Garvan scientists have developed a way to spot rare immune cells, by revealing RNA 'barcodes' of immune cell receptors.[Dr Martin Smith]

Australian scientists have developed a kind of barcode tracking system for cells that can identify rare immune system cells that are produced in response to cancer. The patented Repertoire and Gene Expression by Sequencing (RAGE-Seq) technology generates full-length antigen receptor sequences with high accuracy and sensitivity, claim the Garvan Institute of Medical Research team reporting on the new technology in Nature Communications.

“This method gives us the most detailed view yet of how immune cells behave in the human body,” explained Chris Goodnow, PhD, executive director of the Garvan Institute. “Immune cells play a critical role in the development of disease. This method shows significant potential to help us personalize cancer treatments to the individual.” Goodnow is co-senior author of the researchers’ paper, which is published today, and titled, “High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.”

The immune system’s T and B lymphocytes express different receptors on their surface (B-cell receptors—BCRs—and T-cell receptors—TCRs—respectively). When these receptors recognize an antigen, the cell replicates many times. “… when a B cell or T cell is stimulated by antigen to divide and undergo clonal expansion, the BCR or TCR sequence serves as a unique ‘clonal barcode’ and provides information on antigen specificity and cell ancestry,” the authors explained.

The immune system tends to react poorly to cancer, which develops from the body’s own cells, so there are usually too few immune cells that mount an effective anticancer immune response. “The immune cells that recognize cancer cells are often rare, pointed out Alex Swarbrick, PhD, who heads the tumor progression laboratory at the Garvan. “We have to sort through thousands of cells to find those replicating cells that may make up only a small fraction of all the immune cells present in a tumor.”

Sequencing the BCRs and TCRs of individual lymphocytes in parallel with their transcriptome can give scientist key insights into how the immune system is responding to diseases such as cancer, autoimmune disorders, or infectious diseases. However, while there are methods for reading long sequences of RNA encoding T- and B-cell receptors at the single cell level, existing technologies can’t sort through the thousands of cells present in a tumor in a single run.

“A common approach to link paired antigen–receptor sequences with gene-expression profiles of single lymphocytes is through the use of the full-length single-cell RNA-Sequencing (scRNA-Seq) method, Smart-Seq2,” the authors noted, but this method relies on plate- or well-based microfluidics, “and is therefore limited in the number of cells that can be processed, typically 10–100s.” And while recent developments in long-read sequencing technologies could address the drawbacks of short-read sequencing, to sequence complete BCR and TCR transcripts, such methods “typically suffer from higher error rates and lower sequencing depth than short-read technologies.”

Study authors from the Garvan Institute of Medical Research. [Garvan Institute]
The Garvan Institute researchers have now developed a new method that is built on four different genomic technologies; Oxford Nanopore Technologies, 10X Genomics, Illumina, and CaptureSeq. The scientists first developed a way to enrich the RNA from single cells, targeting the RNAs encoding immune cell receptors. They then developed a computational tool that could accurately read the full-length immune cell antigen receptor sequences. Importantly, the resulting RAGE-seq technology can be applied to high-throughput droplet-based scRNA-Seq workflows, to accurately pair gene-expression profiles with targeted full-length mRNA sequences from large numbers of cells.”

RAGE-Seq works like a barcode tracker, scanning immune cell receptors in thousands of cells to provide an accurate snapshot of how immune cells in a tissue sample are related, and which might be effective against cancer. Importantly, the team noted, the technology negates the need to isolate specific lymphocyte populations using flow cytometry, and so can be used for retrospective characterization of low abundance lymphocytes in tissues.

In a proof-of-principle study, the researchers carried out RAGE-Seq on a sample of 7,138 cells from the tumor and lymph node of a patient with triple-negative breast cancer, to track the transcriptome profiles of expanded lymphocyte clones, effectively those immune systems cells that are reacting and replicating in response to the cancer. “An important application of RAGE-Seq is the ability to track clonally related T or B cells across tissues, to gain systems-level insights into the evolution of immune responses,” the team stated. “One such application is the analysis of lymphocytes in a tumor and its draining lymph node, the presumptive site of antigen presentation and source of tumor infiltrating lymphocytes (TILs)”.

Using the method they were able to identify a number of related cells in both the tumor and lymph node tissues, which indicated specific genetic signatures of immune response to the patient’s tumor. This ability to identify such rare immune system cells in tumor tissue could help guide personalized therapy. The researchers indicate that the pharmaceutical industry is also looking to have a better understanding of how the immune system responds to cancer, at the level of detail now possible using RAGE-seq. “We hope RAGE-seq will be implemented in clinical trials, providing crucial information that will help potential cancer therapeutics get to the right patients more quickly,” Goodnow said.

“Here, we report a generalizable experimental workflow and computation pipeline to integrate single cell gene expression with targeted characterization of full-length mRNA transcripts,” the Garvan Institute team concluded. “We have found RAGE-Seq to be robust in its ability to sample across both Illumina and Nanopore sequencing platforms and highly sensitive and accurate in providing full-length BCR and TCR sequences across immortalized and primary human B and T cells.”

While the researchers’ published study was focused on antigen receptor sequences, they pointed out that any transcripts of interest can be targeted using variations of RAGE-seq. “Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity, and cancer.” The high accuracy and single nucleotide precision achieved by RAGE-Seq is particularly well suited to identifying somatic mutations in cancer. “The adaptability of RAGE-Seq across multiple scRNA-Seq platforms and the flexibility to target a range of genes offers a new genomic toolkit for advanced single cell analysis.”

The team is now using RAGE-Seq to analyze samples from melanoma patients, with a view to gaining new insights into why this type of cancer responds so poorly to immunotherapy. They do acknowledge that the method has some limitations, but anticipate that these will be addressed in future developments.




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