Pch101

CD3, CD25 (SK7 and M-A251 respectively; BD Biosciences) and CD127 (eBioRDR5; eBiosciences Hatfield, UK) antibodies were used for cell surface phenotyping. Cells were then further stained for intranuclear Foxp3 (PCH101; eBiosciences) using the Foxp3 staining kit as per the manufacturer's instructions (eBiosciences).
For intracellular cytokine staining on day 7, cells were restimulated for 4 hr with 5 ng/ml PMA and 500 ng/ml ionomycin, with 2 μm monensin (Sigma-Aldrich, St Louis, MO) added for the final 2 hr. Cells were washed, fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and then stained with fluorescently labelled monoclonal antibodies to IL-2 (MQ1-17H12; eBiosciences). Dead cells (7-aminoactinomycin D-positive; Sigma-Aldrich, Gillingham, UK) were gated out.
For phospho-STAT5 staining two different buffer kits were used with the same antibody clone: 47/Stat5[pY694] obtained from BD Biosciences. The buffer set used for phospho-STAT5 staining on total CD4⁺ cell populations was PerFix EXPOSE Kit (Beckman Coulter, High Wycombe, UK). The buffer set used for phopspho-STAT5 staining on sorted Treg cell and effector T cell populations was CytoFix/CytoPerm Buffer and Perm Buffer III (BD Biosciences). Both buffer sets were used as per the manufacturer's instructions.

PBMCs (1 × 10⁶) were stimulated with an anti-CD3 antibody with IL-15, IL-2, and retinoic acid. After incubation for six days, CD8⁺ T cells were purified by negative selection with the CD8⁺ T Cell Isolation Kit II (Miltenyi Biotec) followed by separation of CD8⁺CCR7⁺ T cells using PE-CCR7 antibody (BD Biosciences) and anti-PE microbeads (Miltenyi Biotec). The cells were then sorted using a FACS Aria device (Becton, Dickinson) or a MoFlo cell sorter (Beckman Coulter) to isolate CCR7⁺CD8⁺ cells. The cells were incubated with a monoclonal antibody against Foxp3 (fluorescein isothiocyanate [FITC], PCH101, IgG2a, κ; eBioscience), PD-1 (fluorescein isothiocyanate [FITC], J43, IgG1, κ; eBioscience), CD25 (APC, CD25-4E3, IgG2b, κ; eBioscience), Granzyme B (FITC, GB11, IgG1; eBioscience), GITR (APC, CD25-4E3, IgG2b, κ; eBioscience), or Granzyme B (FITC, 621, IgG1, κ; Biolegend). Isotype controls were monitored non-specific binding. The expression of FOXP3 in sorted CD8⁺CCR7⁺ T cells was compared across the different protocols.

Immunohistochemistry was performed on 4-μm tissue sections from each TMA block using the Bond Polymer Intense Detection system (Vision BioSystems, Victoria, Australia), according to the manufacturer's instructions with minor modifications as previously reported.^{[16 (link),18 (link)]} In brief, 4-μm formalin-fixed, paraffin-embedded tissue sections were deparaffinized using a Bond Dewax Solution (Vision BioSystems), and an antigen retrieval procedure was performed using Bond ER Solution (Vision BioSystems) for 30 minutes at 100°C. Endogenous peroxidase activity was quenched by incubating the tissue with hydrogen peroxide for 5 minutes. The sections were incubated for 15 minutes at room temperature with primary polyclonal antibodies to SKP2 (1:100, Abcam, Cambridge, UK), Beclin-1 (1:100, Abcam), and FOXP3 (1:100, PCH101, eBioscience, Cambridge, UK) using a biotin-free polymeric horseradish peroxidase-linker antibody conjugate system and a Bond-max automatic slide stainer (Vision BioSystems). The nuclei were counterstained with hematoxylin. The negative control was treated in an identical manner using mouse IgG instead of primary antibody.

For analysis of radioactivity in harvested tissues, tissues were weighed, and radioactivity was quantified using a γ- counter (1282-Compugamma, LKB-Wallac) together with calibration standards. Data were expressed as %ID/g.
Skin grafts were harvested alongside normal mouse skin of similar size (from the lower back) for histology and frozen in Optimal Cutting Temperature (OCT) compound followed by tissue sectioning (8 μm). Thawed sections were fixed in 4% paraformaldehyde/PBS for 10 min, permeabilized (0.2% [v/v] Triton X-100/PBS), washed, and blocked (20% [v/v] goat serum/PBS containing 0.1% fish skin gelatin and 0.25% [v/v] Tween 20) before being stained with the following primary antibodies (2 μg/mL) overnight at 4°C: anti-human CD3 (polyclonal rabbit, A0452, Dako), anti-FOXP3 (monoclonal rat, clone PCH101, eBioscience), anti-Gr-1 (monoclonal rat, RB6-8C5, eBioscience), and anti-GFP (monoclonal mouse, 3E6, Invitrogen). After washing, sections were stained with secondary antibodies: goat anti-mouse-AlexaFluor 555, anti-rabbit-AlexaFluor 568 (Invitrogen), and anti-rat-Cy5 (Jackson ImmunoResearch). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; 10 μg/mL PBS). Samples were mounted using fluorescence mounting medium (Dako) and imaged on a NikonA1 confocal microscope. Fiji/ImageJ v.1.5 software was used for analysis.

Isolated PBMCs were analyzed by cell-surface staining using fluorescent antibodies against CD3 (SP34-2 and SK7), CD4 (L200), CD8 (SK1), CD19 (SJ25C1), CD24 (ML5), CD25 (M-A251), CD27 (M-T271), CD28 (CD28.2), CD38 (HIT2), CD45RO (UCHL1), CD56 (B159), CD57 (NK-1), CD69 (FN50), CD138 (MI15), PD1 (NAT105), HLA-DR (G46-6) (BD Pharmingen, San Diego, CA, USA), and CCR7 (G043H7) (BioLegend, San Diego, CA, USA). To assess the intracellular protein expression of FOXP3, INF-γ, IL-4, and IL-17A, the cells were permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) and stained with fluorescent antibodies against FOXP3 (PCH101, eBioscience), IFN- γ (B27, BD Pharmingen), IL-4 (8D4-8, BD Pharmingen), and IL-17A (eBio64DEC17, eBioscience). The cells were analyzed on the FACSverse (BD Biosciences, San Diego, CA, USA), and FlowJo software (BD Biosciences) was used for data analysis.