Fig 2: TNFR2 blockade and PTX combination therapy. A Schematic representation of 4T1-transplanted murine model and therapeutic procedure. B Tumor growth curves. C Primary tumors harvested from mice. D Representative histogram (left) and quantification (right) of PD-L1 expression on tumor ECs (CD45−CD31+PD-L1+) isolated from mice. E, F Gating scheme for (E) CD45+CD3+CD8+ T cells and (F) PD-1+ T cells (CD45+CD3+CD8+PD-1+) from tumor-bearing mice. G Quantification of the proportion of PD-1+CD8+ T cells. H Schematic illustration showing immune features of CD8+ T cells and ECs from responders and non-responders following PTX therapy. The expression of the ligands PD-L1, PVR, and MHC class II molecules on ECs and the corresponding receptors PD-1 and LAG3 on CD8+ T cells increase in non-responders but decrease in responders. Endothelial TNF–TNFR2 signaling enables inhibition of CD8+ T cell through PD-L1/PD-1 axis via regulating cellular NF-κB–glycolysis pathway on the basis of IFNγ-mediated immune feedback. Schematic illustration was created on the website of “Home for Researchers”. *P < 0.05, ***P < 0.001, ****P < 0.0001

Fig 3: Endothelial immunosuppression is enhanced by TNFR2 but not TNFR1. A Representative histograms and quantification of PD-L1 expression by sEND.1 cells following administration of either human or mouse TNFα with mouse IFNγ; n = 3. B Representative histograms and quantification of PD-L1 expression by ECs incubated with IFNγ, TNFα and anti-TNFR2; n = 3. C Representative histograms and quantification of PD-L1 expression by sEND.1 cells incubated with IFNγ, TNFα, and anti-TNFR1; n = 3. D Proliferation of OT-I CD8+ T cells detected by FCM using the FITC channel after incubation with IFNγ-stimulated sEND.1 cells treated by TNFα with or without anti-TNFR2. E Quantification of CD8+ T cell proliferation; n = 3. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant

Fig 4: TNFR2-decreased endothelial glycolysis is NF-κB-dependent. A Significantly enriched inflammatory pathways by GO analysis from the RNA-seq dataset. B Bar graphs showing expression of NF-κB (RELA) in EC_sub2. C Expression and phosphorylation state of NF-κB in sEND.1 cells treated with or without TNFα assessed by WB. n = 3. D Expression and phosphorylation state of NF-κB in sEND.1 cells treated with or without TNFα and anti-TNFR1 (left) or anti-TNFR2 (right). n = 3. E Expression of glycolysis-associated proteins by sEND.1 cells treated with or without TNFα and NF-κB (RELA) inhibitor (SC75741) measured by WB. n = 3. F, G Time course (F) and quantification (G) of glycolytic function of sEND.1 detected by the Seahorse Analyzer; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: TNFR2 + ECs present spatial proximity to exhausted CD8+ T cells. A Images of immunofluorescence staining for TNFR2 (red) and CD31 (green); Yellow arrows indicate TNFR2 and CD31 co-localization. Scale bar: 1 mm (left) and 200 µm (right). B Representative immunohistochemistry images of TNFR2 distribution in breast cancer tissues from HPA; Black arrows indicate TNFR2 expression on blood vessels. C Spatial visualization of ECs, TNFR2, CD8+ T cells and exhausted CD8+ T cells in the representative sample 094D. D Spatial visualization of TNFR2+ and TNFR2− ECs, exhausted and non-exhausted CD8+ T cells, and their overlapping regions. Colored by cell type assignment. E A summary of the resulting z-scores of cell subtype pairs represented in a box plot calculated by JCA. Vertical line represents two extreme points of the z-values in a wide spectrum; a vertical line in the box marks the mean; the box shows the interquartile range of the z-value. Z > 0, positive dependency, indicating spatial aggregation; Z < 0, negative dependency, indicating spatial dispersion. Z value reflects the degree of spatial deviation of the observed join count between cell pairings; when it is close to 0, z value indicates no spatial dependency, which is a state of near-random distribution. The z-value presenting significance of the results was also selected at cutoff of + 3 visualized between the dotted lines