Foxp3 pe

Spleen tissues were isolated from mice in both the group at 14 weeks after curdlan injection. To examine the populations of Th17 and Treg cells, the tissues were stained with Abs against CD4–fluorescein isothiocyanate (FITC), IL-17–phycoerythrin (PE), CD25–allophycocyanin, and FOXP3–PE (all from eBioscience, San Diego, CA, USA). To determine the population of cells expressing STAT, the tissues were stained with Abs against CD4–FITC, p-STAT3–Y705–PE, and p-STAT3–S727–PE (eBioscience, San Diego, CA, USA). The stained tissue sections were analyzed using a confocal microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany). We counted cell number using Pannoramic MIDI and Pannoramic viewer (3D HISTECH Ltd, Hungary).

To systematically investigate the in vivo antitumour immune responses against mimic distant tumours, the tumours were harvested and treated with the tissue dissociation kit (Miltenyi Biotec, Germany) to produce a single-cell suspension according to the specified procedures. The harvested cells were further stained with several fluorochrome-conjugated antibodies: CD45-FITC (BD, Catalog: 561088), CD3-PerCP-Cy5.5 (BD, Catalog: 551163), CD4-BV510 (BD, Catalog: 563106), CD8-BV421 (BD, Catalog: 563898), NKp46-APC (Biolegend, Catalog: 137608), B220-PE-Cy7 (BD, Catalog: 552772) and Foxp3-PE (eBioscience, Catalog: 12–4771) and then analysed by FCM. For the analysis of the memory T cells, spleen cells of mice were harvested and stained with anti-CD3-FITC (Biolegend, Catalog: 100306), anti-CD4-PE-Cy7 (eBioscience, Catalog: 25–0041), anti-CD8-PerCP-Cy5.5 (eBioscience, Catalog: 45–0081), anti-CD44-PE (eBioscience, Catalog: 12–0441) and anti-CD62L-APC (eBioscience, Catalog: 17–0621) antibodies and then analysed by FCM. All antibodies were diluted ~100 times.

T cells along with untreated or lactate treated pDCs from 5 day old co-cultures were either stained with BDCA2 APC or CD4 BUV395 to differentiate the T cells from pDCs during flow cytometry. This was followed either only by intracellular staining with FoxP3 PE (clone: 259D/C7, BD Biosciences) or in some cases by surface staining with CD25 APC followed by intracellular staining with FoxP3 PE (clone: PCH101, eBioscience) and acquisition in a flow cytometer.

To determine the Th1, Th2, Th17, and Treg cells in mice, flow cytometric analysis was performed on isolated DRG cells using CD4, Foxp3, IL-17A, TGF-β, and IL-4 antibodies as reported previously (18 (link)). Briefly, the cells suspension was transferred into 1 mL phosphate buffer saline (PBS) and centrifuged at 350 g for 5 min. After centrifugation, the supernatant was removed and the cells were resuspended with 500 μL fixation/permeabilization then centrifuged at 350 g for 5 mice after standing at room temperature for 30 min. The resuspension was repeated for twice. The cells were then incubated with monoclonal antibodies, including CD4-FITC, FOXP3-PE, IL-17A-PE, IL-4-PE, and IFN-γ-PE antibodies (eBiosciences, San Diego, California, USA) at dark for 30 min. After washing with PBS, the cells were resuspended in 150 μL PBS and then tested by using Beckman counter flow cytometer (USA). The data were analyzed using FlowjoX software. The lymphocytes were gated by FSC and SSC. CD4⁺IL-17A⁺, CD4⁺IL-4⁺, CD4⁺ IFN-γ⁺, and CD4⁺ FOXP3⁺ lymphocytes were identified as Th17, Th2, Th1, and Treg respectively.

Expression of B7-1, ICAM-1, and LFA-3 was determined by flow cytometry. Treated MC38 cells were stained with FITC-labeled antibodies to CD80 (B7-1), CD54 (ICAM-1) and CD48 (LFA-3) (BD Biosciences, San Jose, CA). Cells were incubated with antibodies for 30 min at 4°C. Samples were acquired on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The effect of MVA-TWIST/TRICOM on splenic immune cell populations was examined in tumor bearing and non-tumor bearing BALB/c mice 17 days after receiving two vaccinations with MVA-TWIST/TRICOM or being left untreated (n = 5/group). Vaccination of tumor bearing mice began 4 days post-implantation of 5 × 10⁴ 4T1 mammary tumor cells. Spleens were prepared and stained as described previously [60 (link)], using the following antibodies: CD3e-V500, t-APC, CD8a-Pacific Blue, CD25-FITC, CD44- PerCP-Cy5.5 CD11b-V500, Gr-1-APC, CD11c-PerCP-Cy5.5, CD40-FITC (BD Biosciences); CD62L-PE-Cy7, FoxP3-PE, MHC II-efluor450 (eBioscience, San Diego, CA); and CD49b-PE-Cy7 (Biolegend, San Diego, CA). Tetramer staining (Beckman Coulter, Pasadena, CA) was performed on splenocytes from non-tumor bearing mice following 7 days of in vitro stimulation with 1.0 μg/mL Twist peptide (BALB/c - LYQVLQSDEL). All samples were acquired on a BD Verse flow cytometer. All marker expression was determined using FlowJo software (TreeStar, Inc., Ashland, OR).

The same procedure was followed as described in DC‐T cell co‐culture procedure and maintained for up to 5 days for induction of T regulatory cells (Tregs). On the 5th day, cells were harvested and stained for Treg‐specific markers; anti‐mouse CD4‐FITC (# 11‐0042‐82), CD25‐PE‐Cy7 (# 25‐0251‐82), and FOXP3‐PE (# 12–5773‐82) all from eBioscience (San Diego, CA); and CD127‐APC (# 158205) Biolegend (San Diego, CA) fluorochrome‐tagged monoclonal antibodies.

To evaluate Treg polarization, PBMC-derived lymphocytes cocultured with cASC-EVs were harvested. Obtained cells (1 × 10⁶⁾ were suspended in 100μL DPBS and 1 μL of each primary antibody against the following proteins: FOXP3-PE (eBioscience, San Diego, CA, USA; 1:100), CD3-FITC (MCA1774F; Bio-Rad, San Diego, CA, USA; 1:100), CD206-FITC (eBioscience, San Diego, CA, USA; 1:100) and CD11c-PE (eBioscience, San Diego, CA, USA; 1:100). After incubation for 1 h at 23 ± 2°C, the cells were washed with DPBS. Unstained cells were used as controls for autofluorescence. Cell fluorescence was analyzed with a flow cytometer (FACS Aria Ⅱ; BD bioscience). The results were analyzed using FlowJo 7.6.5 software (Tree Star, Inc., Ashland, OR, USA).