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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Cytometry A. 2010 Jun;77(6):571–579. doi: 10.1002/cyto.a.20875

Association of two clones allows for optimal detection of human FOXP3

Pietro Presicce 1, Maria E Moreno-Fernandez 1, Celine S Lages 1, Kris I Orsborn 1, Claire A Chougnet 1
PMCID: PMC2903740  NIHMSID: NIHMS215034  PMID: 20162533

Abstract

FOXP3 is a key transcription factor expressed by regulatory T cells (Treg cells). However, differences in staining and analysis protocols have led to conflicting results. Moreover, the transient upregulation of FOXP3 that follows activation in non-Treg cells renders the interpretation of FOXP3 data more difficult in humans than in mice.

Human Peripheral Blood Mononuclear Cells (PBMCs), isolated CD25− or CD25+CD4+ T cells were stained with three different anti-FOXP3 clones (PCH101, 206D and 259D) alone or in combination, and using different permeabilization methods. FOXP3 expression was evaluated following T cell activation by several pathways.

Gating based on a population that did not express FOXP3 (such as CD3−CD4− T cells) allowed for the optimal characterization of Treg cells. The 206D clone detected a lower percentage of cells than PCH101 or 259D. In contrast, 259D stained a population of activated T cells that PCH101 did not. Staining with two clones together consistently increased the proportion of FOXP3+ cells. However, it is likely that only the double positive cells are Treg cells, as they expressed the highest CD25 and lowest CD127 levels.

Our results emphasize that the choice of staining protocol leads to very different results concerning the frequency of Treg cells in humans. A more consistent identification of these cells will improve the knowledge of their biology, particularly during disease processes.

Key terms: FOXP3, Treg cells, PCH101, 259D, 206D

INTRODUCTION

Regulatory T cells (Treg cells) play a major role in the homeostasis of the immune system (1–5). Treg cells are a subpopulation of CD4+ T cells that represent approximately 1–10 % of circulating CD4+ T cells (6). They were first characterized by the constitutive expression of the IL-2Rα chain (CD25) (1). More recently, it was shown that they usually express low levels of the IL-7Rα chain (CD127) (7,8). The transcriptional factor FOXP3 (Forkhead box P3) plays a crucial role in Treg differentiation, function and biology in both mice and humans (9–13). However, because FOXP3 is an intracellular protein, surrogate surface markers must be used to purify Treg cells. Additional markers are frequently associated with human Treg function, including cytotoxic T lymphocyte associated antigen (CTLA-4) (14), L-selectin (CD62L) (15), αE-integrin (CD103) (16,17), and the glucocorticoid-induced tumor necrosis factor receptor (GITR) (18). However, none of these markers are selectively expressed by Treg cells, as they are also transiently up-regulated in recently activated effector T cells (Teffs) (18–21).

The first commercially available monoclonal antibody (mAb) for studies of FOXP3 in human Treg cells (hFOXY) was quickly supplanted by the more specific mouse mAbs 206D, 236A and 259D (22) and the rat anti-FOXP3 PCH101 clone (eBioscience) in flow-cytometry applications. PCH101 targets the N-terminal region of the 431 amino acid FOXP3 protein, while 206D, 236A, and 259D bind en epitope within the remaining N-terminus (aa 105–235), near the zinc finger region of FOXP3 (23). All four of these antibodies recognize both full-length and alternatively spliced human FOXP3 (13,24). Recent studies reported discrepant findings about FOXP3 expression in unstimulated and activated human T cells. These differences could come from the use of different anti-FOXP3 clones (25–29), different methods of cell permeabilization (25,30) and/or the different gating strategies used to identify FOXP3+ cells (26,28,31).

For this purpose, we compared the proportion of FOXP3+ cells detected when stained with different anti-FOXP3 clones, conjugated with different fluorochromes, used alone or combined. We analyzed FOXP3 expression in both unstimulated and activated PBMCs, as well as in CD4+ T cells purified by magnetic bead negative selection or sorted Treg cells. We also determined the optimal gating strategy.

