Cxcl13

Ex vivo chemotaxis assays were performed as described previously (33 (link), 95 (link)). Briefly, MLN cells in RPMI medium with 5% fetal calf serum (Invitrogen) were placed in the upper chamber of a 5-μm Transwell insert in a 24-well plate (Corning, Corning, NY). The lower chamber contained 200 ng of either CXCL13 or CCL21 (Peprotech, London, UK)/ml. Five technical replicates were performed. After 24 h, the upper chamber was discarded, and the cells in the lower chamber were collected and immunostained with anti-CD3 (clone 17A2) to detect T cells, anti-B220 (clone RA3-2GB) to detect B cells, and anti-CD11c (clone N418; BioLegend, London, UK) and resuspended in 500 μl of fluorescence-activated cell sorting buffer. The number of migrated cells was counted for 60 s using a LSRFortessa flow cytometer (BD Biosciences).

WT host mice were anesthetized by intraperitoneal injection of xylazine (12.5 mg/kg) and ketamine hydrochloride (125 mg/kg) (Sanofi, Mexico-City, Mexico). The inguinal lymph node was then inoculated with 100 μl CXCL13 (25 ng/ml) (PeproTech) to promote cell recruitment. One hour later, 1 × 10⁷ Hoechst 33342-labeled, LPS + IL-4 activated B cells were injected via the cannulated carotid artery. Venules of the inguinal lymph node were recorded using an intravital upright microscope (Axioscope, Model A1, Zeiss, Jena, Germany) with a 40 × and 0.75 saline immersion objective (Zeiss). Videos and images were analyzed using ImageJ (NIH, Bethesda, MD. USA) and Zen Blue Edition 2.5 software (Zeiss, Microscopy). The venules' diameter, the number of adherent cells, the number of transmigrated cells, and the cells' velocity were measured using ImageJ. Cell flux, blood flow, and rolling velocity were analyzed by Zen Blue Edition 2.5 software (Zeiss, Microscopy). This methodology was carried out following a previously published protocol³² .

Previously activated or retrovirally transduced T cells were deprived of serum for 3 h. Approximately 2 × 10⁶ T cells were suspended in serum-free RPMI-1640 medium, incubated with purified anti-ICOS (Biolegend) or CXCL13 (PeproTech) with or without PD-L1-Fc (R&D) of indicated concentration for 30 min in 37°C. Stimulated cells were quickly spun down and lysed by 2% SDS and loading buffer and then boiled at 100 °C for 15 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with TBS containing 5% milk and 0.1% Tween-20. Antibodies for immunoblotting included rabbit anti–pAKT (S473), anti-AKT, anti-actin, anti-pSHP-2 (Y542), anti-SHP2 (Cell Signaling Technology). Appropriate HRP-conjugated secondary reagents were from Jackson Immunoresearch Laboratories.

The human chemokines CCL1, CCL5, CCL7, CCL11, CCL17, CCL19, CCL20, CCL25, CCL27, CXCL8, CXCL11, CXCL12, CXCL13, CXCL16, and CX₃CL1 were purchased from PeproTech, whereas XCL1 was purchased from R&D Systems. All chemokines were reconstituted in a buffer containing 0.1% (w/v) bovine serum albumin (BSA) and 1 mM acetic acid.