Cd4 fitc

The following fluorochrome-conjugated antibodies (mAb) were used. For the in vitro assay, Dead Cell Marker-FarRed (Life Technologies), CD3-DAPI (Biolegend, San Diego, CA, USA), CD45-BV510 (Biolegend), CD4-FITC (eBiosciences, Thermo Fisher Scientific, Waltham, MA, USA), CD8-PE (eBiosciences), CD11c-PE-Cy7 (eBiosciences), and CellTrace Far Red-APC (Invitrogen, Waltham, MA, USA) were used. For the in vivo studies, Dead Cell Marker-Aqua (Life Technologies), CD3-DAPI (Biolegend), CD45.2-PE (eBiosciences), CD45.1-eFluor780 (eBiosciences), CD4-FITC (eBiosciences), CD8-BV421 (BD), CD11c-PE-Cy7 (eBiosciences), CD25-PE Cy7 (eBiosciences), CD19-BV711 (BD) and CellTrace Far Red-APC (Invitrogen) were used. For cell surface analysis of proliferation, CD4+ T cells were collected after the co-culture, washed two times with FACS Stain Buffer (BD), and stained with the indicated antibody mastermix for 20 min at 4 °C. Then, the cells were harvested and fixed with Fix/Perm (eBioscience) and finally analyzed for Celltrace dilution with flow cytometry. Samples were acquired on a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed using FlowJo v10 (Tree Star Inc., San Carlos, CA, USA). Calculations of percent proliferated T cells were essentially performed according to [30 (link)].

T cells were isolated from the spleen and fluorescent conjugated antibodies including APC‐CD3 (eBioscience, San Diego, CA, USA, 17‐0032‐80), FITC‐CD4 (eBioscience, 11‐0041‐82), and PE‐CD8 (eBioscience, 12‐0081‐82). Treg cells were obtained from spleen and stained with FITC‐CD4, PE‐CD25 (eBioscience, 12‐0259‐42), and Alexa‐Foxp3 (BD, 560047). BMDCs were generated from bone marrow and incubated with the specific antibody. Antibodies used included FITC‐CD11c (eBioscience, 17‐0114‐82), APC‐CD86 (eBioscience, 11‐0862‐82), PE‐MHC‐II (eBioscience, 11‐5321‐82), Alexa‐CCR7 (eBioscience, 12‐1971‐82), and PE‐PD‐L1 (eBioscience, 12‐5982‐82). Analysis was performed on a flow cytometer (Becton Dickinson, Mountain View, CA, USA), and the FMO controls were used to set gating (Fig. S1A,B), MHC‐II‐FMO stained with CD11C and CD86, CD86‐FMO with CD11C and MHC‐II, CD4‐FMO with CD3 and CD8, CD8‐FMO with CD3 and CD4, CD25‐FMO with CD4 and Foxp3, and Foxp3‐FMO with CD4 and CD25. The cell populations were gated on forward versus side scatter pulse area and side scatter width versus height to exclude cell debris and clumps to select live singlet cells. Prepared cell samples were detected by flow cytometer immediately to make sure accurate cell counts.

On day 28 p.i., mice were sacrificed and spleens were removed under aseptic conditions. MNCs were prepared as above described stained for 20 minutes at RT in 1% BSA‐PBS buffer with the following panel of antibodies: FITC‐CD4 (eBioscience, San Diego, CA, USA) and PE‐CD25 (eBioscience), Alexa Fluor 488‐anti‐CD11b (eBioscience) and PE‐CD16/32, PE‐CD206, PE‐CD11c, PE‐CD40, PE‐CD8a, PE‐CD14, PE‐CD200 (eBioscience). For intracellular staining, MNCs were stained for 20 minutes at RT in 0.3% saponin/1% BSA‐PBS buffer with the following panel of antibodies: FITC‐CD4 and PE‐IL‐10, PE‐transforming growth factor‐β (TGF‐β), PE‐IFN‐γ, PE‐IL‐17 (eBioscience), Alexa Fluor 488‐anti‐CD11b and PE‐IL‐12, PE‐chemokine receptor7 (CCR7) (eBioscience). At least 10 000 events were collected using flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and data were analysed using CellQuest software (BD Biosciences).