MATERIALS AND METHODS

Cell isolation

Blood samples from healthy subjects were obtained from the Hoxworth Blood Bank Center (Cincinnati, OH). PBMCs were isolated using gradient centrifugation (Ficoll–Hypaque, GE Healthcare, Piscataway, NJ). CD4+ T cells were purified by negative selection using the CD4+ T cell Isolation Kit II (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions, resulting in a 95% purity of the selected cells, as determined by post purification flow cytometry analysis.

To enrich Treg cells (CD4+CD25+ cells) or non-Treg cells (CD4+CD25- cells), purified CD4+ T cells were selected according to their expression of CD25 with anti-CD25 beads (Miltenyi Biotec), according to the manufacturer’s instruction.

To obtain a highly purified population of Treg cells, bead-selected CD4+ T cells were stained with anti-CD8 FITC-, anti-CD25 APC- and anti-CD127 PE-conjugated mAbs, and sorted using a FACS Vantage™ (BD Bioscience, San Diego, CA). Treg cells were defined as CD25hiCD127lowCD8− and Teff cells as CD25−CD127hi CD8−.

CD14+ monocytes were purified by positive selection using CD14+ MicroBeads (Miltenyi Biotec), according to the manufacturer’s instruction.

Cell culture

RPMI-1640 supplemented with L-glutamine, penicillin and streptomycin, 10mM HEPES and 10% fetal calf serum (Life Technologies, Carlsbad, CA,) was used in all experiments. Cells were stimulated in three different ways to examine the effect of activation on FOXP3 expression. PBMCs (1×106 cells/well) were incubated for 3 and 5 days in the presence of 2 μg/ml phytohemagglutinin (PHA). CD4+ T cells (2×106 cells/well) were stimulated with anti-CD3/CD28 beads (3 beads/cell; Invitrogen, Carlsbad, CA) + IL-2 (100 U/ml; Hoffman-LaRoche, NIH AIDS Research and Reference Program, Germantown, PA) for 3 and 5 days. CD4+ T cells (2×106 cells/well) were also cultured in the presence of allogeneic monocytes (4×105 cells/well. Ratio 1 monocyte: 5 T cells) for 3 and 5 days. For the cytokines (IL-17A and TNF-α) with FOXP3 studies, PBMCs were stimulated with 50 ng/ml Phorbol Myristate Acetate (PMA; Sigma-Aldrich, Saint Louis, MO) and 750 ng/ml ionomycin (Calbiochem, San Diego, CA) for 5 hours. 10μg/ml brefeldin A (BFA; Sigma-Aldrich) and 1μl/ml monensin (eBioscience) were added for the last 4 hours.

Antibodies

Anti-CD3 (SK7) Peridin Chlorophyll Protein Cyanin 5.5- (PerCPCy5.5), anti-CD4 (RPA-T4) Alexa Fluor 700- (AF700), anti-CD8 (RPA-T8) Fluorescein isothiocyanate- (FITC), anti-CD25 (MA25) Allophicocyanin- (APC), anti-CD25 (M-A25) Allophicocyanin-H7-(APC-H7) or Phycoerythrin-Cyanin7- (PE-Cy7) and anti-TNF-α (MAB11) Phycoerythrin- (PE) conjugated mAbs were obtained from BD Biosciences (San Diego, CA). Anti-CD127 (R34.34) PE-conjugated mAb was obtained from Beckman Coulter (Miami, FL). Anti-IL17A (eBio64DEC17) PE-, anti-CD127 (eBioRDR5) FITC-, anti-FOXP3 (PCH101) FITC-, PE-, Pacific Blue- (PB), Alexa Fluor 647- (AF647) and rat IgG2a, k FITC-, PE-, PB-, AF647-conjugated mAbs were obtained from eBioscience (San Diego, CA). Anti-FOXP3 (259D) AF647-, anti-FOXP3 (206D) AF647-, mouse IgG2a, k isotype control (MOCP-21) AF647-, anti-FOXP3 (206D) PB- and mouse IgG2a, k isotype control (MOCP-21) PB-conjugated mAbs were obtained from BioLegend (San Diego, CA). All antibodies had previously been titrated for optimal detection of positive populations and arithmetic mean fluorescent intensity (MFI). All isotype control mAbs were used at the same concentration as the corresponding anti-FOXP3 mAbs.

