Generation of EBV-specific T cells for adoptive immunotherapy: a novel protocol using formalin-fixed stimulator cells to increase biosafety.
Adoptive immunotherapy with in vitro generated Epstein-Barr virus (EBV)-specific T cells is a safe and effective treatment in patients with EBV-related complications after transplantation. More frequent use of EBV-specific T cells is held back by their cost and time-intensive generation under good manufacturing practice (GMP) conditions. Currently, EBV-specific T cells are produced by repetitive stimulation of peripheral blood mononuclear cells with EBV-infected lymphoblastoid cell lines (LCLs), a protocol that requires several open GMP-handling steps. The aim of the present study was to improve T-cell generation under GMP conditions. We introduce a novel generation protocol that replaces repetitive with short-term LCL stimulation of PMBCs. Vital and formalin-fixed LCLs were used to further increase biosafety. Stimulated T cells were selected by the clinically approved cytokine secretion assay followed by nonspecific expansion. Sufficient numbers of EBV-specific T-cell lines were generated with all protocols. Specific recognition and killing of EBV-infected targets was found and was independent of the generation protocol applied. The novel protocol based on formalin-fixed cells, selection, and expansion reduced open GMP-handling steps and increased biosafety. Furthermore, fixation will allow the use of transgenic LCLs (eg, with cytomegalovirus or tumor antigens) and thereby facilitate the generation of antigen-specific T cells directed against pathogens other than EBV.
Interdisciplinary Transplant Laboratory Nephrology/ Medical Immunology, Charité-Universitätsmedizin Berlin, Germany.
Markus H
MH
Hammer
Gordon
G
Brestrich
Alexa
A
Mittenzweig
Andy
A
Roemhild
Sandra
S
Zwinger
Marion
M
Subklewe
Carola
C
Beier
Andreas
A
Kurtz
Nina
N
Babel
Hans-Dieter
HD
Volk
Petra
P
Reinke
D017252:CD3 Complex / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D018920:Coculture Techniques / Q000379:methods; D003601:Cytotoxicity Tests, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000276:immunology; D005557:Formaldehyde / Q000737:chemistry*; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007371:Interferon-gamma / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D009369:Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy; D009842:Oligopeptides / Q000276:immunology; D013601:T-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*; D014763:Viral Matrix Proteins / Q000276:immunology
D017252:CD3 Complex; C508151:EBNA-3A antigen; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D018984:Epitopes, T-Lymphocyte; D019309:Epstein-Barr Virus Nuclear Antigens; D009842:Oligopeptides; D014763:Viral Matrix Proteins; C403195:glycyl-leucyl-cysteinyl-threonyl-leucyl-valyl-alanyl-methionyl-leucine; D005557:Formaldehyde; D007371:Interferon-gamma