MHC-Dependent and -Independent Activation of Human Nickel-Specific CD8+ Cytotoxic T Cells from Allergic Donors1 Corinne Moulon, Doris Wild, Hans Ulrich.

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MHC-Dependent and -Independent Activation of Human Nickel-Specific CD8+ Cytotoxic T Cells from Allergic Donors1 Corinne Moulon, Doris Wild, Hans Ulrich Weltzien Journal of Investigative Dermatology Volume 111, Issue 3, Pages 360-366 (September 1998) DOI: 10.1046/j.1523-1747.1998.00306.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 In vitro culture of PBMC with NiSO4 stimulates both CD4+ and CD8+ cells and CD8+ T cell number increases with the number of antigenic stimulations. Hapten-specific T cell lines were produced by invitro stimulation of PBMC from three nickel-allergic donors with NiSO4. (a) Percentages of CD4+versus CD8+ T cells were assessed in nickel-specific T cell lines generated from three allergic donors, after one, two, or three rounds of invitro stimulation with irradiated autologous PBMC and NiSO4. nd, not determined. (b) Nickel-specificity of a T cell line from donor IF was assessed by stimulation with NiSO4 without APC, with irradiated autologous PBMC, or with EBV-B cell lines. The proliferative response was determined by incorporation of [3H]thymidine in triplicate and is expressed in cpm±SD. Representative data are shown for a nickel-specific T cell line after the second invitro stimulation. (c) CD4, CD8, HLA-DR, and αβTcR phenotypes of the nickel-specific T cell lines were investigated by fluorescence-activated cell sorter analysis and the percentages of CD4+ and CD8+ T cells were determined. Black histograms represent the staining with the appropriate antibody and white histograms represent the isotype control. Results are shown for a representative cell line from donor IF. Journal of Investigative Dermatology 1998 111, 360-366DOI: (10.1046/j.1523-1747.1998.00306.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Nickel-specific CD8+ T cell clones display both proliferating capacities and specific cytotoxic activities. The two representative T cell clones L1.1 and 3.3, isolated from donor IF, are shown. (a) CD4, CD8, and αβTcR phenotypes were investigated by fluorescence-activated cell sorter analysis. Black histograms represent the staining with the appropriate antibody and white histograms represent the isotypic control. (b) Proliferative responses to nickel in the presence of autologous EBV-B cells were determined by incorporation of [3H]thymidine in triplicate and are expressed in cpm. Stimulation with other metals such as Cr, Co, Cu, Pd, and Zn was also tested. The horizontal bold line in each figure represents the proliferation observed in the absence of metal salts. (c) Cytotoxic activities were determined in a 51Cr-release assay and data are expressed as the percentage of specific lysis of autologous EBV-B cell targets in the absence or in the presence of nickel or phytohaemaglutinin at different effector/target ratios. Journal of Investigative Dermatology 1998 111, 360-366DOI: (10.1046/j.1523-1747.1998.00306.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Nickel-specific CD8+ T cell clones require the constant presence of nickel and living APC for activation. (a–b) Proliferative responses to nickel in the presence of autologous EBV-B cells were determined by incorporation of [3H]thymidine in triplicate and are expressed in cpm±SD. NiSO4 was added to the well (+Ni) or APC were pulsed with NiSO4 and washed as indicated in Materials and Methods. APC were either irradiated (a) or fixed (b) and representative data are shown for clone 5C from donor AE. (c–d) Cytotoxic activities were determined in a 51Cr-release assay and representative data, expressed as the percentage of specific lysis, are shown for clone L1.1 from donor IF. Autologous EBV-B cells were used as targets in the presence of either nickel or PHA at different effector/target ratios during the 4h assay (c) or were pulsed with NiSO4 and then washed (d) before the incubation with T cells. (e) Proliferative response of the DR1-restricted clone 12F to 25μg per ml of the peptide HA309–319 (±HA) in the presence of DR1 homozygous EBV-B cells was determined by incorporation of [3H]thymidine in triplicate and is expressed in cpm±SD. APC were either irradiated or fixed. Journal of Investigative Dermatology 1998 111, 360-366DOI: (10.1046/j.1523-1747.1998.00306.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The majority of the CD8+ clones, but not clone L1.1, only exhibit cytotoxic activities towards autologous EBV-B cell lines. Cytotoxic activities of the CD8+ T cell clones were determined in a 51Cr-release assay using autologous (AE-, CM-, and IF-B cells) and allogeneic (Sweig B cells) EBV-B cell lines as targets. Representative experiments are shown for the three T cell clones, 5C from donor AE, 5K from donor CM, and L1.1 from donor IF. Data are expressed as the percentage of specific lysis of EBV-B cell targets in the absence or in the presence of nickel at different effector/target ratios. Journal of Investigative Dermatology 1998 111, 360-366DOI: (10.1046/j.1523-1747.1998.00306.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The clone L1.1 is neither MHC class I nor MHC class II restricted. Cytotoxic activities of the CD8+ T cell clone L1.1 were determined in a 51Cr-release assay using MHC class I-(721.221) and class II-(T2) deficient cell lines as targets. Representative data are shown and are expressed as the percentage of specific lysis of targets in the absence (empty symbols) or in the presence of nickel (black symbols) or phytohaemaglutinin (dotted line) at different effector/target ratios. Journal of Investigative Dermatology 1998 111, 360-366DOI: (10.1046/j.1523-1747.1998.00306.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions