Anti cxcr5

Anti-CD16/CD32 (BD, 2.4G2 [anti-FcγR]) was used as a blocking reagent and PI-PECF594 (Invitrogen, CAT P3566) was used to identify dead cells. The following antibodies and reagents were used for germinal center B-cell staining: anti-IgD-BV-605 (BioLegend,11-26c.2a), anti-B220-APCeFlour780 (eBioscience, RA3-6B2), anti-CD38-Alexa 700 (eBioscience, 90), and anti-CD95-PE-CY7 (BD, Jo2). For T follicular helper cell (Tfh) staining, anti-CXCR5 (BD, 2G8), anti-rat IgG (fab’) Alexa-647 (Jackson Immuno Research, Code:712-606-153). 2W-specific T-cells were identified using a 2W:I-Ab tetramer labeled with PE (50959 I-A(b)) or BV421(50960 I-A(b) (NIH Tetramer Core Facility), and the following antibodies: anti-CD3-Alexa flour 700 (BioLegend, 17A2), anti-CD4-FITC (BioLegend, RM4-5), anti-B220-APCeFlour780 (BioLegend, RA3-6B2), and anti-PD1-PE-CY7 (eBioscience, J43). The Click-iT™ Plus EdU Alexa fluor 647 Flow cytometry kit was used for EdU staining of newly synthesized DNA (Invitrogen, CAT C10634). Porcine cardiac myosin (Sigma-Aldrich, CAT: M0531) and a cardiac myosin monoclonal antibody (ThermoFisher, CAT: MA1-26180) was used for investigation of myosin-reactive antibody responses.

Single-cell suspensions were prepared from spleens by standard gentle mechanical disruption. Antibodies for surface staining, anti-B220 (RA3-6B2), anti-CD44 (IM7), anti-SLAM (TC15-12F12.2), anti-IgD (11-26c.2a) were purchased from Biolegend. Anti-CD95/Fas (Jo2), anti-T-and B-cell activation antigen (GL7), anti-CD19 (ID3), anti-CD4 (RM4-5) were purchased from BD Biosciences. Anti-PD1 (J43) and anti-IgM (Il/41) were purchased from eBioscience. CXCR5 staining was done using either purified anti-CXCR5 or biotin labeled anti-CXCR5 (BD Biosciences) and performed as previously described [51] (link). Anti-CD8a (5H10) and Alexa Fluor 647 labeled streptavidin was purchased from Life Technologies. GC were detected with PNA-FITC (Vector Laboratory) and all surface stains included viability dye, 7-AAD (eBioscience). For intracellular staining, splenocytes were first stained with surface makers followed by staining with live/dead fixable cell staining from Life Technologies. Cells were then fixed and permeabilized using Foxp3 Staining Buffer set purchased from eBioscience following manufacturer's protocol. Cells were stained for intracellular proteins with anti-Bcl-6 (K112-91, BD Biosciences) and anti-Ki67 (B56, BD Biosciences) and Foxp3 (FJK-16s, eBioscience).

One week-old (8 mice/group) and adult CB6F1 mice (6 mice/group) were immunized s.c. at the base of the tail with either HA/CAF01 or HA/GLA-SE. Ten days post vaccination the 2 inguinal draining LNs were collected, samples from each group were pooled and CD4⁺ T cells were enriched by using the EasySep™ Mouse CD4⁺ T Cell Isolation Kit (Stemcell Technologies). Total CD4⁺ T cells were stained with fluorescently labeled antibodies to CD4, B220, PD-1, CXCR5, and streptavidin. CXCR5 staining was performed using purified anti-CXCR5 (BD Pharmingen), followed by FITC anti-rat IgG (Southern Biotech), and normal rat serum (Invitrogen). DAPI was added before sorting as a dead cell discriminator dye.
Highly pure CD4⁺CXCR5^highPD-1^high Tfh cells, CD4⁺CXCR5^dimPD-1^dim effector T cells and the CD4⁺CXCR5^negPD-1^neg resting T cells (non- Tfh) were simultaneously isolated from the enriched CD4⁺ T cells by flow-cytometry sorting using a MoFlo^® Astrios™ flow cytometer (Beckman Coulter); (purity ≥99%).