A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells

Graphical abstract Lymphocytes were isolated from human tissue samples by enzymatic and mechanical methods. Treg and Tconv were cell sorted from the tissue cell suspension by gating the lymphocytes forward scatter–side scatter region into a CD4–Live/Dead dot plot. CD4+ live cells were then gated into a CD127–CD25 dot plot. This last dot plot was used to define Treg (Streams 1 and 2) and Tconv cells (Stream 3). Treg were gated into a CCR5 histogram to define CCR5low and CCR5high Treg. Thus, specific Treg subsets were isolated for use in downstream suppression assays. Histograms depicting percentage Foxp3 expression by colorectal cancer-isolated Treg and Tconv show representative data from 9 separate experiments. Treg isolated from colorectal cancer therefore express high amounts of Foxp3 whereas Tconv do not.


Method details
Regulatory T cells (Treg) suppress the proliferation of conventional T cells, maintaining peripheral tolerance. Surface markers used to define Treg are CD4, CD25 and CD127 and CD4 + CD25 + CD127 low cells express high levels of the master regulatory transcription factor, Foxp3 [1]. Most reports of suppression assays use peripheral blood-isolated Treg, or spleen-derived Treg in the case of murine studies, as these sources provide a high yield of lymphocytes [2][3][4]. Isolation of lymphocytes from other tissues is more complex, resulting in lower yields of viable cells [5] and access may be limited to small tissue biopsies. In our method, lymphocytes were isolated from human tissue by an enzymatic and mechanical method. Specific lymphocyte subsets were then isolated by cell sorting and a suppression assay was set up as detailed herein.

Samples
Peripheral blood and tissue samples of CRC were obtained from patients undergoing a bowel resection as part of their treatment for CRC at the QEHB. Ethical approval had been obtained from the local research and ethics committee (LREC South Birmingham 2003/242, renewed 2012) and all patients donating tissue had given full prior consent.
Furthermore, the importance of suppressive subpopulations of Treg favours their isolation by fluorescentactivated cell sorting. Here we describe a method to isolate Treg from human tissues, using colorectal cancer tissue as an example. Treg suppressive capacity was further examined by expression of CCR5 to demonstrate the ability of our method to assess the suppressive capacity of regulatory T cell subsets.
To optimise the standard suppression assay to achieve our research aims, the following modifications were made: Treg, isolated from tissues, were sorted directly into a well-plate. Responder T cells, which had been fluorescently-labelled prior to sorting, were added directly into the wellplate. Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd., UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2. This method quantified the suppression of responder T cell proliferation by small numbers of strictly-defined Treg populations isolated from tissues. 4. The mononuclear cell band was aspirated and the cell suspension was washed and resuspended in 1 ml of RPMI 1640 media containing 1% foetal calf serum (FCS, 10270-106, Life Technologies Ltd., UK) and antibiotics, as above. 5. 10 ml peripheral blood from healthy volunteers was layered on top of an equal volume of Lymphoprep TM (07851, Stemcell Technologies Ltd., UK) and centrifuged at 300 Â g for 25 min. The mononuclear cell band was aspirated, washed and resuspended in RPMI 1640 media containing 1% FCS. Cell sorting 1. Cell sorting was used to add Treg or Tconv to a 96-well plate followed by Tresp. The Treg population was defined by gating on live CD4 + CD25 + CD127 low cells while Tconv were defined by gating on live CD4 + CD25 À cells (see Graphical Abstract).
2. Each well of a round-bottomed 96-well plate was filled with 100 ml of RPMI + 10% FCS. An equal number of CCR5 low Treg, CCR5 high Treg and Tconv were 3-way sorted from labelled CRC-isolated cell suspension into separate wells using a MoFlo XDP High-Speed Cell Sorter (Beckman Coulter Inc., USA) in purity mode. In some cases, sorted lymphocytes were analysed on a flow cytometer to assess purity (see Fig. 1). Typical yields were between 10 000 and 60 000 Treg, according to the sorter event counter. In cases with high yields, 10 000 CCR5 low Treg, CCR5 high Treg and Tconv were 3-way sorted into separate Eppendorfs containing 1 ml of PBS in preparation for later DNA extraction and analysis of the TSDR (Treg-specific demethylated region). 3. Violet-labelled Tresp (peripheral blood-isolated CD4 + CD25 À cells) were plate-sorted into the same wells as the previously sorted Treg and Tconv, yielding Treg/Tconv:Tresp ratios of 1:1, 1:2 or 1:4.

