Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor

      High-throughput sequencing (HTS) of the T-cell receptor (TCR) is a rapidly advancing technique that allows sensitive and accurate identification and quantification of every distinct T-cell clone present within any biological sample. The relative frequency of each individual clone within the full T-cell repertoire can also be studied. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or under pathologic conditions, as well as to understand the kinetics of antigen-specific T-cell responses that lead to protective immunity (i.e., vaccination) or immune-related disorders (i.e., autoimmunity and cancer). HTS can be tailored for personalized medicine, having the potential to monitor individual responses to therapeutic interventions and show prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances, and limitations of HTS of the TCR and describe emerging applications of this technique in the field of investigative dermatology. We highlight studying the pathogenesis of T cells in allergic dermatitis and the application of HTS of the TCR in diagnosing, detecting recurrence early, and monitoring responses to therapy in cutaneous T-cell lymphoma.

      Abbreviations:

      CDR3 (complementarity determining region 3), CTCL (cutaneous T-cell lymphoma), HTS (high-throughput sequencing), LAM-PCR (linear amplification-mediated PCR), nrLAM-PCR (nonrestrictive linear amplification-mediated PCR), TCR (T-cell receptor), TRM (resident memory T cells), V(D)J (variable, diversity, joining)
      CME Activity Dates: 19 May 2017
      Expiration Date: 18 May 2018
      Estimated Time to Complete: 1 hour
      Planning Committee/Speaker Disclosure: All authors, planning committee members, CME committee members and staff involved with this activity as content validation reviewers have no financial relationship(s) with commercial interests to disclose relative to the content of this CME activity.
      Commercial Support Acknowledgment: This CME activity is supported by an educational grant from Lilly USA, LLC.
      Description: This article, designed for dermatologists, residents, fellows, and related healthcare providers, seeks to reduce the growing divide between dermatology clinical practice and the basic science/current research methodologies on which many diagnostic and therapeutic advances are built.
      Objectives: At the conclusion of this activity, learners should be better able to:
      • Recognize the newest techniques in biomedical research.
      • Describe how these techniques can be utilized and their limitations.
      • Describe the potential impact of these techniques.
      CME Accreditation and Credit Designation: This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education through the joint providership of William Beaumont Hospital and the Society for Investigative Dermatology. William Beaumont Hospital is accredited by the ACCME to provide continuing medical education for physicians.
      William Beaumont Hospital designates this enduring material for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
      Method of Physician Participation in Learning Process: The content can be read from the Journal of Investigative Dermatology website: http://www.jidonline.org/current. Tests for CME credits may only be submitted online at https://beaumont.cloud-cme.com/RTMS-June17 – click ‘CME on Demand’ and locate the article to complete the test. Fax or other copies will not be accepted. To receive credits, learners must review the CME accreditation information; view the entire article, complete the post-test with a minimum performance level of 60%; and complete the online evaluation form in order to claim CME credit. The CME credit code for this activity is: 21310. For questions about CME credit email [email protected] .

      Introduction

      T lymphocytes form an essential component of the adaptive immune system. Each individual T cell expresses a unique T-cell receptor (TCR) that specifically recognizes a single antigenic determinant. Taking the whole T-cell population together, the adaptive immune system has a spectacularly large and highly diverse TCR repertoire at its disposal, capable of identifying unlimited numbers of antigens, hence providing protection against constant diverse pathogenic threats. TCRs are heterodimer molecules consisting of either a combination of α and β chains, the most common TCR, or a combination of γ and δ chains (Figure 1). The superb specificity and diversity occurs during lymphocyte development by randomized combinations of DNA from distinct variable, diversity, joining (V(D)J) gene segments and by deletion and/or insertion of nucleotides at the junctions of these segments, which in particular takes place in the hypervariable complementarity determining region 3 (CDR3). This hypermutation process of TCR genes can lead to over 1018 different αβ TCRs, making it highly improbable that two TCRs with an identical nucleotide CDR3 sequence will be generated (
      • Murphy K.
      Janeway’s immunobiology, 8th ed.
      ). Consequently, the TCR nucleotide sequence of each T cell is akin to a barcode that enables recognition and tracking of each specific T-cell clone (Figure 1).
      Figure 1
      Figure 1The T-cell receptor (TCR) can function as a unique identifying bar code of T cells. (a) In the germline genome, the multiple gene segments of the TCR have not yet been rearranged. Specificity and diversity occurs during lymphocyte development by combining the distinct variable (V), diversity (D), and joining (J) gene segments and by deletion and/or insertion of nucleotides at the junctions of those segments (C, Constant gene segments). This randomized process makes it highly improbable that two T-cell receptors with the same complimentary determining region 3 (CDR3) nucleotide sequence will be generated. Thus, the unique TCR nucleotide sequence of each T cell is akin to a bar code that enables recognition and tracking of each specific T-cell clone. The unique mRNAs are translated into the peptide chains of the TCR. (b) TCR proteins heterodimerize to form either a combination of α and β chains or γ and δ chains.

