MHC-Dependent and -Independent Activation of Human Nickel-Specific CD8+ Cytotoxic T Cells from Allergic Donors11This work was presented in part at the 26th Annual Meeting of the European Society of Dermatologic Research, Amsterdam, September 19–22, 1996
1998; Elsevier BV; Volume: 111; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.1998.00306.x
ISSN1523-1747
AutoresCorinne Moulon, Doris Wild, Hans Ulrich Weltzien, Anne Dormoy,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoT lymphocytes are critical effectors in the pathogenesis of contact hypersensitivity. Nickel is the most common contact sensitizer in humans and nickel-specific CD4+ T helper cells have been extensively characterized. Because recent observations have suggested the activation of CD8+ T cells in murine models of contact hypersensitivity,we investigated the existence of CD8+ hapten-specific T lymphocytes in patients with allergy to nickel. Nickel-specific T cell lines were generated from the peripheral blood of three allergic donors. The T cell lines were composed of a majority of CD4+ T cells, but CD8+ T cells were also present and their percentage increased with repeated invitro stimulations. In addition to nickel-reactive helper T cell-0-type or helper T cell-2-type CD4+ T cell clones, CD8+ T cell clones could be derived from these cell lines and a total of 15 clones were further studied. Cytokine production was evaluated for 11CD8+T cell clones that were either cytotoxic T cell-0- or cytotoxic T cell-1-type clones. Additional effector functions were investigated on the complete panel of T cell clones. These CD8+T cells did not only display hapten-specific proliferation, but also specific cytotoxic activities towards autologous EBV-B cells in the presence of nickel. Two different types of CD8+ T cells were characterized. Most of the clones lysed only autologous targets in the constant presence of nickel; however, one clone was able to lyse numerous targets in the presence of NiSO4, irrespective of the expression of either major histocompatibility complex class I or class II molecules. The characterization of nickel-specific cytotoxic CD8+ T cells with different requirements for nickel-specific target lysis, may have important implications in the development or in the control of human contact hypersensitivity reactions to nickel invivo. T lymphocytes are critical effectors in the pathogenesis of contact hypersensitivity. Nickel is the most common contact sensitizer in humans and nickel-specific CD4+ T helper cells have been extensively characterized. Because recent observations have suggested the activation of CD8+ T cells in murine models of contact hypersensitivity,we investigated the existence of CD8+ hapten-specific T lymphocytes in patients with allergy to nickel. Nickel-specific T cell lines were generated from the peripheral blood of three allergic donors. The T cell lines were composed of a majority of CD4+ T cells, but CD8+ T cells were also present and their percentage increased with repeated invitro stimulations. In addition to nickel-reactive helper T cell-0-type or helper T cell-2-type CD4+ T cell clones, CD8+ T cell clones could be derived from these cell lines and a total of 15 clones were further studied. Cytokine production was evaluated for 11CD8+T cell clones that were either cytotoxic T cell-0- or cytotoxic T cell-1-type clones. Additional effector functions were investigated on the complete panel of T cell clones. These CD8+T cells did not only display hapten-specific proliferation, but also specific cytotoxic activities towards autologous EBV-B cells in the presence of nickel. Two different types of CD8+ T cells were characterized. Most of the clones lysed only autologous targets in the constant presence of nickel; however, one clone was able to lyse numerous targets in the presence of NiSO4, irrespective of the expression of either major histocompatibility complex class I or class II molecules. The characterization of nickel-specific cytotoxic CD8+ T cells with different requirements for nickel-specific target lysis, may have important implications in the development or in the control of human contact hypersensitivity reactions to nickel invivo. contact hypersensitivity human serum albumin peripheral blood mononuclear cells cytotoxic T cell helper T cell Contact hypersensitivity (CHS) reaction is a skin disease involving the proliferation of T cells in response to low molecular weight compounds. Nickel represents the most common contact sensitizer in humans with about 10% of the Caucasian population sensitized (Basketter et al., 1993Basketter D.A. Briatico-Vangosa G. Kaestner W. Lally C. Bontinck W.J. Nickel, cobalt and chromium in consumer products: a role in allergic contact dermatitis?.Contact Dermatitis. 