Suppression assay
Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd., UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2. The total volume of each well was made up to 200 ml with RPMI 1640 media + 10% FCS. The plate was incubated at 37 8C and 5% CO 2 for 3 days. Violet dye wash-out was analysed by flow cytometry (Cyan ADP flow cytometer, Beckman Coulter). Violet-labelled cells cultured alone, in the absence of bead stimulation, were used as a negative control, enabling a gate to be set around the proliferating cell population. The percentage of Tresp proliferating is thus easily determined. However a small number of proliferating Tresp undergoing multiple divisions will have the effect of under-representing the suppressive capacity of Treg, an effect that can be overcome by measuring the division index (DI). The DI is defined as the average number of divisions for all Tresp in the culture and was calculated using Flowjo version 8.7 (Tree Star Inc.) [6]. Results were reported as percent suppression [3]: Percent suppression ¼ 100 À

Percentage of proliferating cells ðor DIÞ with Treg present Percentage of proliferating cells ðor DIÞ without Treg present Â100
Differences in percent suppression, calculated using both percentage of proliferating cells and DI, between CRC-isolated Treg and CD4 + CD25 À cells were tested for statistical significance using the Wilcoxin signed-rank test.
Representative results from one experiment, in which enough Treg were isolated to perform three different Treg:Tresp ratios, are shown in Fig. 2. Following 72 h of culture, 75% of Tresp proliferated in response to Treg Suppression Inspector beads alone. Both CRC-isolated CCR5 low Treg and CCR5 high Treg suppressed proliferation of Tresp in a dose-dependent manner, while CRC-isolated Tconv did not. The differences in percent suppression by CCR5 low Treg, CCR5 high Treg and Tconv were compared using pooled data for Treg:Tresp ratios of 1:1 and 1:2 over a series of five experiments (see Table 1). Both CCR5 low Treg and CCR5 high Treg suppressed Tresp proliferation significantly more then Tconv, P = 0.043 and P = 0.018, respectively. Also, CCR5 high Treg suppressed Tresp proliferation significantly more than CCR5 low Treg (P = 0.018), an effect that has been noted previously in patients with psoriasis [7].

TSDR analysis
TSDR analysis was performed on sorted Treg to show that Treg isolated from human tissues were naturally-occurring Treg as opposed to induced Treg.
1. Eppendorfs containing sorted Treg and Tconv were pelleted by centrifugation in a microfuge at 300 Â g for 10 min then resuspended in 200 ml of PBS. DNA was extracted from the cell suspensions using the DNeasy Blood & Tissue Kit (69504, Qiagen) according to the manufacturer's instructions. DNA samples were bisulphite treated using the Epitect Bisulfite kit (59104, Qiagen) and the quantity and quality of recovered bisulphite-treated DNA was assessed by measurement of UV absorbance at 260/280 nm. 2. The TSDR region was amplified using primers designed to amplify the TSDR region irrespective of its methylation status. A Taqman 1 probe labelled with 6-carboxyfluorescein (FAM) was designed to specifically bind to an unmethylated sequence while another Taqman 1 probe labelled [ ( F i g . _ 1 ) T D $ F I G ] [ ( F i g . _ 2 ) T D $ F I G ]  with 4,7,2 0 -trichloro-7 0 -phenyl-6-carboxyfluorescein (VIC) was designed to specifically bind to a methylated sequence. To achieve maximum specificity, each probe was designed to span 3 CpGs within the TSDR. The primers, probes and method have been previously published [8]. Primers and probes were obtained from Applied Biosystems Inc., USA. 3. The following programme was run using the LightCycler 480:  Fig. 3).

Author contributions
STW was the primary researcher who developed, executed and analysed the performed experiments. STW drafted the manuscript. KKL assisted in obtaining and analysing data. KKL and SMC edited the manuscript.