Anti cd4

Cellular subsets and cytokines were depleted by intraperitoneally administering depleting antibodies (BioXCell) beginning 1 d before therapy as we previously reported (Moynihan et al., 2016 (link)): CD8 T cells with anti-CD8-α (clone 2.43, 400 μg every 3 days), CD4 T cells with anti-CD4 (clone GK1.5, 400 μg every 3 days), NK cells with anti-NK1.1 (clone PK136, 400 μg every 3 days), neutrophils with anti-Gr-1 (clone RB6–8C5, 400 μg every 2 days), macrophages with anti-F4/80 (clone CI:A3–1, 200 μg every day) (Lin et al., 2017 (link)), IFN-γ with anti- IFN-γ (clone XT3.11, 200 μg every 3 days), TNF-α with anti-TNF-α (clone XMG1.2, 500 μg every 2 days) and CXCL9 with anti-CXCL9 (clone MIG-2F5.5, 300 μg every 2 days). VEGFR2 was blocked by anti-VEGFR2 (clone DC101, 500 μg every 3 days). Apoptosis of intratumoral T cells were induced with anti-CD3ε F(ab’)2 (clone 145–2C11, 50 μg for intratumoral injection or 100 μg for systematic i.p. injection every day) (Besançon et al., 2017 (link)) to avoid toxicity associated with full anti-CD3 antibodies in treated mice (data not shown). Cellular depletions of CD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, neutrophils, macrophages and NK cells were confirmed by flow cytometry of PBMCs (Figures S3A and S4A).

Depletion of neutrophils and T-cell subsets was achieved by i.p. injection of 200 μg antibody (BioXCell, Lebanon, NH, USA) every 3 days, beginning 3 days prior to cell transplantation, using the following antibodies: anti-Ly6G (clone 1A8), anti-CD8α (clone 2.43), anti-CD4 (clone GK1.5), and anti-Vγ2 TCR (clone UC3-10A6). For macrophage and NK-cell depletion, 200 μg of Clodronate liposomes (Liposoma BV, Amsterdam, Netherlands) and anti-asialo GM1 (Cedarlane, Burlington, ON, Canada), respectively, was injected 24 hours before cell transplantation and 50 μg every 3 days after. Depletion of each cell type was confirmed by flow cytometry of peripheral blood mononuclear cell (PBMC) before injecting each new dose of antibodies (data not shown). Antibodies used for depletion were from different clones than those used in flow cytometry to confirm the depletion.

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).