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http://immunol.nature.com • december 2000 • volume 1 no 6 • nature immunology

Angus M. Moodycliffe1*, Dat Nghiem1,2, Gavin Clydesdale1 and Stephen E. Ullrich1,2

Ultraviolet (UV) radiation is carcinogenic and immunosuppressive. UV-induced immune suppression ismediated by antigen-specific T cells, which can transfer suppression to normal recipients.These cellsare essential for controlling skin cancer development in the UV-irradiated host and in suppressingother immune responses, such as delayed-type hypersensitivity. Despite their importance in skincancer development, their exact identity has remained elusive.We show here that natural killer T cellsfrom UV-irradiated donor mice function as suppressor T cells and play a critical role in regulating thegrowth of UV-induced skin cancers and suppressing adaptive immune responses in vivo.

1The Department of Immunology,The University of Texas, M. D.Anderson Cancer Center and 2The Graduate School of Biomedical Sciences, 1515 Holcombe Blvd., Houston,TX 77030-4009, USA. *Present address:The Department of Nutrition, Nestlé Research Center, PO Box 44, 1000 Lausanne,Vers-Chez-Blanc, Switzerland.

Correspondence should be addressed to S. E. U. ([email protected]).

Immune suppression and skin cancerdevelopment: regulation by NKT cells

The American Cancer Society estimates approximately 1,300,000 newcases of skin cancer will be diagnosed in the year 2000, making it themost prevalent type of cancer reported in the United States (www.can-cer.org/statistics). In addition to being the primary cause of non-melanoma skin cancer1,2, the ultraviolet (UV) radiation present in sun-light is immunosuppressive. Three major lines of evidence support thishypothesis. First, epidemiological studies report that there is a substan-tially increased incidence in skin cancer of the sun-exposed body partsof immune-suppressed transplant patients as compared to those of thegeneral population. This indicates a link between immunosuppressionand skin cancer induction3. Second, studies with biopsy-proven skincancer patients have indicated that UV-induced immune suppression isa major risk factor for skin cancer induction4. Finally, exposing mice tosubcarcinogenic doses of UV radiation suppresses their immuneresponse and permits the outgrowth of highly antigenic UV-inducedskin tumors. The development of UV-induced skin cancers in the pri-mary UV-irradiated host and the ability to transfer immune suppressionfrom UV-irradiated animals to normal recipients is controlled by CD4+

T cells5,6. Despite having been identified 23 years ago7,8, the exact iden-tity of the cells that transfer suppression of tumor immunity remainsunknown. The immunosuppressive mechanism is also unclear, butinterleukin 4 (IL-4) secretion is involved9.

Two types of CD4+ T cells secrete IL-4: type 2 T helper cells (TH2)and natural killer T (NKT) cells. NKT cells, unlike conventional Tcells, express intermediate amounts of T cell receptor (TCR) molecules(TCRαβint) on their surface and coexpress surface antigens normallyfound on natural killer cells (NK1.1, DX5 and Ly49a)10,11. They alsosecrete high concentrations of immunomodulatory cytokines, especial-ly IL-4, within hours of primary CD3 stimulation in vivo12 and in vitro13.Unlike conventional T cells, naïve NKT cells exhibit an activated phe-notype (CD44highCD62Llow) and are present at very low frequencies inperipheral lymphoid organs such as the spleen where they account for2–3% of the total T cell population10,11. They are positively selected andrestricted by CD114,15 and exhibit a very restricted TCR (Vα14-Jα281,Vβ2, Vβ7 or Vβ8) usage10,11.

Because NKT cells can act as negative immunoregulatory cells16–18,in part through their rapid and substantial release of IL-4, we tested the

hypothesis that NKT cells mediate UV-induced immune suppression.Our findings demonstrate a critical role for NKT cells in mediating UV-induced immune suppression. We show that UV-induced suppressor Tcells are NKT cells that can suppress delayed type hypersensitivity(DTH) and tumor immunity.