      Overview of Methodology

      A recently developed methodology, known as immunosequencing, combines bias-controlled multiplexed PCR with high-throughput sequencing (HTS) of the CDR3 of the TCR. Subsequent innovative bioinformatic analysis enables the simultaneous identification, quantification, and tracking of each individual lymphocyte and of the entire repertoire within any sample of interest (Figure 2). To describe HTS of the TCR briefly, DNA or mRNA is extracted from the biologic sample of interest (e.g., blood or skin), and the CDR3 region is amplified from the isolated DNA or cDNA (synthetized by reverse transcription of mRNA). Then, in a second PCR step, bias-controlled V and J gene primers are used to further amplify the rearranged V(D)J segments, which finally are subjected to HTS. Clustering algorithms are subsequently used to correct the raw data for sequencing errors (Figure 3). Lastly, the unique CDR3 segments and the V(D)J genes within each rearrangement are identified and quantified (Figure 2), based on previously described sequences, which can be accessed within the IMGT data bank (http://www.imgt.org), offering a common standard for nomenclature, numbering, and annotation (
      • Lefranc M.P.
      • Giudicelli V.
      • Ginestoux C.
      • Jabado-Michaloud J.
      • Folch G.
      • Bellahcene F.
      • et al.
      IMGT, the international ImMunoGeneTics information system.
      ,
      • Robins H.S.
      • Campregher P.V.
      • Srivastava S.K.
      • Wacher A.
      • Turtle C.J.
      • Kahsai O.
      • et al.
      Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells.
      ). These results allow identification of somatic allelic mutations, study of the lymphoid clonality and diversity in healthy and malignant tissues, and tracking of each clone over time (e.g., as a biomarker during disease progression or therapy) among different anatomical sites or within defined populations.
      Figure 2
      Figure 2High-throughput TCR sequencing. (a) The biological sample of interest is collected. (b) DNA is extracted or cDNA is synthetized. (c) Bias-controlled multiplexed PCR amplifies and sequences the CDR3 from the DNA or cDNA. Then, bias-controlled V and J gene primers are used to amplify the rearranged V(D)J segments. (d) Bioinformatics can then be used to identify, quantify, and track each individual lymphocyte and the entire repertoire within any sample of interest. It is possible to identify and quantify the unique CDR3 segments and the V(D)J genes within each rearrangement, based on previously described sequences that can be accessed within data banks. CDR3, complementarity determining region 3; TCR, T-cell receptor; V(D)J, variable, diversity, joining.
      Figure 3
      Figure 3High-throughput TCR CDR3 sequencing captures entire T-cell diversity. (a) Comparison of standard TCRβ spectratype data and calculated TCRβ CDR3 length distributions for sequences using representative TCR Vβ gene segments. CDR3 length is plotted along the x-axis, and the number of unique CDR3 sequences with that length or the relative intensity of the corresponding peak in the spectratype is plotted along the y-axis. The length of the differently colored segments within each bar of the histograms indicates the fraction of unique CDR3 sequences that was observed 1–5 times (black), 6–10 times (blue), 11 –100 times (green), or more than 100 times (red). (b) A representative spectratype of TCRβ CDR3 cells that use the Vβ10 gene segment. The CDR3 sequences were sorted by CDR3 length into a frequency histogram, and the sequences within each length were then color-coded on the basis of their Jβ use. The inset represents CDR3 sequences having a length of 39 nucleotides (nt), as well as the number of times that each of these sequences was observed in the data. The origin of the nucleotides in each sequence is color-coded as follows: Vβ gene segment, red; template-independent N nucleotide, black; Dβ gene segment, blue; Jβ gene segment, green. (c) Shown are the results of TCRγ HTS of lesional skin from a patient with stage III cutaneous T-cell lymphoma. The two rearranged TCRγ allele sequences of the malignant clone are indicated by asterisks. A healthy diverse population of benign infiltrating T cells was present. Parts a and b were adapted from
      • Robins H.S.
      • Campregher P.V.
      • Srivastava S.K.
      • Wacher A.
      • Turtle C.J.
      • Kahsai O.
      • et al.
      Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells.
      , with permission from the American Society of Hematology. Part c was adapted from
      • Kirsch I.R.
      • Watanabe R.
      • O’Malley J.T.
      • Williamson D.W.
      • Scott L.L.
      • Elco C.P.
      • et al.
      TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.
      , with permission from The American Association for the Advancement of Science. CDR3, complementarity determining region 3; D, diversity; HTS, high-throughput sequencing; J, joining; TCR, T-cell receptor; V, variable.