Immunostaining

For FOXP3 staining, 0.5×106 cells were incubated with 20 μg/ml of human IgG to block Fc receptors, and stained for surface markers for 30 min at 4°C, in PBS containing 2% fetal calf serum and 0.1% sodium azide (FACS Buffer). Cells were then washed and fixed with Fixation/Permeabilization Buffer (eBioscience), indicated as buffer#1 in the text, following manufacturer’s instructions for 30 min at 4 °C. Alternatively, they were fixed with 2% formaldehyde (Fisher Scientific, Pittsburgh, PA) for 30 min at 4°C and permeabilised for 30 min at 4°C using different types of buffer, as detailed in the Results section. After 15 min of preincubation with rat or mouse serum, depending on the clone used, cells were stained by FOXP3 mAbs for 30 min at 4°C. To detect IL-17A and TNF-a production, Cytofix/Cytoperm (BD) or lab-derived fix/perm were used.

Calibration and compensation

Instrument setup was standardized to reduce experiment to experiment variation. Cytometer Settings & Tracking (CST) beads (Becton Dickinson) were run daily at the preoptimized detector PMT voltages to ensure that the predetermined baseline PMT voltages were appropriately set and that predetermined target channels were met.

Compensation for 6 and 8 color stain sets was accomplished based on either antibody-capture beads (CompBeads, BD) (32,33) or PBMCs stained with the above described antibodies. Before experiments, PMT values were adjusted running unstained cells to exclude autofluorescence and to control the intensity of background. Then, single-stained cells with different mAbs used in the study were run to ensure that each stain was the brightest in its own channel before acquisition of 10,000 events per tube.

Manual compensation was used, as it allowed for an optimal adjustment of the spectral overlap between the different fluorochromes otherwise not allowed by software compensation (Supplementary Figure 1).

Acquisition

After washing in FACS buffer, cells were analyzed using a flow cytometry LSRII™ with FACS DIVA™ (Becton Dickinson) or Flowjo software. 150,000 events/tube were routinely collected. Dead cells were excluded by analyzing forward scatter (FSC) vs. side scatter (SSC) dot plots and using Live/Dead Fixable Dead Cell Stain kit (Invitrogen). Doublets were excluded by FSC-H vs., FSC-A dot plots.

Statistical analysis

The statistical significance of differences between the groups was determined by paired t-test. P values lower than 0.05 were considered significant.

RESULTS

The use of CD3+CD4− T cells as reference allows the optimal detection of FOXP3+ cells

Defining a positive population when phenotyping human cells always constitutes a challenge. The most commonly used strategy is to use an isotype matched irrelevant control or a Fluorescence Minus One (FMO), a staining control that combines all reagents except the one of interest. The second strategy is to define the positivity in comparison to a negative biological population, i.e. a cell population that has been reported to not express the marker of interest.

The choice of reference is particularly crucial when rare cell subsets such as Treg cells are analyzed. Recent studies have reported discrepant results about the percentage of FOXP3+ cells, so we compared three approaches to optimize our gating strategy for FOXP3+CD4+ T cells: 1) matched isotype control mAbs for FOXP3; 2) CD3+CD4−(mainly CD8+ T cells); and 3) CD3−CD4− cells (mainly B cells), which are thought to not express FOXP3. We also tested whether these gating strategies gave consistent results when different anti-FOXP3 mAbs labeled with different fluorochromes were used For all experiments, cell viability was checked either by trypan blue exclusion test or by fixable viability dyes and was consistently higher than 95%.

We first defined the lymphocyte region on the basis of their size (FSC) and internal complexity (SSC), excluding monocytes and debris. Moreover, doublets were excluded by FSC-H vs. FSC-A dot plots. Second, we created a FOXP3+ gate within the lymphocyte region using the populations described above, by excluding 97% of the chosen negative population (outer line of a 3% contour plot). We chose a cutoff of 97% because it provided the highest level of consistency from one experiment to another (31). The percentage of FOXP3+ cells in the FOXP3− population was consistently less than 0.8% (data not shown). A higher percentage of FOXP3+CD4+ T cells was observed if either the CD3+CD4− CD3−CD4− population was used as negative reference than when an isotype control was used, as shown in Figure 1A for PE-conjugated PCH101. Similar results were obtained with PB- or AF647-conjugated PCH101 (Figure 1B).

