Foxp3

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Foxp3 is a transcription factor that plays a critical role in the development and function of regulatory T cells. It is a key marker used to identify and study this important cell population.

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307 protocols using foxp3

Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes

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TILs were stained with fluorescently labeled antibodies to CD45, CD4, CD8, CD3, CD11c, CD11b, PD1, PDL-1 and Foxp3 (eBiosciences). For Foxp3 staining, cells were permeabilized and fixed using a commercially available kit (eBioscience) according to the manufacturer’s instructions. FACS acquisition was performed with the cytometer Cytomics FC500 (Beckman Coulter), and the LSRFortessaTM cytometer (BD Biosciences) and data were analyzed using the FlowJo software. The antibodies used for the analyses were the following and were purchased by: CD45 (clone: 30-F11, Invitrogen), CD4 (clone: GK1.5,eBioscience), CD8 (clone: 53–6.7, BioLegend), CD3 (clone: 17A2, BioLegend),CD11c (clone: N418, eBioscience), CD11b (clone: M1/70, BioLegend), PD1 (clone: J43, eBioscience), PDL-1 (clone: MIH1, BD Biosciences), Foxp3 (clone: FJK-165, eBioscience).

Skavatsou E., Semitekolou M., Morianos I., Karampelas T., Lougiakis N., Xanthou G, & Tamvakopoulos C. (2021). Immunotherapy Combined with Metronomic Dosing: An Effective Approach for the Treatment of NSCLC. Cancers, 13(8), 1901.

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Multiplex Immunohistochemistry for Tissue Analysis

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Patient tissue slides were rehydrated in xylene, 100% ethanol, 95% ethanol, then running deionized water sequentially. Antigen retrieval was performed with sodium citrate, pH 6.0. Tissue was blocked in 10% donkey serum overnight at 4°C. Primary antibodies (PVR/CD155 (1:100, Cell Signaling Technology), VE-Cadherin/CD144 (1:250, R&D), FOXP3 (1:100, Cell Signaling), Vimentin (1:100, Cell Signaling), CD163 (1:100, Novus Biologicals), or Pan Cytokeratin-488 (1:250, Thermo Fisher Scientific) were diluted in 5% donkey serum in PBST (DS/PBST) and incubated overnight at 4°C. For tissue co-stained for TIGIT-FOXP3 and PVR-Vimentin: the tyramide signal amplification kit with Alexa Fluor 488 (Thermo Fisher Scientific) was used following the manufacturer’s recommendation to enhance signaling for PVR and FOXP3. Samples underwent a second citrate antigen retrieval and were then multiplexed with TIGIT and Vimentin following the aforementioned standard IFC protocol. A 1% BSA block was used throughout the TSA protocol and subsequent multiplex staining. Tissue was mounted with DAPI ProLong^™ Gold Antifade Mountant (Thermo Fisher Scientific) and subsequently imaged by confocal microscopy on a Leica SP5. Supplementary Table 4 lists the antibodies used.

Steele N.G., Carpenter E.S., Kemp S.B., Sirihorachai V., The S., Delrosario L., Lazarus J., Amir E.A., Gunchick V., Espinoza C., Bell S., Harris L., Lima F., Irizarry-Negron V., Paglia D., Macchia J., Chu A.K., Schofield H., Wamsteker E.J., Kwon R., Schulman A., Prabhu A., Law R., Sondhi A., Yu J., Patel A., Donahue K., Nathan H., Cho C., Anderson M.A., Sahai V., Lyssiotis C.A., Zou W., Allen B.L., Rao A., Crawford H.C., Bednar F., Frankel T.L, & Pasca di Magliano M. (2020). Multimodal Mapping of the Tumor and Peripheral Blood Immune Landscape in Human Pancreatic Cancer. Nature cancer, 1(11), 1097-1112.