UAMC-1110 was generously provided by Dr. Pieter Van der Veken (University of Antwerp). Unless otherwise specified, UAMC-1110 was administered in high molecular weight PEG at a concentration of 20mg/kg by oral gavage twice per day. Fluorescently-conjugated antibodies CD3-e450, CD8-PerCP, CD4-FITC, CD4-e450, CD4-PerCP, CD25-APC, IFNγ-APC, IL-2-PE, TNFa-PE-Cy7, CD11b-PE-Cy7, Ly6G-FITC, Ly6C-PerCP-Cy5.5, Gr1-PE-Cy7, and MHCII-EF450, PDGFRβ-APC, CD31-PE, CD45-BV510, and EpCam-BV605 were purchased from Thermo Fisher Scientific (Lafayette, CO) or BD Biosciences (San Jose, CA). CD8-PE-TxRD was purchased from Invitrogen (Carlsbad, CA). SIY, SIINFEKL, and β-galactosidase peptides were obtained from Integrated DNA Technologies (Coralville, Iowa). β-galactosidase (βgal) for the ICPMYARV peptide and SIINFEKL peptide tetramers were obtained from the NIH Tetramer core facility (Atlanta, GA). Antibodies to CD31 (Thermo Fisher Scientific), αSMA (Sigma-Aldrich, St Louis, MO), PDGFRα (Thermo Fisher Scientific), Vimentin (Cell Signaling Technology, MA), Ki67 (Abcam), Cleaved Caspase 3 (Cell Signaling Technology) were used for immunofluorescent staining and hypoxyprobe Omni Kit (Burlington, MA) was used for hypoxia staining. Anti-PD1 antibody clone RPM1-14 (BioXcell, West Lebanon, NH) was used where indicated for treatment studies.

Flow cytometry was used to identify the immune cell subsets from the fresh spleen tissues of mice. Briefly, the spleen tissues were dissociated into single cells by trituration and cell lysis with red blood cell lysis buffer (Cat. No: 420301; Biolegend, San Diego, CA, USA). After centrifugation, the density of the single-cell suspension was adjusted to 1 × 10⁶ cells/mL. The CD3⁺CD8⁺T cells, CD3⁺CD4⁺T cells, CD4⁺IFNγ⁺T cells, and Treg cells were stained with antibodies for labeling. Next, the percentage of the different immune cell subsets was identified by flow cytometry (FACSFortessa X20). The antibodies used were as follows: CD3 eF450 (Cat. No: 11-0042-82, Thermo), CD4 PE-Cy7 (Cat. No: 100528; BD Biosciences, San Jose, CA, USA), CD8a PerCP-Cy5.5 (Cat. No: 45-0081-82; Thermo), CD4 FITC (Cat. No: 11-0042-82; Thermo), and IFN eF450 (Cat. No: 48-7311-82; Thermo).

After isolation and re-stimulation, the splenocytes were collected, washed and immunophenotyped by using the following anti-mouse mAbs: CD3-eFluor 450, CD4-FITC (Thermo Fisher Scientific), and CD8a-APC-H7 (BD Biosciences). Appropriate isotype controls were used for comparison. After staining, the splenocytes were assessed by BD FACSAria™ II Flow Cytometer (BD Biosciences, San Jose, CA, USA). Dead cells were excluded by using LIVE/DEAD Fixable Dead Cell Stain (Invitrogen). Antigen specific CD4⁺ or CD8⁺ T cells were analyzed with FlowJo software (Tree Star Inc., Ashland, OR) [43 (link)]. All experiments were performed in triplicate.

Before immunofluorescence staining of cells, Fc receptors were blocked using FcR Blocking Reagent (Miltenyi Biotec). Subsequently, the cells were stained with fluorochrome-labeled mouse antibodies and analyzed using a MACSQuant^® Analyzer (Miltenyi Biotec). Dead cells identified by propidium iodide (Miltenyi Biotec) staining were excluded from the analysis. All antibodies were purchased from Miltenyi Biotec except where noted. The following antibodies were used to stain tumor infiltrating CD8⁺ T cells: CD45-VioBlue, CD4-FITC, CD8a PE-Cy7 (Thermo Fisher Scientific, MA, U.S.A.), MHC class II-APC and APC/Cy7-TCRβ (BioLegend, CA, U.S.A.). The following antibodies were used for detailed analysis of CD8⁺ T cells: CD45-VioBlue, CD4-VioGreen, CD44-FITC, CD8a PE-Cy7 (Thermo Fisher Scientific), CD25-APC, CD69-APC-Vio770 and CD62L-PE. The following antibodies were used in the anti-CD8 antibody experiment: CD45-VioBlue, CD8a-PE-Vio770 and APC/Cy7-TCRβ (BioLegend).