ResultsIsolation of splenic NKT cellsSplenic CD4+ T cells enriched on the basis of DX5 expression areTCRαβint and rapidly secrete high concentrations of IL-4 upon primaryCD3 stimulation in vitro, which distinguishes them from conventionalCD4+ T cells13. We report here that CD3+ or CD4+ T cells isolated fromUV-irradiated mice, and enriched on the basis of DX5 expression19, arealso TCRαβint (Fig. 1a). In addition, they secrete higher quantities ofIL-4 compared to conventional T cells (UV CD3+DX5– versus UVCD3+DX5+; UV CD4+DX5– versus UV CD4+DX5+) (Fig. 1b).However, compared to normal age-matched splenic CD3+ NKT cells(NR CD3+DX5+), CD3+ NKT cells from chronically UV-irradiatedmice (UV CD3+DX5+) produced significantly more IL-4 upon primaryCD3-stimulation in vitro (P<0.01), which implied that chronic UVexposure promotes their activation in vivo.

Antigen-specific suppression of DTHTo test the hypothesis that NKT cells mediate UV-induced immunesuppression, we determined whether these cells could transfer UV-induced suppression of DTH. Mice were exposed to a single dose ofUV radiation and then immunized with Candida albicans or sheep redblood cells (SRBCs). After confirming that UV irradiation completelysuppressed the induction of DTH in the donor mice (data not shown),spleens from the donors were removed and the NKT cells (CD3+DX5+)were separated from the conventional T cells (CD3+DX5–) by stainingwith monoclonal antibody to DX5 followed by magnetic bead enrich-ment13. These cells were then transferred into recipient mice that wereimmunized with the same antigen as the donors (Fig. 2a) and DTH inthe recipients was measured. Although injecting 2×107 purified T cellsfrom the UV-irradiated donors (UV-T) significantly suppressed DTH inthe recipient mice, all suppressive activity was lost when 2×107

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UV-NKT cell–depleted (CD3+DX5–) lympho-cytes were injected into the recipient mice.Conversely, when the DX5+ cells were recoveredfrom the magnetic beads and 2×106 NKT cellswere injected into mice, the suppression observedwas equal to that found when 2×107 T cells wereinjected (Fig. 2a). These findings show that NKTcells transfer UV-induced immune suppression.Because NKT cells represent only 2–3% of thetotal splenic T cell population, these data implythat UV-activated NKT cells function in vivo aspowerful negative immune regulators.

One of the hallmarks of UV-induced immunesuppression of DTH, and the suppressor cellsinvolved, is antigen specificity. To determinewhether UV-induced NKT cells transfer immunesuppression in an antigen-specific manner, NKTcells from UV-irradiated C. albicans –immu-nized mice were injected into two sets of recipi-ents. One set was immunized with C. albicans and the second set wasimmunized with SRBCs (Fig. 2b). Immune suppression was notedwhen whole spleen cells from UV-irradiated C. albicans–immunizedmice (UV-SCs) were transferred into recipients immunized with C. albicans. Similarly, injecting NKT cells (UV CD4+DX5+), but notconventional T cells (UV CD4+DX5–) from UV-irradiated C. albi-cans–immunized donors, into C. albicans–immunized recipient micesuppressed DTH. No immune suppression was observed, however,when whole spleen cells or NKT cells from UV-irradiated C. albi-cans–immunized donors were injected into SRBC-immunized recipi-ents. In a reciprocal experiment, the donor mice were exposed to UVradiation and immunized with SRBCs. Transferring UV-SCs or NKT

cells (UV CD4+DX5+) from UV-irradiated donor mice that wereimmunized with SRBCs into SRBC-immunized recipient mice signif-icantly suppressed the induction of DTH (Fig. 2c). No immune sup-pression was found when UV NKT cells from donors immunized withSRBCs were transferred into recipients immunized with C. albicans.These findings demonstrate that UV-induced NKT cells suppress DTHin an antigen-specific fashion.

UV-induced NKT cells are CD1-restrictedNKT cells are CD1-restricted10,15, both the number and function of NKTcells are severely reduced in CD1-deficient mice14. If CD1-restrictedNKT cells mediate UV-induced immune suppression then exposing

CD1-deficient mice to UVradiation should have littleor no effect on DTH.Despite the generation of avigorous DTH reaction inboth the wild-type (WT)and CD1-deficient mice,which confirmed thatCD1-deficient mice have anormal complement ofconventional T cells14, onlythe DTH response of theWT mice was suppressedby UV exposure (Fig. 3a).

