Anti cd8

For NK and CD8+ T cell depletion in C57BL/6 mice, 300 μg anti-NK1.1 (BioXCell) and anti-CD8 (BioXCell) were injected intraperitoneally at days 0, 2, 4, and 6, respectively. Depletion was verified by flow cytometry.

We performed in vivo CD8 + T-cell depletion studies using intraperitoneal (IP) injection of monoclonal anti-mouse CD8 (YTS 169.4, BioXCell, Lebanon, NH, USA) in addition to treatment with either IgG or ICI. IP injections of anti-CD8 (BioXCell, 300 µg/animal) or IgG2b (BioXcell, 300 µg/animal) were performed at day 0 and day 3 prior to IgG1 and 2 (IgG) or anti-CTLA4 and anti-PD-L1 (ICI) injections, and once a week thereafter. The four treatment groups in this experiment were anti-CD8 + ICI, anti-CD8 + IgG, IgG2b + ICI, and IgG2b + IgG. CD8 + T-cell depletion was confirmed by FACS analysis of primary splenocytes of treated mice.

Anti-CD8, anti-IFN-γ, and isotype-matched control antibodies were purchased from Bio X Cell. For CD8+ T-cell depletion, 200 μg of antibody in 100 µl of PBS was delivered into each animal intranasally or intraperitoneally at 1 day before infection and repeated at day 1 and day 3 post infection. IFN-γ neutralization was performed by intranasal inoculation of 200 μg of antibody in 50 μl of PBS at indicated time points after infection.

The mouse colon carcinoma cell lines MC-38 was obtained from Korean Cell Line Bank (KCLB) and CT-26 was obtained from ATCC. Cells were routinely cultured in RPMI and DMEM medium containing 10% fetal calf serum. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. KMRC011, a TLR5 agonist, was developed and supplied by Connext ([Daegu], Korea). KMRC011 is a biologically recombinant protein derived from Salmonella enterica flagellin. While it retains the TLR5 binding domain of Salmonella enterica flagellin, the N-terminal ancillary tail has been removed to prevent unnecessary immune responses. Anti-PD-1, anti-CD8 and anti-CD4 were sourced from Bio X Cell (Lebanon, NH, USA).

All mouse experiments were performed at OHSU in ABSL3 laboratories in compliance with OHSU IACUC protocols. The small lab animal unit at OHSU is accredited by the Association for the Accreditation and Assessment of Laboratory Animal Care (AALAC) International. Animals were housed in ventilated racks and monitored daily by veterinary staff. C57BL/6J mice were vaccinated as indicated with MCMV delivered intraperitoneally (10⁶ PFU, i.p.), and/or AdV injected intramuscularly in the thigh (10⁸ PFU, i.m.). Mice were challenged with 10³ PFU CHIKV in a 20 μl volume in the footpad (f.p.), or they were challenged (i.m.) with 10³ or 10⁴ PFU in a 20 μl volume in the calf muscle. Footpad measurements were taken with calipers. For T cell depletion experiments, mice were administered T cell depleting antibodies diluted in PBS in a 100 μl volume (i.p.). Vaccinated groups were injected with 300 μg anti-CD4 (GK1.5, BioXCell), 300 μg anti-CD8 (2.43, BioXCell), 300 μg Rat IgG2b Isotype Control (LTF-2, BioXCell), or a combination of 300 μg anti-CD4 and 300 μg anti-CD8. T cell depletions were confirmed by flow cytometry. To confirm T cell depletions, splenocytes were stained with fluorophore-conjugated antibodies specific for mouse CD3, CD4, CD8, and CD19. Fluorescent markers were detected on an LSRII instrument (BD Pharminogen) and data was analyzed using FlowJo (TreeStar).