Generation of adult human T-cell progenitors for immunotherapeutic applications Laura Simons, MD, PhD, Kuiying Ma, MSc, Corinne de Chappedelaine, PhD,

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Generation of adult human T-cell progenitors for immunotherapeutic applications Laura Simons, MD, PhD, Kuiying Ma, MSc, Corinne de Chappedelaine, PhD, Ranjita Devi Moiranghtem, PhD, Elodie Elkaim, MD, Juliette Olivré, Bc, Sandrine Susini, PhD, Kevin Appourchaux, PhD, Christian Reimann, MD, PhD, Hanem Sadek, MSc, Olivier Pellé, MSc, Nicolas Cagnard, MSc, Elisa Magrin, MD, Chantal Lagresle-Peyrou, PhD, Tom Taghon, PhD, Antonio Rausell, PhD, Marina Cavazzana, MD, PhD, Isabelle André-Schmutz, PhD Journal of Allergy and Clinical Immunology Volume 141, Issue 4, Pages 1491-1494.e4 (April 2018) DOI: 10.1016/j.jaci.2017.10.034 Copyright © 2017 The Authors Terms and Conditions

Fig 1 Generation of T-cell progenitors from mPB CD34+ HSPCs. A-C, Phenotype, frequency, and cell numbers of day 3 and day 7 DL-4 cultures. D, Quantification of T-cell potential by T-cell differentiation on OP9/DL-1 cells in limiting dilution conditions. E-I, Gene expression profile of day 0 CD34+ cells and day 3 and day 7 CD7+ cells by using quantitative RT-PCR (Fig 1, E-G) or day 3 and day 7 CD7− and CD7+ cells (Fig 1, H and I). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 1491-1494.e4DOI: (10.1016/j.jaci.2017.10.034) Copyright © 2017 The Authors Terms and Conditions

Fig 1 Generation of T-cell progenitors from mPB CD34+ HSPCs. A-C, Phenotype, frequency, and cell numbers of day 3 and day 7 DL-4 cultures. D, Quantification of T-cell potential by T-cell differentiation on OP9/DL-1 cells in limiting dilution conditions. E-I, Gene expression profile of day 0 CD34+ cells and day 3 and day 7 CD7+ cells by using quantitative RT-PCR (Fig 1, E-G) or day 3 and day 7 CD7− and CD7+ cells (Fig 1, H and I). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 1491-1494.e4DOI: (10.1016/j.jaci.2017.10.034) Copyright © 2017 The Authors Terms and Conditions

Fig 2 In vivo reconstitution potential of T-cell precursors derived from mPB. A, Human thymic engraftment evaluated by the frequency of human CD45+ (hCD45) and mouse CD45+ (mCD45) lymphocytes in NSG recipients 6 weeks after transplantation with CD34+ cells before (day 0) or after 3 or 7 days of culture in DL-4 conditions. B, Presence of mature CD4+CD8+ double-positive cells in thymi of recipients transplanted with day 7 DL-4–cultured progenitors. The picture shows a representative photograph of recipient thymi injected with day 7 DL-4 cultured cells and day 0 HSPCs 8 weeks after transplantation. C, Presence of CD3+TCRαβ+ T cells in thymi and spleens of recipients injected with day 7 DL-4 cultured progenitors (gated on hCD45+ cells 8 weeks after transplantation). D, TCRβ VJ diversity in splenocytes from NSG mice transplanted with day 7 DL-4–cultured cells (8 weeks after transplantation). Journal of Allergy and Clinical Immunology 2018 141, 1491-1494.e4DOI: (10.1016/j.jaci.2017.10.034) Copyright © 2017 The Authors Terms and Conditions

Fig E1 Phenotype of adult CD34+ HSPCs exposed to 3 or 7 days of DL-4–Fc fusion protein compared with Fc alone. Journal of Allergy and Clinical Immunology 2018 141, 1491-1494.e4DOI: (10.1016/j.jaci.2017.10.034) Copyright © 2017 The Authors Terms and Conditions

Fig E2 Presence of human CD25+CD4+Foxp3+CD127− regulatory T cells in the spleens of NSG recipients 8 weeks after transplantation of adult day 7 DL-4 precursors. Cells were first gated on 7AAD− live human CD45+ cells (left panel) and then on CD4+CD25+ cells (right panel). Journal of Allergy and Clinical Immunology 2018 141, 1491-1494.e4DOI: (10.1016/j.jaci.2017.10.034) Copyright © 2017 The Authors Terms and Conditions