Immunosequencing Methods: Which is Right for You?

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    There are numerous ways to study the immune system. Serological tests can detect the presence of antibodies reactive to a given antigen. Cell counting can provide information on the quantity and type of circulating immune cells. Functional assays can evaluate the activity of immune cells, such as the ability of B cells to produce antibodies or T cells to produce cytokines.

    Immunosequencing, also known as immune repertoire profiling, is a relatively new technique wherein the genes encoding T-cell receptors (TCRs) and B-cell receptors (BCRs) are sequenced to provide a snapshot of the entire immune repertoire. Generating such a profile was nearly impossible before the advent of high-throughput sequencing. (1) But now, sequencing the full immune repertoire can enable a better understanding of how the immune system functions and how it responds to disease and treatment.

    Immunosequencing has found applications in numerous fields, including oncology and infectious disease. It has been used to explore changes in the immune system relating to disease progression and response to therapies or vaccines. In addition, immunosequencing can identify T-cell signatures that can be used to assess past infection or to monitor vaccine-induced immune responses. Immunosequencing has also shed light on the etiologies of several autoimmune disorders, such as multiple sclerosis, rheumatoid arthritis, ulcerative colitis, lupus, and type I diabetes. (1, 2)

    Several technologies enable researchers to sequence the immune repertoire. These generally rely on sequencing the complementary determining region (CDR). BCRs and TCRs contain three CDRs (CDR1, CDR2, and CDR3). CDR3 is the most variable CDR as it is created from the combination of the noncontiguous variable (V), diversity (D), and joining (J) region gene segments. Because of this exceptionally high diversity, CDR3 can be used to identify and track unique T- and B-cell populations. While the underlying concept for most of these technologies is similar, they have distinct differences, including the following:

    • Sample requirements.
    • Hands-on and total workflow time.
    • Degree of polymerase chain reaction (PCR) bias.
    • Quantifiability.
    • Immune repertoire coverage.
    • Ease of data analysis.
    • Cost.

    Understanding each technique’s limitations and advantages is key to choosing the right immunosequencing method for your research.

    Methods for immunosequencing

    Spectratyping

    Spectratyping characterizes the immune cell repertoire by separating CDR3 fragments based on length. TCR messenger RNA (mRNA) is reverse transcribed into complementary DNA (cDNA), which is amplified using primers specific for the surrounding constant regions and selected V genes (Figure 1). Amplicons are then separated based on size via gel electrophoresis, and the frequency distribution of CDR3 lengths is visualized via a densitogram. (3) Regarding the workflow, spectratyping is one of the more straightforward methods. It consists of a single PCR step followed by a brief primer extension step, and most labs already have the necessary equipment.

    Spectratyping gives a rough overview of an immune repertoire’s diversity and is useful for studying broad changes. Unlike other techniques, it does not provide any sequence information and cannot, for example, provide information on frequencies of or changes in single T-cell clones.

    Nonetheless, immune repertoire diversity can provide valuable insight. Many disease states have a markedly less diverse immune repertoire than a healthy immune system. For example, spectratyping has been used to show that lower TCR diversity is associated with severe immune-related adverse events in cancer patients receiving immunotherapy. (4) Another recent study used spectratyping in mice lacking regulatory T cells to understand how regulatory T cells affect TCR repertoire diversity among antigen-stimulated T cells. (5)

    Overview of spectratyping.

    Figure 1. Overview of spectratyping. Adapted from Kepler et al. (3)

    5’ RACE PCR

    The sheer diversity of TCRs and BCRs allows them to target a vast array of antigens. However, this diversity also makes them challenging to study, partly because the lack of sequence similarity makes it difficult to design PCR primers that can amplify the full repertoire. 5’ RACE PCR (Rapid amplification of cDNA ends PCR) is a common method to amplify regions of unknown or variable sequence. In this technique, during reverse transcription of mRNA to cDNA, a known adapter sequence is added to the 3’ end of the cDNA (Figure 2).

