RAGE-seq : New method that enables scientists to track how immune cells evolve inside tumour tissue

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Researchers from the Garvan Institute of Medical Research have developed a new method to spot rare immune cells that are reactive against cancer cells, from within a patient’s own immune system.

The patented ‘RAGE-seq’ method enables scientists to track how immune cells evolve inside tumour tissue for the first time, revealing unprecedented insight into how to better arm the immune system to target cancer.

The technique can be likened to a barcode tracker, able to scan detailed information from thousands of immune cells at a time.

“This method gives us the most detailed view yet of how immune cells behave in the human body,” says Professor Chris Goodnow, Executive Director of the Garvan Institute and co-senior author of the published work.

Immune cells play a critical role in the development of disease.

This method shows significant potential to help us personalise cancer treatments to the individual.”

Development of the method, by Dr. Mandeep Singh (Immunogenomics Laboratory) and Ghamdan Al-Eryani (Tumour Progression Laboratory) at Garvan, is published in the journal Nature Communications.

Rare immune cells that ‘see’ cancer

Our immune system helps protect us against foreign pathogens, such as bacteria or viruses.

But it often responds poorly to cancers, which arise from the body’s own cells—usually too few immune cells ‘recognise’ them to mount an effective immune response.

Immune cells come in many different forms—they mix-and-match different types of ‘receptors’ on their cell surface, which monitor the cell’s environment.

When an immune cell’s receptors recognise a potential hazard, the cell replicates to make more copies of itself, able to target the threat more effectively.

“The immune cells that recognise cancer cells are often rare,” says Associate Professor Alex Swarbrick, who heads the Tumour Progression Laboratory at Garvan.

“We have to sort through thousands of cells to find these replicating cells that may make up only a small fraction of all the immune cells present in a tumour.”

Garvan scientists have developed a way to spot rare immune cells, by revealing RNA ‘barcodes’ of immune cell receptors. Credit: Dr. Martin Smith

Building a cellular barcode tracker

Previous methods have made it possible to read the long stretches of genetic output (the RNA) that encodes an immune cell’s receptor, from single cells.

But they have not had the capacity to sort through the thousands of cells present in a tumour, at a single time.

The study authors developed a new method by harmonising four different genomic technologies (Oxford Nanopore Technologies, 10X Genomics, Illumina and CaptureSeq).

They first developed a way to enrich the RNA from single cells, targeting the RNAs encoding the immune cell receptors.

They then developed a computational tool to accurately read full-length sequences of the immune cell receptors.

The resulting Repertoire and Gene Expression by Sequencing, or ‘RAGE-seq’, method works much like a barcode tracker.

By ‘scanning’ the relevant immune cell receptors in many thousands of cells at once it can provide an accurate snapshot of how the immune cells in a tissue sample are related, and which cells may be effective at mounting a response against cancer.

“This high-throughput strategy is really opening the door to a much more detailed understanding of the cellular dynamics of the immune response,” says Dr. Martin Smith, Leader of the Genomic Technologies Group at Garvan’s Kinghorn Centre for Clinical Genomics.

In a proof-of-principle study, the researchers used the method to sample 7,138 cells from the tumour and associated lymph node of a breast cancer patient.

The team pinpointed a number of related cells that were present in both tissues, and which revealed specific genetic signatures of the immune response within the patient’s tumour.

A new look at disease

The researchers say the ability to find and barcode these rare cells of the immune system has the power to guide treatment strategies based on the individual.

Immunotherapy is an emerging form of cancer therapy designed to activate the immune system to better target cancer, but not all patients respond well and current methods used to assess a patient’s response give a poor snapshot of the behaviour of their immune cells.

Professor Goodnow says there is significant interest from pharmaceutical companies to better understand the immune system’s response to cancer, at a resolution now available through the RAGE-seq method.

“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.”

The team is now applying the technique to samples from melanoma patients, to understand why half of patients receiving immunotherapy have a poor response.

The researchers believe the method could also be applied to provide a better understanding of autoimmune and inflammatory diseases.


Cell diversity in humans and other vertebrates arises from complex gene rearrangement and alternative RNA splicing events that are not yet captured by current short-read sequencing technologies for measuring differential mRNA expression in single cells.

A key example of this problem is the need for better ways to trace the response of single cells of the immune system during their response to cancer.

Each newly-differentiated T or B lymphocyte in the immune system carries a different antigen receptor as the result of critical DNA rearrangements that alter the 450 nucleotides at the 5’ end of their T or B cell antigen receptor mRNA.

In the case of B lymphocytes, they use additional DNA rearrangements to ‘isotype switch’ between 9 alternative constant region sequences comprising 1000-1500 nucleotides at the 3’ end of the heavy chain (IgH) mRNA {Di Noia, 2007 #33}, and use alternative mRNA splicing to change the 100-250 nucleotides at the 3’ end of IGH mRNA in order to secrete the encoded receptors as antibody {Alt, 1980 #43}.