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Comprehensive Immunophenotyping by Flow Cytometry

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Immunophenotyping was performed by flow cytometry as detailed in our previous reports (Karumuthil-Melethil et al., 2008 (link); Perez et al., 2008 (link)). Briefly, single cell suspensions of spleens were stained using different fluorochrome labelled antibodies against mouse CD4, CD8, CD19, CD11c. CD11b, and Foxp3 (Invitrogen). Staining for Foxp3 was done using intranuclear staining buffer kit from Invitrogen. The stained samples were acquired using FACSVerse instrument (BD Biosciences and the data was analyzed using FlowJo software (BD Biosciences). The frequencies of various immune cell populations among all spleen cells were quantified as B cells (CD19⁺), helper T cells (CD4⁺), cytotoxic T cells (CD8⁺) regulatory T cells (CD4⁺Foxp3⁺), monocytes (CD11b⁺) and dendritic cells (CD11c⁺). CD4⁺ population was gated for determining Foxp3⁺ cell (CD4⁺Foxp3⁺ regulatory T cells) frequencies. Samples stained using isotype control antibodies were used to gate for each specific population. Differences in numbers of mice used per group were due to processing accidents preventing accurate preparation or interpretation of data, and were each made on blinded samples (N = 1 vehicle control mis-processed, N = 2 Doxil controls mis-processed).

Helke K.L., Gudi R.R., Vasu C, & Delaney J.R. (2022). Combination of Autophagy Selective Therapeutics With Doxil: An Assessment of Pathological Toxicity. Frontiers in Toxicology, 4, 937150.

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Comprehensive Immune Profile Analysis

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Characterization of Tregs, T cell immune activation and senescence, and T
and B cell expression of RANKL and OPG was performed by polychromatic flow
cytometry on frozen/thawed peripheral blood mononuclear cells (PBMCs). To assess
cell viability, PBMCs were stained using the Aqua Live/Dead cell stain kit
(Invitrogen) prior to cell surface staining and intracellular OPG and FoxP3
staining. PBMCs were surface stained with fluorochrome-conjugated monoclonal
antibodies to CD3, CD4, CD8, CD19, CD28, CD38, CD57, HLA-DR, CD25 (Becton
Dickinson, BD), and RANKL (R&D Systems). Cells were subsequently stained
intracellularly for FoxP3 (eBioscience), or OPG Biotin (Leinco) and
Streptavidin-BV421 (BD), and fixed in 1% formaldehyde. PBMCs were acquired on an
LSRFortessa flow cytometer (BD) and analyzed using FlowJo software (TreeStar,
version 9.9.3). Immune activation (CD38⁺/HLA DR⁺),
senescence (CD28⁻/CD57⁺) phenotypes were
characterized on the CD3⁺CD4⁺ and
CD3⁺CD8⁺ T cell subsets. Tregs
(CD4⁺/CD25⁺/FoxP3⁺), and CD19⁺ B
cells phenotypes were compared as the % of parent population. Expression of
RANKL and OPG was measured by Mean Fluorescence Intensity (MFI) in the
CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell
subsets and CD19⁺ B cell populations.

Yin M., Chan E., Brown T., Kinslow J., Martinson J., Landay A., Melbourne K., Ribaudo H, & Overton E. (2019). Vitamin D does not modulate Immune-mediated bone loss during ART initiation. Antiviral therapy, 24(5), 355-362.

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Immunofluorescence Analysis of Intestinal Biopsies

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Cryo-frozen intestinal cross sections from patient colonic biopsies were fixed with 4% paraformaldehyde and stained using the TSA plus kit [Perkin Elmer] according to the manufacturer’s instructions. Alternatively, slides were fixed with methanol at −20°C for 10 min, blocked with 10% fetal calf serum [FCS]/1% bvine serum albumin for 1 h and incubated overnight at 4°C with the primary antibody. Paraffin-embedded tissues were deparaffinized and antigen unmasking was performed using citrate buffer. Thereafter slides were incubated with different antibodies.
For DSS-induced colitis mice, immunofluorescence analysis was performed in intestinal sections, which were taken at the end of the experiment on day 10 for IL10 [Abcam], IL17 [Abcam] and Foxp3 [eBioscience]. Colon biopsies of UC patients were taken before and 28 days after topical cobitolimod or placebo treatment^{21 (link),22 (link)} and the corresponding cross sections were stained for IL10 [Abcam], Foxp3 [eBioscience] or IL17 [Abcam]. From each sample, four to six high power fields [HPFs] per patient were analysed using a 10× objective magnification. Analysis of images was done with a fluorescence microscope [BZ-8100 or BZ-9000, Keyence] or a confocal microscope [LSM, Leica Microsystems] and calculated with ImageJ 1.52a [NIH]