To determine whetherUV-induced suppressor Tcells were CD1-restricted,spleen cells from UV-irra-diated WT mice wereinjected into WT andinbred CD1-deficientrecipients20. The mice wereimmunized with C. albi-cans and the effect that celltransfer had on DTH wasmeasured (Fig. 3b). Asexpected, no immune sup-pression was observedwhen spleen cells from

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Figure 2. NKT cells mediate UV-induced immune sup-pression. (a) 20×106 purified T cells (UV-T), 20×106 conven-tional CD3+ T cells devoid of NKT cells (UV CD3+DX5–) or2×106 NKT cells (UV CD3+DX5+) were isolated from UV-irra-diated mice. Cells from UV-irradiated C. albicans–immunizeddonors were injected into C. albicans–immunized recipients(left) and cells from UV-irradiated SRBC-immunized donorsmice were injected into SRBC-immunized recipients (right). (b)Antigen-specific transfer of UV-induced immune suppression byNKT cells. 50×106 whole spleen cells (UV-SC), 1×106 conven-tional CD4+ T cells devoid of NKT cells (UV CD4+DX5–) or1×106 NKT cells (UV CD4+DX5+) were isolated from UV-irra-

diated donors immunized with C. albicans and injected into recipient mice that were immunized with C. albicans (left) or SRBCs (right).Theeffect that each T cell population had on the induction of DTH in the recipient mice was measured. (c) T cells were isolated from thespleens of mice exposed to UV and immunized with SRBCs. 50×106 whole spleen cells (UV-SC), 1×106 conventional CD4+ T cells that weredevoid of NKT cells (UV CD4+DX5–) or 1×106 NKT cells (UV CD4+DX5+) were injected into recipient mice that were immunized withSRBCs (left) or C. albicans (right).The effect that each T cell population had on the induction of DTH in the recipient mice was measured.Negative control mice were not immunized but were challenged. Positive controls did not receive cell transfer but were immunized andchallenged.As a control for cell transfer, recipient mice were injected with 50×106 whole spleen cells from nonirradiated C. albicans– orSRBC-immunized donor mice (NR-SC). (Results are expressed as mean±s.d.; n=5; ND, not determined; *, P<0.001 compared to the posi-tive control using Student’s t-test. Each experiment was repeated three times.)

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c

Figure 1.TCR expression and IL-4 production byT cells isolated from the spleens of UV-irradiat-ed mice. (a) Conventional CD3+DX5– T cells andCD3+DX5+ NKT cells were isolated from the spleens of UV-irradiated and normal age-matched controls andstained with fluorescein isothiocyante–anti-TCRαβ. (b) 2×105 conventional T cells (CD3+DX5– or CD4+DX5–)or NKT cells (CD3+DX5+ or CD4+DX5+) isolated from chronically UV-irradiated mice and age-matched con-trols were stimulated in vitro with plate-bound anti-CD3 (30 µg/ml). IL-4 secretion (mean±s.d.) was deter-mined 8 h later by ELISA. (Statistical differences between the groups were determined by Student’s t-test.Thisexperiment was repeated three times. UV, UV-irradiated; NR, nonirradiated.)

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nonirradiated mice (NR-SCs) or conventional T cells from UV-irradiat-ed mice (CD4+DX5–) were injected into the WT animals. Immune sup-pression was observed when WT UV-SCs were transferred into WTmice. However, when WT UV-SCs or WT UV CD4+DX5+ T cells wereinjected into CD1-deficient animals they failed to transfer immune sup-pression, which indicated that the UV-induced suppressor cells areCD1-restricted. These data support the hypothesis that NKT cells medi-ate UV-induced immune suppression. In addition, these data suggestthat distinct populations of T cells induce and suppress DTH in UV-irradiated animals.

Suppression of skin cancer rejection by NKT cellsChronic UV exposure induces CD3+CD4+ T cells that permit thegrowth of UV-induced skin cancers in the primary host and suppressthe rejection of highly antigenic UV-induced skin tumors when trans-ferred to normal mice5,6. To test the hypothesis that NKT cells areresponsible for suppressing tumor immunity, 1×106 CD4+DX5+ NKTcells were isolated from the spleens of mice chronically exposed to UVradiation, mixed with normal spleen cells and used to reconstitute theimmune systems of lethally (850 rad) X-irradiated recipients6. Thereconstituted mice were then challenged with an UV-induced regressortumor, UV-2240 (Fig. 4a). The regressive nature of UV-2240 is illus-trated by the fact that this tumor was rejected in mice reconstituted onlywith normal spleen cells but grew progressively in immunocompro-mised RAG-2–/– mice.