    Figure 2. Overview of 5’ RACE PCR. (6)

    In this way, the entire immune repertoire can be amplified using a PCR primer that anneals to the adapter sequence and reverse primers that anneal to the receptor’s constant region. Amplicons can then be sequenced using a variety of sequencing technologies. With the advent of next-generation sequencing, 5’ RACE PCR can enable sequencing of the whole immune repertoire with a single primer. 5’ RACE PCR uses mRNA as a starting material. Reverse transcription of mRNA to cDNA adds an extra step and hands-on time to this method. Since mRNA is far less stable than DNA, sample preparation and handling are essential to prevent RNA degradation. Also, because mRNA levels can vary from cell to cell, 5’ RACE PCR cannot provide quantitative information about the number of T cells and clone frequencies in a sample. (6)

    Large-scale immunosequencing: The immunoSEQ® Assay

    The immunoSEQ Assay uses a two-step bias-controlled multiplex PCR to amplify receptor genes. The inherent diversity of CDR3 makes it highly improbable that the same CDR3 sequence will be independently created twice, effectively making each CDR3 sequence a unique nucleotide tag for any specific T or B cell and its clonal descendants.

    First, CDR3 sequences are amplified with an array of forward and reverse primers targeting the various V and J genes (Figure 3). (7, 8) The primers also attach a universal adapter sequence that can be used in the second PCR step. Only the CDR3 sequences—rather than the entire V(D)J region—are amplified. This shorter amplicon improves sequence recovery from potentially degraded DNA samples, including formalin-fixed, paraffin-embedded samples (FFPE).

    In the second PCR step, a unique sample-specific barcode is added to both ends of each amplicon. The amplicons are then sequenced to capture a comprehensive picture of the TCR or BCR repertoire. The immunoSEQ Assay also incorporates proprietary internal control sequences to correct for any PCR bias and allow for quantification. The immunoSEQ® hs TCRB Assay, which sequences the human TCRB locus, is available via a kit that will enable you to prepare your sequencing libraries with less than 4 hours of hands-on time and takes approximately 8 hours from sample to sequencing. If even 4 hours of hands-on time is too much, you can use our immunoSEQ Service. We’ll send you a customized shipping box and detailed instructions on how to send us your samples. We’ll prepare and sequence your samples in-house and send you access to the data via our immunoSEQ® Analyzer. Our immunoSEQ Service is available for human and mouse TCRB and IGH, and human TCRG and TCRA/D loci.

    The immunoSEQ Assays are optimized for genomic DNA (gDNA) but can be used with cDNA as well. Because each nucleated cell contains the same amount of gDNA, the immunoSEQ Assay can provide quantitative information about specific T- or B-cell clones in a sample. (9) The immunoSEQ Assay can be used to analyze a variety of sample types, including sorted B or T cells, cultured cells, PBMCs, whole blood, bone marrow, fresh/frozen/FFPE tissues, or even previously hematoxylin and eosin (H&E) stained slides.

    Overview of the immunoSEQ Assay

    Figure 3. Overview of the immunoSEQ Assay.

    Analysis of immunoSEQ Data is easily performed with the immunoSEQ Analyzer, a user-friendly suite of analysis tools that can perform complex calculations and comparisons between samples and generate publication-ready figures. The immunoSEQ Analyzer can report on qualities such as T-cell fraction, clonal overlap, gene usage, TCR sequence, and repertoire diversity and clonality. In addition, our immuneACCESS® open-access database contains immunoSEQ Data from >12,000 human and >1000 mouse samples, allowing you to compare your immunoSEQ Data with others’ and identify public clones between datasets.

    The immunoSEQ Analyzer also contains the immunoSEQ® T-MAP™ COVID offering, which allows you to compare your samples with a library containing more than ~2 billion unique TCR sequences, including >160,000 TCR/SARS-CoV-2 antigen pairs, enabling you to identify TCR rearrangements associated with SARS-CoV-2. You can get a picture of the data produced using the immunoSEQ Assay and explore the immunoSEQ Analyzer by signing up for a free analyzer account, where you can access all of the data deposited in the immuneACCESS database.

    Single-cell sequencing

    In single-cell sequencing, live cells are encapsulated into barcoded droplets, cDNA is prepared and sequenced, and quantitative transcriptomics is used to report out TCRs as well as other cell markers (Figure 4). Since each cell is individually barcoded, amplification bias is not an issue. While this technique can provide valuable, quantitative information beyond just TCRs, it generally misses the full diversity of the immune repertoire due to poor encapsulation efficiency and dropout events, in which RNA fails to amplify and is not detected.

    Sample quality is paramount for single-cell sequencing. Because the method requires viable cells, it’s important to treat your samples gently and work quickly. Removing contaminants such as free RNA, dead cells, debris, and clumps is necessary to ensure adequate recovery and data. There are various sample cleaning techniques; however, methods that produce the cleanest data, such as thorough centrifugation, can also cause the most sample loss. Therefore, choosing the appropriate single-cell sequencing protocol for your needs often requires balancing speed and sample integrity while minimizing sample loss.