Schmitt H., Ulmschneider J., Billmeier U., Vieth M., Scarozza P., Sonnewald S., Reid S., Atreya I., Rath T., Zundler S., Langheinrich M., Schüttler J., Hartmann A., Winkler T., Admyre C., Knittel T., Dieterich Johansson C., Zargari A., Neurath M.F, & Atreya R. (2019). The TLR9 Agonist Cobitolimod Induces IL10-Producing Wound Healing Macrophages and Regulatory T Cells in Ulcerative Colitis. Journal of Crohn's & Colitis, 14(4), 508-524.

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Multiparametric Flow Cytometry Analysis

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Cells were stained with CD29, CD44, CD73, CD90, CD105, HLA-ABC, CD14, CD31, CD34, CD45, HLA-DR, CD3, CD4, CD8, CD25, IL-4, IL-17, and FoxP3 (eBioscience). In addition, intracellular IL-4, IL-17 and FoxP3 staining were treated with a FoxP3 staining buffer set (eBioscience). Flow cytometry was carried out on the FACScalibur flow cytometer (BD, San Diego, USA). Data were analyzed using FlowJo software (Treestar, Inc., San Carlos, CA, USA).

Tang R.J., Shen S.N., Zhao X.Y., Nie Y.Z., Xu Y.J., Ren J., Lv M.M., Hou Y.Y, & Wang T.T. (2015). Mesenchymal stem cells-regulated Treg cells suppress colitis-associated colorectal cancer. Stem Cell Research & Therapy, 6(1), 71.

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Flow Cytometric Analysis of Immune Cells

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The following fluorescent mouse Abs from Biolegend (San Diego, CA, USA) were used for flow cytometry analysis: CD3, CD4, CD8, CD103, CD25, CD62L, CD69, CD86, CD138, Foxp3, and Ki-67; from eBioscience (San Diego, CA, USA): B220, granzyme B, perforin; from Santa Cruz (Dallas, TX, USA): granzyme A. Cell subsets were stained with mAbs and isotype control as indicated above and analyzed on BD LSRFortossa™ flow cytometer (BD Biosciences, San Diego, CA, USA) using FACSDiva Software (BD Biosciences). For intracellular staining, such as Foxp3, Granzyme A, GranzymeB, perforin, and Ki-67, cells were first stained with surface marker, and further fixed and permeabilized for intracellular staining using Fix and Perm (eBioscience). Final plmiceot/histogram figures were prepared using FlowJo Software (Tree Star, Ashland, OR, USA).

Zhong H., Liu Y., Xu Z., Liang P., Yang H., Zhang X., Zhao J., Chen J., Fu S., Tang Y., Lv J., Wang J., Olsen N., Xu A, & Zheng S.G. (2018). TGF-β-Induced CD8+CD103+ Regulatory T Cells Show Potent Therapeutic Effect on Chronic Graft-versus-Host Disease Lupus by Suppressing B Cells. Frontiers in Immunology, 9, 35.

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Comprehensive Analysis of T-cell Subsets

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Spleen, peripheral lymph nodes (inguinal, axillary, and brachial) and mesenteric lymph nodes were harvested and crushed through 100 μm filters (BD Falcon) to generate single-cell suspensions. 1×10⁶ cells were surface stained with a combination of the following antibodies: anti-CD4, CD44, CD62L, ICOS, CCR7, CD69, CD25, Nrp-1 (all from eBioscience, San Diego, CA), Vβ1-17 (BD Biosciences, San Diego, CA). Cells were intracellularly stained for Bim (Cell Signaling Technology, Danvers, MA), Bcl-2 (generated in-house), Ki67 (eBioscience), Helios (eBioscience), and FoxP3 (eBioscience) using the eBioscience FoxP3 staining kit and protocol. For surface staining of CCR7, cells were incubated at 37C for 1 hour prior to adding the anti-CCR7 antibody. Data were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences). Histogram overlays were generated using FlowJo software (FLOWJO, LLC, Ashland, OR); the smoothing effect was applied to the histograms, and the y-axis is representing the data normalized to the mode.