1993; 28: 15-25Crossref PubMed Scopus (216) Google Scholar). An abundant literature is available on nickel-induced CHS reactions including the characterization of nickel-specific T cells isolated from either peripheral blood or skin lesions of sensitized patients. Such cells mainly belong to the CD4+ subset and are major histocompatibility complex (MHC) class II restricted (Sinigaglia et al., 1985Sinigaglia F. Scheidegger D. Garotta G. Scheper R. Pletscher M. Lanzavecchia A. Isolation and characterization of Ni-specific T cell clones from patients with Ni-contact dermatitis.J Immunol. 1985; 135: 3929-3932PubMed Google Scholar;Kapsenberg et al., 1987Kapsenberg M.L. Res P. Bos J.D. Schootemijer A. Teunissen M.B.M. Van Schooten W. Nickel-specific T lymphocyte clones derived from allergic nickel-contact dermatitis lesions in man: heterogeneity based on requirement of dendritic antigen-presenting cell subsets.Eur J Immunol. 1987; 17: 861-865Crossref PubMed Scopus (76) Google Scholar, Kapsenberg et al., 1988Kapsenberg M.L. Van der Pouw-Kraan T. Stiekma F.E. Schootemeijer A. Bos J.D. Direct and indirect nickel-specific stimulation of T lymphocytes from patients with allergic contact dermatitis to nickel.Eur J Immunol. 1988; 18: 977-982Crossref PubMed Scopus (40) Google Scholar;Silvennoinen-Kassinen et al., 1991Silvennoinen-Kassinen S. Poikonen K. Ikäheimo I. Characterization of nickel-specific T cell clones.Scand J Immunol. 1991; 33: 429-434Crossref PubMed Scopus (35) Google Scholar). They have been characterized in terms of MHC restriction, processing requirements (Moulon et al., 1995Moulon C. Vollmer J. Weltzien H.U. Characterization of processing requirements and metal cross-reactivities in T-cell clones from patients with allergic contact-dermatitis to nickel.Eur J Immunol. 1995; 25: 3308-3315Crossref PubMed Scopus (152) Google Scholar), and cytokine production (Kapsenberg et al., 1992Kapsenberg M.L. Wierenga E.A. Stiekma F.E.M. Tiggelman AMBC Bos J.D. Th1 lymphokine production profiles of nickel-specific CD4+ T-lymphocyte clones from nickel contact allergic and non-allergic individuals.J Invest Dermatol. 1992; 98: 59-63Crossref PubMed Scopus (182) Google Scholar;Probst et al., 1995Probst P. Kuntzlin D. Fleischer B. T (h) 2-type infiltrating T-cells in nickel-induced contact-dermatitis.Cell Immunol. 1995; 165: 134-140Crossref PubMed Scopus (75) Google Scholar;Werfel et al., 1997Werfel T. Hentschel M. Kapp A. Renz H. Dichotomy of blood-derived and skin-derived IL-4-producing allergen-specific T-cells and restricted Vβ repertoire in nickel-mediated contact-dermatitis.J Immunol. 1997; 158: 2500-2505PubMed Google Scholar). Very little information is available concerning the nature of the antigenic determinants recognized by nickel-specific human T cells. Current data suggest that nickel-modified self peptides presented by MHC class II molecules might be reponsible for the activation of nickel-specific T cells (Romagnoli et al., 1991Romagnoli P. Labhardt A.M. Sinigaglia F. Selective interaction of Ni with an MHC-bound peptide.Embo J. 1991; 10: 1303-1306Crossref PubMed Scopus (132) Google Scholar;Emtestam and Olerup, 1996Emtestam L. Olerup O. On T-cell recognition of nickel as a hapten.Acta Dermatol Venereol. 1996; 76: 344-347PubMed Google Scholar), but the actual antigenic determinants recognized by nickel-activated T cells remain to be identified. No preferential HLA association has been found in subjects allergic to nickel (Emtestam et al., 1993Emtestam L. Zetterquist H. Olerup O. HLA-DR, -DQ and -DP alleles in nickel, chromium, and/or cobalt-sensitive individuals: genomic analysis based on restriction fragment length polymorphisms.J Invest Dermatol. 