    Isolating single cells can also be challenging. While droplet-based instruments can encapsulate thousands of cells, they require a dedicated hardware platform. (10) The encapsulation efficiency of lipid droplet methods varies widely based on the method used. Encapsulation rates for “out-of-the-box” commercial products range between 50% and 65%, meaning they can miss rare clones. (11) Single-cell sequencing is also expensive, and data analysis requires unique tools and expertise regarding the most appropriate analytical approaches.

    Overview of single-cell sequencing via droplet-based encapsulation

    Figure 4. Overview of single-cell sequencing via droplet-based encapsulation.

    Single-cell sequencing provides detailed transcriptomic data at the single-cell level that can complement data obtained from a large-scale immunosequencing technique such as the immunoSEQ Assay. The immunoSEQ Assay can reveal the overall contours of a T-cell repertoire and how clonal populations shift over time or across various tissue samples. Single-cell sequencing can correlate T-cell clonotypes and gene expression changes.

    Table 1 summarizes some of the characteristics of immune repertoire sequencing technologies. You can learn more about the features of each of these techniques in our webinar on ‘Approaches to VDJ Sequencing’.

    Table 1: Immunosequencing technologies

    Table showing characteristics of immune repertoire sequencing technologies comparing Spectratyping, 5' RACE PCR, ImmunoSEQ and single-cell sequencing.

    Applications of the immunoSEQ Assay

    Immune repertoire sequencing via the immunoSEQ Assay has been used to gain key insights into the inner workings of the immune system and better understand the role of the immune system in disease and treatment response. One valuable metric that immunosequencing provides is clonality, a measure of how evenly receptor sequences are distributed among populations. This can quantify how focused the immune repertoire is on a particular set of antigens.

    T-cell clonality was assessed with the immunoSEQ Assay in a recent study of patients undergoing stereotactic ablative radiotherapy (SABR) for recurrent hormone-sensitive oligometastatic prostate cancer. The authors showed that patients who underwent SABR had more expanded clones, i.e. increased clonality, than those who received no treatment. In addition, greater T-cell clonality at baseline was associated with favorable clinical benefit in the SABR group (Figure 5). These data can help reveal the mechanism by which SABR inhibits disease progression. (12)

    Correlation between baseline T-cell clonality and disease progression at 180 days in recurrent hormone-sensitive oligometastatic prostate cancer.

    Figure 5. Baseline T-cell clonality is negatively associated with disease progression at 180 days. Adapted from Phillips et al. (12)

    Immunosequencing can also be used to assess the immune repertoire over time to understand how it changes in response to disease or treatment. The immunoSEQ Assay was used to monitor the remodeling of the T-cell repertoire in a patient with refractory bone-marrow-metastatic rhabdomyosarcoma who experienced a durable remission after receiving several chemotherapy induction treatments and HER2 chimeric antigen receptor (CAR) T-cell infusions. Longitudinal sequencing of TCR CDR3 showed an increase in peripheral T cells and increased clonality after CAR T-cell infusion (Figure 6).

    Overall, several immunodominant clones prior to infusion declined, while unique T-cell clones that were not present prior to infusion or in the infusion product appeared and persisted over the infusion timeline. Of the 20 immunodominant clones identified after the third CAR T-cell infusion, 8 were not detectable before treatment or in the infusion product. Longitudinal analysis of the repertoire overlap between the infusion product and the peripheral T cells after each infusion demonstrated continual remodeling of the T-cell repertoire after each infusion (Figure 6). These data can reveal which features of immune reactivation are important for response to CAR T-cell therapy. (13)

    Increased T-cell clonality following CAR T-cell treatment.
    Heat map representing repertoire overlap (via Morisita’s index) between the CAR T-cell product and longitudinal samples from the peripheral blood (PB).

