Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce T cell paralysis [CITE-seq]

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), is largely unknown. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n=9) and matched healthy donors (n=8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI-resistant. CD14CTX can directly suppress CD4+ T cells and induce SOCS3 expression in CD4+ T cells rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce T cell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance. Blood samples from patients with biliary tract cancer (BTC) and healthy donors were processed using ficoll; after centrifugation, the peripheral blood mononuclear cell (PBMC) layer was isolated and cryopreserved in cell media with human serum and DMSO. Previously frozen PBMCs were thawed using media containing RPMI, heat-inactivated sterile filtered human serum, penicillin-streptomycin, non-essential amino acids, sodium pyruvate, and L-glutamine. Samples were then incubated for DNAse I before washing and counting. 1 x10>6 cells from 16 unique samples were combined and stained with one pooled cocktail containing 99 AbSeq antibody-oligonucleotide conjugates (BD Biosciences) per standard protocols (Olvera JG et al., 2018), following pre-incubation with TruStain FcX (Fc Receptor Blocking Solution). Samples from different individuals and timepoints were randomly mixed across experiments to minimize batch and confounding effects. Droplet-based single cell RNA sequencing (scRNAseq) was performed using the 10x Genomics Chromium Single Cell 3ʹ Reagent Kits v3, according to manufacturer instructions. >>>Submitter states: Raw data not included where identifiable.<<<