Sorting and Downstream Functional
Assessment of Regulatory T Cells
Isolating live cells with the BD FACSMelody™ cell sorter

Features• Sort regulatory T cells (Tregs) and responder T cells (Tresps)• Assess Treg-mediated suppression of CD69 and CD154
activation• Assess intracellular cytokine production by freshly sorted
and stimulated cells• Evaluate in vitro expansion of sorted Tregs and assess viability• Perform immunophenotyping of expanded Tregs• Determine suppression of peripheral blood mononuclear
cell (PBMC) proliferation by expanded Tregs

Tregs play a critical role in maintaining immune homeostasis andself-tolerance. Altered frequencies or impaired functions of Tregs
have been implicated in conditions such as graft-versus-host diseaseand various autoimmune diseases including type I diabetes, systemic
lupus erythematosus, multiple sclerosis and rheumatoid arthritis.
These cells also play a role in immune suppression during tumor
progression and chronic infections. Tregs display a high level of phenotypic complexity, and understanding the biological implication(s)of this phenotypic diversity is critical for harnessing the therapeuticutility of Tregs. Therefore, there is great interest in isolating this cell
population with high purity, yield and viability.
In this data sheet, we demonstrate the use of the BD FACSMelody™
cell sorter to sort Tregs with high purity and viability. The system canalso be used to study the expansion and immunophenotyping ofthese cells, which retain functionality as demonstrated in suppression
and cytokine production assays. The BD FACSChorus™ software guides
researchers throughout the entire sorting process. This advanced
automation technology simplifies the workflow, thereby minimizing
the requirement for cell sorting technical expertise to make sorting
available for more researchers.

Sorting regulatory T cells and responder T cells
The CD4⁺ T-cell compartment is comprised of conventional T helper cells and Tregs. Treg-driven immunosuppression assays typically requireco-culturing of Tregs with responder cells (Tresps) that comprise conventional T cells. Staining human cells for surface CD3, CD4, CD25 and CD127
molecules provides a robust method for identifying and sorting viable Tregs and Tresps. Tregs can be identified as CD25high/+CD127low/− cells
within the CD4⁺ T cell population, as well as from positive staining of the transcription factor FoxP3, which controls Treg development andsuppressive function. However, staining for FoxP3 to identify Tregs requires fixing and permeabilizing cells prior to intracellular staining and isincompatible with live-cell sorting. Tresps can be identified as CD25 low/−CD127high/+ cells within the CD4⁺ T cell population.
To isolate Tregs and Tresps, PBMCs were isolated from the whole blood of a normal healthy donor. BD IMag™ Human CD4 T Lymphocyte
Enrichment Set-DM was used to enrich CD4⁺ T cells prior to staining, thus minimizing sort time. The enriched CD4⁺ T cell population was stainedusing the multicolor panel outlined in Table 1 to identify dead cells, lineage-positive cells, Tregs and Tresps. Cells were then re-suspended in
BD FACS™ Pre-Sort Buffer to minimize cell clumping and preserve viability prior to sort. The BD FACSMelody cell sorter was prepared for cellsorting utilizing the automated setup. Fluorescence compensation was performed using single-color compensation controls and the temperature control setup in the BD FACSChorus™ software maintained the cells at 4oC before and during the sort to preserve cellular viability andfunction.
The staining panel enabled exclusion of dead (7-AAD⁺) and other lineage-positive cells (ie, those expressing CD8, CD14 or CD19) to ensure viability
and enrichment of CD4⁺T cells. Tregs and Tresps (Figure 1A) were sorted into sterile polypropylene collection tubes coated with heat-inactivatedfetal bovine serum (FBS). The purity of sorted Tregs and Tresps was assessed immediately after the sort by analyzing the sorted populationon the BD FACSMelody system. Post-sort purity of both populations was determined to be over 98% pure within the parent population (Figure
1B and 1C). For functional studies, cells were immediately transferred to complete RPMI 1640 media supplemented with 10% heat-inactivated
FBS for culture.
Table 1. Flow cytometry panel for sorting
Treg and Tresp populations
Specificity

