Out-of-the-Box

Tuesday July 03, 2018 from 16:30 to 17:30

Room: Hall 10 - Exhibition

C494.5 Ex-vivo generation of alloantigen-specific immunomodulatory cells: Elucidating the mechanistical synergy of cell components

Ming Han Yao, Sweden

Training surgeon
Department of Transplantation surgery
Karolinska University Hospital

Abstract

Ex-Vivo Generation of Alloantigen-Specific Immunomodulatory Cells: Elucidating the Mechanistical Synergy of Cell Components

Ming Han Yao1, Makiko Kumagai-Braesch1, Masaaki Watanabe1, Kazuaki Tokodai1, Torbjörn Lundgren1, Michael Uhlin1, Bo Göran Ericzon1.

1Division of Transplantation Surgery, Karolinska Institute, Stockholm, Sweden

Introduction: The efficacy of adoptive transfer of ex-vivo generated Tregs has been attempted to induce operational tolerance in preclinical models and early clinical trials. However, Tregs purification strategies require highly specialized facilities using complicated techniques. Recently, Todo et al. reported that ex-vivo generated immunomodulatory cells without any purification processes induced operational tolerance in 7/10 liver transplanted recipients. In this trial, immunomodulatory cells were ex-vivo generated by coculture of recipient PBMCs with irradiated donor cells in the presence of CD28/B7 co-stimulatory blockade. In terms of the wide spread application of tolerance induction strategies, it is crucial to address which cells are responsible for the donor-antigen specific immunomodulatory effects.
Material and Methods: Immunomodulatory cells were generated by 14 days co-culture of recipient peripheral blood mononuclear cells (PBMCs) (50 x 106 cells) with irradiated HLA-mismatched donor PBMCs (20 x 106 cells) with CTLA4-Ig (40 mg/106 recipient PBMCs). At day 14, the generated cells were collected and enriched for CD4+ CD25+ T cells (Tregs) using MACS, or depleted of CD4+ T cells, CD8+ T cells (MACS), CD19+ (B cells), or (CD25+CD127+lo) Tregs using FACS. The suppressive functions of the generated cells (non-sorted), and each enriched or depleted cells were measured in vitro, i.e. the freshly isolated recipient PBMCs were stimulated by irradiated donor or 3rd party PBMCs, with or without varying numbers of generated cells. The cell proliferation and cytokine production was compared.
Results and Discussion: The addition of non-sorted generated cells significantly inhibited proliferation of responder cells against donor antigen in a generated cell-number dependent fashion (Fig.1). Interestingly, non-sorted cells showed significantly stronger donor antigen-specific immunomodulatory effects than Tregs enriched cells (Fig. 2), indicating that non-T regs in generated cells also play important roles for the immunomodulatory effects. In order to examine the hypothesis, we further depleted specific population from the generated cells. The results indicate  that Treg or B cell depleted generated cells show immunomodulatory effects against both donor and 3rd party stimulations (Fig. 3-4). Interestingly, the depletion of CD4+ or CD8+ T cells from the generated cells maintained the donor-antigen specific inhibitory effects.IFN-g was mainly produced by CD4+ T cells in the generated cells. Depletion of B cells or Treg from the generated cells lower the production of IL-10, indicating these cells are major producer of IL-10.
Conclusion: The ex-vivo generated cells without any purification showed stronger donor antigen-specific immunomodulatory effect. In this ex-vivo generation protocol, Tregs don’t need to be purified. The Tregs and B cells seems to be important in generating the donor antigen-specific immunomodulatory effects. 



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