title: Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry.
abstract: Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of "Treg markers". Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.
authors.affiliation: Department of Clinical Oncology, Leiden University Medical Center (LUMC), Leiden, The Netherlands, s.j.a.m.santegoets@lumc.nl.
authors.forename: Saskia J A M
authors.initials: SJ
authors.lastname: Santegoets
authors.forename: Eveline M
authors.initials: EM
authors.lastname: Dijkgraaf
authors.forename: Alessandra
authors.initials: A
authors.lastname: Battaglia
authors.forename: Philipp
authors.initials: P
authors.lastname: Beckhove
authors.forename: Cedrik M
authors.initials: CM
authors.lastname: Britten
authors.forename: Awen
authors.initials: A
authors.lastname: Gallimore
authors.forename: Andrew
authors.initials: A
authors.lastname: Godkin
authors.forename: Cecile
authors.initials: C
authors.lastname: Gouttefangeas
authors.forename: Tanja D
authors.initials: TD
authors.lastname: de Gruijl
authors.forename: Hans J P M
authors.initials: HJ
authors.lastname: Koenen
authors.forename: Alexander
authors.initials: A
authors.lastname: Scheffold
authors.forename: Ethan M
authors.initials: EM
authors.lastname: Shevach
authors.forename: Janet
authors.initials: J
authors.lastname: Staats
authors.forename: Kjetil
authors.initials: K
authors.lastname: Taskén
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
authors.forename: Judith R
authors.initials: JR
authors.lastname: Kroep
authors.forename: Marij J P
authors.initials: MJ
authors.lastname: Welters
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D032921:Consensus; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007391:International Cooperation; D019394:Ki-67 Antigen / Q000378:metabolism; D008213:Lymphocyte Activation; D015166:Monitoring, Immunologic; D010051:Ovarian Neoplasms / Q000276:immunology*; D012015:Reference Standards; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D019139:Tumor Escape
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D019394:Ki-67 Antigen