Foxp3 efluor450

FOXP3‐eFluor 450 (eBioscience, Cat.# 48‐5773‐82, clone: FJK‐16s, 1:333), CD95 (Fas)‐Brilliant Violet 605 (BioLegend, Cat.# 152612, clone: SA367H8, 1:80), CD185 (CXCR5)‐Brilliant Violet 711 (BioLegend, Cat.# 145529, clone: L138D7, 1:40), CD4‐PerCP/Cyanine5.5 (BioLegend, Cat.# 100434, clone: GK1.5, 1:80), CD279 (PD‐1)‐PE (BioLegend, Cat.# 135206, clone: 29F.1A12, 1:20), CD25‐PE/Cy5 (BioLegend, Cat.# 102010, clone: PC61, 1:80), GL7‐eFluor 660 (eBioscience, Cat.# 50‐5902‐82, clone: GL7, 1:80), CD19‐APC/Cyanine7 (BioLegend, Cat.# 115530, clone: 6D5, 1:20), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:7500).

Isolation and analysis of skin lymphocytes [18 (link), 27 (link)] were carried out as described. Briefly, after removing fur with an electric groomer, trunk skin was separated from subcutaneous fat tissue, cut to small pieces and, for lymphocyte analysis, digested for 45 min. at 37°C in RPMI 1640 containing collagenase XI (4000 U/ml), hyaluronidase (260 U/ml) and DNase (0.1mg/ml), all from Sigma-Aldrich. Cells were harvested by filtration, washed and stained, first for surface markers with mAbs CD45.2-PerCP, CD3-APC-eFluor780, CD4-FITC, CD25-PE, CTLA4-APC, ICOS-PE-Cy7, and live/dead-eFluor506, then fixed and permeabilized and stained with FoxP3-eFluor450, all from eBioscience. For DC analysis, digestion was with 150μg/ml Liberase and 120μg/ml in HBSS and cells were stained with mAbs CD11c-PerCPCy5.5 and CD103-PE (both BioLegend) and, after fixation and permeabilization, with anti-langerin-Alexa488 (clone 929F3.01, Dendritics).

TNF‐α‐Brilliant Violet 421 (BioLegend, Cat.# 506328, clone: MP6‐XT22, 1:80), FOXP3‐eFluor 450 (eBioscience, Cat.# 48‐5773‐82, clone: FJK‐16s, 1:300), IL‐2‐Brilliant Violet 711 (BioLegend, Cat.# 503837, clone: JES6‐5H4, 1:40), IFN‐ɣ‐Brilliant Violet 785 (BioLegend, Cat.# 505838, clone: XMG1.2, 1:80), CD8a‐FITC (BioLegend, Cat.# 100706, clone: 53‐6.7, 1:50), CD4‐PerCP‐Cy5.5 (BioLegend, Cat.# 100434, clone: GK1.5, 1:80), CD25‐PE/Cy5 (BioLegend, Cat.# 102010, clone: PC61, 1:80), CD44‐PE/Cyanine7 (BioLegend, Cat.# 103029, clone: IM7, 1:80), IL‐10‐APC (BioLegend, Cat.# 505010, clone: JES5‐16E3, 1:80), CD107b‐Alexa Fluor 647 (BioLegend, Cat.# 108512, clone: M3/84, 1:200), CD3‐APC/Cyanine7 (BioLegend, Cat.# 100222, clone: 17A2, 1:80), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:7500).

Intracellular cytokine staining for T cells was conducted on day 3, after the T cells were cultured for 72 hours. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 hours. Brefeldin A 1000X (eBioscience) was added for the final 4.5 hours. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4°C. T cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17A (FITC; eBioscience), KI67 (PE; eBioscience), or FOXP3 (eFluor450; eBioscience). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD4⁺TCRβ⁺ Live cells or TCRβ⁺ Live cells.

For analysis of in vivo experiments, cells were stained with antibodies against human CD45 APC (Invitrogen), CD4 ECD (Beckman Coulter), CD3 Pacific Blue (eBioscience) CD8 PE and CD25 PE-Cy7 (BD) and the viability dye 7-AAD (eBioscience). To analyse expression of Treg-associated markers, freshly sorted or expanded Tregs were stimulated for 15h with anti-CD3/anti-CD28 beads at a ratio of 1 bead to 5 cells, or left untreated. Cells were stained with 7-AAD and antibodies against GITR FITC (R&D Systems), CTLA-4 PE, CD69 APC-Cy7, CD25 PE-Cy7 (BD), TIGIT PE, OX-40 FITC, TIM-3 APC, CD39 PE, FOXP3 eFluor 450, Perforin APC (eBioscience) Helios AlexaFluor 647 (Biolegend), and CD4 ECD (Beckman Coulter). For intracellular antigens (FOXP3, Helios, Perforin, CTLA-4), cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (eBioscience). Samples were acquired using a BD FACSCanto (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences). For staining for BCL-XL and MCL1, Abcam anti-BCL-XL FITC (7B2.5; ab26148) and anti-MCL-1 Alexa Fluor 488 (Y37, ab197529) antibodies were used, respectively, following manufacturer's instructions. Briefly, the cells were fixed with 4% paraformaldehyde and permeablized with PBS/0.1% Tween. The cells were then blocked with 10% normal goat serum/0.3 M glycine, followed by incubation with the antibody.

Cell surface antigens were stained first. The dendritic cell antibody panel quantified the following: CD11c-APC-eFluor780, CD80-PE-Cy7, CD86-FITC, and CD40-eFluor450 (all mAbs are from eBioscience now Thermo Fisher, Waltham, MA). The CD4+ T_Reg cell panel included the following: CD3ε-PE, CD4-PE-Cy7, and CD25-PerCP-Cy5.5s (all mAbs are from eBioscience). For T_Reg intracellular staining, cells were fixed and permeabilized with the one step eBioscience Fix-Perm Foxp3 Buffer Staining Kit (eBioscience). The panel included Foxp3-eFluor450 and CTLA4-APC (mAbs are from eBioscience). Intracellular staining was also used to detect TGFβ-PE, IL12 p40-PE, IL10-APC, and IFNγ-APC production (mAbs are from eBioscience) after ex vivo nonspecific antigen stimulation with a Leukocyte Activation Cocktail with GolgiPlug (BD Biosciences) for flow cytometry. Mouse INFγ Single-Color ELISPOT to determine antigen-specific response of T cells was from Cellular Technology Limited (CTL), Cleveland, OH. For attempting to assess SIRT1 expression by flow cytometry, we used antibodies from Santa Cruz and Abcam.