Fig. 1: Tumor-specific CD8⁺ T cells upregulate NRP1 and Plexin-A1 allowing for SEMA3A binding.

a, b Representative histogram of flow cytometric analysis of surface NRP1 expression on human NY-ESO-1-specific HLA-A2 restricted CD8⁺ T cells and mouse OT-I CD8⁺ T cells following 48 h stimulation with cognate peptides. Cells are gated on CD45, CD8 and TCRβ. Experiment repeated three times. c Analysis of NRP1 upregulation using peptides with varying TCR affinities. Cells are gated on CD45.1, CD8 and TCRβ. Cells from 3 mice per group, experiment was performed once. d Quantification of surface binding of SEMA3A_S-P on naïve and 48 h stimulated OT-I T cells. Cells are gated on CD8 and CD3. Experiment was repeated three times. e Confocal imaging of 48 h stimulated OT-I T cells stained with AF647-labeled SEMA3A_S-P shows that the protein can bind to the cell membrane (white arrow) and within the cell (black arrow). Scale bar = 10 μm. Representative of two independent experiments. f Flow cytometric analysis of PD-1 and NRP1 expression on OT-I T cells 11 days after adoptive transfer in spleen, non-antigen expressing tumor (B16.F10) and antigen-expressing tumor (B16.F10.Ova) (n = 6 mice). Data representative of two independent experiments. g Schematic of NRP1 interactions partners (left). Flow cytometric analysis of expression of selected NRP1 interactions partners on OT-I T cells 11 days after adoptive transfer (n = 5 mice) (right). Experiment was performed once. Abbreviations: gMFI, geometric mean fluorescence intensity. N4, SIINFEKL. Q4, SIIQFEKL. T4, SIITFEKL. SD, standard deviation. Error bars are means ± SD (c, d, f, g) from representative experiments. Source data are provided as a Source Data file.