CD1a-autoreactive T cells are a normal component of the human αβ T cell repertoire

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1 CDa-autoreactive T cells are a normal component of the human αβ T cell repertoire Annemieke de Jong, Victor Peña-Cruz, Tan-Yun Cheng, Rachael A. Clark, Ildiko Van Rhijn,, D. Branch Moody Division of Rheumatology, Immunology and Allergy, Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA USA Dana Farber Cancer Institute, Boston, MA, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

2 de Jong et al., Supplementary Figure spots /, T cells BC BC7 BC9 BC7 BCT BCT Supplementary Figure. Background IFN-γ production on mock transfected K cells by total T cells. T cells from different donors were purified by depletion of B-cells, monocytes, NK cells, platelets, dendritic cells, granulocytes and erythrocytes (Dynabeads Untouched Human T Cells, Invitrogen). Purified T cells were incubated for hrs with K cells transfected with vector only (mock) at an APC to T cell ratio of : and the number of IFN-gamma producing cellls was measured using IFN-γ Elispot assay. (mean ± S.D.)

3 de Jong et al., Supplementary Figure HT proliferation (x - cpm) CDb T cell line BNf K mock CDb CDb CDb anti-cdb HT proliferation (x - cpm) CDc T cell line BGcr K mock CDc CDc CDc anti-cdc IFN-gamma (ng/ml) CDd CDd CDd NKT cell clone JN. NKT cell clone JL.7 NKT cell clone BMa. K mock CDd CDd CDd anti-cdd IFN-gamma (ng/ml) K mock CDd CDd CDd anti-cdd IFN-gamma (ng/ml) K mock CDd CDd CDd anti-cdd Supplementary Figure. CDb, CDc and CDd restricted T cell responses against CD-transfected K cells. CD-autoreactive T cell lines and NKT cell clones were incubated with K cells transfected with vector only (K mock), transfected with CD or K CD cells pretreated with anti-cd blocking antibody or isotype control. After hrs supernatant was analyzed for IL- release using HT bioassay or IFN-γ using ELISA. (mean ± S.D.)

4 de Jong et al., Supplementary Figure Donor 9 Donor spots / T cells APC: K anti-cda anti-cdb anti-cdc CDa CDb CDc CDa CDb CDc Supplementary Figure. Ex vivo response of total T cells against K cells transfected with CDa, CDb or CDc T cells from donors were purified by depletion of B-cells, monocytes, NK cells, platelets, dendritic cells, granulocytes and erythrocytes (Dynabeads Untouched Human T Cells, Invitrogen). Purified T cells were incubated with K cells transfected with CDa, CDb or CDc in an IFN-gamma Elispot assay (mean ± S.D.). Blocking antibodies to CD or were added to confirm the CD specificity of the response.

5 de Jong et al., Supplementary Figure CDa dependent fold increase cytokine mrna 7 BC cl IFN-γ IL- Bgp cl8 BC cl BC cl BC cl BC cl9 BC cl IFN-γ IL- IFN-γ IL- IFN-γ IL- IFN-γ IL- IFN-γ IL- IFN-γ IL- Supplementary Figure. IFN-γ and IL- production by CDa-autoreactive T cell clones CDa autoreactive T cell clones were incubated at an APC : T cell ratio of : for 8 hrs with K CDa cells pretreated with anti-cda or. RNA was extracted and after transcription the cdna was analyzed by real -time PCR for CDa dependent upregulation of IFN-gamma and IL- message. Samples were normalized to beta-actin.

6 de Jong et al., Supplementary Figure a donor 8 T cell line Dermal T cell line AhR beta-actin Supplementary Figure. Aryl hydrocarbon receptor (AhR) expression in CDa-autoreactive T cell lines Western blot for AhR on cell lysates from two CDa-autoreactive T cell lines (anti-ahr monoclonal antibody, RPT, Abcam 779) (a) Relative expression of AhR was determined by real-time PCR in two CDa-autoreactive T cell lines and IFN-γ producing cells isolated from two donors by IFN-γ secretion assay (b) b mrna AhR / beta-actin (x ) Donor IFN- γ sorted Donor IFN- γ sorted Donor 8 T cell line Dermal T cell line

7 Supplementary Table. Antibodies Target antigen Clone Fluorophore/tag Manufacturer Application CDa OKT none In house Flow cytometry CDb BCDb. none In house Flow cytometry CDc F/A none In house Flow cytometry CDd CDd none In house Flow cytometry Isotype IgG P none In house Flow cytometry L L none In house Flow cytometry W/ W/ none In house Flow cytometry Mouse IgG Polyclonal goat PE BioSource Int. Flow cytometry F(ab ) IFN-γ -D-K none Mabtech Elispot IFN-γ 7-B- biotin Mabtech Elispot CD SK7 FITC BD Bioscience Flow cytometry CD HITa APC BD Pharmingen Flow cytometry CDRO UCHL PE ebioscience Flow cytometry IL-7 ebiodec7 Alexa Fluor 7 ebioscience Flow cytometry IL- 98 PE R&D systems Flow cytometry CLA HECA- FITC BD Pharmingen Flow cytometry CCR G PE-Cy7 BD Pharmingen Flow cytometry CCR PE R&D systems Flow cytometry CCR 88- APC Biolegend Flow cytometry CXCR C/CXCR Alexa Fluor 88 BD Pharmingen Flow cytometry