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Contact Hypersensitivity in MHC Class II-Deficient Mice Depends on CD8 T Lymphocytes Primed by Immunostimulating Langerhans Cells

1998; Elsevier BV; Volume: 111; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.1998.00236.x

1523-1747

Anne Bouloc, Andrea Cavani, Stephen I. Katz,

T-cell and B-cell Immunology

Studies to determine if CD4+ and/or CD8+ T cells are critical for the initiation and propagation of contact hypersensitivity (CHS) reactions have yielded conflicting results regarding their roles. We studied the induction and expression of CHS to trinitrochlorobenzene (TNCB) using major histocompatibility complex class II-deficient mice that display normal numbers of CD8+ T cells but lack CD4+ T cells. CHS to TNCB, detected as an increase in ear thickness 24 h after epicutaneous challenge, was significantly enhanced in major histocompatibility complex class II-deficient mice compared with wild-type controls. Ear swelling responses in major histocompatibility complex class II-deficient mice were decreased by treatment with anti-CD8 antibody or by injection of wild-type CD4+ T cells. To further characterize mechanisms involved in the initiation of CHS responses, phenotypical and functional characteristics of both freshly isolated and cultured Langerhans cells were studied. Like Langerhans cells from wild-type controls, Langerhans cells from major histocompatibility complex class II-deficient mice upregulated B7–1 and B7–2 costimulatory molecules and enhanced major histocompatibility complex class I expression upon short-term culture. Cultured Langerhans cells induced a 3.5-fold increase in the stimulation of autologous hapten-specific CD8+ T cell proliferation compared with fresh Langerhans cells. Finally, TNP-coupled Langerhans cells from major histocompatibility complex class II-deficient mice primed naïve mice to TNCB after transfer. These results demonstrate that hapten-specific CD8+ T cells are sufficient for the expression of CHS and that CD8 priming does not require the presence of CD4+ T cells or major histocompatibility complex class II antigen. Studies to determine if CD4+ and/or CD8+ T cells are critical for the initiation and propagation of contact hypersensitivity (CHS) reactions have yielded conflicting results regarding their roles. We studied the induction and expression of CHS to trinitrochlorobenzene (TNCB) using major histocompatibility complex class II-deficient mice that display normal numbers of CD8+ T cells but lack CD4+ T cells. CHS to TNCB, detected as an increase in ear thickness 24 h after epicutaneous challenge, was significantly enhanced in major histocompatibility complex class II-deficient mice compared with wild-type controls. Ear swelling responses in major histocompatibility complex class II-deficient mice were decreased by treatment with anti-CD8 antibody or by injection of wild-type CD4+ T cells. To further characterize mechanisms involved in the initiation of CHS responses, phenotypical and functional characteristics of both freshly isolated and cultured Langerhans cells were studied. Like Langerhans cells from wild-type controls, Langerhans cells from major histocompatibility complex class II-deficient mice upregulated B7–1 and B7–2 costimulatory molecules and enhanced major histocompatibility complex class I expression upon short-term culture. Cultured Langerhans cells induced a 3.5-fold increase in the stimulation of autologous hapten-specific CD8+ T cell proliferation compared with fresh Langerhans cells. Finally, TNP-coupled Langerhans cells from major histocompatibility complex class II-deficient mice primed naïve mice to TNCB after transfer. These results demonstrate that hapten-specific CD8+ T cells are sufficient for the expression of CHS and that CD8 priming does not require the presence of CD4+ T cells or major histocompatibility complex class II antigen. contact hypersensitivity Contact hypersensitivity (CHS) describes the responses elicited by epicutaneous sensitization and challenge with chemically reactive haptens, small chemically defined molecules capable of binding directly to soluble and cell associated proteins (Polak, 1980Polak L. Immunological Aspects of Contact Sensitivity. Karger, Basel1980: 6-16Google Scholar). Since Landsteiner and Jacobs, 1935Landsteiner K. Jacobs E. Studies on the sensitization of animals with simple chemicals compounds.J Exp Med. 1935; 61: 643-652Crossref PubMed Scopus (343) Google Scholar first reported the phenomenon of CHS in guinea pigs, many investigators have elucidated various mechanisms involved in the generation and expression of CHS to various haptens (Knight et al., 1985Knight S.C. Krejci J. Malkovxky M. Colizzi V. Gautam A. Asherson G.L. The role of dendritic cells in the initiation of immune responses to contact sensitizers.Cell Immunol. 