Immune Cell Depletion and Neutralization Protocols

Depletion and neutralization experiments were based on previously established protocols in mice. Neutralization of the early IFN-γ burst was done by a single high dose (1 mg) injection of an anti-IFN-γ neutralizing rat-anti mouse monoclonal antibody (clone R4-6A2; BioXCell, USA) or an IgG1 isotype control antibody (clone HRPN; BioXCell, USA) one day prior to infection [48 (link),49 (link)]. In C57Bl/6 mice, NK and NKT cells were depleted using anti-NK1.1 depleting rat anti-mouse monoclonal antibody (clone PK136; BioXCell, USA). One hundred μg of the antibody or an IgG2a isotype control antibody (clone C1.18.4; BioXCell, USA) was given intraperitoneally (i.p.) on days -7, -4, -1, 1, 4 and 7 [50 (link),51 (link)]. For neutralization of CD1d presentation to NKT cells, BALB/c and C57Bl/6 mice were treated i.p. with 75 μg neutralizing anti-mouse CD1d antibody (clone 19G11; BioXCell, USA) or an IgG1 isotype control (clone HRPN; BioXCell, USA) on days -7, -4, -1, 1, 4 and 7 [52 (link)]. For the depletion of neutrophils, BALB/c mice received 75 μg of depleting anti-Ly-6G antibody (clone 1A8) (BioXCell, USA) or isotype IgG2a isotype control antibody (clone 2A3; BioXCell, USA) i.p. on days -4, -1, 1, 4 and 7 [53 (link),54 (link)]. Experiments were performed in duplicate with 3 mice per infection group.