Fluorochrome

CD3

APC-H7

CD4

BV786

CD25

APC

CD127

PE

CD8/CD14/CD19

PerCP-Cy ™ 5.5

Dead Cells

7-AAD

Figure 1
A

B

Tresps 99.9%

Tregs

CD25 APC

Tregs
98.6%

CD25 APC

CD127 PE

Tresps

CD127 PE

CD127 PE

TrespsC

Tregs

CD25 APC

Figure 1. Sorting Treg and Tresp populations
CD4⁺ T cells were enriched using the BD IMag Human CD4 T Lymphocyte Enrichment Set-DM and stained using the panel outlined in Table 1. Samples were acquired and sorted
using the BD FACSMelody cell sorter. Single cells were gated based on forward and side light scatter (not shown). Dead (7-AAD⁺) and other lineage-positive cells (ie, those expressing
CD8, CD14 or CD19) were excluded, and CD4⁺ T cells were identified as CD3⁺CD4⁺ (not shown). 1A. Representative final sorting gates of Tregs identified as CD25 high/+CD127 low/−
and Tresps as CD25 low/−CD127 high/+ are shown within CD4⁺ T cells. The purity of sorted Treg and Tresp populations was assessed by re-acquiring the cells using the BD FACSMelody
system. Sorted Tregs (1B) and Tresps (1C) had greater than 98% purity within the CD4⁺ T cell gate. Population percentage statistics are shown.

Treg-mediated suppression of CD69 and CD154 expression by activated Tresps
The BD FastImmune™ Regulatory T-Cell Function Kit enables flow-cytometric assessment of Treg function within 7 hours by analyzing theexpression of well-established activation markers CD69 and CD154 on responder T cells. Using a similar strategy, we assessed the ability ofsorted Tregs to suppress Tresps. Sorted Tresps were stimulated in the presence or absence of sorted Tregs. Upregulated CD69 and CD154
expression on Tresps was assessed by staining the cells using the multicolor panel listed in Table 2. Purified Tregs suppressed the expressionof CD69 and CD154 on freshly-sorted and activated Tresps by approximately 40%, when co-cultured at a ratio of 0.5 Treg per Tresp, andapproximately 25% at a 0.25:1 Treg:Tresp ratio (Figure 2A and 2B), showing that the purified Tregs retained their suppressive function after
sorting with the BD FACSMelody.
Table 2. Panel for assessing Treg suppressionof CD69 and CD154 activation on Tresps
Fluorochrome

CD3

APC-H7

CD4

BV786

CD25

APC

CD127

PE

CD69

BV421

CD154

BB700

40.0

30.0

% Suppression

Specificity

Treg-mediated suppressionof CD69 and CD154 activation

Figure 2A

CD69
CD154
20.0

10.0
0
1.0

1:0.25

1:0.5

Tresp:Treg Ratio
Figure 2B
Tresps only

Tresps:Tregs (1:0.25)

CD69

Tresps:Tregs (1:0.5)

CD69

CD69

CD69 BV421

CD154

49.21%

Normalized %Normalized %Normalized %64.80%

39.16%

CD69 BV421

CD69 BV421

CD154

CD154

CD154 BB700

46.17%

CD154 BB700

Normalized %Normalized %Normalized %60.75%

37.26%

CD154 BB700

Figure 2. Treg mediated suppression of CD69 and CD154 activation on Tresps
Sorted Tresps were stimulated using anti-CD3/CD28 magnetic beads (Gibco™ Dynabeads™ Human T-Activator CD3/CD28) in the presence or absence of sorted BD Horizon™ CFSE-labeled
Tregs (BD Horizon CFSE-labeled) at two different Tresp:Treg ratios (1:0.25 and 1:0.5). Upregulated CD69 and CD154 expression on Tresps was assessed by staining the cells with the–fluorescent antibodies listed in Table 2 prior to acquisition on a BD FACSLyric™ flow cytometer. Tresps were identified as CFSE cells (not shown). 2A. Plotted percent suppression of Tresp
activation, as measured by expression of CD69 and CD154 is shown (% suppression in marker frequency was calculated for CD69 and CD154 separately as: {100 – [(% positive in thepresence of Tregs / % positive in the absence of Tregs) × 100]}. 2B. Histograms of CD69 and CD154 expression on Tresps are shown for each ratio tested (1:0.25 and 1:0.5), and % positive
statistics are presented on the corresponding histograms.

