Anti icos l

Eight- to ten-week old mice were treated daily for 4 days with tamoxifen (Sigma-Aldrich) at a dose of 1.5 mg per mouse and rested for 1 day. YFP⁺CD44^loVα2^hiCD4⁺ naive T cells (OT-II Foxp1-cKO) from OT-II Foxp1^f/f Cre-ERT2⁺Rosa^YFP mice and CD44^loVα2^hi CD4⁺ naive T cells (OT-II Foxp1-WT) from OT-II Foxp1^f/f Rosa^YFP mice (or OT-II Foxp1^+/+Cre-ERT2⁺Rosa^YFP mice) were isolated and sorted with a BD FACS Aria cell sorter (BD Biosciences). Naive Igλ⁺ B1-8i cells from eight- to ten-week old B1-8i mice were enriched by labeling with phycoerythrin-anti-Igκ (RMK-45; Biolegend) and negative selection with anti-CD43 and anti-PE magnetic beads (both from Miltenyi Biotech) following manufacturer’s protocols. Sorted naive OT-II cells, or together with Igλ⁺ B1-8i cells in co-transfer experiments, were transferred into age and sex matched SMARTA mice through tail intravenous injection followed by immunization with 100 μg of NP-OVA emulsified in alum adjuvant (Thermo Fisher Scientific) by intraperitoneal injection. For in vivo antibody blocking, recipient mice were treated with 100 μg anti-CTLA-4 (UC10-4F10-1, Bio-X-cell) or 500 μg anti-ICOSL (HK5.3, Bio-X-cell) monoclonal antibodies or PBS by intraperitoneal injection.

For OT-II transfer experiments, 100 μg of anti-ICOS-L (HK5.3) or isotype control (BioXCell) were given i.v. on day-1, then i.p. every other day until the day of sacrifice. For treatment of wild-type and Pik3cd^E1020K/+ mice, antibodies were given i.v. on day 0 and i.p. on day +2, +4, +6.