1993; 100: 271-274Abstract Full Text PDF PubMed Google Scholar); however, skewing of the nickel-reactive TCR repertoire has been reported (Vollmer et al., 1997Vollmer J. Fritz M. Dormoy A. Weltzien H.U. Moulon C. Dominance of the BV17 element in nickel-specific human T-cell receptors relates to severity of contact sensitivity.Eur J Immunol. 1997; 27: 1865-1874Crossref PubMed Scopus (64) Google Scholar;Werfel et al., 1997Werfel T. Hentschel M. Kapp A. Renz H. Dichotomy of blood-derived and skin-derived IL-4-producing allergen-specific T-cells and restricted Vβ repertoire in nickel-mediated contact-dermatitis.J Immunol. 1997; 158: 2500-2505PubMed Google Scholar). The originally postulated effector role of CD4+ T cells in CHS reactions (Hauser, 1990Hauser C. Cultured epidermal Langerhans cells activate effector T cells for contact sensitivity.J Invest Dermatol. 1990; 95: 436-440Abstract Full Text PDF PubMed Google Scholar;Kondo et al., 1996Kondo S. Beissert S. Wang B.H. et al.Hyporesponsiveness in contact hypersensitivity and irritant contact-dermatitis in CD4 gene targeted mouse.J Invest Dermatol. 1996; 106: 993-1000Crossref PubMed Scopus (61) Google Scholar) has been recently challenged in different mouse models of this disease. In several systems, hapten-specific CD8+ T cells have been described as mediators of CHS reactions, whereas CD4+ T cells were shown to be regulators of the inflammatory reaction (Gocinski and Tigelaar, 1990Gocinski B.L. Tigelaar R.E. Roles of CD4 + and CD8+ T cells in murine contact sensitivity revealed by invivo monoclonal antibody depletion.J Immunol. 1990; 144: 4121-4128PubMed Google Scholar;Anderson et al., 1995Anderson C. Hehr A. Robbins R. Hasan R. Athar M. Mukhtar H. Elmets C.A. Metabolic requirements for induction of contact hypersensitivity to immunotoxic polyaromatic hydrocarbons.J Immunol. 1995; 155: 3530-3537PubMed 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 (270) 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 gamma-producing (Tc1) effector CD8+ T cells and interleukin (IL-) 4/IL-10-producing (Th2) negative regulatory CD4+ T cells.J Exp Med. 1996; 183: 1001-1012Crossref PubMed Scopus (360) Google Scholar). CD8+ hapten-specific T cells have also been described in human CHS reactions (Kalish, 1990Kalish R.S. The use of human T-lymphocyte clones to study T-cell function in allergic contact dermatitis to urushiol.J Invest Dermatol. 1990; 90: 108S-111SCrossref Scopus (18) Google Scholar;Hertl et al., 1993Hertl M. Bohlen H. Jugert F. Boecker C. Knaup R. Merk H.F. Predominance of epidermal CD8+ T lymphocytes in bullous cutaneous reactions caused by β-lactam antibiotics.J Invest Dermatol. 1993; 101: 794-799Abstract Full Text PDF PubMed Google Scholar); however, although CD8+ T cells specific for nickel have been occasionally mentioned in different reports, no detailed studies on CD8+ human nickel-specific T cell clones have been conducted. The aim of this study was thus to investigate the presence of and the activation requirements of nickel-specific CD8+ human T lymphocytes. Three nickel allergic donors participated in this study after giving informed consent. Nickel allergy was evaluated according to the standard procedure using epicutaneous application of NiSO4.6H2O at 5% in vaseline using the Finn chamber method. All patch test areas were read at days 2 and 3 after application. The HLA haplotypes of the donors were as follows: donor AE [HLA-A28, B27, B55(22), Cw2, Cw9(3), DR13(6), DR15(2), DR51, DR52, DQ6(1)], donor CM [HLA-A1, A29(19), B8, Cw7, DR15(2), DR17(3), DR51, DR52, DQ2, DQ6(1)], donor IF [HLA-A1, A66(10), B35, B53, Cw4, DR4, DR13(6), DR52, DR53, DQ6(1), DQ7(3)]. The concentrations of each metal salt used in culture were as follows: NiSO4.6H2O, 10–4–2.5×10–5 M; CoCl2.6H2O, 5×10–5–7.5×10–6 M; PdCl2, 5×10–4–7.5×10–5 M; CuSO4.5H2O, 10–5–2.5×10–6 M; K2Cr2O7, 10–7–2.5×10–8 M; and ZnSO4.7H2O, 7.5×10–6–10–7 M (all obtained from Sigma, Deisenhofen, Germany). For each metal, nontoxic ranges of concentrations were defined as those that did not affect a PHA-induced proliferation of peripheral blood mononuclear cells (PBMC) from nonallergicdonors (data not shown). Stock solutions were