Cxcr4

For flow cytometry after culture, cells were stained in FACS buffer (0.5% BSA + 0,05% NaN3 in PBS) with monoclonal antibodies against CD4, CCR3, CXCR3, CCR2, CXCR4 (BioLegend), CCR6 (R&D Systems), CD45RO, CCR6, CCR7 (BD Biosciences), and CXCR5 (eBioscience). Samples were measured on a FACSCantoII Flow Cytometer.

Cells from CDLNs and the retina were harvested on day 14 after immunization. Cells were stained with live/dead dye (Thermo Fisher Scientific, Waltham, MA, USA) to identify the living cells. Subsequently, these cells were labeled with the following cell surface markers: CD4 (BioLegend, 100434), CD19 (BioLegend, 302244), Cxcr4 (BioLegend, 153805), CD138 (BioLegend, 142505), and B220 (BioLegend, 69-0452-82). For intracellular staining, cells were cultured with 1 µg/mL brefeldin A (Sigma‒Aldrich), 50 ng/mL phorbol myristate acetate (Sigma‒Aldrich, St. Louis, MO, USA), and 500 ng/mL ionomycin (Sigma‒Aldrich) for 5 h. Subsequently, the cells underwent fixation and permeabilization procedures before being stained with antibodies for various markers, including anti-IL-17A (BioLegend, 506930), anti-IFN-γ (BioLegend, 505808), anti-Foxp3 (BioLegend, 11-5773-82), anti-PIM1 (NOVUS, NBP2-67528), anti-phospho-AKT1 (BioLegend, 17-9715-42), anti-phospho-FOXO1 (Thermo Fisher Scientific, PA5-104977), and anti-rabbit IgG (H + L), F(ab’)2 fragment secondary antibody (Cell Signaling Technology, Danvers, MA, USA, 4412). Flow cytometry (BD LSRFortessa) was used for the analysis of these cells. FlowJo software was used for further results analysis.

Fluoroisothiocyanate, PE/dazzle, allophycocyanin, and phycoerythrin-labeled CD3, CD4, CD8, CXCR4, CXCR7, CD45R, HLA-DR, GATA3, Ki-67, Helios, and FOXP3 human monoclonal antibodies, RBC lysis buffer (10X), intracellular staining permeabilization wash buffer (10X), and fixation buffer were purchased from BioLegend (San Diego, USA). GolgiStop was purchased from BD Biosciences (San Diego, USA). RPMI 1640 medium, phorbol myristate acetate (PMA), and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol was purchased from Life Technologies (Paisley, UK). SYBR green and cDNA kits were purchased from Applied Biosystems (Foster City, CA, USA). Primers were synthesized from GenScript (Piscataway, NJ, USA).

Fluorescently-conjugated or biotin-conjugated antibodies to B220, CD5, CD19, CD21, CD23, CD69, CD86, CD93 (AA4.1), CD95 (Fas), CD138, CXCR4, IgA, IgM, IgM[a], IgM[b], IgD, IgD[a], IgG1[a], IgG1[b], Igκ, Igλ, GL-7, MHC Class II, and fluorescently-conjugated streptavidin were from Biolegend, eBiosciences, BD Biosciences, Tonbo, or Life Technologies. NP-PE was from Biosearch Technologies. 100 nm Rhodamine PtC liposomes were from FormuMax. Antibodies for intra-cellular staining, pErk Ab (clone 194g2) and pS6 Ab (2F9), were from Cell Signaling Technologies, and Nur77 Ab (clone 12.14) conjugated to PE was from eBioscience. Goat anti-mouse IgM F(ab’)₂ was from Jackson Immunoresearch. Goat anti-mouse Igκ and goat F(ab’)₂ anti-mouse Igκ were from Southern Biotech. Anti-IgD was from MD Biosciences. CXCR4 ligand (CXCL12/hSDF-1α) was from Peprotech. LPS (Cat. L8274) was from Sigma. CpG DNA (ODN 1826 Biotin; Cat. tlrl-1826b) and Pam3CSK4 (Cat. tlrl-pms) were from InvivoGen.