title: Photodynamic purging of alloreactive T cells for adoptive immunotherapy after haploidentical stem cell transplantation.
abstract: After haploidentical stem cell transplantation immune recovery is inevitably slow and infectious related mortality is about 30-40%. Immune reconstitution could be improved by infusing donor T cells, but the obstacle is graft-versus-host disease. In a mixed lymphocyte reaction, alloantigen-stimulated T cells uptake 4,5-dibromorhodamine methyl ester (TH9402), a compound that is structurally similar to rhodamine. TH9402 preferentially localizes in mitochondria and when exposed to 500- to 600-nm wavelength visible light delivered through the Theralux device (Kiadis Pharma, Amsterdam, The Netherlands), it becomes highly cytotoxic through oxidative damage. This study investigated a range of parameters, and combinations thereof, with the aim of achieving optimal T cell allodepletion and preservation of pathogen-specific responses. We report on 11 clinical scale dry runs which reproducibly yielded the following results. Blood mononuclear cells were stimulated with haploidentical irradiated (20 Gy). Blood mononuclear cells in a mixed lymphocyte reaction. Cells were then incubated with TH9402 and exposed to light delivered through the Theralux device. Optimal conditions for T cell allodepletion emerged as (1) duration of mixed lymphocyte reaction: 24 h; (2) responder cell concentration: 3-5x10(6)/ml; (3) TH9402 concentration: 5 microM; (4) quantity of internalized TH9402, as measured by mean fluorescence intensity (MFI): 20,000-25,000 MFI; (5) energy delivered: 0.1 J/cm(2). Only under these conditions were the frequencies (by limiting dilution analyses) of alloantigen-specific T cells maximally reduced, i.e., 2467+/-639 (mean+/-SD) times, when compared with non-TH9402-treated cells. Pathogen-specific responses to pathogen antigens such as Cytomegalovirus, Adenovirus, Varicella Zoster Virus, Herpes Simplex Virus, Aspergillus fumigatus, Candida albicans, Toxoplasma gondii were retained, although with a 19+/-9.7 times reduction in frequency. This remarkable drop in frequency of alloreactive T cells is expected to allow safe infusion of relatively large numbers of T cells across histocompatibility barriers for adoptive transfer of donor immunity. Consequently, a clinical trial is planned to incorporate infusion of photo-allodepleted donor T cells after haploidentical stem cell transplantation with the aim of decreasing infection-related mortality.
authors.affiliation: Department of Clinical and Experimental Medicine, IRCCS Foundation on Transplantation Biotechnologies, University of Perugia, Via Brunamonti 51, 06123, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Perruccio
authors.forename: Fabiana
authors.initials: F
authors.lastname: Topini
authors.forename: Antonella
authors.initials: A
authors.lastname: Tosti
authors.forename: Alessandra
authors.initials: A
authors.lastname: Carotti
authors.forename: Teresa
authors.initials: T
authors.lastname: Aloisi
authors.forename: Franco
authors.initials: F
authors.lastname: Aversa
authors.forename: Massimo F
authors.initials: MF
authors.lastname: Martelli
authors.forename: Andrea
authors.initials: A
authors.lastname: Velardi
mesh_terms: D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002986:Clinical Trials as Topic; D006239:Haplotypes; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007959:Lymphocyte Culture Test, Mixed; D008212:Lymphocyte Depletion; D017710:Lymphocyte Transfusion / Q000379:methods*; D010777:Photochemistry / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology / Q000637:transplantation*