Comprehensive Immune Profiling of IFNα-Treated PBMCs

PBMCs collected before and after 1 week of IFNa treatment were thawed and divided into multiple samples that were stained with separate antibody panels for myeloid-derived suppressor cell (MDSC), inhibitory/memory, regulatory T cell and dendritic cell (DC) markers, respectively (online supplementary table 1a). Dead cells were stained using Yellow ArC-Qdot585 (ThermoFisher, L34959).
Staining was carried out according to our standard protocols,24 (link) washed with Fluorescence Activated Cell Sorting (FACS) buffer, fixed in 1% paraformaldehyde and analyzed using a LSRFortessa X20 (BD Biosciences).
Staining of the regulatory T cell panel was conducted using the Transcription Buffer Set (BD Biosciences) as previously described.25 (link) FACS results were analyzed with BD FACSDiva software (V.8.02).

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Verdegaal E., van der Kooij M.K., Visser M., van der Minne C., de Bruin L., Meij P., Terwisscha van Scheltinga A., Welters M.J., Santegoets S., de Miranda N., Roozen I., Liefers G.J., Kapiteijn E, & van der Burg S.H. (2020). Low-dose interferon-alpha preconditioning and adoptive cell therapy in patients with metastatic melanoma refractory to standard (immune) therapies: a phase I/II study. Journal for Immunotherapy of Cancer, 8(1), e000166.

Publication 2020

Yellow ArC-Qdot585 LSRFortessa X20 Transcription Buffer Set BD FACSDiva software

Antibody Buffer Cells Dendritic cell Ifna Inhibitory Memory Myeloid derived suppressor cell Paraformaldehyde Regulatory t cell Transcription

Corresponding Organization : Leiden University Medical Center

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Variable analysis

independent variables

IFNa treatment

dependent variables

Myeloid-derived suppressor cell (MDSC) markers
Inhibitory/memory markers
Regulatory T cell markers
Dendritic cell (DC) markers

control variables

Positive and negative controls for staining as described in the standard protocols

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