Epigenetic regulators of T cell exhaustion after immunotherapy revealed by single cell ATAC-seq
In a study published online today in Nature Biotechnology, researchers at Stanford University collaborated with scientists at 10x Genomics to map chromatin landscapes in over 200,000 single cells from blood, bone marrow, and patient solid tumor biopsies (1). Using the Chromium Single Cell ATAC (Assay for Transposase Accessible Chromatin) Solution, the group profiled epigenetic regulation at the single cell level to characterize human immune cell development and regulation of tumor-infiltrating lymphocytes. Their data demonstrate how single cell ATAC-Seq enables discovery of cell types and regulatory DNA elements in healthy and diseased tissues.
"This method can reveal several layers of gene regulation in a single assay and enable epigenomic profiling of primary samples with newfound precision," the team wrote. "We developed a bottom-up, data-driven approach to iteratively group single cells together based on their accessible genomes."
The group analyzed primary tumor biopsies from patients with basal cell carcinoma taken before and after PD-1 blockade immunotherapy, and generated high-quality single cell ATAC-seq profiles from over 30,000 cells.
"We asked whether chromatin landscapes in the tumor microenvironment could identify epigenetic regulators of the anti-tumor T cell response," the researchers explained. "Our goal was to identify cell types that were responsive to therapy and the regulatory mechanisms controlling their activity in responder versus non-responder patients."
They found previously unrecognized regulatory programs controlling tumor-infiltrating lymphocytes after checkpoint inhibitor immunotherapy. Notably, patients responding to treatment showed increases in two specific T cell populations—exhausted CD8+ T cells and CD4+ follicular helper T cells—in similar proportion. Reconstructing the epigenetic transitions revealed that these two cell types also share the same core set of accessible transcription factor binding motifs during differentiation.
"For the first time, we can look at the epigenetic signatures of exhausted tumor-infiltrating lymphocytes in patients and follow their differentiation trajectory from a naive CD8+ T cell to a terminally exhausted T cell," said Ansuman Satpathy, assistant professor of pathology at Stanford and member of the Parker Institute for Cancer Immunotherapy, and lead author on the study. "This is an example of how you can use single-cell epigenetic information to understand the molecular pathways driving disease-relevant cell states."
The authors suggest that future studies targeting these regulatory pathways may identify new therapeutic interventions that could synergize with PD-1 blockade in cancer.
Control experiments with bone marrow and blood samples from healthy donors demonstrated how data from the Chromium Single Cell ATAC Solution can recapitulate the regulatory pathways of immune cell development.
"Widespread adoption of this technique for single cells has been hindered by the difficulty and cost of performing the assay at scale while maintaining high data quality," the authors noted. "We anticipate that droplet-based single cell chromatin accessibility will provide a broadly applicable means to identify regulatory factors and elements that underlie cell type and function."
The Chromium Single Cell ATAC Solution enabled high library quality while also scaling to 10,000 single cell chromatin profiles per reaction (up to 80,000 per chip). Library quality was assessed by examining the number of unique ATAC-seq nuclear fragments in each single cell. Each fragment represents unique Tn5 cut sites in the nucleus. More complex libraries will have a higher number of unique nuclear fragments per cell and the number detected will continue to increase as sequencing depth increases, until all of the unique fragments in the library have been sequenced. For samples such as the cell line GM12878, which have a high proportion of accessible chromatin, the unique nuclear fragments per cell can reach ~100,000 (if all of the unique molecules in the library are sequenced).
To determine if the signal in the single cell ATAC library is biologically meaningful, the authors also examined the fraction of fragments overlapping called peaks. They observed upwards of 60% of total fragments overlapped a peak in some samples, comparable to other high-quality ATAC libraries. In addition, the fraction of fragments overlapping a transcription start site was also high. TSS enrichment scores of up to 30 in the immune cell populations were reported in the study.
Importantly, the authors noted "the quality of scATAC-seq profiles was highly uniform across individuals, samples, and cell types" and thus allowed them to characterize biopsy samples longitudinally across patients and treatments.
The Chromium Single Cell ATAC Solution also enabled profiling of limited patient samples (down to starting with 2,000 cells) and has been validated on fresh and frozen tissues, including fragile samples, such as biopsies. With low multiplet rates (~1% per 1000 cells), and partitioning of a single cell with a single barcode, no computational deconvolution was necessary to identify single cell profiles.
1. Satapathy et al., Nature Biotechnologies. Aug 2019