Publication protocol
The cryopreserved cell samples were thawed according to SOPs and as described before [19], and Treg subsets were assessed by flow cytometry staining. To this end, one million PBMCs or ~250,000–750,000 TDLN or tumor sample cells was used per condition. Since it has been described that Foxp3 staining can be highly variable and depend on the choice of antibody (clone), buffer, and/or fluorochrome [22–24] and the performance of a specific antibody is optimized by the manufacturer using their own permeabilization procedures, optimal Foxp3 staining was determined first. We selected four different Foxp3 antibodies on the basis of in-house availability, compatibility with the rest of our panel and with the LSR Fortessa optical configuration, and two different intranuclear staining kits. Optimal staining was determined by the analysis of the percentage of positive cells and at the strength of the positive signal (compared to the negative fluorescence minus one (FMO) signal). Antibodies and intranuclear staining kits used for Foxp3 staining setup were AF700-labeled Foxp3 (clone PCH101, eBiosciences), PE-labeled Foxp3 (clone PCH101, eBiosciences, and clone 206D, R&D systems), PE-CF594-labeled Foxp3 (clone 259D/C7, BD), AmCyan-labeled CD3 (clone SK7, BD), V500-labeled CD3 (clone UCHT1, BD), PE-CF594- or AF700-labeled CD4 (both clone RPA-T4, BD), PE-CY7-labeled CD25 (clone 2A3, BD), BV650-labeled CD127 (clone HIL-7R-M21, BD), the Foxp3/transcription factor staining buffer set (eBiosciences), and the BD Pharmingen Transcription Factor Buffer set (BD). Cell surface antibody staining was performed in PBS/0.5 % BSA/0.02 % sodium azide (PBA) buffer for 30 min at 4 °C. Intranuclear Foxp3 staining was conducted with the BD or eBiosciences Transcription Factor Buffer sets according to the manufacturers’ protocol. Analysis revealed that Foxp3 could be detected with all used clones when using the eBiosciences kit. Yet, staining intensity (and thus discrimination between negative and positive) was lower with the PCH101 clones when compared with the 206D (PE) clone (Supplementary figure 1a–c), which may be due to fluorochrome choice. Staining pattern and positive-to-negative signal ratio [i.e., staining index (SI)] of the 259D/C7 (PE-CF594) clone were most optimal with the BD TF kit (not shown) and were comparable to the staining pattern of the 206D clone using this kit, indicating that both antibodies could be used in our Treg panel (Supplementary figure 1d–f). After selection of the best Foxp3 antibody and intranuclear staining buffer set, all additional antibodies in the final panel were titrated, and spillover profiles were generated to ascertain that there was no spectral overlap of the selected antibodies into the secondary detectors. Optimal antibody concentrations were determined based on the following criteria: (a) frequency and (b) highest SI (positive mean divided by negative mean), and spillover profiles were generated as described by Murdoch et al. [25]. Antibodies and kits used in the final panel were V500-labeled CD3 (clone UCHT1, BD), AF700-labeled CD4 (clone RPA-T4, BD), PE-CY7-labeled CD25 (clone 2A3, BD), BV650-labeled CD127 (clone HIL-7R-M21, BD), APC-H7-labeled CD45RA (clone HI100, BD), PerCP-Cy5.5-labeled CD8 (clone SK1, BD), PE-CF594-labeled Foxp3 (clone 259D/C7, BD), BV421-labeled CTLA-4 (clone BNI3, BD), FITC-labeled Ki67 (clone 20Raj1, eBiosciences), APC-labeled Helios (clone 22F6, Biolegend), PE-labeled CD39 (clone ebioA1, eBiosciences), LIVE-DEAD® Fixable yellow dead cell stain kit (Q-dot585, Life technologies), and the BD Pharmingen Transcription Factor Buffer set. Stained cells were acquired on a LSR Fortessa (BD) and analyzed using DIVA software version 6.2. Events collected were generally >200,000 per sample, except for one tumor-infiltrating lymphocyte (TIL) sample (~35,000 cells). In the latter, still adequate numbers (~400) of Tregs could be detected.
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