The suppressive activity of UV spleen cells was demonstrated byprogressive growth of UV-2240 in mice reconstituted with a mixtureof normal spleen cells (NR-SCs) and UV spleen cells (P<0.0001, UV-SCs versus NR-SCs, Fisher’s exact test). Similarly, tumor rejectionwas suppressed in mice injected with a mixture of NR-SCs and UVCD4+DX5+ T cells (P<0.0007 versus NR-SCs), which demonstratedthat NKT cells isolated from the spleens of chronically UV-irradiatedmice are powerful suppressor cells. No suppressive activity was noted,however, when the same number of conventional CD4+ T cells, devoidof NKT cells, were injected into the recipient mice (P<0.001, UVCD4+DX5– versus UV CD4+DX5+). Neither did the transfer of normalCD4+DX5+ T cells isolated from age-matched control mice suppresstumor immunity (P<0.0004, NR CD4+DX5+ versus UV CD4+DX5+),which confirmed that a chronic course of UV exposure induces NKTcells to acquire suppressive activity. We repeated these experimentswith another UV-induced regressor tumor (UV 5497) and obtainedidentical results (data not shown).

UV-induced suppressor T cells are specific for UV-induced skin can-cers as a group and fail to inhibit the rejection of chemically inducedregressor tumors5,6. To determine whether NKT cells from chronicallyUV-irradiated mice recognize non-UV–induced regressor tumors, thesame cells were used to reconstitute a different set of X-irradiatedrecipient mice. These mice were challenged with a methylcholan-threne-induced regressor tumor, MCA-113 (Fig. 4b). Progressivetumor growth was only found in the RAG-2–/– mice (P<0.0001 versustumor growth in mice reconstituted with normal spleen cells).Reconstitution with a mixture of whole normal spleen cells and UV-SCs or UV CD4+DX5+ T cells failed to suppress the rejection of theMCA-induced regressor tumor. These data indicate that UV-inducedNKT cells recognize UV-induced regressor tumors as a class, presum-ably by recognizing the “UV-common antigen”5,6 and suppress tumorrejection in an antigen-specific manner.

DiscussionIt has been suggested that NKT cells should be categorized as a thirdclass of T cells. In addition to CD4 “helper” and CD8 “killer” T cells,

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Figure 3. UV-induced NKT cells are CD1-restricted. (a) WT mice and CD1-deficient mice were exposed to UV and immunized with C. albicans. DTH was measured9 days later. (*, P<0.001 versus the positive control using Student’s t-test.) (b) 50×106 whole spleen cells (UV-SC), 1×106 conventional CD4+ T cells devoid of NKT cells (UVCD4+DX5–) or 1×106 NKT cells (UV CD4+DX5+) isolated from UV-irradiated WT mice were injected into WT or CD1-deficient recipient animals.The effect that each Tcell population had on the induction of DTH in the recipient mice was measured. Negative-control mice were not immunized but were challenged. Positive-control mice didnot receive cell transfer but were immunized and challenged.As a control for cell transfer, recipient mice were injected with 50×106 whole spleen cells from nonirradiateddonor mice (NR-SC). (Results are expressed as the mean±s.d.; n=5; ND, not determined; *, P<0.001 compared to the positive control using Student’s t-test.)

a b

Figure 4. CD4+ DX5+ T cells transfer antigen-specific suppression of tumorimmunity. Two groups of lethally X-irradiated C3H mice were reconstituted with50×106 normal spleen cells (�) or a mixture of normal spleen cells + 50×106 wholeUV spleen cells (�), 106 UV CD4+DX5+ NKT cells (�), 106 UV CD4+DX5– conven-tional T cells (�) or 106 normal CD4+DX5+ NKT cells (�). After 24 h, mice wereinjected with (a) 5×105 UV-2240 tumor cells or (b) 5×105 MCA-113 cells.As a pos-itive control for tumor growth both regressor tumors were injected into RAG-2–/–

mice (�). (Tumor incidence was scored on a weekly basis. Each experiment wasrepeated twice.)