Raynor J., Karns R., Almanan M., Li K.P., Divanovic S., Chougnet C.A, & Hildeman D.A. (2015). IL-6 and ICOS antagonize Bim and promote Treg accrual with age. Journal of immunology (Baltimore, Md. : 1950), 195(3), 944-952.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry analysis was performed with fluorescein-labeled monoclonal antibodies to mouse CD4, CD8a, CD25, H-2K^b, H-2K^d, FoxP3, IFN-γ, CXCR3, KLRG, CD44, CD62L, CD69, CD127, granzyme B, and IL-17A (eBioscience, San Diego, CA), as described previously (42 (link)). For intracellular staining (FoxP3, IFN-γ, and IL-17A), permeabilization buffer (eBiosciences) was used. IFN-γ and IL-17A production by donor T cells was assessed following in vitro re-stimulation for 5 hours with Cell Stimulation Cocktail (eBioscience, San Diego, CA), according to manufacturer’s instructions. To measure mitochondrial mass and matrix oxidant burden, cells, following MLR, were incubated for 15 min at 37°C in phosphate-buffered saline (PBS) supplemented with 25nM Mitotracker Green FM and 1 μM CellRox Deep Red (Invitrogen, Carlsbad, CA), respectively. Fatty acid transport was assessed by staining with BoDipy_C1-C12 (ThermoFisher Scientific, Waltham, MA). Cells were analyzed using a BD Accuri C6 (BD Biosciences, San Jose, CA) or an Attune NxT (Invitrogen) flow cytometer. Annexin V (Biolegend), 7-AAD (Biolegend), and CellRox Deep Red were used to stain tissue culture cells according to manufacturer protocol.

Toubai T., Tamaki H., Peltier D.C., Rossi C., Oravecz-Wilson K., Liu C., Zajac C., Wu J., Sun Y., Fujiwara H., Henig I., Kim S., Lombard D.B, & Reddy P. (2018). Mitochondrial Deacetylase SIRT3 Plays an Important Role in Donor T Cell Responses after Experimental Allogeneic Hematopoietic Transplantation. The Journal of Immunology Author Choice, 201(11), 3443-3455.

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Multiparametric Flow Cytometry Analysis

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After Fc blocking, cells were stained with immunofluorescence (fluorescein isothiocyanate [FITC], Alexa Fluor 488, PE, PE-Cy5.5, APC, or Alexa Fluor 647)-conjugated antibodies specific for mouse and/or human CD45 (BD Biosciences), ALCAM (eBioscience or LifeSpan Biosciences), DIP2A (Bioss Antibodies), FOXP3 (eBioscience), CD3 (BD Biosciences), CD4 (BD Biosciences), CD8 (BD Biosciences), FOXP3 (eBioscience), PD1 (BD Biosciences), TIM3 (R&D Systems), CD11b (BD Biosciences), Gr1 (eBioscience), tetramer for gp70 (MBL International), CD44 (BD Biosciences), GZMB (eBioscience), EOMES (R&D Systems), Ki67 (Invitrogen), IFN-g (BD Biosciences), or the appropriate isotype control antibodies. For intracellular staining, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) before antibody staining. The immunofluorescence was analyzed and compared with the isotype controls by CellQuest software using a FACSCalibur cytometer (BD Biosciences). In the in vivo study, tumor-infiltrating cells were analyzed after gating CD45 + cells or GFP À cells to exclude GFP + tumor cells.

Kudo-Saito C., Ishida A., Shouya Y., Teramoto K., Igarashi T., Kon R., Saito K., Awada C., Ogiwara Y, & Toyoura M. (2018). Blocking the FSTL1-DIP2A Axis Improves Anti-tumor Immunity. Cell reports, 24(7).

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