PD-L1 AND ICOSL DISCRIMINATE HUMAN SECRETORY AND HELPER DENDRITIC CELLS IN CANCER, ALLERGY AND AUTOIMMUNITY

In this study, we define a novel phenotypic and functional dichotomy of human dendritic cells (DC), with strong relevance to cancer, allergy, and autoimmunity. Using 16 different stimuli in vitro (130 observations), we describe two states of human activated DC. PD-L1highICOSLlow Secretory DC produced large amounts of inflammatory cytokines and chemokines but induced very low levels of T helper (Th) cytokines following DC-T co-culture; conversely PD-L1lowICOSLhigh Helper DC produced low levels of secreted factors but induced high levels and a broad range of Th cytokines. Secretory DC were aligned with mature migratory LAMP3+ DC in three different cancer types, atopic dermatitis, and lupus nephritis by single-cell transcriptomics. They were associated with T cell inflammation, overexpressed stimulatory and inhibitory checkpoints, acquired increased cell-cell communication, and were associated with good prognosis in head and neck squamous cell carcinoma, and with response to checkpoint blockade in Melanoma. This functional dichotomy of DC has broad implications in inflammation and immunotherapy. Fresh samples of HNSCC tumor tissues and blood of untreated patients with head and neck cancers were obtained from the pathology departments of the Institut Curie and the Pitié-Salpêtrière hospital. Single Cell RNA Purification Kit (Norgen Bioteck) was used for RNA extraction, including on-column DNase digestion (Qiagen), as described by the manufacturer's protocol. RNA integrity was controlled with Agilent RNA 6000 Pico Kit (Agilent Technologies) in BioAnalyzer. cDNA was generated with SMARTer Ultra Low input RNA for Illumina Sequencing-HV (Clontech), following manufacturer’s protocol with 14 cycles for amplification. Quality controls were performed with Qubit dsDNA high sensitivity (Thermofisher) and an Agilent RNA 6000 Nano Kit (Agilent Technologies). Multiplexed paired-end libraries 50nt in length were obtained using Nextera XT kit (Clontech). Sequencing was performed in a single batch with Illumina HiSeq 2500 using an average depth of 15 million reads. Library, sequencing, and quality controls were performed by the NGS facility at the Institut Curie. Reads were mapped to the human genome reference (hg19/GRCh37) using Tophat2 version 2.0.14. Gene expression values were quantified as read counts using HTSeq-count version 0.6.1. Genes with less than one read count in at least one sample were filtered out and.