Anti cd4

Flow cytometry analysis was performed to determine the relative amount of Treg cells as described earlier [20 (link)]. Briefly, the cells were first stained by the anti-CD4 (1:100, Abcam, Shanghai) and anti-CD25 (1:100, Abcam, Shanghai) antibodies. Subsequently, the cells were fixed as well as permeabilized using the fixation/permeabilization solution kit (BD Cytofix/Cytoperm, U.S.A.) followed by being stained with the anti-Foxp3 antibody (1:50, Cell Signaling Technology). Samples were sorted on apparatus and the relative amount of Treg cells was obtained. Treg cells were measured by flow cytometer with Foxp3+ as the marker.

MCF-7 cells were purchased from the Pasture Institute of Iran (Tehran, Iran). Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS), penicillin, and streptomycin were purchased from Life Technologies Co. (Gibco Life Technologies, Carlsbad, CA). Ketamine and xylazine were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The Cobalt-60 facility was provided by the Cancer Institute of Imam Khomeini hospital Complex of Iran (Tehran University of Medical Sciences, Tehran, Iran). Mouse monoclonal antibodies of anti-CD19, anti-CD4, and anti-CD8 were purchased from Abcam CO. INF-γ Mouse ELISA Kit (Abcam; ab46081), IL-4 Mouse ELISA Kit (Abcam; ab100710), and IL-17 Mouse ELISA Kit (Abcam; ab100702) were used for IFN-γ, IL-4, and IL-17 serum level assessment. Total RNA was isolated with a Total RNA extraction kit (GRM1002, VIOGENE, Sunnyvale, CA, USA). Template cDNAs were synthesized using SuperScript III (18080-051, Invitrogen, Carlsbad, CA). Quantitative RT-PCR was performed with a quantitative PCR mix (QPK-201T, Toyobo, Osaka, Honshu, Japan) using a real-time PCR detection system (CFX96; Bio-Rad Laboratories).

Sections stained with immunofluorescence were incubated overnight in a cold room with the antimouse primary antibody (as required), rabbit anti-CD11b (1:500, Abcam), rabbit anti-CD4/80 (1:500, Abcam), anti-CD4 (1:500, Abcam), or rabbit anti-CD8 (1:200, Cell Signaling Technology, Danvers, MA, USA). The sections were subsequently immunohistochemically stained with specific fluorophore-conjugated goat anti-rabbit (F-2765, 1:500, Invitrogen). Fluorophore FITC (488 nm excitation wavelength), CY3 (555 nm excitation wavelength), or CY5 (670 nm excitation wavelength) antibody. Sections were then incubated with DAPI, and the slides were scanned (×40) using a Panoramic Midi Scanner (3DHISTECH, Budapest, Hungary). High-magnification images were obtained using Case Viewer software, and intensity of fluorescence was measured using Fiji/ImageJ software following standard protocol. Corrected total fluorescence (CTF) was analyzed following the standard formula [CTF = Integrated density − (Area of selected cell × Mean fluorescence of background readings)], and average intensity was calculated.

Briefly, peripheral elbow venous blood (5 ml) was obtained from all patients within 24 h after admission. The samples were collected in tubes with heparin and added with 5 ml phosphate buffer (PBS) and then centrifuged at 1500 g for 10 min. The cell layer was then collected, added with 5 ml PBS, following with centrifugation at 1200 g for 5 min. After removing the supernatant and washing with PBS, the samples were further centrifuged at 1000 g for 2 min to obtain the peripheral blood mononuclear cells. The cells with density of 1 × 10⁶/ml were maintained in RPMI-1640 Medium (Sigma-Aldrich, St. Louis, MO, USA).
For measurement of PD-1⁺ and LAG-3⁺ T cells, flow cytometry was performed as described elsewhere [17 (link), 18 (link)]. Antibodies used in this study included anti-CD4, anti-CD8, anti-PD-1 and anti-LAG-3 (all purchased from Abcam, USA). The measurement was conducted on a FACS Calibur flow cytometry analyzer (BD Biosciences) using Diva software (version 6.1, BD Pharmingen).

The paraffin-embedded tissue sections on slides were deparaffinized. The sections were then incubated with a primary antibody (1:100 dilution) for 1 h at 37 °C. The primary antibodies were anti-CD4 and anti-CD11b (Abcam, Cambridgeshire, UK). The signal was visualized using an Envision System (DAKO, CA, USA) for 30 min at 37 °C; 3,3′-diaminobenzidine tetrahydrochloride was used as the coloring reagent, and hematoxylin was used as the counter-stain. The slides were examined with a Pannoramic MIDI slide scanner, and the integrated optical density was analyzed with iSolution DT software.

Paraffin-embedded tissue sections of 4 μm thicknesses were deparaffinized with xylene, dehydrated in gradually diminishing concentrations of ethanol, and treated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. The tissue sections were immersed in a 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95 °C. The last step was repeated using a 10 mM sodium citrate solution (pH 6.0). The sections were allowed to stay in the same solution while cooling for 20 min, following which they were rinsed in PBS. Then, the sections were incubated with a primary antibody (1:100 dilution) for 1 h at 37 °C. The primary antibody was as follows: anti-neutrophil gelatinase-associated lipocalin (NGAL, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-kidney injury molecule-1 (Kim-1, formerly called Tim-1, Abcam, Cambridge, MA, USA), anti-Mac-2 (formerly called Galectin-3, Abcam), and anti-CD4 (Abcam). The signal was visualized using an Envision System (DAKO, Carpinteria, CA, USA) for 30 min at 37 °C. 3,3′-Diaminobenzidine tetrahydrochloride (DAB) was used as the coloring reagent, and hematoxylin was used as the counter-stain. The slides were examined using a slide scanner (Pannoramic MIDI) and analyzed with iSolution DT software (IMTechnology, Vancouver, BC, Canada).