins of recipient mice. The recipient mice were then immunized with C. albicans and the DTH reaction was measured 11 d later. The data from this experiment are found in Figure 3b. Significant (p <0.005) immune suppression was observed when spleen cells from donor mice, exposed to solar-simulated UV radiation (UVA + B-SC), were injected into the recipient mice. In addition, significant immune suppression (p <0.005) was observed when spleen cells from donor mice exposed to UVA were injected into recipient mice (UVA-SC). As before no immune suppression was observed when cells from non-UV-irradiated control