Figure 1.

Figure 1

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Use of a biologically negative FOXP3 population allows for a better characterization of FOXP3+ cells than isotype control. A. Percentage represents FOXP3+ cells stained by PE-conjugated anti-FOXP3 clone PCH101. Bold, solid and dashed arrows indicate the percentage of FOXP3+ cells, after gating on CD3+CD4−, CD3−CD4− T cells or isotype control, respectively. Data from one of five representative experiments are shown. B. Similar analysis was performed with PCH101 labeled with either PB, AF647 or FITC. Data shown come from the same cells as those shown in A. C. Gating based on Fluorescence Minus One (FMO) control was compared with gating using CD3+CD4− or CD3− CD4− T cells as negative population. Bold, solid and dashed arrows indicate the percentage of FOXP3+ cells gating on CD3+CD4−, CD3−CD4− T cells or FMO control, respectively, after staining with AF647-conjugated PCH101. Data from one experiment are shown, representative of data obtained with cells from five donors, stained with three different clones conjugated with several fluorochromes.

Our results also showed that, for this particular application, gating based on FMO data overestimated FOXP3+ cells using AF647-conjugated PCH101 (Figure 1C). Similar results were obtained with PE- or PB-conjugated PCH101 (data not shown). Thus, we used the CD3+CD4− T cell population to define the FOXP3+ population in all subsequent experiments.

The choice of optimal fluorochrome depends on the application

The FITC-conjugated PCH101 was consistently the least sensitive of all forms of PCH101 we tested and the proportion of FOXP3+ cells was substantially overestimated when the FITC-conjugated isotype control was used (Figure 1B). The latter finding is in agreement with results recently shown by Law et al. (34). In addition, staining with FITC-conjugated PCH101 did not clearly differentiate a positive population within PBMCs, leading to an underestimation of the frequency of FOXP3+ cells. However, FITC-conjugated PCH101 clearly detected FOXP3+ cells in sorted Treg cells and could therefore be used for this particular application (Figure 2).

Figure 2.

Figure 2

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FITC-conjugated anti-FOXP3 mAb allows a clearly detection of a FOXP3+ population post-sorting. CD4+ T cells were stained with anti-CD127 PE- and anti-CD25 APC-conjugated. A. CD25hiCD127low Treg cells and CD25lowCD127hi effector T cells (Teff) subsets were identified as shown. B. sorted Treg and Teff cells were then stained with FITC-conjugated PCH101 to confirm their phenotype and analyzed by flow cytometry. Data from one representative experiment are shown (n=20).

Buffer#1 allows for optimal FOXP3 detection

We also compared different methods of cell fixation/permeabilization (fix/perm). The highest percentage of FOXP3+ cells was detected when PBMCs were stained using the buffer#1, compared to any other buffer tested (Table 1). Consistent with previous experiments, the percentage of cells expressing IL-17 and TNF-α in unstimulated cells was consistently lower than 0.01% (data not shown). Our results show that buffer#1 allowed for a better detection of FOXP3 in conjunction with cytokines (IL-17A and TNF-a) in stimulated PBMCs than other fix/perm methods. However, it must be noted that PMA/ionomycin stimulation down-regulated FOXP3 expression. Law et al. recently reported similar data, i.e. that PCH101 clone used with buffer#1 led to the highest detection of FOXP3+ cells (34). Therefore, such buffer was used in all subsequent experiments.

Table 1.

The use of commercial buffer#1 allows detecting the highest percentage of FOXP3+ cells.