Intracellular cytokine production by freshly sorted and stimulated cells
Analogous to conventional CD4⁺ T helper cell lineages, Tregs can be categorized into Th1-, Th2-, Th17- and Th22-like subsets based on theexpression patterns of chemokine receptors, transcription factors and the cytokines they produce. The diverse cytokine milieu that drives
conventional T helper cell specialization during inflammation also drives the polarization of Th-like Tregs that can suppress the specificinflammatory conditions in which they arise.
Similar to T helper cells, Th1-like Tregs have been shown to produce IFN-γ, Th2-like Tregs produce IL-4, IL-5 and IL-13 and Th17-like Tregs
produce IL-17A. To assess post-sort cytokine production capabilities, intracellular cytokine profiles of Tresps and Tregs from autologous
fresh PBMCs and freshly sorted Tresps and Tregs after stimulation were assessed (Figure 3) using the fluorescent antibody panel listed inTable 3. Both sorted and fresh PBMC Tresps had comparable intracellular cytokine responses. Similarly, sorted and fresh PBMC Tregs showedcomparable cytokine responses, thereby confirming that the sorted cells were functionally similar to their freshly-isolated counterparts.
Table 3. Intracellular cytokine assessment panel
Specificity

Fluorochrome

CD3

APC-H7

CD4

BV786

CD25

APC

CD127

PE

IFN-γ

BV605

IL-17A

APC-R700

IL-17A APC-R700

Sorted Tresps

IL-17A APC-R700

Unsorted Tresps

Figure 3

IFNγ BV605

IFNγ BV605

Sorted Tregs

IL-17A APC-R700

IL-17A APC-R700

Unsorted Tregs

IFNγ BV605

IFNγ BV605

Figure 3. Assessment of intracellular cytokines in freshly isolated PBMCs and sorted cells upon PMA/ionomycin stimulation
Freshly isolated/unsorted total PBMC Tresps (top left) and Tregs (bottom left), sorted Tresps (top right) and sorted Tregs (bottom right) from the same healthy donor were used to assess
intracellular cytokine production (IFN-γ and IL-17A). 100,000 total PBMCs, sorted Tresps or sorted Tregs were seeded at a density of 0.5 × 106 cells/mL and treated with 0.05 μg/mL
phorbol 12-myristate 13-acetate (PMA) and 0.5 μg/mL ionomycin (Stemcell Technologies) for 8 hours in the presence of BD FastImmune™ Brefeldin A and BD GolgiStop™ Protein
Transport Inhibitor containing Monensin. Following stimulation, cells were stained with fluorescently labeled antibodies listed in Table 3 in combination with the BD Cytofix/Cytoperm™
Fixation/Permeabilization Solution Kit, per the manufacturer’s instructions. Samples were acquired using the BD FACSLyric flow cytometer. Freshly isolated total PBMCs (unsorted
Tresps, unsorted Tregs), sorted Tresps and sorted Tregs were gated as previously described (Figure 1) and intracellular cytokine profiles were analyzed. The percentage of cells positivefor each marker is indicated in each quadrant. Sorted Tresps and Tregs had comparable cytokine production to freshly isolated counterparts from the same donor.

In vitro expansion of sorted Tregs and assessment of viability
Tregs are relatively rare, comprising approximately 5-10% of the total CD4⁺ T cell population in healthy donors. We tested the expansion abilityof sorted Tregs over the course of 2 weeks. Viability and expansion measurements were performed after expansion for 7 and 14 days. Over thecourse of 14 days as shown in Figure 4, the sorted Tregs expanded approximately 55-fold in culture while maintaining >98% viability.
Phenotypic characterization of expanded Tregs
We next performed phenotypic characterization of the expanded Treg population to ensure that the expanded cells maintained their
Treg phenotype prior to functional assessment of these cells. Tregs are known to express high levels of CD25 in the normal CD4⁺ T cellcompartment with low-to-no expression of CD127. The most reliable marker for Tregs is FoxP3, the key transcription factor controlling Treg
development and function.
To evaluate the expanded Tregs phenotype, freshly isolated PBMCs and Tregs expanded post-sort from the same healthy donor were
phenotypically characterized with the multicolor panel listed in Table 4. The expanded sorted Treg population showed a high level of purity(data not shown), as well as a FoxP3⁺ ratio similar to that of fresh PBMCs from the same donor indicating that cells sorted with the BD
FACSMelody system maintained their viability and high purity after in vitro expansion (Figure 5).
Table 4. Panel for Treg phenotypic characterization

