Simrit Parmar1, Xiaoying Liu2, Shawndeep S Tung2, Simon N Robinson2, Gabriel Rodriguez2, Laurence J N Cooper3, Hui Yang4, Nina Shah2, Hong Yang2, Marina Konopleva4, Jeffery J Molldrem2, Guillermo Garcia-Manero4, Amer Najjar5, Eric Yvon2, Ian McNiece2, Katy Rezvani2, Barbara Savoldo6, Catherine M Bollard7, Elizabeth J Shpall2. 1. Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA. Electronic address: sparmar@mdanderson.org. 2. Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA. 3. Division of Pediatrics, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA. 4. Department of Leukemia, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA. 5. Department of Experimental Diagnostic Imaging, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA. 6. Department of Pediatrics and Immunology, Baylor College of Medicine, Houston, Texas, USA. 7. Department of Pediatrics, Medicine and Immunology, Baylor College of Medicine, Houston, Texas, USA.
Abstract
BACKGROUND AIMS: Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1-5% of total nucleated cells in cord blood (CB) (<3 × 10⁶ cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed. METHODS: Several methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model. RESULTS: With the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⁺ cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo-expanded Treg were CD4⁺25⁺ FOXP3⁺127(lo) and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4⁺25⁻ cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro. CONCLUSIONS: In our NOD-SCID IL-2Rγ(null) xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo-expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.
BACKGROUND AIMS: Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1-5% of total nucleated cells in cord blood (CB) (<3 × 10⁶ cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed. METHODS: Several methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHDmouse model. RESULTS: With the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⁺ cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo-expanded Treg were CD4⁺25⁺ FOXP3⁺127(lo) and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4⁺25⁻ cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro. CONCLUSIONS: In our NOD-SCID IL-2Rγ(null) xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo-expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.
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