Expression profiling by high throughput sequencing Other
Summary
While T cell accumulation in tumors is associated with response to immune checkpoint blockade (ICB), many T cell-rich tumors fail to respond to ICB. Here we leveraged a large neoadjuvant PD-1 blockade trial in hepatocellular carcinoma (HCC) to search for correlates of ICB response within T cell-rich tumors. Paired scRNAseq/TCRseq of nearly one million immune cells revealed that tumor responses to ICB correlated with significant clonal expansion of intratumoral CH25H+CXCL13+IL-21+CD4 T cells and GranzymeK+PD-1+CD8 effector T cells, whereas terminally exhausted CD39hiToxhiPD-1hi CD8 clones dominated in non-responders. Notably, proliferating and progenitor CD8 T cells clones were found in tumors of responders and non-responders. However, tumors from responders were highly enriched in mregDCs, a DC state triggered by capture of tumor debris, which formed physically interacting cellular triads with Tfh-like CD4 and progenitor-like CD8 T cells. Receptor-ligand analysis revealed unique interactions within these triads that may promote progenitor CD8 T cell differentiation into effector cells upon PD-1 blockade. These results suggest that discrete cellular niches that include mregDCs and Tfh-like CD4 T cells might control the differentiation of tumor-specific progenitor CD8 T cell clones into effective anti-tumor T cells in human tumors.
Overall design
Samples of tumor and non-involved liver were obtained from surgical specimens of patients undergoing resection at Mount Sinai Hospital (New York, NY) after obtaining informed consent in accordance with a protocol reviewed and approved by the Institutional Review Board at the Icahn School of Medicine at Mount Sinai. Cells were counted using the Nexcelom Cellaca. An aliquot of 400,000 cells from each sample were centrifuged at 350g for 5 minutes at 4°C. The supernatant was discarded and the pellets were resuspended in a hashtag antibody solution and incubated on ice for 20 minutes. Hashtag antibodies were conjugated as per New York Genome Center hashing protocol (https://citeseq.files.wordpress.com/2019/02/cell_hashing_protocol_190213.pdf). Stained cells were washed three times in 1mL wash buffer (PBS + 0.5% BSA) by 4°C centrifugation at 350g to remove unbound antibodies. Washed cells were resuspended in 150µl wash buffer and counted using a Nexcelom Cellaca. Hashed samples were pooled and centrifuged at 350g for 5 minutes at 4°C. Supernatant was removed and pellet was resuspended in 100µl of antibody cocktail (Table S4) and incubated at 4°C for 30 minutes. Stained cells were washed three times in 1mL PBS + 0.5% BSA by 4°C centrifugation at 350g to remove unbound antibodies. The washed cells were resuspended in wash buffer to achieve a target concentration of 4 million cells/mL. Sample pool was counted and loaded on the 10x Genomics NextGem 5’v1.1 assay as per the manufacturer’s protocol with a targeted cell recovery of 25,000 cells per lane (CITEseq) or 10,000 cell per lane (direct load). Gene expression and Feature Barcode libraries were made as per the 10x Genomics demonstrated protocol. Hashtag oligonucleotides (HTO) were enriched during cDNA amplification with the addition of 3 pmol of a HTO Additive primer (5’GTGACTGGAGTTCAGACGTGTGCTC). This PCR product was isolated from the mRNA-derived cDNA via SPRISelect size selection, and libraries were made as per the New York Genome Center Hashing protocol. All libraries were quantified via Agilent 2100 hsDNA Bioanalyzer and KAPA library quantification kit (Roche) Gene expression libraries were sequenced at a targeted depth of 25,000 reads per cells. ADT libraries were sequenced at a targeted read depth of 10,000 reads per cell. HTO libraries were sequenced at a targeted read depth of 1,000 reads per cell. Paired-end sequencing was performed on Illumina NovaSeq 6000 for RNA-seq and CITE-seq libraries (for 3’ libraries, used Read 1 28 bp for UMI and cell barcode, Read 2 80-bp for transcript read, with 8-bp i7 and 0-bp i5 reads; for 5’ libraries, used Read 1 26 bp for UMI and cell barcode, Read 2 80-bp for transcript read, with 8-bp i7 and 0-bp i5 reads). V(D)J libraries were also sequenced on Illumina NovaSeq 6000 (Read 1 150-bp, 8-bp i7, 0-bp i5, Read 2 150-bp). Hashtag libraries were sequenced on Illumina NextSeq500.
Human liver tumor samples were collected at baseline biopsy without treatment and after PD-1 Antibody (Cemiplimab) treatment at resection. Tumor tissus were sequenced for TCR.
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