stored at –20°C. The culture medium was RPMI 1640 supplemented (all from Gibco BRL, Eggenstein, Germany) with 2mM L-glutamine, 100μg kanamycin per ml, 5×10–5 M 2-mercaptoethanol, 1mM sodium pyruvate, 1×mixture of nonessential amino-acids, and 5% pooled human AB serum (RPMI-HS) or 10% heat-inactivated fetal calf serum (RPMI-FCS). To support the antigen-independent growth of T cell clones, this medium was supplemented with 100U recombinant human IL-2 per ml (Eurocetus, Ratingen, Germany). PBMC (106 per ml) isolated by Ficoll-Hypaque (Pharmacia, Freiburg, Germany) were cultivated invitro with 10–4 M NiSO4 in RPMI-HS for 7d in a final volume of 5ml in 6 well culture plates. Cells were then washed and expanded in IL-2-containing RPMI-HS for another 5d. These lines were stimulated a second time with the hapten and irradiated (3000 rads) autologous PBMC (106 per ml) and, after 3d, expanded again by adding IL-2. In some cases, T cell lines were further stimulated using the same procedure. T cell clones were generated from these nickel-specific T cell lines after the second stimulation of PBMC by the sensitizing hapten. For cloning, T cell blasts were seeded at 0.3 cells per well in Terasaki plates (Nunc, Wiesbaden, Germany) in the presence of 1μg PHA-P per ml (Murex Diagnostics, Dartford, U.K.), 100U IL-2 per ml, and 104 irradiated allogeneic fresh PBMC. The obtained T cell clones were expanded and maintained in culture by periodic stimulation in the presence of irradiated allogeneic PBMC, PHA, and IL-2. The CD4+ clones reported here are all specific for NiSO4, but some of them (called HSA.Ni clones) were induced using nickel-modified human serum albumin (HSA) as previously reported (Vollmer et al., 1997Vollmer J. Fritz M. Dormoy A. Weltzien H.U. Moulon C. Dominance of the BV17 element in nickel-specific human T-cell receptors relates to severity of contact sensitivity.Eur J Immunol. 1997; 27: 1865-1874Crossref PubMed Scopus (64) Google Scholar). The other T cell clones were all induced using NiSO4. PBMC and autologous EBV-transformed B cell lines (EBV-B cells) were used as antigen-presenting cells (APC). Sweig 007 [Bcell line IHW 9037 with the following HLA haplotype: HLA-A29(19), B61(40), Cw2, DR11(5), DR52, DQ7(3)], and T2 cell lines were obtained from Dr. P. Cresswell (Howard Hughes Medical University, New Haven, CT) (Newcomb and Cresswell, 1993Newcomb J.R. Cresswell P. Characterization of endogeneous peptides bound to purified HLA-DR molecules and their absence from invariant chain-associated αβ dimers.J Immunol. 1993; 150: 499-507PubMed Google Scholar). Jesthom is a DR1 homozygous B cell line (IHW 9004). The 721.221 cell line was obtained from Dr. P. van Endert (INSERM, Paris, France) (Shimizu and DeMars, 1989Shimizu Y. DeMars R. Production of human cells expressing individual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C null human cell line.J Immunol. 1989; 142: 3320-3328PubMed Google Scholar). For immortalization of B cells, PBMC (107) were resuspended in 10ml RPMI-FCS containing 30% supernatant of the EBV-producing marmoset cell line B95–8 [American Type Culture Collection (ATCC)] and 600ng cyclosporine A per ml (Sandoz, Basel, Switzerland). After incubation overnight, cells were washed and cultured in RPMI-FCS. T cells (2×104) were cocultured in triplicate with 2×104 irradiated (3000 rads) PBMC or irradiated (6000 rads) EBV-B cells in 200μl of RPMI-FCS with the different metal salts, without hapten or with 25μg peptide HA307–319 per ml. After 48h, cultures were pulsed with [3H]thymidine and incorporation was measured after another 18h. Triplicates were harvested on GF/A-filters and the isotope incorporation was measured in an automatic β-counter (Inotech, Asbach, Germany). In some experiments, APC were fixed as described byShimonkevitz et al., 1983Shimonkevitz R. Kappler J. Marrack P. Grey H. Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing.J Exp Med. 1983; 158: 303-316Crossref PubMed Scopus (511) Google Scholar. Briefly, EBV-B cells were resuspended in medium without serum and fixed in 0.05% glutaraldehyde for 45s at room temperature. The reaction was stopped by adding 1ml of 0.2M L-Lysine for an additional 45s. Cells were then washed and in some experiments pulsed for 90min at 37°C with the hapten and washed again before being used as APC as described above. A DR1-restricted T cell clone (12F), specific for the hemagglutinin peptide HA307–319 (PKYVKQNTLKLAT), was used as positive control. EBV-B cells were used as targets in a standard 4h 51Cr-release assay. Briefly, targets were labeled with Na251CrO4 (NEN Life Science Products, Boston, MA) for 90min at 37°C and extensively washed. The number of target cells was maintained constant (2×103 cells per 200μl) and effector cells wereused at effector:target ratios between 30:1 and 1:1 in RPMI-FCS. NiSO4 (10–4 M) or PHA (1μg per ml) were added to the round-bottom microwells at fixed concentrations if not otherwise indicated. In some experiments, target cells were pulsed for 90min at 37°C with the hapten and washed before addition to the effector cells. After 4h, 51Cr-release in the supernatants was measured in triplicate in an automatic γ-counter (Packard, Dreieich, Germany). Specific lysis was calculated according toShearer, 1974Shearer G.M. Cell-mediated cytotoxicity to trinitrophenyl-modified syngenic lymphocytes.Eur J Immunol. 1974; 4: 527-533Crossref PubMed Scopus (426) Google Scholar. All data represent the mean of triplicates with SD<10%. Mouse monoclonal antibodies used for flow cytometry were as follows: CD4 (13B8.2, Immunotech, Krefeld, Germany), CD8 (B9.11, Immunotech), TcR Panα/β (BMA031, Immunotech), and anti-DR (L243, ATCC). The rat monoclonal antibody HECA-452 against the cutaneous-associated-antigen (CLA) was kindly provided by Dr. L.J. Picker (University of Texas Southwestern Medical Center, Dallas, TX) (Picker et al., 1990Picker L.J. Michie S.A. Rott L.S. Butcher E.C. A unique phenotype of skin-associated lymphocytes in humans. Preferential expression of the HECA-452 epitope by benign and malignant T cells at cutaneous sites.Am J Pathol. 1990; 136: 1053-1068PubMed Google Scholar). Fluoroscein isothiocyanate-conjugated goat anti-mouse IgG+M (H+L) and PE-conjugated goat anti-rat IgM were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). T cells were stained at 4°C in 96 well plates (2×105 per well) with monoclonal antibody and fluoroscein isothiocyanate- or PE-conjugated secondary antibody. Fluorescence was measured in a FACScan instrument (Becton Dickinson, San Jose, CA). Cloned T cells (2×105) were cocultured in triplicate with 105 irradiated (6000 rads) autologous EBV-B cells in 200μl of RPMI-HS with or without hapten. After 48h, culture supernatants were used to measure IFNγ and IL-4 production by enzyme-linked immunosorbent assays (Rogge et al., 1997Rogge L. Barberis-Maino L. Biffi M. Passini N. Presky D.H. Gubler U. Sinigaglia F. Selective expression of an interleukin-12 receptor component by human T helper 1 cells.J Exp Med. 1997; 185: 825-831Crossref PubMed Scopus (662) Google Scholar). In order to investigate the nature of the T cells activated by NiSO4, PBMC from three donors, whose allergy to nickel was assessed by positive epicutaneous reactions to this metal, were cultivated invitro in the presence of NiSO4. Activated T cells were expanded after 1 wk by addition of IL-2 and the phenotype of the cell lines was investigated by fluorescence-activated cell sorter analysis. In all three cases we found more CD4+ T cells (74%–92%) than CD8+ T cells (6%–17%) after the first invitro antigenic stimulation. When these T cell lines were stimulated repeatedly with NiSO4 and autologous PBMC, we found that the fraction of CD8+ cells increased to values of 35%–60% of the total T cells (Figure 1a). The nickel-specificity of these cell lines was tested by proliferation assays and representative data are shown for donor IF in Figure 1(b). T cells alone proliferated weakly in response to nickel in the absence of APC. This could be explained by the expression of MHC class I and II molecules by human T cells that might allow them to act as APC (Barnaba et al., 1994Barnaba V. Watts C. de Boer M. Lane P. Lanzavecchia A. Professional presentation of antigen by activated human T cells.Eur J Immunol. 