L10119

Cells were stained for surface markers (such as CD4⁺ CD8⁻ T cells, B220⁺ CD3⁻ B cells, and CD11b⁺ Ly6G⁺ neutrophils) in PBS containing 1% bovine serum albumin with 0.5% sodium azide (PBA) for 30 min at 4°C and fixed with IC fixation buffer (eBioscience) as described previously (31 (link)). Nonviable cells were excluded using a fixable near-infrared dead cell staining kit with excitation at 633 or 635 nm (L10119; Invitrogen). Antibodies were purchased from BD Pharmingen or eBioscience.
Data acquired on a BD Fortessa III with 20,000 live lymphocytes or myeloid events were analyzed with the FlowJo (TreeStar) analysis program. Data are shown as a percentage of live myeloid or lymphocyte gates. Myeloid and lymphocyte gates are determined by their position on the live/dead versus forward scatter plots generated by the cytometer. A fluorescence-minus-one (FMO) control was included for each fluorescent marker, and the expression of a particular marker was calculated by subtracting FMO fluorescence values from fluorescent antibody levels.

CEM were counted and resuspended at a concentration of 2–3 × 10⁶ cells/mL in ice-cold FACS wash buffer (PBS, 10% FCS, 2 mM EDTA) containing 1.25 µL anti-human CD71 clone OKT9 on FITC (Invitrogen 14-0719-82) used at 0.6 µg/mL for 1 h at 4 °C then moved to 37 °C and incubated for 1 h with or without 100 µM cytochalasin D (Sigma-Aldrich C8273), 100 µM jasplakinolide (Abcam ab141409) or 1 µM ikarugamycin^{55 (link)} (Abcam ab143408). Cells were acid-washed (0.1 M glycine and 150 mM NaCl at pH 3) for 1 min, washed twice in cold FACS buffer, and labeled with near-IR fixable viability dye used at 1:1000 (Invitrogen L10119) before fixation and flow cytometric analysis. Gating on the relevant cell population was set according to Forward Scatter (FSC) and Side Scatter (SSC) before doublet and dead cell exclusion. Internalization was represented by the geometric mean fluorescence intensity (GMFI) of CD71 normalized to non-internalized sample.

To quantify T cells in ear skin, dermal sheets were separated, and finely minced in RPMI 1640 media (ThermoFisher 11875093) complemented with 10% fetal bovine serum (R&D Systems S11150) (cRPMI) containing 100 μg/mL of Liberase TL (Roche 5401020001) and 50 μg/mL of DNase I (Sigma-Aldrich DN25). Minced tissue was incubated with shaking at 37°C for 1 h and then strained through a 70 μm filter into a new tube containing 1 mL cRPMI. Cells were stained with cell surface stains and live-dead stain at 4°C for 15 min in PBS. Flow cytometry was then performed using an LSR II or LSR Fortessa instrument (BD Biosciences). Compensation was performed using compensation beads (BD Biosciences 552845). Flow cytometry data was analyzed using FlowJo software (BD Biosciences). Staining antibodies used included CD45.2 (mouse, PE fluorochrome, clone 104, BD Biosciences 560695, 1:200 dilution), TCRβ (mouse, PE-Cy7 fluorochrome, clone H57-597, BioLegend 109222, 1:200 dilution), CD4 (mouse, FITC fluorochrome, clone RM4-5, BioLegend 100510, 1:200 dilution), CD8a (mouse, PerCP-Cy5.5 fluorochrome, clone 53–6.7, BioLegend 100734, 1:200 dilution) and Live/Dead Near-IR (ThermoFisher L10119, 1:1000 dilution). CountBright beads were used for counting cells and normalization (ThermoFisher C36950).

5 × 10⁵ cells were requested per condition and were diluted at 5 × 10⁶ cells/ml in PBS 2% FBS. Near-IR staining (#L10119, Thermo Fisher, Waltham, MA, USA) was performed during 20 min at 4 °C to detect dead cells. The cells were washed once in PBS 2% FBS and stained for 1 h at 4 °C with PD-L1 antibody or BB515 isotype control at the indicated dilutions (Additional file 1). BB515 IgG1 isotype control is used as a negative control and binds specifically to KLH antigen which is not expressed in human cells. Flow cytometry analyses were conducted on a BD FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).