Anti ly6g ab

WT and JAM-A^–/– mice received an i.p. injection of anti-Ly6G Ab (250 μL of 1 mg/mL, clone 1A8; BioXcell) 24 hours before CLP.

Mice were treated for the indicated days with injections of cytokine or cytokine: anti-cytokine monoclonal antibody complexes. For IL-2 complexes (IL-2C), mice received 2 μg per day of recombinant IL-2 (eBioscience) premixed with 20 μg of anti-mouse IL-2 monoclonal antibody clone S4B6-1 (S4B6) (BD Pharmingen). In certain experiments, the amount of IL-2 in the complexes was varied, as indicated. Complexes were incubated at room temperature for 20 minutes (min.) before intraperitoneal (i.p.) injection in 200 μL of PBS. IL-2C in 50 μL of PBS were also administered intranasally (i.n.). When IL-2 was administered as free cytokine, animals were treated with 20 μg per day in 200 μL of PBS injected i.p.
For some experiments, mice were treated as indicated with 0.25 mg per day of anti-CD122 (IL-2 Rβ) antibody (5H4) to block IL-2 signaling, 0.25 mg per day anti-IL-2 antibodies (S4B6 and JES6-1A12) to neutralize IL-2, 0.5 mg per day of anti-CD70 antibody (FR-70) to block CD70 signaling, 0.25 mg per day of anti-NK1.1 (PK136) to deplete NK cells, 0.5 mg of anti Ly-6G Ab to deplete neutrophils (1A8), or with appropriate isotype control antibody (all Bioxcell). Antibody was delivered by i.p. injection in 200 μL of PBS.

Neutrophil depletion was achieved with intraperitoneal injection of 150 μg anti-Ly6G Ab (BioXCell) or IgG isotype control (BioXCell) at day −1 and day 0 (day of infection). Macrophage depletion was performed with intravenous administration of 1 mg Clodronate liposomes (Clo-L) or 1 mg PBS liposomes (PBS-L) as control (Liposoma) at 1 day prior to infection.