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.

Purpose Despite aggressive conventional therapy, glioblastoma multiforme (GBM) remains uniformly lethal. Immunotherapy, in which the immune system is harnessed to specifically attack malignant cells, offers a treatment option with less toxicity. The expression of cytomegalovirus (CMV) antigens in GBM presents a unique opportunity to target these viral proteins for tumor immunotherapy. Although the presence of CMV within malignant gliomas has been confirmed by several laboratories, its relevance as an immunological target in GBM has yet to be established. The objective of this study was to explore whether T cells stimulated by CMV pp65 RNA-transfected dendritic cells (DCs) target and eliminate autologous GBM tumor cells in an antigen-specific manner. Experimental Design T cells from patients with GBM were stimulated with autologous DCs pulsed with CMV pp65 RNA, and the function of the effector CMV pp65-specific T cells was measured. Results In this study, we demonstrate the ability to elicit CMV pp65-specific immune responses in vitro using RNA-pulsed autologous DCs generated from patients with newly diagnosed GBM. Importantly, CMV pp65-specific T cells lyse autologous, primary GBM tumor cells in an antigen-specific manner. Moreover, T cells expanded in vitro using DCs pulsed with total tumor RNA demonstrated a 10–20 fold expansion of CMV pp65-specific T cells as assessed by tetramer analysis and recognition and killing of CMV pp65-expressing target cells. Conclusion These data collectively demonstrate that CMV-specific T cells can effectively target glioblastoma tumor cells for immunologic killing and support the rationale for the development of CMV-directed immunotherapy in patients with GBM.

Previous results from two proficiency panels of intracellular cytokine staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling.