title: Quantification of CD4(+) T Cell Alloreactivity and Its Control by Regulatory T Cells Using Time-Lapse Microscopy and Immune Synapse Detection.
abstract: Assays designed to select transplant recipients for immunosuppression withdrawal have met with limited success, perhaps because they measure events downstream of T cell-alloantigen interactions. Using in vitro time-lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD4(+) T cells forming prolonged interactions with donor dendritic cells. By blocking CD4-MHC class II and CD28-B7 interactions, we defined immunologically relevant interactions as those ≥500 s. Using this threshold, T cell-dendritic cell (T-DC) interactions were examined in rejection, tolerance and T cell control mediated by regulatory T cells. The frequency of T-DC contacts ≥500 s increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. Regulatory T cells reduced prolonged T-DC contacts. Importantly, this effect was replicated with human polyclonally expanded naturally occurring regulatory T cells, which we have previously shown can control rejection of human tissues in humanized mouse models. Finally, in a proof-of-concept translational context, we were able to visualize differential allogeneic immune synapse formation in polyclonal CD4(+) T cells using high-throughput imaging flow cytometry.
authors.affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK.|Toronto Lung Transplant Program and Division of Respirology, Department of Medicine, University Health Network and University of Toronto, Toronto, Ontario, Canada.
authors.forename: S C
authors.initials: SC
authors.lastname: Juvet
authors.affiliation: NIHR BRC Translational Immunology Laboratory, Nuffield Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: S
authors.initials: S
authors.lastname: Sanderson
authors.affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: J
authors.initials: J
authors.lastname: Hester
authors.affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: K J
authors.initials: KJ
authors.lastname: Wood
authors.affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: A
authors.initials: A
authors.lastname: Bushell
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D003713:Dendritic Cells / Q000276:immunology; D006084:Graft Rejection / Q000175:diagnosis* / Q000209:etiology; D006085:Graft Survival / Q000276:immunology; D016027:Heart Transplantation / Q000009:adverse effects*; D007108:Immune Tolerance / Q000276:immunology; D054992:Immunological Synapses / Q000276:immunology*; D007519:Isoantigens / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D059008:Time-Lapse Imaging / Q000379:methods*
chemical_list: D007519:Isoantigens