title: Optimization of methodology for production of CD25/CD71 allodepleted donor T cells for clinical use.
abstract: OBJECTIVE
  Immunotherapy with allodepleted donor T cells improves immunity after T cell-depleted hematopoietic stem cell transplantation. We developed a methodology for selective depletion of alloreactive T cells after activation with host antigen-presenting cells by targeting T cells up-regulating CD25 and CD71. Combined depletion of these cells yields a pool of allodepleted donor T cells with antiviral properties with minimal capacity to cause graft-versus-host disease.
  
  
  METHODS
  Mature dendritic cells were irradiated and used to stimulate donor peripheral blood mononuclear cells for 4 days. The co-culture was stained with anti-CD71-biotin followed by CliniMACS CD25 and Anti-Biotin Reagents (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany) before depletion on the CliniMACS Plus (Miltenyi Biotec GmbH). Residual alloreactivity was tested by flow cytometry, a secondary mixed lymphocyte reaction and limiting dilution analysis, and specific anti-viral immunity with pentamer staining. The large-scale protocol was tested under current good manufacturing practice conditions in five donor-recipient pairs of human leukocyte antigen-matched volunteer donors.
  
  
  RESULTS
  We developed a closed-system methodology using cell differentiation bags for cell culture and the COBE2991 Cell Processor (CaridianBCT, Lakewood, CO, USA). We also validated an anti-CD71-biotin generated for ex vivo clinical use. In five large-scale runs, the depleted fraction demonstrated excellent viability (99.9%), minimal residual expression of CD3/CD25 and CD3/CD71 (<0.2%) and passed tests for Mycoplasma, endotoxin, bacterial and fungal sterility. In secondary mixed lymphocyte reaction assays, the median response to host after allodepletion was 0%, whereas responses to third-party peripheral blood mononuclear cells were preserved (median, 105%; range 37%-350%). Limiting dilution analysis assays also demonstrated a reduction in response to host (median, -1.11 log) with preservation of third-party responses, and testing with human leukocyte antigen-restricted pentamers showed that populations of Epstein-Barr virus-specific and cytomegalovirus-specific CD8(+) T cells were retained after depletion.
  
  
  CONCLUSIONS
  We optimized a protocol for the combined immunomagnetic depletion of alloreactive CD25/CD71 T cells under current good manufacturing practice conditions and tested the efficacy in five donor-recipient pairs.
authors.affiliation: Molecular Immunology Unit, UCL Institute of Child Health, London, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Albon
authors.forename: Christoph
authors.initials: C
authors.lastname: Mancao
authors.forename: Kimberly
authors.initials: K
authors.lastname: Gilmour
authors.forename: Geoffrey
authors.initials: G
authors.lastname: White
authors.forename: Ida
authors.initials: I
authors.lastname: Ricciardelli
authors.forename: Jennifer
authors.initials: J
authors.lastname: Brewin
authors.forename: Gertjan
authors.initials: G
authors.lastname: Lugthart
authors.forename: Rebecca
authors.initials: R
authors.lastname: Wallace
authors.forename: Persis J
authors.initials: PJ
authors.lastname: Amrolia
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000276:immunology*; D005434:Flow Cytometry; D006086:Graft vs Host Disease / Q000276:immunology; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008212:Lymphocyte Depletion; D011990:Receptors, Transferrin / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; C068126:CD71 antigen; D053645:Interleukin-2 Receptor alpha Subunit; D011990:Receptors, Transferrin