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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1729-x) contains supplementary material, which is available to authorized users.

Reduced frequency of circulating CD4+CD25brightCD127lowFOXP3+ regulatory T cells in primary myelofibrosis

“…To determine whether Tregs from patients with PMF maintained the same antiproliferative activity on effector T cells, we cocultured CD4 21 Normal subjects had a median blood Treg frequency of 1.99% (range, 0.48-6.51). In contrast, subjects with PMF had a median Treg frequency of 0.80% (range, 0-10.2; Mann-Whitney U test, P , .001).…”

The blood mMDSC to DC ratio is a sensitive and easy to assess independent predictive factor for epithelial ovarian cancer survival

“…Foxp3 expression within the isolated Tregs was determined after subsequent staining with PE-CF594-labeled Foxp3 monoclonal antibody (clone 259D/C7, BD) using the BD Pharmingen Transcription Factor Buffer set as described before. 45 Gating strategies for FACS sorting, purity check and characteristics of the isolated populations are given in suppl. Fig.…”

Low-dose cyclophosphamide depletes circulating naïve and activated regulatory T cells in malignant pleural mesothelioma patients synergistically treated with dendritic cell-based immunotherapy

“…24 and Santegoets, et al .. 25 Cryopreserved PBMCs were thawed and washed twice in cold PBS. Dead cells were stained using LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen life technologies, Cat# L34957).…”

“…Santegoets et al . 25 showed that the frequency of aTregs and proliferating Ki67 + FoxP3 + CD25 + CD127 low Tregs prior to treatment were associated with worse survival in recurrent ovarium carcinoma patients undergoing chemo-immunotherapeutic treatment, whilst frequencies of classically defined Tregs prior to treatment were not associated with survival. In mesothelioma, Tregs contribute to an impaired T cell function 26,27 and are associated with tumor progression and poor prognosis.…”