Similarly, complex gene rearrangements and alternative splicing events create pathological cell diversity amongst cancer cells. Hence there is a critical need for single cell resolution methods that capture these sequence changes occurring throughout the length of mRNA molecules, and integrate that information with gene expression features.

The extraordinary diversity of antigen receptors on B and T lymphocytes governs the development, survival and activation of these cells.

T cells express on their cell surface a T cell receptor (TCR) heterodimer composed of either α and β or γ and δ chains, each the product of a different germline TRA, TRB, TRG or TRD gene loci, respectively.

B cells express a B cell receptor (BCR) hetero-tetramer composed of two identical membrane immunoglobulin heavy chains encoded by the IGH gene locus and two identical immunoglobulin kappa or lambda light chains encoded by the IGK or IGL genes, respectively.

Each of these gene loci comprise in their germline configuration a cluster of separate variable (V), diversity (D) and joining (J) gene segments, one member of each cluster becoming joined through irreversible somatic DNA rearrangements during T or B lymphocyte development in a process known as V(D)J recombination {Bassing, 2002}.

Further diversity between cells is created by random addition or removal of nucleotides at the V(D)J junctions, which lie ~400 nucleotides from the 5’ end of the mRNA and encode complementarity determining region 3 (CDR3) in the antigen binding site of the receptor. The resulting diversity of the lymphocyte antigen receptor repertoire is estimated at >1012 different TCR or BCR proteins governed by the rule of “one cell clone – one receptor sequence” {Calis, 2014; Laydon, 2015}.

Consequently, it is extremely unlikely that two cells descended from different lymphocytes will carry the same antigen receptor sequence or ‘clonotype’.

As a result, 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.

Sequencing the BCR or TCR of individual lymphocytes in parallel with their transcriptome provides high-resolution insights into the adaptive immune response in a range of disease settings such as infectious disease, autoimmune disorders and cancer.

A common approach to link paired antigen receptor sequences with gene expression profiles of single lymphocytes is through the use of the full-length scRNA-seq method SmartSeq2 {Picelli, 2013}, where computational methods can reconstruct paired TCRαβ sequences or paired IgH and IgL sequences from Illumina short-reads {Afik, 2017; Eltahla, 2016; Rizzetto, 2018; Stubbington, 2016; Upadhyay, 2018}. However, SmartSeq2 generally relies on plate- or well-based microfluidics and is therefore limited in the number of cells that can be processed, typically 10s to 100s. Additionally, a large number of sequencing reads are generally required to

computationally reconstruct paired antigen receptors {Rizzetto, 2017}. As such, the cost per cell is relatively high ($50-$100 USD) {Ziegenhain, 2017}.

Moreover, assembly of short reads makes it difficult or impossible to decipher critical alternative splicing of mRNA segments separated by more than >500 nucleotides, as occurs in IGH genes.

Recent technological advancements in high-throughput scRNA-seq methods allow thousands of cells to be captured and sequenced in a relatively short time frame and at a fraction of the cost {Ziegenhain, 2017}.

Such methods rely on capture of polyadenylated (polyA) mRNA transcripts followed by cDNA synthesis, pooling, amplification, library construction and Illumina 3’ cDNA sequencing.

The combination of fragmentation and short-read sequencing fails to sufficiently sequence the V(D)J regions of rearranged TCR and BCR transcripts, which are located in the first 500 nucleotides at the 5’ end of the transcript. Consequently, 3’-tag scRNA-seq platforms have limited application for determining clonotypic information from large numbers of lymphocytes.

Variations on this approach employing 5’ cell barcodes enable the V(D)J sequences and global gene expression to be measured {Azizi, 2008}, but don’t solve the need to integrate this information with the diversity of switching and mRNA splicing involving the 3’ end of IGH mRNA.

Recent advances in long read sequencing technologies present a potential solution to the shortcomings of short-read sequencing.

Full- length cDNA reads can encompass the entire sequence of BCR and TCR transcripts, but typically suffer from higher error rates and lower sequencing depth than short read technologies {Byrne, 2017}.

Here, we describe a rapid high-throughput method to sequence full-length transcripts using targeted capture and Oxford nanopore sequencing and link this with short-read transcriptome profiling at single cell resolution.

This novel method, termed Repertoire and Gene Expression by sequencing (RAGE-Seq), can be applied to high-throughput droplet-based scRNA-seq workflows to accurately pair gene expression profiles with targeted full-length cDNA sequences from a large number of cells. We demonstrate the power of this method by combining transcriptome profiling with full-length antigen receptor sequence characterization from thousands of human tumour-associated lymphocytes.

Using de novo assembly of nanopore reads, complete antigen receptor sequences were recovered at high accuracy and sensitivity, including the identification of somatic mutations from immunoglobulin full- length heavy and light chains allowing for the inference of B cell clonal evolution.


More information:Nature Communications (2019). DOI: 10.1038/s41467-019-11049-4

Journal information: Nature Communications
Provided by Garvan Research Foundation

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