Antibody depletion of immune cell subsets

For NK cell depletion, anti-NK1.1 (BioXCell clone PK136; 200 μg) or an isotype control (BioXCell clone C1.18.4; 200 μg) was administered to mice by intraperitoneal injection one day prior to and 3 dpi. For monocyte and neutrophil depletion, anti-Ly6G/Ly6C (Gr-1; BioXCell clone RB6–8C5; 500 μg) or an isotype control (BioXCell clone LTF-2; 500 μg) was administered to mice by intraperitoneal injection one day prior to and 1, 3, and 5 dpi. For neutrophil depletion, anti-Ly6G (BioXCell clone 1A8; 250 μg) or an isotype control (BioXCell clone 2A3; 250 μg) was administered to mice by intraperitoneal injection one day prior to and 1, 3, and 5 dpi. For monocyte depletion, anti-CCR2 (clone MC-21; 25 μg) (56) or an isotype control mAb (BioXCell clone LTF-2; 25 μg) was administered to mice by intraperitoneal injection on 1, 3, and 5 dpi. Anti-CHIKV mAbs or an isotype control was administered to mice by intraperitoneal injection on 3 dpi. Foot swelling was monitored using digital calipers. Following extensive perfusion, ipsilateral ankles were collected, homogenized, and processed for CHIKV infection as described above.

For analysis of immune cell depletion, peripheral blood was collected on the day of harvest. Red blood cells were lysed with ACK lysis buffer (Gibco) and resuspended in RPMI supplemented with 10% heat inactivated FBS. For NK1.1 depletion, single cell suspensions were blocked for FcγR binding and stained with CD45 BUV395, CD3 APC-Cy7, NKp46 BV421 (BioLegend clone 29A1.4; 1:50), and fixable viability dye (e506). For Ly6G/Ly6C, Ly6G, or CCR2 depletion, single cell suspensions were blocked for FcγR binding and stained with antibodies against CD45 BUV395, CD11b PerCP-Cy5.5, Ly6C Pacific Blue, Ly6B FITC, Ly6G PE-Cy7, CD11c APC, and fixable viability dye (e506).