METHOD Unstimulated Stimulated
FOXP3 FOXP3 IL-17 TNF-α
Buffer#1 2.36 1.17 0.16 19.87
 2% formaldehyde (methanol free) + permeabilization buffer from buffer#1 0.04 0.38 0.25 11.57
Cytofix/Cytoperm buffer (BD) 0.34 0.10 0.25 16.66
2% formaldehyde + Saponin 0.03% 0.06 0.13 0.26 15.85
2% formaldehyde + Saponin 1% 0.03 0.03 0.22 17.51
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Unstimulated or stimulated PBMCs with PMA/ionomycin were stained with anti-CD3 PerCP-Cy5.5-, anti-CD4 AF700-, anti-IL-17 PE-, anti-TNF-α PE- and anti-FOXP3 (PCH101) AF647-conjugated. The numbers represent the percentage of FOXP3+, IL-17+ or TNF-α+ cells in CD3+CD4+ T cells. In unstimulated PBMCs, less than 0.1% of cells were expressing IL-17 and TNF-α (data not shown). Data are representative of two independent experiments.

Compensation based on CompBeads or cells provides similar results

The use of beads to compensate not only allows for a clear separation of a positive and a negative population necessary for optimal compensation, but also saves cells, an important point when the size of the samples is limiting, such as in HIV-infected patients, pediatric samples or tissue biopsies. However, whether similar results are obtained using these two methods of compensation needs to be determined. We first determined the optimal photomultiplier tube (PMT) settings using primary cells, because these levels cannot be determined using an artificial system such as beads. No difference in the percentage of FOXP3+ cells was found when compensation was based on CompBeads or cells, whatever the fluorochrome or the clone used (Supplementary figure 2). Thus, beads were used for compensation in all subsequent experiments.

PCH101 and 259D detect the highest percentage of FOXP3+ cells of any single antibody in unstimulated PBMCs, but a combination of two clones detects more FOXP3+ cells than PCH101 alone

To determine the anti-FOXP3 clone with the optimal balance of specificity and sensitivity, we compared the staining provided by PCH101, 206D and 259D, using the same PBMCs and the same fluorochrome. Our results show that PCH101 and 259D detected a similar percentage of FOXP3+ cells. In contrast, 206D consistently detected the lowest percentage of FOXP3+ cells (Figure 3), as shown also by Law et al. (34).

Figure 3.

Figure 3

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206D is the least sensitive clone to detect FOXP3+ cells. Unstimulated PBMCs were stained with anti-CD3 PerCP-Cy5.5-, anti-CD4 AF700-, Live/Dead Fixable Dead Cell Stain kit PE-Texas Red-, anti-CD127 FITC-, anti-CD25 APC-H7- and with 206D AF647- or PCH101 AF647- or 259D AF47-conjugated. Data show mean±SE from three different donors. P values correspond to paired t-tests.

PCH101 binds an epitope within N-terminal region on the FOXP3 protein (aminocids 1-50), while 206D and 259D recognize human FOXP3 epitopes in the region of amino acids 105–235 (23). For this reason, we also investigated whether staining cells with 2 clones used together would increase the sensitivity of the technique, or conversely, whether the different clones would interfere with each other. Figure 4A shows that the percentage of PCH101+ cells was similar when PCH101 was used either alone or in association with another anti-FOXP3 clone, suggesting that there was no staining interference. PE-conjugated PCH101 was the most sensitive clone to detect FOXP3+ cells in unstimulated PBMCs when used in combination with another clone (Figure 4B). However, the use of 206D or 259D in combination with PCH101 allowed detection of a minor FOXP3+ population otherwise lost by staining with PCH101 alone. In contrast, the combined staining by 206D and 259D did not detect as many FOXP3+ cells as PCH101 used alone (Figure 4B).

Figure 4.

Figure 4

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The combination of two clones allows for a recognition of a FOXP3+ population not detected when each clone is used singularly. A. Percentage of PCH101+ cells was similar when PCH101 was used alone or in association with another anti-FOXP3 clone. B. The simultaneous use of 206D or 259D with PCH101 reveals a minor FOXP3+ population, unstained by PCH101. The percentage of FOXP3 + cells detected by the combination of 206D and 259D is lower than that detected by each clone used in combination with PCH101. Data from one representative experiment are shown (n=3).

The combination of two clones allows for a better definition of the Treg cell population in unstimulated PBMCs

Treg cells express high levels of CD25 and low levels of CD127 (7,8). To better characterize the FOXP3+ cells detected by the three different clones, CD25 and CD127 levels were measured in FOXP3+ PBMCs. Our results show that the cells stained by two anti-FOXP3 clones had higher CD25 expression and lower CD127 expression than cells stained by only one clone, whatever the combination of clones used (Table 2). Therefore, combining two clones increased the chance of optimally detecting Treg cells. Inclusion of PCH101 in this combination increased the percentage of double positive (DP) cells detected (Table 2).