1985; 94: 427-434Crossref PubMed Scopus (105) Google Scholar;Macatonia et al., 1987Macatonia S.E. Knight S.C. Edwards A.J. Griffiths S. Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate: functional and morphological studies.J Exp Med. 1987; 166: 1654-1667Crossref PubMed Scopus (520) Google Scholar;Kripke et al., 1990Kripke M.L. Munn C.G. Jeevan A. Tang J.M. Bucana C. Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization.J Immunol. 1990; 145: 2833-2838PubMed Google Scholar;Enk and Katz, 1992Enk A.H. Katz S.I. Early molecular events in the induction phase of contact sensitivity.Proc Natl Acad Sci USA. 1992; 89: 1398-1402Crossref PubMed Scopus (594) Google Scholar,Enk and Katz, 1995Enk A.H. Katz S.I. Contact sensitivity as a model for T-cell activation in skin.J Invest Dermatol. 1995; 105: 80S-83SAbstract Full Text PDF PubMed Scopus (76) Google Scholar). According to the current paradigm, during the afferent or sensitization phase, hapten-specific T cells are primed by Langerhans cells and/or dermal dendritic cells that migrate from the area of antigen exposure to the skin-draining lymph nodes and present hapten-major histocompatibility complexes (MHC) to naïve T cells. Subsequently, the reapplication of the same hapten to skin results in the influx of hapten-specific T cells that react to the hapten locally, release cytokines, and attract other inflammatory cells. The resultant swelling can be measured and reflects the ongoing immune response. CHS is a T cell-mediated immune response; studies to define the cellular elements that are responsible have yielded conflicting results. Both CD4+ T cells and CD8+ T cells can recognize haptenated peptides (Martin et al., 1993Martin S. von Bonin A. Fessler C. Pflugfelder U. Weltzien H.U. Structural complexity of antigenic determinants for class I MHC-restricted, hapten-specific T cells. Two qualitatively differing types of H-2Kb-restricted TNP epitopes.J Immunol. 1993; 151: 678-687PubMed Google Scholar;Cavani et al., 1995Cavani A. Hackett C.J. Wilson K.J. Rothbard J.B. Katz S.I. Characterization of epitopes recognized by hapten-specific CD4+ T cells.J Immunol. 1995; 154: 1232-1238PubMed Google Scholar). In vivo and in vitro cell depletion studies from several laboratories have indicated the importance of CD4+ T cells as the effector cells (Miller and Jenkins, 1985Miller S.D. Jenkins M.K. In vivo effects of GK1.5 (anti-L3T4a) monoclonal antibody on induction and expression of delayed type hypersensitivity.Cell Immunol. 1985; 92: 414-426Crossref PubMed Scopus (57) Google Scholar;Gautam et al., 1991Gautam S.C. Matriano J.A. Chikkala N.F. Edinger M.G. Tubbs R. L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants.Cell Immunol. 1991; 135: 27-41Crossref PubMed Scopus (46) Google Scholar), and others have demonstrated the ability of CD8+ T cells to mediate CHS (Gocinski and Tigelaar, 1990Gocinski B. Tigelaar R. Roles of CD4+ and CD8+ T cells in murine contact sensitivity revealed by in vivo monoclonal antibody depletion.J Immunol. 1990; 144: 4121-4128PubMed Google Scholar;Bour et al., 1995Bour H. Peyron E. Gaucherand M. et al.Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively mediate and regulate contact sensitivity to dinitrofluorobenzene.Eur J Immunol. 1995; 25: 3006-3010Crossref PubMed Scopus (280) Google Scholar;Xu et al., 1996Xu H. DiIulio N.A. Fairchild R.L. T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon γ-producing (Tc1) effector CD8+ Tcells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells.J Exp Med. 1996; 183: 1001-1012Crossref PubMed Scopus (373) Google Scholar). In this study, we used MHC class II-deficient mice that were generated by disruption of the MHC class II Ab β gene in embryonic stem cells. The mice lack cell surface expression of class II molecules and are depleted of mature CD4+ T cells (Grusby et al., 1991Grusby M.J. Johnson R.S. Papaioannou V.E. Glimcher L.H. