Fluorochrome conjugated monoclonal antibodies mabs

All fluorochrome-conjugated monoclonal antibodies (mAbs) were purchased from Biolegend Inc. All the reagents for detection of mouse immunogobulins were purchased from Southern Biotech Inc. The reagents for detection of TGFβ were from R&D Systems. The reagents for isolation of bacterial DNA and pyrosequencing were from Qiagen and Roche, respectively.

Fluorochrome-conjugated monoclonal antibodies (mAbs) were purchased from BioLegend (San Diego, CA, USA; for list of antibodies used, see Supplemental Table S3). Single-cell suspensions (1 × 10⁶ cells per sample) were prepared in fluorescence-activated cell sorting (FACS) buffer (phosphate buffered saline [PBS], 2% fetal bovine serum [FBS], 1% sodium azide, and 0.4% EDTA) and Fc receptors were blocked by incubation with purified rat anti-mouse CD16/CD32 (mouse Fc block; BD Biosciences, San Jose, CA, USA; #553141 used at 1:50 dilution) before proceeding to surface marker staining. Surface markers were detected by incubating cells with respective antibody cocktails for 15 min at room temperature in the dark; intracellular markers were detected using a Fixation/Permeabilization kit (Thermo Fisher Scientific, Waltham, MA, USA) with incubation at 4°C for 30 min in the dark. Samples were fixed in 4% paraformaldehyde for 5 min before acquisition on a FACS Canto II cytometer (BD Biosciences). Data were analyzed and compensated using FlowJo software (version 9.9.6; FlowJo, LLC, Ashland, OR, USA) and using single-stained antibody-capture bead controls (UltraComp eBeads Compensation Beads; Thermo Fisher Scientific).

B6 mice were injected iv or sc with ALT-803 or PBS as described above. Mice were humanely sacrificed and spleens were harvested at 0, 16, 24, 48, and 72 h following injection. For splenic immune cell phenotyping, splenocytes were isolated and stained with the fluorochrome-conjugated monoclonal antibodies (mAbs) against mouse CD4, CD8, NK1.1, NKp46, KLRG1, CD25, and intracellular Foxp3 and granzyme B molecules (Biolegend, San Diego, CA). The stained cells were analyzed on a FACSverse with FACSuite software (BD Biosciences, San Jose, CA). Serum cytokine levels of ALT-803-treated mice were also assessed using a Cytometric Bead Array, Mouse Inflammation Kit (BD Biosciences, San Jose, CA) according to manufacturer’s instructions. The data were analyzed using Flow Cytometric Analysis Program (FCAP) Array Software (BD Biosciences, San Jose, CA).