Proliferation of Tregs

Figure 460

Fluorochrome

CD3

APC-H7

CD4

FITC

CD25

BV421

CD127

BB700

FoxP3

Alexa Fluor® 647

50

Fold Expansion

Specificity

40
30
20
10
0
7

14

Days
Figure 4. Expansion of Tregs in vitro and assessment of viability
Sort-purified Tregs were cultured at a density of 0.5 × 106 cells/mL in the presence of anti-CD3/CD28 coated magnetic beads at a ratio of 1 bead per every 2 cells in complete RPMI
medium supplemented with 10% heat-inactivated human serum AB male (Sigma-Aldrich) and 300 Units/mL of recombinant human IL-2 protein (Roche). Viability and expansion
measurements were performed using Trypan blue-exclusion staining and subsequent cell counting after expansion on days 7 and 14. The graph displays fold expansion of sorted
Tregs which maintained >98% viability.

Figure 5
PBMCs

Expanded Tregs

Tregs

Tregs
20.5%

77.5%

Normalized %81.6%

Normalized %16.1%

FoxP3 Alexa 647

FoxP3 Alexa 647

Figure 5. Assessment of Treg phenotype in freshly isolated PBMCs and expanded Tregs
Freshly prepared PBMCs (left) and Tregs expanded post-sort (right) were stained with the multicolor panel outlined in Table 4 using the BD Pharmingen™ Transcription Factor
Buffer Set according to the manufacturer’s recommendations. Lymphocytes were gated based on forward and side light scatter (data not shown). Tregs were identified within the
CD3⁺CD4⁺ cell gate as CD25 high/+ CD127 low/− cells as described previously and the percentage of cells positive for FoxP3 was determined (histograms). Numbers displayed represent
the percentages of FoxP3⁻ and FoxP3⁺ cells.

Secreted cytokine production by expanded Tregs
While Tregs are known to produce signature inflammatory cytokines similar to their conventional T helper cell counterparts, Tregs also secreteanti-inflammatory cytokines such as IL-10, TGF-β and IL-35 that are capable of suppressing pro-inflammatory responses of T cells, NK cells,
B cells, dendritic cells and macrophages. To evaluate the functionality of sorted and expanded Tregs, we evaluated the cytokine productionof these cells during a three-week in vitro expansion time course. Sorted Tregs maintained their ability to produce IL-17A, IFN-γ, IL-4 and IL-10
throughout the expansion process, indicating that the proliferating Tregs retained cytokine production capabilities (Figure 6).
IL-17

Picograms/mL

Figure 6

IFNγ

5.0
4.0
3.0

IL-4

40.0

80.0

30.0

60.0

IL-10
100.0
80.0
60.0

20.0

40.0

10.0

20.0

40.0

2.0
1.0
0

0
Week 1

Week 220.0

0Week 3

Week 1

Week 2

Week 30
Week 1

Week 2

Week 3

Week 1

Week 2

Week 3Figure 6. Cytokine production by expanding Tregs
Tissue culture media was collected at multiple time points (1, 2 and 3 weeks) during Treg expansion and cytokine levels were measured using the BD™ Cytometric Bead Array (CBA)
Enhanced Sensitivity Flex Set for IL-17A, IFN-γ, IL-4 and IL-10 according to manufacturer’s instructions. Samples were acquired using the BD FACSLyric flow cytometer and analysis
was performed using FCAP Array™ 4 software.