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice.Science. 1991; 253: 1417-1420Crossref PubMed Scopus (613) Google Scholar). CHS to TNCB was significantly enhanced in these mice compared with wild-type controls. Our data indicate that Langerhans cells from MHC class II-deficient mice undergo functional activation upon short-term culture, stimulate hapten-specific CD8+ T cells in vitro, and can prime T cells in vivo. MHC class II-deficient mice that have a disruption of the Ab β gene and wild-type controls (C57BL/6) were obtained from the Taconic Laboratory (Germantown, NY). Mice were used at ages ranging from 8 to 12 wk. All animals were used in accordance with institutional guidelines. Cell culture and cell proliferation assays were performed in RPMI 1640 supplemented with 10% fetal calf serum (Biofluids, Rockville, MD), 2 mM glutamine, 1% penicillin/streptomycin/fungizone, 10 mM HEPES buffer, 1% nonessential amino acids, 1 mM sodium pyruvate (all from Life Technologies, Chagrin Falls, OH), and 5 × 10–5 M β2-mercapto-ethanol (Sigma, St. Louis, MO). The following monoclonal antibodies were produced by hybridomas obtained from the American Type Culture Collection (ATCC, Rockville, MD) GK1.5 (anti-CD4), 53–6.72 (anti-CD8). Anti-CD45, B7–1, B7–2, and MHC class I (H-2Kb/H-2Db) antibodies were purchased from Pharmingen (San Diego, CA). TNCB was obtained from Polysciences (Warrington, PA), TNBS and fluoroscein isothiocyanate (FITC) were obtained from Sigma. Epidermal cell suspensions were obtained from the ears of nontreated mice as previously described (Tamaki et al., 1979Tamaki K. Stingl G. Gullino M. Sachs D.H. Katz S.I. Ia Ag in mouse skin are predominantly expressed on Langerhans cells.J Immunol. 1979; 123: 784-787PubMed Google Scholar). Epidermal cell suspensions were applied to Lympholyte M density gradients (Cedarlane, Ontario, Canada) and centrifuged at 800 ×g for 10 min at room temperature. Interface cells, containing 5–10% Langerhans cells referred to as fresh Langerhans cells (fresh epidermal cells), were washed extensively in complete medium and used as antigen-presenting cells in the proliferation assays. Epidermal cells were suspended at 2 × 106 cells per ml in complete medium and cultured for 72 h in 25 ml T flasks. Non-adherent cells were collected by vigorous pipetting and enriched in Langerhans cells by Lympholyte M gradient centrifugation as described. Such suspensions contained 20–30% Langerhans cells and are referred to as cultured epidermal cells. TNP-coupled fresh epidermal cells and TNP-coupled cultured epidermal cells were obtained by incubating cell suspensions with 1 mM TNBS for 10 min at 37°C, and were washed extensively in complete medium. FITC-coupled fresh epidermal cells were obtained by incubating cell suspensions with 1 mM FITC for 10 min at 37°C, and were washed extensively in complete medium. Mice were sensitized with 100 μl of 3% TNCB solution (4:1, acetone:olive oil) (both from Sigma) painted onto shaved abdominal skin. Mice were challenged on day 6 with 20 μl 1% TNCB solution on the right ear and ear swelling was quantitated with a spring-loaded micrometer (Mitutuyo, Japan) 24 h after challenge. Mice that were ear-challenged without prior sensitization served as negative controls. The results are presented for each group (four mice) as the average difference (±SD) in ear thickness before and after the challenge. In some experiments, CD4+ T cells or CD8+ T cells were depleted in vivo by intraperitoneal injection of 100 μg of anti-CD4 or anti-CD8 antibodies, on three consecutive days (–3, –2, –1) prior to sensitization as described (Cobbold et al., 1985Cobbold S.P. Jayasuriya A. Nash A. Prospero T. Waldmanm H. Therapy with monoclonal antibodies by elimination of T cell subsets in vivo.Nature. 