OMIP-006: Phenotypic subset analysis of human T regulatory cells via polychromatic flow cytometry

Authors


Abstract

This panel was optimized for the enumeration and phenotypic characterization of T regulatory cells (Tregs) within the CD4+ T-cell pool using human peripheral blood mononuclear cells (PBMC) using intranuclear and intracellular staining methods. The panel was optimized for HIV+ clinical trial specimens through the use of HIV-infected and normal donor PBMC. Because the panel is to be used in the context of testing cryopreserved PBMC obtained from multiple sites participating in clinical trials, it was essential to develop an assay that performed well using cryopreserved PBMC. Other tissue types have not been tested. © 2012 International Society for Advancement of Cytometry.

INTRODUCTION

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

Table 1. Summary table for OMIP-006
PurposeEnumerating and phenotyping of T regulatory cells and subsets
SpeciesHuman
Cell TypesCryopreserved PBMC
Cross referencesNone
Table 2. Reagents used in OMIP-006
SpecificityCloneFluorochromePurpose
  1. V500, Horizon V500; PerCP, peridinin chlorophil protein complex; Cy, cyanin; FITC, fluorescein isothiocyanate; ECD, energy coupled dye; PE, phycoerythrin; vAmine, LIVE/DEAD fixable violet dead cell stain.

CD3UCHT1V500T cell subset
CD4SK3PerCP-Cy5.5 
CD45ROUCHL1FITCMaturation/ Differentiation
CD25B1.49.9ECDT cell activation/ Treg identification
FoxP3PCH101PETreg identification
Helios22F6Alexa647Treg subsets
CD39eBioA1PE-Cy7 
CD49d9F10PE-Cy5 
Dead cellsvAmine (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.

Figure 1.

Example staining and gating for a normal donor. A: Identification of CD4+ FoxP3+ Tregs. After selecting singlets, live cells are selected using FCS-A versus vAmine dead cell exclusion. This gating strategy often results in the least amount of nonspecific staining from debris/dead cells. CD3+ lymphocytes are then derived from CD3+ versus SSC. Tregs are identified via CD25 versus FoxP3, which yields a demarcated Treg cluster through which Treg frequency within CD4+ lymphocytes is best visualized for manual analysis. B: Identification of Treg subsets. Using dot plots and the remaining Treg maturational markers identifies unique clustering within the CD4+ FoxP3+ Tregs. C: Using data derived from Boolean analysis of all Treg subset markers, simplified presentation of incredibly complex evaluations (SPICE) analysis(3) of CD4+ FoxP3+ cells is shown in D). 39 + = CD39+ cells; o + = CD45RO+ cells; d + = CD49+ cells; h + = Helios+ cells.

Similarity to Published OMIPs

This OMIP is similar to OMIP-004 in its objective to phenotype human Tregs.

Acknowledgements

The authors would like to acknowledge Jennifer Enzor, Tao Wang, Jianling Shi, and Twan Weaver of the Duke Center for AIDS Research Flow Cytometry Core, without whose work this panel would not have been possible. They would also like to acknowledge Dr. Cliburn Chan's important contributions to the panel development through innovative mixture modeling techniques. They would also like to thank Dr. Nina Bhardwaj and Sonia Jimenez Baranda of the NYU Langone Medical Center for their collaborative efforts in optimizing the panel for interlaboratory use. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health.

Ancillary