    Figure 6. Top: Increased T-cell clonality following CAR T-cell treatment. Bottom: Heat map representing repertoire overlap (via Morisita’s index) between the CAR T-cell product and longitudinal samples from the peripheral blood (PB). The overlap index has values ranging from 0 to 1, depicting a low to a high degree of overlap, respectively. Adapted from Hegde et al. (13)

    Another study on CAR T-cell therapy investigated what happened to CAR T cells after infusion in a group of patients with B-cell malignancies. The immunoSEQ Assay showed that the clonal diversity of the infused cells decreased over time; single-cell transcriptional analysis showed that rare clones in the infusion product expanded after infusion and came to dominate the immune response and that different clones expanded and receded at different rates. Thus, the two complementary techniques conspired to give a complete picture of the dynamics dictating the expansion of different T-cell clones. This approach therefore gave insights into the make-up of the entire T-cell repertoire during and after treatment. (14)

    Immunosequencing methods summarized

    There are several ways to sequence BCRs and TCRs and analyze immune repertoires. Understanding each method’s requirements and your experimental goals is key to choosing the right technique for you. The immunoSEQ Assay provides a robust, scalable, reproducible, and quantitative profile of the T- or B-cell repertoire. It can analyze a variety of sample inputs, including but not limited to gDNA, cDNA, blood, and FFPE. The immunoSEQ hs TCRB Assay, which sequences the human TCRB locus, is available via a kit to allow you to perform your experiments in your lab. You can also send your samples directly to us for sequencing. Both the kit and the service options give you access to the immunoSEQ Analyzer, a user-friendly data analysis and visualization suite, as well as to the open-access immuneACCESS database. Contact us for more information. A representative can help you determine if the immunoSEQ Assay is the right approach for your research needs and walk you through the best way to set up your experiments, analyze data, and generate publication-ready figures.

    For Research Use Only. Not for use in diagnostic procedures.

    References

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    2. Liu X, Wu J. History, applications, and challenges of immune repertoire research. Cell Biol Toxicol 2018;34(6):441–57.

    3. Kepler TB, He M, Tomfohr JK, Devlin BH, Sarzotti M, Markert ML. Statistical analysis of antigen receptor spectratype data. Bioinforma Oxf Engl 2005;21(16):3394–400.

    4. Arakawa A, Vollmer S, Tietze J, Galinski A, Heppt MV, Bürdek M, et al. Clonality of CD4+ Blood T Cells Predicts Longer Survival With CTLA4 or PD-1 Checkpoint Inhibition in Advanced Melanoma. Front Immunol 2019;10:1336.

    5. Fontaine M, Vogel I, Van Eycke Y-R, Galuppo A, Ajouaou Y, Decaestecker C, et al. Regulatory T cells constrain the TCR repertoire of antigen-stimulated conventional CD4 T cells. EMBO J 2018;37(3):398–412.

    6. Mulhardt C. Rapid Amplification of cDNA Ends, In: Molecular Biology and Genomics. Academic Press, 2007. pp. 70-80.

    7. Carlson CS, Emerson RO, Sherwood AM, Desmarais C, Chung M-W, Parsons JM, et al. Using synthetic templates to design an unbiased multiplex PCR assay. Nat Commun 2013;4:2680. https://doi.org/10.1038/ncomms3680.

    8. Montagne JM, Zheng XA, Pinal-Fernandez I, Milisenda JC, Christopher-Stine L, Lloyd TE, et al. Ultra-efficient sequencing of T Cell receptor repertoires reveals shared responses in muscle from patients with Myositis. EBioMedicine 2020;59:102972.

    9. Rosati E, Dowds CM, Liaskou E, Henriksen EKK, Karlsen TH, Franke A. Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol 2017;17(1):61.

    10. Chen G, Ning B, Shi T. Single-Cell RNA-Seq Technologies and Related Computational Data Analysis. Front Genet 2019;10:317.

    11. Salomon R, Kaczorowski D, Valdes-Mora F, Nordon RE, Neild A, Farbehi N, et al. Droplet-based single cell RNAseq tools: a practical guide. Lab Chip 2019;19(10):1706–27.

    12. Phillips R, Shi WY, Deek M, Radwan N, Lim SJ, Antonarakis ES, et al. Outcomes of Observation vs Stereotactic Ablative Radiation for Oligometastatic Prostate Cancer: The ORIOLE Phase 2 Randomized Clinical Trial. JAMA Oncol 2020;6(5):650–9.

    13. Hegde M, Joseph SK, Pashankar F, DeRenzo C, Sanber K, Navai S, et al. Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma. Nat Commun 2020;11(1):3549.

    14. Sheih A, Voillet V, Hanafi L-A, DeBerg HA, Yajima M, Hawkins R, et al. Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy. Nat Commun 2020;11(1):219.

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