Appropriate isotype controls

Differentiated/activated MDMs were detached by treatment with trypsin (1.6 µg/ml) in PBS. Cells were treated with Fc receptor blocking agent (Miltenyi Biotec) and Aqua Dead Cell Stain kit (Thermo Fisher Scientific) for 1 hr. They were then washed and stained for 1 hr at room temperature with appropriate antibodies (see Figure 5—figure supplement 4) and appropriate isotype controls (BD biosciences). Samples were analyzed on a Beckman Coulter Gallios flow cytometer.

Detection of activation in CD4⁺ or CD8⁺ T cells were done by freshly isolated PBMCs (10⁶/ml) from eight HVL and eight naive individuals within recruited individuals for this study, and incubated with TSLA (10 μg/ml) in a 96-well flat-bottom plate for 120 hrs at 37°C. After 120 hrs, the cells were harvested, washed with staining buffer (0.02 M PBS, 1% FBS, and 0.01% sodium azide), and surface stained with fluorochrome-conjugated antibodies to CD3-FITC, CD4-PerCP-Cy5.5, CD8-PerCP-Cy5.5 and CD69-APC, along with appropriate isotype controls (BD Biosciences) for 30 min at 4°C. Cells were then washed and finally suspended in 500 μl staining buffer (BD Biosciences). Samples were acquired and analyzed on flow cytometer, BD FACSCalibur using BD CellQuest Pro software on at least 10,000 events. Analysis gates were set for lymphocytes using forward and side scatter properties and the frequencies of activated CD4⁺ and CD8⁺ T cells were acquired onCD3⁺ T cells. Cell viability using 7AAD staining (BD) of a limited number of samples confirmed that the gated lymphocytes were >99% viable for both HVL and naive groups.

Cells isolated at passage 6 or 7 were resuspended in PBS, and then incubated with antibodies conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) at 4 °C for 30 minutes. Cells were analyzed using a FACSCalibur system (BD Biosciences) and data were analyzed using CellQuest™ (BD Biosciences). Dead cells were excluded by forward scatter (FSC)/side scatter (SSC) parameters, and viable cells were further gated as a singlet population. Antibodies against CD29, CD90, CD73, CD31, CD45, nestin (BD Biosciences), CD34, CD105 (Abcam), and CD44 (R&D Systems, Minneapolis, MN, USA) were used in conjunction with appropriate isotype controls (BD Biosciences). Isotype-matched control antibodies were used in each antibody analysis.