(DOCX) Click here for additional data file.(14K, docx) S4 MethodsFlow cytometry analysis (FCM). construction Mouse anti-CTSL monoclonal antibody (mAb) and rabbit anti-CTSL polyclonal antibody (pAb) were produced by our lab at China Agricultural University. The concentrations of CTSL were analyzed with sandwich ELISA as described in S2 Methods. Isolation and culture of DC cells and B Melagatran cells For isolation of DCs, peripheral blood mononuclear cells (PBMC) were isolated and cultured (S3 Methods) firstly. Then, monocytes were enriched by MACS separation. CD11b+ cells were positively selected with magnetic microbeads accordingly to manufacturers instructions (Miltenyi Biotec, Auburn, CA), and cultured in a complete RPMI 1640 medium with 20 ng/ml of recombinant porcine granulocyte-macrophage colony-stimulating factor (rpGM-CSF, Invitrogen, CA, USA) Melagatran and 10 ng/ml of recombinant porcine interleukin 4 (rpIL-4, Invitrogen, CA, USA). Half of the culture medium was replaced with fresh medium every two days. At 7 d, monocytes differentiated to immature monocyte-derived DC (Mo-DC), then become mature after stimulated with 2 g/ml of lipopolysaccharide (LPS, Sigma) for 24 h. For isolation of B cells, pig spleens were disaggregated in RMPI-1640 using a mortar and pestle and sifted through a cell filter, followed by depletion of RBC with ammonium chloride solution. CD19+ B cells were separated by MACS Melagatran with mouse anti-pig CD19 antibody. B cells were cultured for 5 days in RPMI-1640 medium with 10% FBS and 25 g/ml of rpIL-4, 50 g/ml interferon (IFN-, Invitrogen, CA, USA) and 50 g/ml CD40L (DAKEWE, China). Half of the medium was replaced by SERPINE1 fresh complete medium every two days. At 7 d, B cells were stimulated for 24 h with 100 ng/ml CpG-ODN 2006 (Invitrogen, CA, USA). Flow cytometric analysis Surface markers of DCs and B cells were analyzed by flow cytometry. Procedures of flow cytometry can be seen in online Supplementary Methods. For DCs, labeling strategies were as follows:(1) anti-CD1 antibody (FITC) [clone 76-7-4], mouse anti pig CD172a antibody [clone BL1H7]; (2) anti-CD4 antibody (FITC) [clone 74-12-4], anti-CD8 antibody (Phycoerythrin) [clone MIL-12]; (3) anti-CD11b antibody (FITC) [clone 2F4/11], anti-porcine MHC Class II DQ antibody [clone K274.3G8]; (4) anti-CD14 antibody (Phycoerythrin) [clone TK4], anti-CD21 antibody (Alexa Fluor647) [clone LT21]; (5) anti-CD163 antibody (Phycoerythrin) [clone 2A10/11 ]. B cells were labeled with strategy: (1) anti-CD1 antibody (FITC) [clone 76-7-4], anti-CD20 antibody [clone MEM-97]; (2) anti-CD4 antibody (FITC) [clone 74-12-4], anti-CD8 antibody (Phycoerythrin) [clone MIL-12]; (3) anti-CD11b antibody (FITC) [clone 2F4/11], anti-CD79a antibody (PerCP/Cy5.5) [clone HM47]; (4) anti-CD21 Santi-porcine MHC Class II DQ antibody [clone K274.3G8]. The presence of CD4+ T cells and CD8+ T cells in PBMC was also analyzed with mouse monoclonal anti-porcine CD4-FITC [clone 74-12-4] and mouse monoclonal anti-pig CD8- PE/Cy5 [clone 76-11-2] separately, as described in S4 Methods. Co-culture of DCs and B cells for 2 h. In group 2, cells were treated with rCTSL protein for 1 h followed by infection with for 2 h. In group 3, cells were transfected with eukaryotic plasmid CTSL-GFP for 48 h followed by infection with for 2 h. In group 4, cells Melagatran were treated with E64 for 1 h followed by infection with for 2 h. In group 5, cells were transfected with px458-cas9-CTSL vector for 48 h followed by infection with for 2 h. At the same time, B cells and DCs were cultured separately as controls. The cell Melagatran supernatants were collected for the detections of various cytokines and SIgA, and the pellets were collected for the detections of CTSL and Ii chain. Cells transfection The eukaryotic expression vector CTSL-GFP was constructed as described in S5 Methods and used for transfection. Knockdown vector was conducted with CRISPR-Cas9 plasmid. According to the gene of porcine CTSL (Gene ID: 396926, S1 Methods), sgRNAs were designed by Biomics Biotechnologies Company. We choose as double targets and constructed the plasmid px458-253r.