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NKT cells may represent the regulatory arm of the T cell response10. Thedata we have reported here support this view. What is different about ourfindings, however, is the antigen-specificity of the immune suppressiontransferred by NKT cells. Because of their germ line–encoded specifici-ty for self, NKT cells are viewed as a bridge between the innate andadaptive immune systems, which generally act in an antigen-nonspecif-ic manner to rapidly provide help to effector cells during the early stagesof the immune response11. In contrast, our findings provide direct evi-dence for antigen-specific regulation of adaptive immunity (tumorimmunity and DTH) by UV-induced NKT cells.

The data reported here show that there was no immune suppressionin UV-irradiated CD1-deficient mice, which supports a critical role forCD1-restricted NKT cells in mediating UV-induced immunosuppres-sion. At first glance, CD1 restriction of NKT cells and antigen-specificimmune suppression may appear to be inconsistent because CD1 pref-erentially binds unconventional antigens such as glycolipids, whosehydrophobicity and lack of a defined shape suggest they are poor tar-gets for specific T cell recognition21. Molecular mapping studies, how-ever, suggest a recognition model in which the alkyl chains of glycol-ipids fit into the hydrophobic cleft of CD1, which positions the glyco-sylated hydrophilic cap of the molecule to interact with the TCR. Thespecificity of T cell recognition is determined by the carbohydratestructure of the hydrophilic cap. Modification or removal of the carbo-hydrate residues diminishes or destroys specific T cell recognition22.This may explain the ability of UV-induced NKT cells to transfer anti-gen-specific immune suppression. Alternatively, CD1 will bind pep-tides containing a hydrophobic motif and CD1-restricted T cells havebeen raised to these antigens23,24. It is possible that the antigen-specificimmune suppression described here is NKT cell recognition of ahydrophobic peptide bound to CD1. Regardless, both models providean explanation for the antigen-specific recognition reported here.

Although our data show that UV-induced NKT cells mediate anti-gen-specific immune suppression, at the present time we cannot defin-itively identify the ultimate suppressor cell. It is possible that UV-induced IL-4–secreting NKT cells directly suppress effector cell func-tion. Alternatively, UV-induced NKT cells may initiate a cascade ofevents in which recipient T cells are activated to secrete IL-4 and sup-press DTH and tumor immunity20. In addition, although we inject ahighly purified population of NKT cells (>96% of the DX5+ cells areCD4+) into recipient mice, activated CD8+ T cells can up-regulate sur-face expression of NK cell–associated antigens25. It is possible that theimmune suppression seen here results from the transfer of a small pop-ulation of conventional antigen-specific DX5+ T cells. However, twoobservations suggest that this possibility is unlikely. First, we havenever been able to transfer UV-induced immune suppression with CD8+

T cells6,26,27. Second, failure of WT UV-SCs or UV CD4+DX5+ T cellsto suppress DTH in CD1-deficient mice indicates that conventionalDX5+ T cells do not transfer UV-induced immune suppression.Regardless of the identity of the ultimate suppressor cell, our dataclearly show that UV-induced NKT cells are involved, directly or indi-rectly, in mediating antigen-specific immune suppression.

The data presented here provide new insights into immune regulationof sunlight-induced skin carcinogenesis. Although it has been known formore than 20 years that CD4+ T cells suppress immunity in the primaryUV-irradiated host and are responsible for the transfer of immune sup-pression5,6, their exact identity has, until now, remained unknown. Ourfindings identify a critical role for NKT cells in the mediation of UV-induced immune suppression and in the regulation of sunlight-inducedskin carcinogenesis. Our data also imply a wider role for NKT cells inregulating the immune response. The observation that NKT cells

transfer antigen-specific immune suppression challenges the widelyaccepted notion that NKT cells are primarily members of the innateimmune system. These data argue that NKT cells represent a powerfulimmunoregulatory arm of the adaptive immune response. This insightmay lead to the design of strategies to modulate the development of skincancer and perhaps of other diseases that are controlled by NKT cells.

MethodsAnimals. Specific pathogen-free female C3H/HeNCr (MTV–), and RAG-2–/– mice werefrom the National Cancer Institute Frederick Cancer Research Facility Animal ProductionArea (Frederick, MD). Inbred CD1–/– and WT mice, backcrossed onto a C57BL/6 back-ground were obtained frm L. Van Kaer, Vanderbilt University Medical Center (Nashville,TN). Genetic identity was confirmed by skin grafting experiments. The animals weremaintained in facilities approved by the Association for Assessment and Accreditation ofLaboratory Animal Care International in accordance with current regulations and stan-dards of the US Department of Agriculture, the Department of Health and HumanServices and the National Institutes of Health. All animal procedures were reviewed andapproved by the Institutional Animal Care and Use Committee. Within each experimentall the mice were matched for age and sex. The mice were 8–10-weeks-old at the start ofeach experiment.

