title: Detailed kinetics of the direct allo-response in human liver transplant recipients: new insights from an optimized assay.
abstract: Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4+ and CD8+ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4+ and CD8+ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3rd party allo-antigens was observed. One year after LTx numbers of CD4+ and CD8+ T cells reacting to donor antigens, as well as those reacting to 3rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4+ and CD8+ T cells responding to donor-derived, as well as those reacting to 3rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.
authors.affiliation: Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Tapirdamaz
authors.forename: Shanta
authors.initials: S
authors.lastname: Mancham
authors.forename: Luc J W
authors.initials: LJ
authors.lastname: van der Laan
authors.forename: Geert
authors.initials: G
authors.lastname: Kazemier
authors.forename: Kris
authors.initials: K
authors.lastname: Thielemans
authors.forename: Herold J
authors.initials: HJ
authors.lastname: Metselaar
authors.forename: Jaap
authors.initials: J
authors.lastname: Kwekkeboom
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D019013:CD40 Antigens / Q000096:biosynthesis; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002469:Cell Separation; D006801:Humans; D007158:Immunologic Techniques; D007371:Interferon-gamma / Q000378:metabolism; D007519:Isoantigens / Q000737:chemistry*; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000166:cytology; D016031:Liver Transplantation / Q000379:methods*; D051379:Mice; D013154:Spleen / Q000166:cytology; D014184:Transplantation, Homologous / Q000379:methods*
chemical_list: D019013:CD40 Antigens; D007519:Isoantigens; D007371:Interferon-gamma