Mouse b cell isolation kit

HSCs were treated with or without 100U IFN-γ for 48 hr. For negative controls, CD8⁺ T cells were isolated from WT C57BL/6 mouse splenocytes using a mouse CD8⁺ T cell isolation kit (Miltenyi Biotec, San Diego, CA), and for positive controls, mouse splenocytes after B cell depletion using a mouse B cell isolation kit (StemCell, Vancouver, Canada) were used. Cell lysates were prepared using NP-40 buffer (Sigma, MI) and total protein concentrations were determined by using a BCA protein concentration assay kit (Pierce, IL) following manufacturer provided protocols. Blotted membrane was incubated with 1 µg/ml of the sheep anti-GARP Ab (R&D, Minneapolis, MN) at 4°C overnight, then incubated with 2 µg /ml of the HRP-conjugated Rabbit anti-sheep IgG (Southern Biotech, Birmingham, AL) for 30min at room temperature. After washing, ECL western blotting regent (GE Healthcare Bio-Sciences, Pittsburg, PA) was applied for the detection. The same membrane was stripped and probed again using an anti-mouse actin antibody to serve as a loading control.

B cell development in bone marrow and spleen of sex-matched Fam72a^−/− and Fam72a^+/+ littermates (6–8 weeks old) was evaluated (Supplementary Fig. 2) as described^{33 (link)}. To induce ex vivo CSR to different immunoglobulin isotypes, splenic B cells were purified with a mouse B cell isolation kit (StemCell Technologies), and then stimulated with LPS (Sigma-Aldrich) in combination with various cytokines, as previously described^{33 (link)}. Cells were collected at day 4 after stimulation and were stained with antibodies against mouse IgG1 (PE, BD Pharmigen, cat. no. 550083; 1: 150 dilution), IgG2b (PE, SouthernBiotech, cat. no. 1090–09S; 1:150), IgG3 (FITC, BD Pharmigen, cat. no. 553403; 1:100), IgE (FITC, BD Pharmigen, cat. no. 553415; 1:100) or IgA (PE, SouthernBiotech, cat. no. 1040–09; 1:150) to assess CSR. For the cell cycle analysis, splenic B cells were stimulated with LPS for 2.5 days and analysed with the Click-iT Plus EdU Alexa Fluor 647 kit (ThermoFisher Scientific) as described^{33 (link)}. IgG1 CSR was induced in carboxyfluorescin diacetate succinimidyl ester (CFSE)-pulsed splenic B cells as we previously described^{33 (link)}. The apoptosis of LPS stimulated splenic B cells was assayed using an APC-conjugated Annexin V Apoptosis Detection Kit (eBioscience). The flow cytometric data were analysed with a FlowJo X10 software.

In vitro co-cultures were modeled after previously reported systems (Bruno et al., 2017 (link); Kolenbrander et al., 2018 (link); Lapointe et al., 2003 (link)). KP and KP-HELLO cells had been cultured at 37°C and 5% CO2, in complete RPMI-1640 (10% HI-FBS + 1% Penicillin & Streptomycin + 1x L-Glutamine + 55μM β-mercaptoethanol) for 2 days. Supernatant of KP and KP-HELLO were filtered with 0.45μm filters. Naïve CD4⁺ T cells and B cells were separated from spleens by negative selection with EasySep Mouse CD4⁺ T Cell Isolation Kit and Mouse B Cell Isolation Kit, respectively (STEMCELL technologies), according to the manufacturer’s protocol. 1 × 10⁶/mL B cells were plated with CD4⁺ T cells in a ratio of B:T=1:2, and co-cultured with the supernatant of KP or KP-HELLO, or complete RPMI-1640 supplemented with LCMV GP_61-80 peptide GLKGPDIYKGVYQFKSVEFD (5μg/mL, Anaspec) or HEL (0.5μg/mL, Sigma-Aldrich). BAFF (10ng/mL, R&D Systems) were added in all the co-culture experimental conditions. Flow cytometric analysis were performed after 48 hours of co-culture.