Background Despite the success of PD1 blockade in various cancers, overcoming resistance to cancer immunotherapy remains challenging. Targeting CD8⁺ T cell-associated alternative immune-checkpoints is anticipated to overcome this issue. However, such immune-checkpoints are also expressed on myeloid cells, but their therapeutic and clinical impact remains enigmatic. Thus, the aim of this study was to reveal underappreciated TAMs-ontology enriching immune-inhibitory receptors, to design biomarker-driven immunotherapy.

Methods We used reverse translational methodologies starting from human tumour multi-omics bioinformatics to inform pre-clinical experimental research, culminating into human multi-omics prognostic/predictive validation

Results We identified a unique niche of tumour-associated macrophages (TAMs), preferentially co-expressing the TIM3 and VISTA immune-checkpoints, that dominated the human and mouse tumours resistant to PD(L)1 blockade. Subcutaneous epithelial-origin tumours and orthotopic melanoma in mouse showed that TIM3⁺VISTA⁺TAMs were sustained by IL4/IL13-enriching tumours with low (neo)antigenic and non-immunogenic milieu. TIM3/VISTA were instrumental in sustaining a hyper-efferocytotic and anti-inflammatory TAM phenotype, and blunting type I interferon (IFN) sensing, thereby fuelling immune subversion. This was established with cancer cells succumbing to immunogenic cell death (ICD). Herein, while dying cancer cells triggered autocrine type I IFN production, yet they also exposed extracellular HMGB1 and surface VISTA as ligands to engage TIM3 and VISTA on TAMs respectively, to suppress paracrine IFN responses. Consequently, TIM3/VISTA blockade preferentially synergized with paclitaxel, an ICD inducing chemotherapy in vivo, to replace the anti-inflammatory TIM3⁺VISTA⁺TAMs with pro-inflammatory TAMs-driven cytotoxicity, thus blunting the immuno-resistant tumours. In vivo macrophage-specific genetic knockout of TIM3/VISTA confirmed this synergism, while immune/genetic ablation of type I IFN sensing, macrophages (but not CD8⁺T cells), or cancer cell associated HMGB1/VISTA disrupted it. Finally, TIM3⁺VISTA⁺TAM signature exhibited pan-cancer negative prognostic impact and predicted resistance to immunotherapy in patients.

Conclusions We discovered that as-yet-uncharacterized TIM3⁺VISTA⁺TAMs, enriched by human and mouse non-immunogenic tumours, mediate chemo-immunotherapy resistance. Thus, targeting TIM3⁺VISTA⁺TAMs is a novel and conserved strategy to overcome low neo-antigenic, CD8⁺T cells independent, tumours.

Ethics Approval Mouse Experiments were approved by the animal ethics committee at KU Leuven (project P114/2019 and p195/2020) following the European directive 2010/63/EU as amended by the Regulation (EU) 2019/1010 and the Flemish government decree of 17 February 2017.