Table 2.

FOXP3+ cells detected by the association of two clones exhibit the highest CD25 MFI and the lowest CD127 MFI.

combination % of positive cells MFI in FOXP3+ cells
CD127 CD25
259D+206D 259D+ 3±0.5 133±2 723±16
206D+ 3.7±0.5 134±1 675±10
DP 2.3±0.5 103±3 764±13
DN - 487±3 463±28
PCH101+259D PCH101+ 4.7±0.5 101±10 480±7
259D+ 3.3±0.5 71±12 514±9
DP 2.6±0.7 66±13 540±9
DN - 473±2 91±5
PCH101+206D PCH101+ 6±0.4 67±16 591±11
206D+ 3.9±0.3 60±18 597±6
DP 3.5±0.4 21±33 679±9
DN - 496±3 138±10
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Unstimulated PBMCs were stained with anti-CD3 PerCP-Cy5.5-, anti-CD4 AF700-, anti-CD25 APC-H7-, anti-CD127 FITC- and anti-FOXP3 (PCH101 PE-, 206D PB- or 259D AF647-conjugated). CD25 and CD127 MFI were determined in gated FOXP3+CD4+CD3+ T cells. Percentage of cells labeled by 259D (259D+), 206D (206D+) or PCH101 (PCH101+) alone, or by two clones together (double positive “DP”) are indicated in the column “% of positive cells”. Results show the mean±SE of 5 donors DP double positive cells; DN double negative cells

Clone 259D stains a FOXP3+ population that is not detected by clone PCH101 in activated CD4+ T cells

It has been shown that human CD4+CD25− T cells upregulate FOXP3 expression upon activation. However, this upregulation is transient in comparison with FOXP3 expression in natural Treg cells (35–37). Different methods of activation might influence FOXP3 structure and therefore its detection by the different clones. As 206D was the least sensitive clone to detect FOXP3+ cells in unstimulated cells, we decided to use only 259D and PCH101 to measure FOXP3 expression upon activation. Several methods of activation were used and cells were stained at 3 and 5 days post activation. Cell activation did not affect the gating strategy as the percentage of CD3+CD4− T cells or monocytes used to define the FOXP3+ gate was similar (data not shown).

In PHA-activated PBMCs, the percentage of CD4+FOXP3+ cells detected by clone 259D was higher than that detected by clone PCH101, at both 3 or 5 days post activation (10.8% vs. 6.0% after 3 days of stimulation; 8.0% vs. 5.9% after 5 days of stimulation) (Figure 5A). A similar percentage of FOXP3+ cells was observed in purified CD4+ cells stimulated with anti-CD3/CD28 beads and IL-2 for 3 days, regardless of the clone used. However, after 5 days of stimulation, the percentage of PCH101+ cells declined, while the percentage of 259D+ cells remained stable (Figure 5A). Allogeneic stimulation of purified CD4+ cells led to a modest increase of 259D+ cells, but not of PCH101+ cells, at day 5. When used alone, 259D also detected a higher percentage of FOX3+ cells in unstimulated cells than PCH101 cultured for 3 or 5 days, although the percentage of 259D+ and of PCH101+ cells was higher after 5 days than after 3 days (Figure 5A). Consistent with these results, when the two clones were used together to stain stimulated cells, 259D detected a higher percentage of FOXP3+ cells than PCH101, and detected a FOXP3+ population not detected by PCH101 in all cases except after 3 days of stimulation with allogeneic monocytes (Figure 5B). 259D used with PCH101 also detected a higher percentage of FOXP3+ cells at day 5 in unstimulated PBMCs (Figure 5B).

Figure 5.

