Clone c1

Monocyte depletion in marmoset monkeys was initiated 14 days after immunization by twice weekly i.v. injections of 5 mg/kg DOC-2 Fr-2 (marmoset IgG1-chimeric humanized mouse anti-human CCR2 antibody). Controls received 5 mg/kg marmoset IgG1-chimeric isotype control antibody. The administration frequency was reduced to once weekly from day 28 to the end of the experiment. In mice, all depletion and blocking experiments started at disease onset. NK cells were depleted in Th/+ mice by daily i.p. injections of 300 µg of the mouse monoclonal anti-NK1.1 antibody (Clone PK136, Bio X Cell, BE0036). Control Th/+ mice received 300 µg i.p. of the isotype control antibody C1.18.4 (Clone C1.18.4, Bio X Cell, BE0085). To block the formation of the membrane attack complex (MAC) 2 µg of the BB5.1 monoclonal antibody against mouse complement component C5 (Hycult biotech, HM1073) [23 (link)] or a mouse IgG1 control antibody (BioLegend, Clone MOPC-21) was injected intracerebrally at the time point of stereotactic cytokine injection.

THP-1 cells were treated with 5 µg/mL α-IFNAR2 antibody (Clone MMHAR-2 Mab, PBL assay science, Piscataway, NJ, USA; cat. no. 21385) or IgG2a isotype control antibody (Clone C1.18.4, Bio X Cell, Lebanon, NH, USA; cat. no. BE0085) prior to normoxic or hypoxic incubation for 24 h.

For lung tumor induction, 5x10⁵ cells resuspended in 200 µl DMEM medium (without supplements) were injected into the tail vein of 6-8 weeks old female mice. At the indicated time points, mice were weighed and injected intraperitoneally (i.p.) with luciferin (0.15 mg per 1 g body weight; Promega, Cat#P1043). Luciferase activity was measured after 20 min by the IVIS Spectrum In Vivo Imaging System (PerkinElmer) as previously described (5 (link)). Briefly, mice were anaesthetized using isoflurane and luciferase activity was measured by detecting luminescence intensity (photons per second). Analyses were performed in a logarithmic scale mode. Mice were sacrificed at day 14-23 after tumor cell injection. For the inhibition of IL-9 in vivo, we used two different protocols. In the first protocol, mice were treated i.p. with 200 µg of anti-IL9 antibody (BioXCell, Clone 9C1, Cat# BE0181) or IgG2a Isotype control (BioXCell, Clone C1.18.4, Cat# BE0085) dissolved in 100 µl PBS at day 1, 3, 6, 9, 13 and 16 after tumor induction with an experiment termination at day 21 to 23 depending on the lung tumor load. In the second protocol, mice were treated i.p. with 20 µg anti-IL9 antibody or IgG2a Isotype control resolved in 200 µl PBS at day 6, 9, 10 and 13 after tumor induction with an experiment termination at day 20.

Cellular subsets were depleted by administering 0.200 mg of depleting antibody by intraperitoneal injection on days 6, 8, 11 and 15 following tumour inoculation. For CD8 T-cell depletion, αCD8a (clone 2.43, rat IgG2b, BioXCell) was compared to an isotype control antibody (clone LTF-2, rat IgG2b, BioXCell). For NK cell depletion, αNK1.1 (clone PK136, rat IgG2a) was compared to an isotype control antibody (clone C1.18.4, rat IgG2a, BioXCell).