In recent years, perhaps no area of modern immunology has received as much attention as the regulatory T cell (Treg). Tregs constitute a small subset of lymphocytes, and are thought to predominantly exist within the CD4+ lymphocyte population, representing 5–10% of CD4+ T cells and 1–5% of all lymphocytes. Initially described and studied as CD4+ CD25+ T cells, Treg identification was advanced by the use of antibody to the forkhead box protein (FoxP3), a relatively specific marker for Tregs. Since then, Treg immunology has rapidly expanded with the description of distinct Treg subsets capable of differing functions (1, 2). Thus, considerable interest exists in phenotyping and enumerating Tregs in a variety of human diseases.
To date, Treg assays have routinely included a highly subjective analysis method for CD25hi gating. The existence of various subsets of Tregs combined with the highly subjective analysis method of CD25hi gating makes the historical analysis of Tregs difficult to measure accurately in the context of clinical trials, where assay reproducibility is critical to interpretation of the results. Therefore, we employed an approach that addressed both specific subsets of Tregs as well as instituted highly standardized methods for data analysis that circumvent CD25hi gating.
Markers for the Treg panel were evaluated based on applicability to the overall project goals (Table 1). First, since FoxP3+ cells are relatively infrequent in cryopreserved PBMC, a viable dye was necessary. Second, basic gate markers include CD3 to identify T-cells, CD4 to identify T-helper cells, as well as FoxP3, and CD25 for gating Tregs. Last, specific Treg markers were evaluated and selected based on the ability of each marker to add information to the panel by further classifying Tregs into subsets (see Table 2 and Supporting Information Table 3). To facilitate the application of this Treg panel across laboratories and studies, only commercially available reagents were used in constructing the panel. All mAbs were tittered for optimal staining and minimal spillover into neighboring detectors (see Supporting Information Fig. 1). Importantly, some Treg markers of interest required abbreviated “in-panel” titration method to optimally determine mAb concentration and in-panel performance (see Supporting Information Figs. 1 and 2). To measure the degree of spillover for each reagent, we employed a novel method called Spillover Profile and Assessment (see Supporting Information).1
|Purpose||Enumerating and phenotyping of T regulatory cells and subsets|
|Cell Types||Cryopreserved PBMC|
|CD3||UCHT1||V500||T cell subset|
|CD25||B1.49.9||ECD||T cell activation/ Treg identification|
|Dead cells||–||vAmine (ViViD)||Exclusion|
Optimal intranuclear staining for FoxP3 required a thorough evaluation of conjugates, clones, and methods. We identified optimal FoxP3 staining as follows: use of eBioscience Fix/Perm for intranuclear FoxP3 staining, use of PE-conjugated FoxP3 clone PCH101 and PE-conjugated isotype, and reduction of FoxP3 PE background by adding a blocking step prior to the FoxP3 staining as well as adding additional washes pre- and postintranuclear staining (see Supporting Information Fig. 7).
The Treg assay requires a number of staining and biological controls. Staining controls are employed for all Treg-specific markers as follows: FMO controls for CD25, CD39, CD45RO, CD49d, and Helios and a PE-conjugated isotype gating control for FoxP3. Methodological improvements combined with the gating control were optimal for the FoxP3 signal. For sample testing, a normal donor biological control was employed across all testing (see Supporting Information Fig. 8).
During panel development, reagent titrations, spillover assessments, and full panel performance were all tested using consistent numbers of total cells (2 × 106 per test), total staining volume of 200 μL, and all staining was performed on ice. A lysing agent was added to remove any residual RBCs and an additional wash step was included following intracellular staining. There were no further deviations from the eBioscience Fix/Perm procedure.
The sequence of gates and combination of dot plots used in the Treg panel gating strategy reflect several analysis exercises designed to identify a manual gating method that yielded the least amount of background and optimal FoxP3 discrimination for positive and negative events following procedures outlined in Figure 1A. Manual gating and Boolean analysis of Treg subsets are presented in Figure 1B and 1C. Subsequent analysis of Treg markers and potential Treg subsets may be performed employing Boolean gating and simplified presentation of incredibly complex evaluations (SPICE) analysis(3) (see Supporting Information). Further examples of gating analysis and the use of the optimized Treg staining panel and biologic controls are presented in Supporting Information Figures 8 and 9.
Similarity to Published OMIPs
This OMIP is similar to OMIP-004 in its objective to phenotype human Tregs.