Gentlemax

Hepatic immune cells were isolated using Miltenyi Biotec Gentlemax technology followed by Percoll gradient. Briefly, whole liver was homogenized using Miltenyi Gentlemax C tubes using RPMI + 10% fetal bovine serum. After resuspension, cells were centrifuged at 2000 rpm for 10 minutes. Cell pellets were homogenized in a 33% Percoll solution (Sigma-Aldrich) diluted in RPMI medium 1640 (Gibco). Following gradient separation, and lysing of red blood cells, hepatic infiltrating immune cells were subsequently analyzed by flow cytometry (3 (link), 25 (link), 26 (link)).

For the analysis of MHC molecules expression by SARC-L1, cells were stained with the
following fluorescent conjugated monoclonal antibodies: Anti-2K^b, (clone
AF6-88.5.5.3,); anti-H-2K^dand anti-I^A/I^E(Ebiosciences, France); anti-HLA-A2 (BD Biosciences, France); anti-HLA-DR (Diaclone,
France) or with their respectively isotype control. For tumor infiltrating immune
cells analysis, tumor cells were mechanically dissociated (GentleMax, Miltenyi,
France) tumor infiltrating lymphocytes were separated on a Percoll (Sigma-Aldrich,
France) density gradient. Lymphocytes layer was collected and stained with the
following fluorescent conjugated monoclonal antibodies: anti-CD3, anti-CD4, anti-
CD8, anti-Gr1, anti-PD-L1 (Biolegend, France), anti-CD11b, anti- FoxP₃,
anti-CD25 (eBiosciences, France), anti-PD-1, and anti-TIM-3 (Miltenyi, France),
fixable viability stain (BD, France). The tumor cells fraction was stained
withanti-CD45, anti-PD-L1, anti-HLA-A2 and anti-HLA-DR (BD Biosciences, France) and
fixable viability stain (Biolegend, France). Samples were acquired on a FACS Canto II
(BD Biosciences, France) and analyzed with the BDFacsDIVA or FlowJo software.

Peyer's patches were collected from the ileum of 4- to 6-month-old healthy Large White pigs. The mucus, intestinal villus, and serosal surface were removed before the separation of lymphocytes. Mesenteric lymph nodes (MLNs) were also collected. These tissues were washed with cold sterile PBS, minced into small pieces and homogenized in a cell separator GentleMax (Miltenyi Biotec). The cell suspension was gently mixed with PBS and passed through a 100 μm cell strainer to exclude the tissue debris. Cells were collected by centrifugation and resuspended in 40% Percoll plus medium (GE healthcare) and laid on the top of 67.5% Percoll plus medium. The lymphocytes were harvested from the interface between the two Percoll layers after centrifugation at 1,800 rpm for 30 min at room temperature. After being passed through a 40 μm cell strainer, the lymphocyte suspension was stained with porcine IgM monoclonal antibody (Washington State University Monoclonal Antibody Center) and then incubated with anti-mouse IgG Microbeads (Miltenyi Biotech). The IgM⁺ B cells were collected through a magnet-based column following the manufacturer's manual.
All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China.