Na?ve CD8+ T cells were isolated from PBMCs based on a two-step process using specific microbeads (Miltenyi Biotec)
Na?ve CD8+ T cells were isolated from PBMCs based on a two-step process using specific microbeads (Miltenyi Biotec). T cells. Spots count for na?ve CD4+ T cells specific to nickel from donors PR4, PR5, PR6, PR12, PR13, PR15, PR19, PR20, PR29, and PR43. Purified CD4+ T cells from each donor were seeded in multiple wells and stimulated weekly by autologous DCs previously loaded with nickel. After three rounds of activation, the specificity of the T-cell lines (each well-containing the expanded T cells) was tested by IFN- ELISpot assays. Dashed collection represents the minimum required spots count (count = 30) for the analysis to be considered acceptable. Each graph represent one donor and donor number are present on the top of each graph. Data_Sheet_1.PDF (1.2M) GUID:?B09F828A-2452-4D59-B9C2-4B64A8F3EBD9 Supplementary Data 3: Spots count for nickel-recognizing na?ve CD8+ T cells. Spots count for na?ve CD4+ T cells specific to nickel from donors PR13, PR16, PR18, PR31, PR37, PR42, and PR44. Purified CD8+ T cells from each donor were seeded in multiple wells and stimulated weekly by autologous DCs previously loaded with nickel. After three rounds of activation, the specificity of the T-cell lines (each well-containing the expanded T cells) was tested by IFN- ELISpot assays. Dashed collection represents the minimum required spots count (count = 30) for the analysis to be considered acceptable. Each graph represent one donor and donor number are present on the top of each graph. Data_Sheet_1.PDF (1.2M) GUID:?B09F828A-2452-4D59-B9C2-4B64A8F3EBD9 Supplementary Data 4: IFN- ELISPOT response of na?ve T-cells. Purified na?ve CD4+ or CD8+ T cells from each donor were seeded in multiple wells and stimulated weekly by autologous DCs previously loaded with nickel. After three rounds of activation, the specificity of the T-cell lines (each well-containing the expanded R18 T cells) was tested by IFN- ELISpot assays (a) IFN- ELISpot response of na?ve CD4+ T cells from donors PR5, PR6, and PR20 stimulated with unloaded DC, DC loaded with NiSO4 or DC loaded with CoCl2. (b) IFN- ELISpot response of na?ve CD8+ T cells from donors PR18, PR19, and PR44 stimulated with unloaded DC, DC loaded with NiSO4 or DC loaded with CoCl2. Data_Sheet_1.PDF (1.2M) GUID:?B09F828A-2452-4D59-B9C2-4B64A8F3EBD9 Supplementary Data 5: TCR V gene usage of HECs based Rabbit Polyclonal to CDK1/CDC2 (phospho-Thr14) on clonal frequency. (a) Scatter dots plot representing the clonal distribution of the TCR repertoire for the six nickel-specific T-cell lines analyzed. Each dot represents a single clone and its frequency in the total repertoire is usually depicted around the y-axis as percentage of total reads. The gray dotted line indicates the frequency cut-off of 0.5% for the definition of highly expanded clones (HECs). (b) Impact on the total repertoire of clones with clonal frequency below 0.1% (light gray bars), between 0.1 and 0.5% (dark gray bars) and above 0.5% (black bars) in the six nickel-specific T-cell lines analyzed. TCR variable gene usage of the HECs in the pre- and post-stimulation repertoire in donor PR19 (c) and PR20 (d). The percentage of HECs transporting a particular TCR variable gene (TRBV) is usually depicted around R18 the y-axis. All the genes names follow the IMGT nomenclature. Data_Sheet_1.PDF (1.2M) GUID:?B09F828A-2452-4D59-B9C2-4B64A8F3EBD9 Supplementary Data 6: Diversity index of the TCR repertoire at baseline and in wells after coculture with nickel-loaded DCs. The diversity in the pre- (baseline) and post-stimulation (well) repertoire in donor PR19 and PR20 measured as Richness (alpha = 0), Shannon entropy (alpha = 1) and Simpson index (alpha = 2). Around the x-axis, Well for donor PR19 includes results from PR19.03, PR19.09, and R18 PR19.30. Well for donor PR20 includes results from PR20.52 and PR20.56. Data_Sheet_1.PDF (1.2M) GUID:?B09F828A-2452-4D59-B9C2-4B64A8F3EBD9 Supplementary Data 7: CD69 expression on na?ve CD4+ T cells cocultured with NiSO4-loaded DCs or unloaded R18 DCs. Purified na?ve CD4+ T cells were seeded in multiple wells and stimulated weekly by autologous DCs previously loaded with nickel R18 to enrich the co-cultures in antigen-specific T cells. After 2 rounds of activation, each impartial T-cell collection was co-cultured with NiSO4-loaded DCs or unloaded DCs as a negative control. CD69 expression was evaluated by circulation cytometry. 76 na?ve CD4+ T-cell lines were evaluated for CD69 expression upon nickel stimulation and the majority of them showed an increase in CD69 expression upon coculture with NiSO4-loaded DCs. Data_Sheet_1.PDF (1.2M) GUID:?B09F828A-2452-4D59-B9C2-4B64A8F3EBD9 Abstract Allergic contact dermatitis caused.