Fig. 1: Rational design of immunostimulatory fusion proteins (IFP) based on extracellular domains.

a Schematic representation of the original and fusion receptors employed in this study. From left to right: PD-1, CD28, PTM (short for PD-1 transmembrane domain), CTM (short for CD28 transmembrane domain), CTM + 41EC (short for CD28 transmembrane domain and 41 amino acids (aa) from the extracellular [EC] portion of CD28), CTM + 39EC (short for CD28 transmembrane domain and 39 aa from the EC portion of CD28), CTM + 12EC (short for CD28 transmembrane domain and 12 aa from the EC portion of CD28) and CTMΔ12EC (short for CD28 transmembrane domain and 12aa from the EC portion of CD28; additionally, 12 aa were removed from the membrane-proximal part of the PD-1 portion). The putative extracellular length of PD-1 is indicated by the dashed line and CTM + 41EC, CTM + 39EC, and CTM + 12EC constructs are predicted to exceed this length. b PD-L1 binding of HEK293 cells after plasmid transfection, detected through flow cytometry after incubation with recombinant Human PD-L1/B7-H1 Fc Chimera Protein (R&D Systems) and subsequent IgG labelling (Zenon™ Human IgG Labelling Kit, R-Phycoerythrin, Invitrogen). The experiment depicted is representative of 3 independent assays. c Transduction efficiency of primary human T cells from healthy donors quantified by flow cytometry through staining with a human PD-1 antibody. Three independent experiments were pooled together; each was performed with a different donor. d Stimulation assay of IFP-transduced human T cells with anti-CD3 (100 ng/ml) and recombinant human PD-L1 (5 µg/ml). IFN-γ concentration in the supernatant was measured as a surrogate cytokine for T-cell activation. The experiment depicted is representative of 3 independent assays with different donors. P values for (d) are based on a two-tailed unpaired t-test.