Overcoming primary and acquired resistance to anti-PD-L1 therapy by induction and activation of tumor-residing cDC1s

T-cell exclusion from the tumor microenvironment (TME) is a significant obstacle in cancer immunotherapy. Evidence indicates crucial roles of Batf3-dependent dendritic cells for inducing antitumor T-cell immunity. However, strategies to maximize the engagement of Batf3-dependent dendritic cells into such 'cold tumors' remain elusive. Using multiple syngeneic orthotopic mouse models of non-T cell-inflamed tumors, we hypothesized that in situ induction and activation of tumor-residing Batf3-dependent dendritic cells overcomes poor T-cell infiltration. In situ immunomodulation with Flt3L, radiotherapy, and TLR3/CD40 stimulation induces an influx of stem-like Tcf1+Slamf6+CD8+ T cells, triggers robust regression not only of primary, but also untreated distant tumors, and renders non-T cell-inflamed tumors responsive to anti-PD-L1 therapy. Furthermore, serial ISIM reshapes repertoires of intratumoral T cells, overcomes acquired resistance to anti-PD-L1 therapy, resulting in eradication of tumors, and establishes tumor-specific immunological memory. These findings provide new insights into Batf3-dependent dendritic cells biology as a critical determinant to the formation of a T cell-inflamed TME. Overall design: To investigate treatment effects on tumor infiltrating immune cell populations, we sorted live tumor-infiltrating CD45+ cells from subcutaneous (s.c.) AT-3 tumors, which were treated with either ISIM (I), aPD-L1 (P), both (IP) or left untreated (NT) and performed scRNA-seq. Droplet-based 3' end massively parallel single-cell RNA sequencing was performed by encapsulating sorted live CD45+ cells into droplets and libraries prepared using Chromium Single Cell 30 Reagent Kits V3 according to manufacturer's protocol (10x Genomics).