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Nature Methods: New Progress in Single Cell TCR Research
introduction:
In March of this year, Dr. Michael Stubbington, a British scientist, and Dr. TapioLönnberg, based on Fluidigm's C1 mRNA sequencing samples and corresponding data, developed a set of open analysis tools for studying T cell gene sequence information, TraCeR. Transcriptome data analysis of T cell subsets further reveals the deeper T cell function specificity implicated in different immune responses. The research was published in the famous "nature methods" magazine.
Original link: http://
Research background and results
When the body is exposed to an exogenous invasion, the immune system recruits all immune cell subpopulations to fight the pathogens by generating an immune response. Due to the wide variety of pathogens, the corresponding immune response and the type of cells involved in each stage are also different. The random rearrangement of the V(D)J gene region in T cell receptors leads to a complex diversification of T cell species; this diversification makes T cells faster and more accurate in identifying and killing specific exogenous antigens. And effective.
One of the main methods of identifying T cell receptors (TCRs) is to sequence their alpha and beta chains. However, in order to further fully understand the T cell response mechanism, it is necessary to more accurately identify the phenotypic differences within and between different T cell subsets. In the traditional immunology research, the data obtained by sequencing of population cells is difficult to analyze the heterogeneity of cells and the characteristics of different cell subpopulations. Therefore, immunocyte phenotypic analysis at the single cell level is more and more widely used in immunology. researching.
Recently, EMBL-EBI and Dr. Stubbington and Dr. Lönnberg of the Wellcome Trust Sanger Institute at Cambridge University used Fluidigm's C1 single-cell mRNA Seq system to rapidly and high-throughput single-cell full transcriptome sequence samples, and obtained a large number of singles by further sequencing. Cell TCR sequence data, and based on this, developed a set of open analysis software for analysis of T cell gene sequences and phenotypes - TraCeR, used to study the transcription and relationship between different T cell clones, thereby Reveal deeper T cell functional specificity.
The TraCeR analysis method not only extracts the TCR sequence information of each single cell, but also analyzes the gene expression of each cell in parallel; the researchers recombine the TCR sequences closely related to different immune responses and disease occurrences. In- depth study of potential molecular mechanisms and cellular functions revealed that TCR sequences possess a "trace-to-source" capability similar to the two-dimensional code structure, closely related to TCR and cell subpopulations, and revealing the health and disease state of the body. relationship. Using this information, it is possible to identify and target T cell subsets associated with pathogenicity, providing a wealth of valid data for complex antibody, vaccine development, tumor immunotherapy, and autoimmune disease research.
The value and advantages of single-cell TCR research
For each organism, there is a wide diversity of TCR sequences. "Almost every cell has a different sequence, so analyzing individual cells is a powerful research method," explains Stubbingto. "Unlike population cell analysis, single-cell research has changed the way we think." This is crucial in immunology research; it can provide us with a new and more effective means of solving those unknown and well-received problems."
“I believe that the impact of single-cell analysis will be far-reaching, and we will only see a beginning. Single-cell analysis goes deep into each single cell, so I can understand their 'ideas' and mechanisms, and bring me very Big inspiration.†Dr. Lönnberg, one of the developers of TraCeR, who has been working on T-cell research for a long time.
“ The activity and differentiation processes of T cells occur at the single-cell level. The value of using single-cell analysis of TCR sequences is that for each specific reaction, we can find the exact sequence corresponding to it, not just It is simply a measure of the expression level of a TCR gene fragment, which is not possible with traditional cell-based research methods." - Dr. Lönnberg
Dr. Stubbington pointed out that although some methods for identifying TCR using single cells have been published, this new assay does not require sequencing of specific TCR targeting sequences; due to the use of Fluidigm's C1 platform, single-cell transcription can be achieved quickly. The full-length sequencing samples were prepared, so subsequent sequencing results can be analyzed by TraCeR; that is, when performing single T cell sequencing, additional scRNASeq data can be obtained in another dimension based on the original experiment. In addition, he added: "Through the detection of thousands of expressed genes, researchers can also fully understand the transcriptional status of each cell."
This is critical for the promotion of single-cell sequencing methods and the reduction of experimental costs. "Using the TraCeR method, TCR data can be analyzed even for previously obtained T cell sequencing results," Dr. Lönnberg pointed out.
Clinical application
TraCeR's new approach is likely to change our previous understanding of T cell gene expression and related immune responses. The importance of T cells as a core factor in cell-cell interactions plays an irreplaceable role in the development and treatment of diseases.
Dr. Stubbington and Dr. Lönnberg believe that in the near future, single-cell detection based methods for patients with different diseases and their different states will be widely used and will be expected to guide clinical treatment and care. E.g:
• Cells with specific antigens can react with the vaccine and produce corresponding T cell memory.
· Important in tumors, autoimmune, and infectious diseases: In-depth study of lymphocyte heterogeneity at the single-cell level can provide a large amount of unknown information.
· Micro sample study: This method can also help researchers to study micro or trace cell samples that are difficult to obtain in clinical samples, such as rare T cell subsets, circulating tumor cells, etc., so as to develop more targeted Sexual, more effective personalized treatment options.
Dr. Lönnberg also predicted that the reduction in T cell diversity caused by aging will receive more attention.
· At present, two scientists have applied the TraCeR method to the research project on the differentiation of helper T cells during infection.
Prospects
The existing TraCeR method allows rapid analysis of human and mouse TCR sequence information; and they are further developing analytical modules for other species based on sequencing of single-cell full-length transcriptome sequences obtained from C1, The V/J region gene sequences in TCRs known from different species were recombined to construct a synthetic genomic data and analysis module. They also plan to extend the method to B cells in the near future.
This method innovatively combines the Fluidigm C1 single cell automated preparation system, the second generation sequencing and the new sequencing data analysis tools, greatly improving the yield of effective data in a single experiment, and studying T cells for scientists. In-depth revealing the mechanism of immune response in different disease stages provides new ideas.
In order to enable users to obtain and use this new method for TCR research more quickly and easily, Fluidigm has recently further optimized its application in the C1 system. Users can download the standard C1 mRNA seq experimental program and TraCeR analysis software from the Script Hub section of Fluidigm's homepage for in-depth analysis of TCR phenotypes and related gene expression.
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