1985; 312: 548-551Crossref Scopus (781) Google Scholar). Cell depletion was assessed by staining for CD4 and CD8 bearing cells in peripheral blood. In some experiments, 2 × 106 CD4+ T cells from sensitized mice were injected intravenously 6 d after sensitization immediately prior to challenge. In others experiments, Langerhans cell-enriched epidermal cell suspensions (2 × 106) modified with TNBS (1 mM) or with FITC or unmodified control cells were injected in the footpads (day 0) as a procedure of sensitization. Inguinal, axillary, and brachial lymph nodes were obtained 1 wk after in vivo sensitization. T cells were enriched by nylon wool column filtration (Polysciences). CD4+ and CD8+ T cells were isolated by negative selection using either rat monoclonal antibodies to CD8 or rat monoclonal antibodies to CD4 followed by incubation with sheep anti-rat Ig-coated magnetic beads (Dynal, Great Neck, NY) as described (Levin et al., 1993Levin D. Constant S. Pasqualini R. Flavell R. Bottomly K. Role of dendritic cells in the priming of CD4+ T lymphocytes to peptide antigen in vivo.J Immunol. 1993; 151: 6742-6750PubMed Google Scholar). Purity of the recovered CD4+ or CD8+ T cells was ≥95% as determined by staining with anti-CD4 and anti-CD8 antibodies. Cells were washed in flow cytometry buffer (PBS, 5% fetal calf serum, 0.02% sodium azide) and were stained for 30 min at 4°C with the following antibodies: FITC-anti-CD45, PE-anti-CD45, FITC-anti-B7–1, FITC-anti-B7–2, PE-anti-MHC class I (H-2Kb/H-2Db) (Pharmingen, San Diego, CA). The cells were then washed three times with flow cytometry buffer and analyzed on a Becton Dickinson FACScan. T cells were plated at 2 × 105 cells per well in 96 flat-bottomed microplates (Costar, Cambridge, MA) and cultured in the presence of different antigen-presenting cells for 5 d. T cell proliferation was determined by the incorporation of [3H]thymidine during the last 16 h of culture using a gas ionization counter (Packard, Meriden, CN). Results are presented as the mean (±SD) of assays performed in triplicate. The significance of differences among groups was examined by the Student's t test; p < 0.05 was considered significant. To test the role of MHC class II and CD4+ T cells in CHS responsiveness, we examined CHS reactions in wild-type and MHC class II-deficient mice after sensitization with TNCB. Sensitization of wild-type mice with 100 μl 3% TNCB led to antigen-specific swelling at the site of challenge 24 h after painting the ear with 1% TNCB. Mice deficient in MHC class II regularly showed significantly increased ear swelling responses (Figure 1a). The kinetics of CHS responses with different challenging doses are shown in Figure 1(b). To confirm that CHS in MHC class II-deficient mice is due to hapten-specific CD8+ T cells, we injected anti-CD8 monoclonal antibody on three consecutive days prior to sensitization. This treatment with anti-CD8 antibody resulted in depletion of CD8+ T cells and abrogated CHS responses to TNCB, indicating that the effector cells in these MHC class II-deficient mice are the CD8+ T cells (Figure 2). To investigate the role played by CD8+ T cells in CHS to TNCB in wild-type mice, we injected anti-CD8 monoclonal antibody before sensitization. This resulted in inhibition of CHS to TNCB, showing that CD8+ T cells play a major role in CHS response initiation. The increased CHS to TNCB in MHC class II-deficient mice and the role of effector cells played by CD8+ T cells suggested that CD4+ T cells could be downregulators of the CHS responses. Consistent with this hypothesis, depletion of CD4+ T cells with anti-CD4 antibody resulted in enhancement of CHS to TNCB compared with the non-injected wild-type mice (Figure 3). The increased CHS response in MHC class II-deficient mice and the results of the in vivo depletion of CD4+ T cells with anti-CD4 monoclonal antibody on CHS in wild-type mice suggested that CD4+ T cells downregulate CHS responses to TNCB. Hapten-specific CD4+ T cells were therefore obtained from lymph nodes of TNCB-painted mice and were injected immediately before challenge into MHC class II-deficient mice, which had been previously sensitized on the abdomen with TNCB 3%. The mice exhibited a diminution of CHS responses to TNCB (Figure 4). These data confirm that the increased CHS observed in MHC class II-deficient mice is due to their lack of CD4+ T cells. Transfer of hapten-specific CD8+ T cells from wild-type mice