Figure 5

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In stimulated T cells, 259D detects a FOXP3+ population that is not stained by PCH101. Unstimulated or activated PBMCs with different stimuli were stained at 3 and days. FOXP3+ cells were gated using CD3+CD4− cells or monocytes as a reference when PBMCs or purified CD4+ T cells were used, respectively. A. Percentages of FOXP3+ cells are shown. In unstimulated and in PHA-stimulated cells, 259D alone detected a higher percentage of FOXP3+ cells than PCH101. No consistent difference were observed between the two clones after anti-CD3/CD28 beads + IL-2 stimulation at day 3, although 259D detected a higher percentage of FOXP3+ cells at day 5. Similar results were observed with allogeneic monocytes stimulation. Data from one representative experiment are shown (n=3). B. When the two clones were used in combination in the same donor as shown in A, 259D detected a higher percentage of FOXP3+ cells, except in unstimulated PBMCs and purified CD4+ T cells stimulated with allogeneic monocytes at day 3. Data from one representative experiment are shown (n=3).

DISCUSSION

Treg cells act as key regulators in the maintenance of immune tolerance and prevention of autoimmunity. Expression of FOXP3 has proven to be a reliable marker for Treg cells. However, staining with different anti-FOXP3 mAb clones has led to different results and whether one clone is better than the other is still debated. Herein, we performed an extensive study of FOXP3 staining using different staining conditions and gating definitions, to determine the optimal strategy.

Our findings clearly demonstrate that, in agreement with previous studies (34,38,39), using an isotype control to set the gate could lead to misleading results because their use either underestimated or overestimated the FOXP3+ cell population in both unstimulated and stimulated cells. Using CD3+CD4− or CD3+CD4− populations instead of an isotype control mAb or a FMO control to define the limits of the positive gate constituted the most reliable gating strategy, and that for all anti-FOXP3 clones. Our data are thus in agreement with those reported by Pillai et al. (26). In these experiments, the PBMCs were purified from healthy subjects, and no difference was found when using either cell population as the FoxP3− population. It should be noted that in some circumstances, including HIV infection (40–45), there is an up-regulation of FOXP3 in CD8+ T cells and therefore data are expected to be different when CD3+CD4− or CD3−CD4− cells are used to define the FOXP3− population. The inclusion of both CD3 and CD4 antibodies is therefore important in staining of human PBMCs, to permit a better characterization of these CD8+FOXP3+ cells. Although it has been shown that FMO controls allow to accurately determine positivity and set regions in samples containing multilabeled subpopulations (32,33,46–48), our results indicate that it can lead to overestimation of the percentage of FOXP3+ cells.

The comparison between different fluorochromes indicated that the anti-FOXP3 FITC-conjugated mAb (clone PCH101) detected low levels of FOXP3+ cells. The limited brightness of FITC compared to that of PE, AF647 or PB may explain these results. For this reason, FITC-conjugated mAbs should not be used to measure FOXP3+ cell frequency in complex cell populations such as PBMCs, although they worked well for Treg cell phenotyping after sorting. Comparing the three clones conjugated with the same fluorochrome, we found that 206D was the least sensitive clone. This finding is in accordance with the data reported by Law et al. (34). However, those authors also showed that Alexa fluor 488- (AF488) conjugated 259D detected a higher percentage of FOXP3+ cells than PCH101 FITC-conjugated clone. Although absorption and emission of AF488 and FITC are close, these two fluorochromes are not identical, which may explain the difference between the two studies. In contrast to our findings, Grant et al. (25) found that 206D detected a higher percentage of FOXP3+ cells than PCH101 in unstimulated PBMCs. The different gating strategy used by these authors could explain these opposite results. Indeed, Grant et al. used an isotype control mAb to define FOXP3 staining within the CD3+CD4+CD25+ cells (25), whereas we used the CD3+CD4− cells as the FOXP3− population.

When different cell fixation/permeabilization methods were compared, our results showed that buffer#1 was the best buffer to investigate FOXP3 expression in conjunction with cytokine detection in stimulated PBMCs. However, it should be noted that this buffer does not permit the detection of some other intracellular proteins, such as the HIV core protein in infected cells (data not shown). These findings, which are in agreement with the results from a recent paper (34), emphasize the fact that staining procedures should be carefully evaluated depending on the application. Moreover, a short PMA/ionomycin stimulation (6 hours) decreased FOXP3 expression, in contrast to the other types of stimulation, and this down-regulation should be taken into consideration when stimulated FOXP3+ cells are characterized in this context.