Isolation of NKT cells. The shaved dorsal skins of adult female C3H/HeNCr (MTV–) micewere exposed to 10 or 15 kJ/m2 of UV radiation supplied by a bank of 6 FS-40 sunlamps(National Biological, Twinsberg, OH). Splenic CD3+ and CD4+ T cells were purified bynegative selection using antibody cocktails and magnetic microbeads (Stem CellTechnologies, Vancouver). They were then stained with rat monoclonal antibodies to mousepan NK cells (DX5, IgM, PharMingen, San Diego, CA) followed by mouse anti-rat IgM(IgG). After staining, the T cells were mixed with magnetic beads coated with anti–mouseIgG, at a cell:bead ratio of 4:1 (Dynal, Great Neck, NY) and the solution was enriched forDX5+ cells by passing over a magnetic column. The relative purity of each population wasdetermined by flow cytometry using monoclonal antibodies specific for TCRαβ, CD8, CD3and CD4. Typically the CD3+DX5– T cell population was ≥95% TCRαβ+; 65% were CD4+

and 30% were CD8+. The CD4+DX5+ T cells were ≥95% positive when stained for TCRαβand ≥95% positive when stained for CD4 and did not stain with anti-CD8. Compared toconventional splenic T cells, intermediate TCRαβ expression was exhibited by theCD3+DX5+ and CD4+DX5+ NKT cells13.

Suppression of DTH. Five days after adult female C3H/HeNCr (MTV–) mice were exposedto 10 or 15 kJ/m2 of UV radiation, they were immunized with either C. albicans (107 for-malin-fixed organisms injected into each flank) or SRBCs (2.5×107 erythrocytes into eachflank), respectively. Six days later the SRBC-immunized mice were challenged by injecting2.5×107 erythrocytes into each hind footpad. Mice immunized with C. albicans were chal-lenged 9 days after immunization by injecting 50 µl of Candida antigen (Alerchek Inc.Portland, ME) into the footpads. Footpad swelling was measured 18–24 h after challengeand compared to the value recorded immediately before challenge. There were at least fivemice per group, statistical differences between experimental and control groups were deter-mined by Student’s t-test and P<0.05% was considered to be significant. Suppressor T cells(CD3+DX5+ or CD4+DX5+) were isolated from the spleens of these mice by antibody andmagnetic bead enrichment and injected into syngeneic recipient mice (intravenous injectionvia the tail vein). The induction of DTH in the recipient mice was measured in a blindedmanner as described27.

Suppression of tumor immunity. The spleens of UV-irradiated, tumor-susceptible mice(10 kJ/m2 per UV exposure, three treatments per week for 12–16 weeks) or age-matchedcontrols were removed and CD4+DX5+ NKT cells isolated as described. One million NKTor conventional T cells from UV-irradiated mice were mixed with 5×107 NR-SCs and inject-ed into the tail veins of lethally X-irradiated (850 rads) syngeneic recipients. After 24 h, onegroup of mice was injected with a UV-induced regressor tumor (5×105 UV-2240, subcuta-neous injection) and a second group injected with a chemically induced regressor tumor(5×105 MCA-113). The tumor dose used was determined in preliminary experiments inwhich different numbers of MCA 113 and/or UV 2240 cells were injected into normal andRAG-2–/– mice. The regressor nature of the tumors was confirmed by 100% growth of 5×105

cells in RAG-2–/– mice and complete rejection when an equal number of cells was injectedinto immune competent animals. Tumor size and incidence were monitored weekly.Generally there were eight to ten mice per group Statistical differences between experi-mental groups and controls were determined by the Fisher exact test and P<0.05 was con-sidered to be significant.

AcknowledgmentsWe thank H.Ananthaswamy and L. Owen-Schaub for critically reviewing this manuscriptand L.Van Kaer for providing inbred CD1-deficient and WT mice. Supported by grantsfrom the NIH (CA75575 and ES07327).The animal facilities at UT MDACC are supportedin part by a NCI Core Grant (CA16672).

Received 8 August 2000; accepted 25 October 2000.

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