or MHC class II-deficient mice in both types of mice did not result in a diminution of CHS to TNCB (data not shown). Using flow cytometry, we evaluated the ability of MHC class II-deficient Langerhans cells to undergo phenotypic change upon short-term culture. Like MHC class II positive cells from wild-type animals, a subpopulation of CD45+ epidermal cells recovered from MHC class II-deficient mice showed upregulation of the costimulatory molecules B7–1 and B7–2 after 3 d of culture, as well as enhanced MHC class I expression (Figure 5, Figure 6). CD45 is expressed on all cells of hematopoietic lineage and is not expressed on keratinocytes. It is used as a marker of Langerhans cells; however, it could also be a marker of dendritic epidermal T cells (DETC). MHC class II-deficient mice have a distribution of T cells and DETC similar to wild-type controls (data not shown). There was no upregulation of MHC class I molecules on the γδ TCR+ population after short-term culture (data not shown), indicating that it was the Langerhans cells that exhibited the enhanced MHC class I expression.Figure 6Epidermal cells from MHC class II-deficient mice exhibit enhanced MHC class I expression upon short-term culture. Detection of MHC class I antigens and CD45 on MHC class II-deficient and wild-type epidermal cell suspensions by two color flow cytometry (data are plotted as intensities of FITC fluorescence on the x axis versus phycoerythrin fluorescence on the y axis). Epidermal cells were studied in freshly prepared suspensions (fEC) and after 3 d of culture (cEC).View Large Image Figure ViewerDownload (PPT) To determine whether Langerhans cells from MHC class II-deficient mice can stimulate hapten-specific CD8+ T cells, we cocultured CD8+ T cells from MHC class II-deficient mice with Langerhans cells: either fresh epidermal cells, fresh epidermal cells coupled with TNBS (TNP-fresh epidermal cells), cultured epidermal cells, or cultured epidermal cells coupled with TNBS (TNP-cultured epidermal cells). Both TNP-fresh epidermal cells and TNP-cultured epidermal cells exhibited antigen-presenting cell function in the stimulation of hapten-specific CD8+ T cells (Figure 7a). TNP-cultured epidermal cells induced a 3.5-fold increase in the stimulation of syngenic hapten-specific CD8+ T cell proliferation compared with TNP-fresh epidermal cells, indicating that MHC class II-deficient Langerhans cells within the epidermal cell population undergo functional maturation upon short-term culture. Similar results were obtained in wild-type mice (Figure 7b). To investigate whether TNP-coupled Langerhans cell-enriched epidermal cells from MHC class II-deficient mice can prime T cells in vivo, we injected TNP-coupled epidermal cells from MHC class II-deficient mice into naïve MHC class II-deficient animals. Mice were sensitized with either 100 μl 3% TNCB on the abdomen or with 2 × 106 TNP-epidermal cells injected in the footpad, and challenged 6 d later on the right ear with 20 μl 1% TNCB. Hapten-derivatized Langerhans cell-enriched epidermal cells from MHC class II-deficient mice induce CHS to TNCB in MHC class II-deficient mice (Figure 8). Uncoupled epidermal cells or FITC-coupled epidermal cells did not induce any significant ear swelling. Similar results were obtained when TNP-coupled epidermal cells from MHC class II-deficient mice were injected into naïve wild-type mice (data not shown). These results indicate that hapten-derivatized class II-deficient Langerhans cells can prime T cells in vivo. In this study, we demonstrate that MHC class II-deficient mice have enhanced CHS reactions to TNCB in vivo compared with wild-type controls. CD8+ T cells appeared to be the effector cells of the CHS as shown by the results of the in vivo CD8 depletion studies. CD4+ T cells seemed to regulate CHS responses as indicated by the transfer of hapten-specific CD4+ T cells from wild-type controls into MHC class II deficient mice that resulted in diminution of CHS. These results confirm experiments reported byGocinski and Tigelaar, 1990Gocinski B. Tigelaar R. Roles of CD4+ and CD8+ T cells in murine contact sensitivity revealed by in vivo monoclonal antibody depletion.J Immunol. 1990; 144: 4121-4128PubMed Google Scholar;Bour et al., 1995Bour H. Peyron E. Gaucherand M. et al.Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively mediate and regulate contact sensitivity to dinitrofluorobenzene.Eur J Immunol. 1995; 25: 3006-3010Crossref PubMed Scopus (280) Google Scholar, and Xu et al., 1996Xu H. DiIulio N.A. Fairchild R.L. T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon γ-producing (Tc1) effector CD8+ Tcells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells.J Exp Med. 1996; 183: 1001-1012Crossref PubMed Scopus (373) Google Scholar. Miller and Jenkins, 1985Miller S.D. Jenkins M.K. In vivo effects of GK1.5 (anti-L3T4a) monoclonal antibody on induction and expression of delayed type hypersensitivity.Cell Immunol. 1985; 92: 414-426Crossref PubMed Scopus (57) Google Scholar have previously reported opposite findings. After injection of anti-CD4 antibody, they had observed inhibition of CHS responses. Gocinski and Tigelaar demonstrated that both CD4+ T cells and CD8+ T cells were sufficient to induce CHS to dinitroflurobenzene and that there were two populations of CD4+ T cells, effector and suppressor cells. Enhanced contact sensitivity reactions to DNFB have been reported as well in MHC class II-deficient mice (Bour et al., 1995Bour H. Peyron E. Gaucherand M. et al.Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively mediate and regulate contact sensitivity to dinitrofluorobenzene.Eur J Immunol. 1995; 25: 3006-3010Crossref PubMed Scopus (280) Google Scholar. Since we began our studies it has been reported by Xu et al. that T cell populations primed by hapten sensitization are distinguished by two polarized patterns of cytokine production: interferon γ producing effector CD8+ T cells and interleukin 4/interleukin 10 producing negative regulatory CD4+ T cells. Altogether these results, consistent with our work, indicate that CD8+ T cells are effector cells in contact hypersensitivity. It should be noted that contact hypersensitivity differs from delayed type hypersensitivity to cellular or protein antigens, where only CD4+ T cells have been reported to be the effector cells (Cher and Mosmann, 1987Cher D.J. Mosmann T.R. Two types of murine helper t cell clone. 2. Delayed-type hypersensitivity is mediated by TH1 clones.J Immunol. 1987; 138: 3688-3694PubMed Google Scholar. Langerhans cell-enriched epidermal cells from MHC class II-deficient mice also induced CHS to TNCB after in vivo transfer, showing that antigen-presenting cells can prime CD8+ T cells in the absence of CD4+ T helper cells. It had been shown that fetal skin derived class I+, class II– dendritic cells can stimulate unprimed CD8+ T cells (Elbe et al., 1994Elbe A. Schleischitz S. Strunk D. Stingl G. Fetal skin-derived MHC class I+, MHC class II- dendritic cells stimulate MHC class I-restricted responses of unprimed CD8+ T cells.J Immunol. 1994; 153: 2878-2889PubMed Google Scholar. It had also been demonstrated that in CD4 deficient mice cytotoxic T cell responses could be observed (Buller et al., 1987Buller R.M. Holmes K.L. Hugin A. Frederickson T.N. Morse H.C. Induction of cytotoxic T cell responses in vivo in the absence of CD4 helper cells.Nature. 1987; 328: 77-79Crossref PubMed Scopus (269) Google Scholar and that the development of CD8+ T cells and the cytotoxic functions were normal (Rahemtulla et al., 1991Rahemtulla A. Fung-leung W.P. Shilham M.W. et al.Normal development and function of CD8+ T cells but markedly decreased helper cell activity in mice lacking CD4.Nature. 1991; 353: 180-184Crossref PubMed Scopus (613) Google Scholar. Another issue that is addressed in this study is the role of CD4+ T cells and MHC class II antigens in Langerhans cells activation. Epidermal Langerhans cells are bone marrow-derived cells and as the primary antigen-presenting cell population in the skin are responsible for initiation of immune responses. They have been implicated as pivotal antigen-presenting cells in the induction and expression of CHS (Haftek, 1988Haftek M. Langerhans cells in cutaneous pathology.in: Thivolet J. Schmitt D. The Langerhans Cell. Libbey Eurotext, Lyon1988: 377-385Google Scholar. They are the first cells of the skin immune system to react with a contact allergen, transporting the antigen to the draining lymph node for presentation to T lymphocytes. Cell surface expression of MHC molecules and costimulatory molecules are central for their function (Stingl and Bergstresser, 1995Stingl G. Bergstresser P.R. Dendritic cells: a major story unfolds.Immunol Today. 