1994; 24: 71-75Crossref PubMed Scopus (107) Google Scholar); however, antigenic stimulation was greatly enhanced by addition of APC (either autologous PBMC or EBV-B cells). Figure 1(c) shows an exemplary phenotypic analysis of these activated T cells expressing an αβTcR and MHC class II molecules and in this particular case comprising 20% of CD8+ cells. In order to characterize the CD8+ populations, T cell clones were produced from T cell lines generated from donors IF, CM, and AE after the second invitro stimulation. The majority of the T cell clones isolated from three different cell lines were again from the CD4+ subset; however, for each donor, CD8+ T cell clones could be derived (three of 35 from donor IF, one of 16 from donor CM, and one of six from donor AE). A new cloning procedure was performed from two other nickel-specific T cell lines of donor IF, providing 10 additional CD8+T cell clones. All tested T cell clones were positive for CLA expression as reported in Table 1. All CD8+ T cell clones were CD4– and TcRαβ+ as shown for two representative clones, L1.1 and 3.3, in Figure 2(a). The T cell clones were specific for nickel, and responded to the antigen as evaluated by different effector functions. As shown in Figure 2(b), they proliferated after addition of NiSO4 in the presence of EBV-B cell lines. Clone 3.3 exhibited a weak cross-reaction to Pd (Figure 1b), but no additional metal reactivities could be detected. As one of the characteristics of CD8+ T cells is their cytotoxic potential, we investigated whether nickel-specific CD8+ T cell clones were cytolytic. We utilized both a nonantigen specific stimulus, the PHA lectin, and an antigen-specific stimulation with NiSO4. As shown in Figure 2(c), CD8+ nickel-specific T cell clones, indeed, lysed autologous B cells in the presence of nickel salts, or in the presence of PHA, but not in their absence. The cross-reactivity of Clone 3.3 towards Pd also occurred in cytotoxic assays (data not shown). The cytokine profile of a total of 11CD8+ independent nickel-specific T cell clones isolated from the peripheral blood of donor IF was studied. For comparison, CD4+T cell clones originating from NiSO4- and HSA-Ni-induced T cell lines of the same donor, or from NiSO4-induced T cell lines of the two other donors, were also included in the assay. In all cases, IL-4 and IFNγ production were determined following activation by NiSO4 in the presence of autologous EBV-B cells. The data are summarized in Table 1. All CD4+ T cell clones produced IL-4 following antigenic stimulation. Five of 21 secreted IFNγ in addition, being thus considered as TH0-type T cells in contrast to the 16 TH2-type clones. All CD8+ T cell clones produced IFNγ, and seven of 11 also produced IL-4 after nickel stimulation. A total of seven TC0- and four TC1-type nickel-specific T cell clones were thus identified. The antigenic requirements for the presentation of NiSO4 to the CD8+ T cell clones were tested in both proliferation and cytotoxic assays. As shown in Figure 3(a) for T cell clone 5C, pulsing of the APC for 90min at 37°C with NiSO4 was not sufficient to allow stimulation of T cell proliferation. Moreover, fixed APC were not able to stimulate T cell proliferation even when nickel was continuously present during the assay (Figure 3b). This is in contrast to Results previously obtained for certain CD4+ T cells (Moulon et al., 1995Moulon C. Vollmer J. Weltzien H.U. Characterization of processing requirements and metal cross-reactivities in T-cell clones from patients with allergic contact-dermatitis to nickel.Eur J Immunol. 1995; 25: 3308-3315Crossref PubMed Scopus (152) Google Scholar). A positive control (Figure 3e) was performed in order to demonstrate that APC fixed by the same method could present an antigen that did not require any processing (peptide HA307–319) to a specifc T cell clone. Pulsing of target cells with NiSO4 in the cytotoxic assay revealed that target cells could only be lysed when nickel was present during the assay (Figure 3c), but not when target cells were pulsed with NiSO4 and washed before addition to the effector cells (Figure 3d). Both kinds of targets were, however, lysed by the T cell clone in the presence of PHA. To investigate the MHC restriction of the CD8+ T cell clones, cytotoxic assays were performed using the MHC-typed EBV-B cell lines from the three