Higher percentages of FOXP3+ cells in unstimulated or stimulated samples were observed by staining with two FOXP3 clones used together than when one clone was used. However, the clone detecting a population of FOXP3 + cells that another clone did not, was not the same for stimulated or unstimulated cells. Indeed, PCH101 stained a minor FOXP3+ population not stained by 259D in unstimulated cells, whereas the inverse was true for stimulated cells. It has been reported that activation of Treg cells leads to the proteolytic cleavage of FOXP3 either at N-terminal or C-terminal sites inducing major topological changes and altering its DNA-binding properties (24). It is possible that such changes generate FOXP3 species better recognized by the 259D clone; such events may explain the differences we observed using 259D and PCH101. Another result deserves attention: cells stained by two clones expressed the highest levels of CD25 and the lowest levels of CD127, in unstimulated PBMCs. Therefore, staining with two clones may help increase the specificity of the staining, which could be important to phenotype Treg cells in disease processes that are accompanied with chronic activation of T cells. Although we could not confirm these data by performing functional assays with FOXP3+ cells, it has previously been shown that sorted human CD25+CD127low and CD25hiCD127low displayed all the characteristics of functional Treg cells such as high expression of FOXP3 and CTLA-4, the ability to suppress proliferation of other T cells, as well as hyporesponsiveness to TCR stimulation (49).

Collectively, our results indicate that the choice of gating strategy is crucial; in particular, the use of an anti-FOXP3 isotype control or FMO to gate could lead to misleading results. Moreover, depending on the activation status of the cells, the optimal detection of FOXP3+ cells was not achieved with the same clone. Finally, the use of two anti-FOXP3 clones in combination may improve both the sensitivity and the specificity of FOXP3 staining in complex cell populations.

Supplementary Material

Suppl 1. Supplementary Figure 1.

Software compensation does not provide an optimal compensation. Manual compensation allows adjusting PE-%FITC spectral overlap until the mean of the positive population has aligned to the mean of the negative. We followed the same procedure for all the fluorochrome combinations used in the study. The mean alignment permits removing the non-specific signal and background and avoids the spectral overlap between fluorochromes.

Suppl 2. Supplementary Figure 2.

The use of beads or cells for compensation does not affect the percentage of FOXP3+ cells. Unstimulated PBMCs were stained with anti-CD3 PerCP-Cy5.5-, anti-CD4 AF700- and anti-FOXP3 (PCH101, 259D or 206D clones) conjugated with different fluorochromes (PE, PB or AF647). PBMCs from a single donor were used to set the PMT values. Compbeads or single color stained PBMCs were used to set up compensation. The histograms represent the mean±SE of the percentage of FOXP3+ cells measured in three independent experiments.

Acknowledgments

This work was supported by grant from NIH AI068524 (to CC). We thank Dr. Barbara Shacklett and Julia M. Shaw for critical review of the manuscript and Tristan Bourdeau for technical support.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl 1. Supplementary Figure 1.

Software compensation does not provide an optimal compensation. Manual compensation allows adjusting PE-%FITC spectral overlap until the mean of the positive population has aligned to the mean of the negative. We followed the same procedure for all the fluorochrome combinations used in the study. The mean alignment permits removing the non-specific signal and background and avoids the spectral overlap between fluorochromes.

NIHMS215034-supplement-Suppl_1.TIF (71.1KB, TIF)
Suppl 2. Supplementary Figure 2.

The use of beads or cells for compensation does not affect the percentage of FOXP3+ cells. Unstimulated PBMCs were stained with anti-CD3 PerCP-Cy5.5-, anti-CD4 AF700- and anti-FOXP3 (PCH101, 259D or 206D clones) conjugated with different fluorochromes (PE, PB or AF647). PBMCs from a single donor were used to set the PMT values. Compbeads or single color stained PBMCs were used to set up compensation. The histograms represent the mean±SE of the percentage of FOXP3+ cells measured in three independent experiments.

NIHMS215034-supplement-Suppl_2.TIF (23.3KB, TIF)

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