1995; 16: 330-333Abstract Full Text PDF PubMed Scopus (104) Google Scholar. MHC class I and class II antigens (Tamaki et al., 1979Tamaki K. Stingl G. Gullino M. Sachs D.H. Katz S.I. Ia Ag in mouse skin are predominantly expressed on Langerhans cells.J Immunol. 1979; 123: 784-787PubMed Google Scholar;Lenz et al., 1989Lenz A. Heufler C. Rammensee H.G. Glassl H. Koch F. Romani N. Schuler G. Murine epidermal Langerhans cells express significant amounts of class I major histocompatibility complex antigens.Proc Natl Acad Sci USA. 1989; 86: 7527-7531Crossref Scopus (9) Google Scholar;Sharrow et al., 1994Sharrow S.O. Barden L.A. Singer A. Katz S.I. Quantitative differences in cell surface expression of class I MHC antigens on murine epidermal Langerhans cells.J Immunol. 1994; 153: 110-116PubMed Google Scholar), as well as costimulatory and adhesion molecules such as ICAM-1 (Tang and Udey, 1991Tang A. Udey M.C. Inhibition of epidermal Langerhans cell function by low dose ultraviolet Radiation: ultraviolet B radiation selectively modulates ICAM-1 (CD54) expression by murine Langerhans cells.J Immunol. 1991; 146: 3347-3355PubMed Google Scholar), LFA-3 (Teunissen et al., 1990Teunissen M.B. Wormmeester J. Krieg S.R. Peters P.J. Vogels I.M. Kapsenberg M.L. Bos J.D. Human epidermal Langerhans cells undergo profound morphologic and phenotypical changes during in vitro culture.J Invest Dermatol. 1990; 94: 166-173Abstract Full Text PDF PubMed Google Scholar), B7–1, and B7–2 (Larsen et al., 1992Larsen C.P. Ritchie S.C. Pearson T.C. Linsley P.S. Functional expression of the costimulatory molecule, B7/BB1, on murine dendritic cell populations.J Exp Med. 1992; 176: 1215-1220Crossref PubMed Scopus (295) Google Scholar;Lee et al., 1993Lee M.G. Borkowski T.A. Udey M.C. Regulation of expression of B7 by murine Langerhans cells: a direct relationship between B7 mRNA levels and the level of surface expression of B7 by Langerhans cells.J Invest Dermatol. 1993; 101: 883-886Abstract Full Text PDF PubMed Google Scholar), have been identified on the surface of Langerhans cells. The increased accessory cell activity of cultured Langerhans cells relative to fresh Langerhans cells is thought to reflect the increased expression of major histocompatibility complex antigens and costimulatory molecules. Here we show that, except from lacking MHC class II molecules, Langerhans cells from MHC class II-deficient mice had otherwise normal phenotypic characteristics. They upregulated B7–1 and B7–2 costimulatory molecules and exhibited enhanced MHC class I expression and increased antigen-presenting cell activity after short-term culture. The full expression of CHS is usually thought to require that epidermal Langerhans cells present antigen in the context of MHC class II molecules to specifically sensitized T lymphocytes. It has been suggested that the CD4+ T cell-derived cytokines may be essential for terminal differentiation of Langerhans cells into lymphoid dendritic cells (Kitajima et al., 1996Kitajima T. Caceres-dittmar G. Tapia F.J. Jester J. Bergstresser P.R. Takashima A. T cell-mediated terminal maturation of dendritic cells: loss of adhesive and phagocytotic capacities.J Immunol. 1996; 157: 2340-2347PubMed Google Scholar). Our data indicate that Langerhans cells from MHC class II-deficient mice had normal functional characteristics in vivo as well as in vitro. One possibility is that terminal differentiation of Langerhans cells in vivo is T cell independant. Another possibility is that both CD8+ T cells and CD4+ T cells may induce these important functional changes. Additional experiments will be required to differentiate between these two alternatives. We thank Jay Linton for expert technical assistance, Harry Schaefer for preparation of the figures, Dr. Mark Udey and Dr. Genevieve Milon for helpful comments and critical review of the manuscript. This work was supported, in part, by the French Ministry of Foreign Affairs, Lilly Corporation, Philippe Fundation, and Fondation Rene Touraine.