Clone w6 32

Macrophages were harvested with a cell scraper and fixed with 4% paraformaldehyde for 20 min, followed by cell staining with antibodies specific for human HLA-ABC, dilution 1:100 (clone W6/32, BioLegend, UK, Cat. No. 311418) or HLA-DR dilution 1:25 (clone L243, BioLegend UK, Cat. No. 307606), for 30 min. Determination of the rate of infection was performed based on the percentage of GFP positive cells, on fixed cells 24 h post-infection, after washing several times with PBS to remove extracellular bacteria. Samples were run on an LSR II flow cytometer and the data analysed using FlowJo (TreeStar Inc, Ashland, USA).
HeLa cells infected with ChAdOx1 and MVA cloned with each selected antigen were used detect antigen expression by western blot, using the iBind Western System (Invitrogen, UK). Antigens were detected with a primary Histidine Tag antibody, diluted 1:1000 BioRad, Cat. No. MCA1396, clone AD1.1.10, followed by a secondary polyclonal antibody, Goat Anti-Mouse IgG Fc (Alkaline Phosphatase), dilution 1:1000 (Abcam, UK, Cat. No. ab98710).

Five osteosarcoma cell lines were stained with antibodies which is pan-reactive for MHC class I (clone W6/32, BioLegend Cat. 311405) or specific for HLA*A2 (clone BB7.2, BioLegend, Cat. 343308). Flow cytometry was performed using BD LSRFortessa cell analyzer (BD Biosciences).

The following antibodies were
used to analyze the cell surface expression of HLA-A2 and HLA-A, HLA-B,
and HLA-C: PE-conjugated antihuman HLA-A2 (clone BB7.2, BioLegend
343306 San Diego, CA), PE-conjugated antihuman HLA-A, HAL-B, and HLA-C
(clone W6/32, BioLegend 311406, San Diego, CA), and Human TruStain
FcX block (BioLegend B247182, San Diego, CA).
The data were
acquired using a BDLSR Fortessa flow cytometer. Flow cytometric analysis
of renal cell carcinoma and bladder tumor-derived organoids was performed
using a BD Accuri 6 plus (BD Biosciences) and analyzed with FlowJo
software (Tree Star, Ashland, OR).

Freshly isolated Tros (Matrigel-coated 24-well plates) or HTR8/SVneo cells, or DSCs were seeded at a density of 2 × 10⁵ cells/ml per well in 24-well plates overnight. The cells were then washed with PBS (HyClone, U.S.A.). Equal numbers of dCD4⁺T cells or pCD4⁺T cells were added to each well. In some wells, dCD4⁺T cells were plated in the upper chamber (0.4 mm poresize cell culture inserts, Millipore, Germany), while Tros were plated in the lower chamber to establish indirect cell contact. In some wells, anti-HLA-G (10 μg/ml, clone 87G; Biolegend, U.S.A.) or/and anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A.) were added. PMA (50 ng/ml), ionomycin (1 μg/ml) and brefeldin A (10 mg/ml) were added 4 h before the end of the 48 h culture for intracellular cytokine analysis. The immune cells were then harvested for flow cytometry analysis.

Freshly isolated Tros were seeded at a density of 2 × 10⁵ cells/ml per well in Matrigel (Coring, U.S.A)-coated 24-well plates overnight. The cells were then washed twice with phosphate-buffered saline (PBS, HyClone, U.S.A). Equal numbers of dCD14⁺ cells or pCD14⁺ cells were added to each well. In some wells, anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A), HLA-G (10 μg/ml, clone 87 G; Biolegend, U.S.A) were added. dCD14⁺ cells were also cultured with HTR8/Svneo cells (ATCC, www.atcc.org) or DSCs for 48 h. In some wells, dCD14⁺ cells (2 × 10⁵ cells) were plated in the upper chamber (0.4 mm pore size cell culture inserts, Millipore, Germany), while Tros were plated in the lower chamber to establish indirect cell contact.
Freshly isolated dCD4⁺ T cells co-cultured with Tim-3⁺dMφs or Tim-3⁻dMφs at a 1: 1 ratio. In some experiments, Tim-3⁺dMφs were pretreated with anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.), or anti-CD132 (10 μg/ml, clone TUGh4, BioLegend, U.S.A.), or anti-IL-4 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.). Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, U.S.A.), ionomycin (1 μg/ml, Biolegend, U.S.A.) and brefeldin A (10 mg/ml, BioLegend, U.S.A.), were added 4 h before the end of the 48 h co-culture. The supernatants were then collected for intracellular cytokine analysis of T cells.

Clathrin-mediated endocytosis was evaluated by measuring the uptake of transferrin from Human Serum, Alexa Fluor 488 Conjugate (T13342, Thermofisher Scientific). Clathrin-independent endocytosis was measured by intake of mouse monoclonal antibodies directed toward MHC-I (major histocompatibility complex I; clone w6/32, Biolegend) or control mouse monoclonal anti-cytokeratin 14 antibody (ab7800; Abcam; antibodies directed against intracellular target), followed by removal of the unbound antibody by low pH acid wash, fixation, and staining with secondary fluorescein isothiocyanate–labeled goat anti-mouse antibody (115-095; Jackson Laboratories).^{16 (link)} In some experiments, control (Adcontrol) and CLIC4-overexpressing (AdCLIC4) cells were treated with 20 mg/L protein synthesis inhibitor cycloheximide (CAS 66-81-9; Santa Cruz Biotechnology), with or without the endocytosis inhibitor Pitsop2 (20 μmol/L; ab120687; Abcam)^{16 (link)} or Pitstop2-negative control (20 μmol/L; ab120688; Abcam,) for 2 hours. Pitsop2-negative control is chemically related to Pitstop2 but does not block receptor-mediated endocytosis. BMPRII expression was analyzed by Western blotting.