, Quantitative PCR amplifications were performed using TaqMan Universal PCRMaster Mix (Applied Biosystems) or SYBR GREEN I Master (Roche Applied Science) and were performed on the LightCycler 480

, Cloning and sequencing of TCRBV rearrangements. TOPO Blunt cloning kit (Invitrogen Life Technologies) was used. Then, PCR amplification was performed and was followed by a second step of elongation using an ABI PRISM Big DyeTerminator kit (Applied Biosystems)

, Applied Biosystems)

, Cell suspensions were prepared in PBS/2% FCS, 5 mM EDTA. For Ag-specific Th and B cells analysis, organs were dissociated, filtered and treated with 2.4G2 for 10 min

. +-t-cells, ), and biotin-Y-Ae (ebioY-Ae, 1:400). The cells were then suspended with Fixable Viability Dye eFluor450 or eFluor660 (eBioscience) for dead cells exclusion. For intracellular staining, cell suspensions were fixed and permabilized using BD Fixation/Permeabilization kit. For intracellular cytokine staining, sorted cells were stimulated with 50 ng ml ?1 PMA (Sigma) and 1.5 ?g ml ?1 ionomycin (Sigma) in the presence of 10 ?g ml ?1 brefeldin A (eBioscience) and 2 ?M monensin (eBioscience) for 4 h, followed by surface and intracellular staining. Data were collected on a BD LSRII? or on a BD LSRII/Fortessa (BD Biosciences) and analyzed using FlowJo software (Tree Star), cells were incubated with PE-1W1K-IA b tetramer (7 ?g ml ?1 ) and APC anti-CXCR5 (REA 215, Miltenyi Biotec, 1:50) for 2 h at room temperature. The tetramer 1W1K-IA b is obtained from NIH Tetramer core facility. To track, antigen-specific B cells were stained for 60 min with NP-PE (Biosearch Technologies Inc.) or OVA-Alexa488 (Invitrogen) at a final concentration of 1 ?g ml ?1 . After tetramer or NP-PE staining, cells were washed and then incubated on ice for 45 min with fluorophore (or biotin)-labeled mAbs. The following mAbs purchased from BD Biosciences were used: anti-Bcl-6 (K112-91, 1:50), anti-CCR7 (4B12, 1:200), anti-CD69 (H1.2F3, 1:100), anti-CXCR3 (CXCR3-173, vol.1, 0200.

, 2 mg ml ?1 NaIO 4 , 10 mg ml ?1 paraformaldehyde), fixed overnight and dehydrated in 30% sucrose prior to embedding in OCT freezing media (Sakura Fineteck), Sections were stained in PBS (0.01% Triton X-100 and 5% goat serum) using the following Abs: anti-B220 (RA3-6B2, 1:200, eBioscience) and anti-CD4

, Differences between variables were evaluated using the non-parametric Mann-Whitney test. All statistical analyses were carried out with Prism 4.0 software (GraphPad). P-values <0

, The data that support the findings of this study are available from the corresponding author upon request

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