Isotype igg

C57BL/6 mice were treated with anti-CD3 antibodies (50 μg/mouse) and then received intraperitoneal injection of neutralizing anti-IL-6 (BioLegend) or isotype IgG (BioLegend) (50 μg/mouse). Blood glucose levels of all mice were monitored at 2, 4, 6, 7, 22, 24, 26, and 27 hours after treatment.

A549, H1299 and NK cells were seeded into six-well plates in the presence or absence of 10–1000 nM of Red-A for 24 h. The pre-treated A549 or H1299 cells were cultured with NK cells at 1:1 ratio while the pre-treated NK cells were incubated in the presence or absence of A549 or H1299 cells at 1:1 ratio. Next, cells were stained with PE/Cy5-conjugated CD107α antibody (Biolegend, San Diego, CA, 555802) or isotype IgG (Biolegend, San Diego, CA, 409304) and incubated at 37 °C for 4 h, following by FITC-conjugated anti-human CD56 antibody (BD Biosciences, Franklin Lakes, NJ, 308304) or isotype IgG (BD Biosciences, Franklin Lakes, NJ, 551497) at 4 °C for 30 min, then the percentage of CD56⁺/CD107α⁺ NK cells was assessed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, Accuri C6).

Reovirus virions (3 × 10¹² particles) were incubated with 20 μM Alexa Fluor 647 NHS ester (Invitrogen, #A20006) and 50 mM NaHCO₃ in a 500 μl total volume rotating at room temperature (RT) for 1.5 h¹⁹ . Unconjugated fluorophore was removed by dialysis overnight in phosphate-buffered saline (PBS) buffer at 4 °C. For reovirus binding assays, cells were dissociated with Cellstripper, incubated with labeled reoviruses (2 × 10⁵ virions/cell) at 4 °C for 1 h, and washed three times with ice-cold 2% FBS DMEM. For PirB-specific antibody blockade assay, CHO cells were incubated with PirA/B-specific monoclonal antibody (mAb) 6C1 (BioLegend, #144101) or isotype IgG (BioLegend, #400402) at 4 °C for 1 h and washed twice with PBS prior to reovirus binding. For cell sorting during the CRISPRa screen, unfixed living MEFs were analyzed using a FACSAria II cell sorter (BD Biosciences) operated by FACSDiva™ Software (BD Biosciences, v6.1.3). For other reovirus binding assays, cells were fixed with 1% paraformaldehyde (PFA, Electron Microscopy Sciences) at 4 °C overnight. Reovirus-binding on cells were measured using an LSR II flow cytometer (BD Biosciences) operated by FACSDiva™ Software (v6.1.3) and analyzed by FlowJo software (v10.8.1).

C57BL/6 mice were treated with anti-CD3 antibodies (50 μg/mouse) and then received intraperitoneal injection of neutralizing anti-IFN-γ (BioLegend) or isotype IgG (BioLegend) (50 μg/mouse). Blood glucose levels of all mice were monitored at 2, 4, 6, 23, and 31 hours after treatment.

LPS (E. coil O111:B4) used for cell culture studies was purchased from Calbiochem (San Diego, CA, USA; cat# 437627), and for animal studies, it was purchased from Sigma-Aldrich (St. Louis, MO, USA; cat# L3012). IL-10, tumor necrosis factor alpha (TNFα), and interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kits and salmeterol (a long-acting selective β2-adrenergic receptor agonist) were obtained from R&D Systems (Minneapolis, MN, USA). Recombinant mouse IL-10, Ultra-LEAF™ Purified anti-mouse IL-10 antibody, and isotype IgG were from Biolegend (San Diego, CA, USA). The anti-mouse IL-10 antibody from Biolegend was used as the detecting/capture antibody for ELISA/ enzyme-linked immunospot (ELISPOT) assay and for neutralization of mouse IL-10 bioactivity in vivo and in vitro (https://www.biolegend.com/en-us/products/ultra-leaf-purified-anti-mouse-il-10-antibody-17764, accessed on 6 August 2021). The specificity and utility of this antibody were further validated in our laboratory. We found that enhanced IL-10 protein level was detected in the supernatant of LPS-stimulated mouse primary cell cultures by the R&D IL-10 ELISA kit, but no significant difference between vehicle and treatment with LPS plus anti-mouse IL-10 antibody was observed.

Mice were sensitized intraperitoneally with 100 µg of ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO, USA) adsorbed in 1.6 mg of aluminum hydroxide on days 1 and 15. Starting on day 22, mice were challenged intranasally with 10 µg of OVA for 5 days each for eight consecutive weeks (Figure 1A). Control mice were injected and challenged with saline. The steroid-treated group received 1 mg/kg dexamethasone (Sigma Aldrich) intraperitoneally into OVA-stimulated WT (WT-OVA) mice and that of Has2^+/− (Has2^+/−-OVA) mice at 24 and 2 h prior to the final intranasal OVA challenge (Figure 6A). The combined treatment group received anti-IL-17A monoclonal antibody (100 μg/body, BioLegend, San Diego, CA, USA) intraperitoneally into WT-OVA mice and Has2^+/−-OVA at 24 and 2 h before final intranasal OVA challenge (Figure 6A). Isotype IgG (BioLegend, San Diego, CA, USA) was used as control.