Cd3 clone 17a2

BM was harvested from femurs, tibias, and ilia, and cells were enriched using anti-CD117 magnetic beads (Miltenyi). The c-kit⁺ fraction was stained with antibodies against CD117 (c-kit APC, clone 2B8, Biolegend, dilution 1/100), Sca-1 (APC-Cy7, clone D7, Biolegend, 1/100), CD135 (Flt3 PE-Cy5, clone A2F10, Life technologies, 1/50), CD150 (Slam Pecy7, clone TC15-12F12.2, Biolegend, 1/100), CD48 (Pacific Blue, HM48-1, Biolegend, 1/100), CD16/32 (PercPCy5.5, clone M1/702.4G2, eBioscienceBD Bioscience, 1/100), CD34 (Alexa 700, 1/100), and a lineage cocktail (PE, CD3ε clone 145-2C11; Ly-6G/Ly-6C clone RB6-8C5; CD11b clone M1/70; CD45R/B220 clone RA3-6B2; TER-119, Biolegend, 1/200). The c-kit⁻ fraction was stained with antibodies against CD11b (PercPCy5.5, clone M1/70, eBioscience, 1/100), Ly6C (APC, HK1.4, Thermofisher, 1/200), Ly6G (BV510, RUO, Biolegend, 1/100), Siglec F (PE CF594, RUO, BD 1/100), F4/80 (alexa 700, clone BM8, Ozyme, 1/100), in addition to a lineage cocktail in PeCy7 (B220 clone RA3-6B2 Biolegend, CD3, clone 17A2 Biolegend, CD11c clone N418, eBioscience, NK1.1 clone PK136 Biolegend, Ter119 clone TER-119 BD Biosciences) and DAPI (1/1000) as live/dead marker. All cells were analyzed on a Ze5 (Bio-Rad) plate-reader cytometers. Data analysis was performed using FlowJo v10.2 software (TreeStar) and Prism v9.

Tissues were obtained from some of the animals previously used for intravital microscopy. Cryosections of 5 μm of the brain encephalic region were obtained and fixed with ice-cold acetone (Merck, Rahway, NJ, USA) for 10 min. The blockage of non-specific binding was made using a goat serum (Vector Laboratories, Newark, NJ, USA) for 20 min. F4/80 clone BM8 and CD3 clone 17A2 (Biolegend, San Diego, CA, USA) were used in the analysis and were incubated overnight after dilution on PBST. The ImmPRESS^® HRP Goat Anti-Rat IgG Polymer Detection Kit with DAB (Dako, Santa Clara, CA, USA) was used to detect the antibodies. The counterstain used was Harris’s Hematoxylin Solution (EasyPath-São Paulo, Brazil) for 1 min. The slides were rinsed with tap water and then dehydrated chemically and mounted with Erv-mount mounting media (EasyPath). The counting of CD3+ and F4/80+ cells was performed manually. Representative images were acquired in Motic Panthera L Microscope Built-in Smart CAM & ImagingOnDevice System (Motic-Xiamen, Xiamen, China).

Mice were s.c. immunized with alum-adsorbed NP–OVA (50 µg) and TLR7–alum or TLR7-NP (equivalent gardiquimod dose, 20 µg) in 100 µl PBS at tail base. Inguinal dLNs were excised at day 4, day 7, day 14 and day 22 to prepare single-cell suspensions. For flow cytometry analysis of GC B cells, follicular T cells (TFH) and plasmablasts, cells from the dLNs were stained with Ghost Dye Violet 510 (Tonbo Biosciences). Cells were then washed and blocked with Fc-blocker (clone 2.4G2, BD Bioscience) prior to staining with markers, including CD19 (clone 1D3/CD19, Biolegend), CD38 (clone 90, BD Biosciences), CD95 (clone Jo2, BD Biosciences), CD138 (clone 281-2, BD Biosciences), CD44 (clone IM7, BioLegend), CD3 (clone 17A2, BioLegend), CD4 (clone GK1.5, BioLegend), CXCR5 (clone L138D7, BioLegend) and PD1 (clone 29F.1A12, BioLegend). After staining, cells were washed and fixed with 1.5% PFA. Stained cells were collected using BD FACS Diva v8.01 software associated with a BD LSRII flow cytometer. Data were analysed with FlowJo 10 software. The gating strategy consisted of gating GCBC on live single CD3⁻CD19⁺CD95⁺CD38⁻ cells, TFH on live single CD19⁻CD3⁺CD4⁺CXCR5⁺PD1^hi, and plasmablasts on live single CD138⁺CD44⁺ cells.

Mice were perfused intracardially with PBS before the brain dissection and homogenization. Brain mononuclear cells (BMCs) were isolated using 37–70% (v/v) Percoll gradients. Three weeks after the peak of the disease, EAE BMCs were separated by magnetic microbeads in a two-step procedure: positive selection of memory CD19⁺ cells (mCD19⁺) (Memory B Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) followed by negative selection of CD3⁺ cells from the remaining unlabeled cells (Pan T cell isolation kit II, Miltenyi Biotec, Bergisch Gladbach, Germany). Four brain tissues were pooled for each experiment. BMC purity and phenotype were analyzed by LSRII flow cytometry (BD) using conjugated anti-B220 (clone RA3-6B2, BioLegend, San Diego, CA, USA), TCR-β (clone H57-597, BioLegend, San Diego, CA, USA), CD3 (clone 17A2, BioLegend, San Diego, CA, USA), CD19 (clone 1D3, Abcam, Cambridge, UK), and appropriate isotype control monoclonal antibodies (mAbs).

Splenic CD4⁺ or CD8⁺ T cells were isolated from 8-week-old B6 mice or PD-1 KO mice. Tissue was harvested, mechanically disrupted and filtered through a 70uM cell strainer. Red blood cells were lysed using Red Blood Cell lysis buffer (Sigma). Cells were stained with surface markers for CD3 (clone 17A2, BioLegend), CD4 (clone GK1.5, BioLegend) and CD8a (clone 53-6.7, BioLegend) for sorting using a BD FACSAria III (BDBiosciences). 100,000 cells were seeded in 96 round u-wells and treated with CD3/28 Dynabeads Mouse T-activator (Thermo Fisher Scientific) in the presence or absence of WT NETs or PD-L1 KO NETs.