donors as well as a donor-unrelated B cell line (Sweig). Representative results shown in Figure 4 indicate that most of the CD8+ T cell clones exclusively lysed their respective autologous targets in the presence of nickel, but none of the three other B cells used as APC (e.g., when nickel was added, clone 5C was only able to lyse autologous AE targets and clone 5K specifically lysed autologous CM targets); however, one T cell clone, namely clone L1.1 from donor IF, could lyse all of the tested targets in the presence of nickel. All of the B cell lines tested to date (from 10 different donors) were antigen-specifically lysed by clone L1.1 independently of MHC haplotype (data not shown). These results thus raised the question whether clone L1.1 was at all MHC restricted. In order to investigate the MHC requirements for activation of the L1.1CD8+ T cell clone, two cell lines lacking either MHC class I molecules (721.221 cells) or MHC class II molecules (T2 cells) were used as target cells (Shimizu and DeMars, 1989Shimizu Y. DeMars R. Production of human cells expressing individual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C null human cell line.J Immunol. 1989; 142: 3320-3328PubMed Google Scholar;Newcomb and Cresswell, 1993Newcomb J.R. Cresswell P. Characterization of endogeneous peptides bound to purified HLA-DR molecules and their absence from invariant chain-associated αβ dimers.J Immunol. 1993; 150: 499-507PubMed Google Scholar). As shown in Figure 5, both targets were lysed either in the presence of nickel or in the presence of the lectin, thereby indicating that neither MHC class I nor MHC class II expression was required for activation of this T cell clone by nickel. CHS has long been described as a typical delayed-type hypersensitvity (DTH) reaction, differing from the latter only by the nature of the inducing antigens, which in the case of CHS are compounds of low molecular weight (Dupuis and Benerza, 1982Dupuis G. Benerza C. Allergic Contact Dermatitis to Simple Chemicals. A Molecular Approach. New York, Marcel Dekker1982Google Scholar) called haptens. Recent studies conducted in different mouse models of CHS have documented other differences between DTH and CHS. Thus, in contrast to DTH, representing a typical TH1CD4+ T cell-mediated disease (Cher and Mosmann, 1987Cher D.J. Mosmann T.M. Two types of murine helper T cell clones. II. Delayed-type hypersensitivity is mediated by TH1 clones.J Immunol. 1987; 138: 3688-3694PubMed Google Scholar), evidence is now accumulating that a major effector role is played by CD8+ T cells in mouse CHS. This has been suggested byGocinski and Tigelaar, 1990Gocinski B.L. Tigelaar R.E. Roles of CD4 + and CD8+ T cells in murine contact sensitivity revealed by invivo monoclonal antibody depletion.J Immunol. 1990; 144: 4121-4128PubMed Google Scholar and has been recently confirmed by different groups (Anderson et al., 1995Anderson C. Hehr A. Robbins R. Hasan R. Athar M. Mukhtar H. Elmets C.A. Metabolic requirements for induction of contact hypersensitivity to immunotoxic polyaromatic hydrocarbons.J Immunol. 1995; 155: 3530-3537PubMed 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 (270) Google Scholar). Moreover,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 gamma-producing (Tc1) effector CD8+ T cells and interleukin (IL-) 4/IL-10-producing (Th2) negative regulatory CD4+ T cells.J Exp Med. 1996; 183: 1001-1012Crossref PubMed Scopus (360) Google Scholar have reported that CD8+ effector T cells are producing IFNγ (TC1 cells), whereas CD4+ downregulator T cells belong to the TH2-type, producing IL-4 and IL-10. In humans, reports have also described the activation of CD8+ T cells, either as the major T cell population, e.g., in CHS reactions to urushiol (Kalish, 1990Kalish R.S. The use of human T-lymphocyte clones to study T-cell function in allergic contact dermatitis to urushiol.J Invest Dermatol. 1990; 90: 108S-111SCrossref Scopus (18) Google Scholar) or to β-lactam antibiotics (Hertl et al., 1993Hertl M. Bohlen H. Jugert F. Boecker C. Knaup R. Merk H.F. Predominance of epidermal CD8+ T lymphocytes in bullous cutaneous reactions caused by β-lactam antibio
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