Summary Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine if NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis, even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti–Ang-2–neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti–colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

†, # These authors contributed equally Ethical Compliance Animal experiments were in compliance with all relevant ethical regulations approved by the IVD committee (Utrecht, the Netherlands). Blood samples from healthy donors was collected after informed consent. The use of human blood samples was in compliance with all relevant ethical regulations approved by the Sanquin Ethics Advisory Council of Sanquin Blood Supply (Amsterdam, the Netherlands). Reporting summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article. Data availability All sequencing datasets have been deposited in the NCBI Sequence Read Archive under accession number SRP144590. In addition, all processed screen results are accessible in an interactive database (https://phenosaurus.nki.nl/). All data presented in this manuscript are available from the corresponding authors upon reasonable request Author contributions M.E.W.L. conceived the project, designed and performed experiments, interpreted data and co-wrote the manuscript. M.R., A.F. an T.R.B. designed, performed and interpreted the haploid genetic screens. M.T. and J.N. designed, performed and interpreted biochemical data. J.H.M.J., A.M.B. and J.H.W.L. designed, performed and interpreted anti-Her2 in vitro and in vivo data, and J.H.W.L. co-wrote the manuscript. K.F., H.L.M. and T.K.v.d.B. designed, performed and interpreted in vitro data with human effector cells. S.v.d.S. supported and performed flow cytometry analyses. R.G.-E. and N.A.M.B. designed, performed and interpreted in vitro studies with human T cells. J.H.v.d.B. and J.B.A.G.H. supervised analyses of T cell reactivity. K.A.M. performed and interpreted experiments. M.V. designed experiments and provided reagents. F.A.S. and T.N.S. conceived the project, designed experiments, interpreted data and co-wrote the manuscript.

Immunotherapy with PD-1/PD-L1-blocking antibodies is clinically effective for several tumor types, but the mechanism is not fully understood. PD-L1 expression on tumor biopsies is generally regarded as an inclusion criterion for this cancer therapy. Here, we describe the PD-L1-blocking therapeutic responses of preclinical tumors in which PD-L1 expression was removed from cancer cells, but not from immune infiltrate. Lack of PD-L1 expression on malignant cells delayed tumor outgrowth in a CD8+ T cell-mediated fashion, showing the importance of this molecule in immune suppression. PD-L1 expression was evident on myeloid-infiltrating cells in the microenvironment of these tumors and targeting stromal PD-L1 with blocking antibody therapy had additional antitumor effect, demonstrating that PD-L1 on both malignant cells and immune cells is involved in the mechanism of immunotherapeutic antibodies. Importantly, comparable results were obtained with PD-1-blocking therapy. These findings have implications for inclusion of cancer patients in PD-1/PD-L1 blockade immunotherapies.