Mingqian Li1,2, Judith Eckl3,4, Jan-Michael Abicht5,6, Tanja Mayr5,6, Bruno Reichart6, Dolores J Schendel3,4, Heike Pohla1,2,3. 1. Laboratory of Tumor Immunology, LIFE Center, Ludwig-Maximilians-Universität, Munich, Germany. 2. Department of Urology, University Hospital, Ludwig-Maximilians-Universität, Munich, Germany. 3. Institute of Molecular Immunology, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany. 4. Medigene Immunotherapies GmbH, Planegg-Martinsried, Germany. 5. Department of Anaesthesiology, Ludwig-Maximilians-Universität, Munich, Germany. 6. Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität, Munich, Germany.
Abstract
BACKGROUND: Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo-organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen-specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non-human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno-specific Treg would benefit research on immune tolerance in xenotransplantation using this model system. METHOD: Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti-inflammatory cytokines. After loading with porcine-specific (PS) in vitro-transcribed RNA (ivtRNA), tolDC were used to induce CD4+ T cells to become porcine-specific Treg (PSTreg) in cocultures supplemented with IL-2 and rapamycin for 10 days. Anti-inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine-specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity. RESULTS: TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL-10 and TGF-β1, whereas IL-12p40 and IFN-γ were not expressed. PSTreg were successfully generated in cocultures of CD4+ T cells and PS ivtRNA-loaded tolDC. They exhibited a CD3+ CD4+ CD25+ CD127low/- CD45RAlow Foxp3+ phenotype and were characterized by high expression of IL-10 and TGF-β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL-10 and TGF-β1 cytokine upon interaction with PSTeff and suppressing IFN-γ expression on PSTeff. CONCLUSION: In this study, a fast 3-day method to generate baboon-derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine-antigen specificity and expression of IL-10 and TGF-β1. Porcine-specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.
BACKGROUND: Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo-organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen-specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non-human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno-specific Treg would benefit research on immune tolerance in xenotransplantation using this model system. METHOD:Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti-inflammatory cytokines. After loading with porcine-specific (PS) in vitro-transcribed RNA (ivtRNA), tolDC were used to induce CD4+ T cells to become porcine-specific Treg (PSTreg) in cocultures supplemented with IL-2 and rapamycin for 10 days. Anti-inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine-specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity. RESULTS: TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL-10 and TGF-β1, whereas IL-12p40 and IFN-γ were not expressed. PSTreg were successfully generated in cocultures of CD4+ T cells and PS ivtRNA-loaded tolDC. They exhibited a CD3+ CD4+ CD25+ CD127low/- CD45RAlow Foxp3+ phenotype and were characterized by high expression of IL-10 and TGF-β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL-10 and TGF-β1 cytokine upon interaction with PSTeff and suppressing IFN-γ expression on PSTeff. CONCLUSION: In this study, a fast 3-day method to generate baboon-derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine-antigen specificity and expression of IL-10 and TGF-β1. Porcine-specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.
Authors: Amber N Carrier; Anjali Verma; Muhammad Mohiuddin; Manuel Pascual; Yannick D Muller; Alban Longchamp; Chandra Bhati; Leo H Buhler; Daniel G Maluf; Raphael P H Meier Journal: Front Immunol Date: 2022-06-09 Impact factor: 8.786