Suppression of PBMC proliferation by expanded Tregs
To assess the functional capabilities of expanded sorted Tregs, we assessed their ability to suppress TCR-stimulated PBMC proliferation as
measured by CFSE-dilution of target responder cells. Following target cell activation, approximately 60% of the PBMC population underwent
proliferation as measured by CFSE-dilution using FlowJo™ software (Figure 7). This proliferative response was almost completely suppressed following the addition of expanded Tregs (Figure 7), indicating the maintenance of suppressive capabilities after sorting with the BD FACSMelody.

PBMCs + Tregs

PBMCs only

Figure 7

59.3%

Division Index

Proliferation Index

0.32

1.91

0.07

1.77

5.9%

CFSE
Figure 7. Suppression of proliferation by in vitro expanded Tregs
Freshly isolated and BD Horizon CFSE-labeled autologous PBMCs were cultured in complete RPMI media supplemented with 10% heat-inactivated human serum in the presenceof 100 Units/mL of recombinant human IL-2 and anti-CD3/CD28 coated magnetic beads at a ratio of 1 bead per 30 cells. Tregs were added at a 1:0 or 1:1 ratio. The cells were
cultured for 5 days and acquired using the BD FACSLyric flow cytometer. CFSE dilution in PBMCs for both conditions is shown as PBMCs only (top histogram) or PBMCs + Tregs(bottom histogram). The percentage of cells that have undergone division is indicated on the histograms. Proliferation analysis was performed using FlowJo™ software. Divisionand proliferation indexes are shown (Division index: average number of cell divisions that a cell has undergone. Proliferation index: the total number of divisions divided by the
number of cells that went into division).

It is often necessary to identify and purify distinct viable cell subpopulations of interest from heterogeneous cell mixtures, such as humanperipheral blood. This capacity allows researchers to study the biology and function of the specific subpopulations of interest at the single
cell level, thus enabling deep scientific insights. As presented in this data sheet, cellular subpopulations purified by the BD FACSMelody
cell sorter are phenotypically and functionally similar to their freshly isolated or cultured counterparts, thereby making them suitable forfunctional studies designed to characterize the nature of the isolated cell types.

Ordering information
Systems and software
Description
BD FACSMelody™ 9-Color Cell Sorter Blue, Red, and Violet Laser Configuration,with Plate Sorting
BD FACSChorus™ software v1.1 (or later)

Reagents
Description

Clone

Cat. No.

APC-H7 Mouse Anti-Human CD3

SK7641397FITC Mouse Anti-Human CD4SK3340133

BD Horizon™ BV786 Mouse Anti-Human CD4

SK3563877

BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD8

SK1565310BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD14MψP9562692

BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD19

HIB19561295APC Mouse Anti-Human CD25

2A3340939BD Horizon™ BV421 Mouse Anti-Human CD25

2A3564033

BD Horizon™ BV421 Mouse Anti-Human CD69

FN50

562884

BD Pharmingen™ PE Mouse Anti-Human CD127

HIL-7R-M21

557938

BD Horizon™ BB700 Mouse Anti-Human CD127

HIL-7R-M21

566398

BD OptiBuild™ BB700 Mouse Anti-Human CD154

TRAP1745814

BD Pharmingen™ Alexa Fluor® 647 Mouse anti-Human FoxP3

259D/C7

560045

BD Horizon™ BV605 Mouse Anti-Human IFN-γ

B27562974

BD Horizon™ APC-R700 Mouse Anti-Human IL-17A

N49-653

565163

BD Horizon™ CFSE

565082

BD Pharmingen™ 7-AAD

559925

BD IMag™ Human CD4 T Lymphocyte Enrichment Set-DM

557939BD FastImmune™ Brefeldin A Solution

347688BD GolgiStop™ Protein Transport Inhibitor (Containing Monensin)

554724BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit

554714

BD Pharmingen™ Transcription Factor Buffer Set

562725

BD™ Cytometric Bead Array (CBA) Human IL-4 Enhanced Sensitivity Flex Set

561510BD™ Cytometric Bead Array (CBA) Human IL-10 Enhanced Sensitivity Flex Set

561514

BD™ Cytometric Bead Array (CBA) Human IFN-γ Enhanced Sensitivity Flex Set

561515

BD™ Cytometric Bead Array (CBA) Human IL-17A Enhanced Sensitivity Flex Set

562143

BD FACS™ Pre-Sort Buffer

563503

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