      Variants of PCR and Other Techniques to Study the TCR

      Multiple variants of PCR can be applied to analyze the CDR3, and for a better understanding of the literature, some relevant methods will be briefly explained. Most PCR protocols require an initial treatment with restriction enzymes to reduce the DNA length or to enable linking of defined oligonucleotides and use double-strand DNA synthesis during amplification, known as linear amplification-mediated PCR (LAM-PCR). As an alternative approach, nonrestrictive LAM-PCR (nrLAM-PCR) avoids using restriction enzymes while the single DNA strands are amplified directly by competitive exponential PCR (
      • Gabriel R.
      • Eckenberg R.
      • Paruzynski A.
      • Bartholomae C.C.
      • Nowrouzi A.
      • Arens A.
      • et al.
      Comprehensive genomic access to vector integration in clinical gene therapy.
      ). Because the time span of the linear DNA synthesis step is limited, the resultant nrLAM-PCR products display substantial variability in length, because size is no longer determined by restriction enzymes. As a consequence, analysis by gel electrophoresis is not possible (PCR products appear as a smear instead of a sharp band), but nrLAM-PCR allows unbiased HTS by restriction site motifs. The overall sensitivity and efficiency of nrLAM-PCR is superior compared with other LAM-PCR techniques if the amount of input DNA is not a strict limitation (
      • Gabriel R.
      • Eckenberg R.
      • Paruzynski A.
      • Bartholomae C.C.
      • Nowrouzi A.
      • Arens A.
      • et al.
      Comprehensive genomic access to vector integration in clinical gene therapy.
      ).
      Rapid amplification of cDNA ends, also known as one-sided PCR or anchored PCR, starts with the generation of the first strand of cDNA with a specific primer targeting a known sequence within the mRNA transcript (e.g., C-gene region of the TCR), and the other terminus is covalently linked to an “anchor” oligonucleotide sequence. Then, double-strand cDNA synthesis by PCR is performed by using a primer that specifically binds to the anchor and to a second primer of interest.
      Multiplex PCR uses multiple primer sets within a single PCR mixture, resulting in multiple PCR products (amplicons) of varying sizes. For example, multiple primers covering all known V genes can be used when studying the lengths and sequences of the CDR3 of the TCR repertoire. The design of the primers for multiplex PCR is of utmost importance: the primer length (18–22 bases) and the annealing and melting temperature must be optimized to act accurately within a single reaction mixture. In addition, binding of primers to each other (primer dimers) must be excluded, and a linear amplification protocol must be applied to prevent bias between the primers.
      The TCR repertoire can also be analyzed by other techniques besides PCR, including flow cytometry or mass cytometry (Table 1). However, comprehensive analysis of the TCR repertoire by cytometry is restricted because of the limited availability of anti-TCR antibodies.
      Table 1Comparison of approaches for analysis of the TCR
      Type of InformationFlow CytometryMass CytometryHTS of the TCR
      Protein/mRNA/DNA levelProteinProteinmRNA/DNA
      Tissue sample preparationObligatory to prepare single-cell suspension from tissueObligatory to prepare single-cell suspension from tissueDirect extraction of mRNA/DNA from tissue
      Cellular throughput (approximate maximum)25,000 events/second2,000 events/secondNot applicable
      Combined analysis of other markersUp to ±20Up to ±40Not possible
      Possible only when starting with sorted single cells or cloned cells.
      Dependent on availability of specific antibodiesYesYesNo
      Antigen specificity possibleUp to ±30 (combination of tetramers)Up to ±100 (combination of tetramers)No
      TCR repertoire informationLimited
      Availability of antibodies to TCR repertoire is incomplete.
      Limited
      Availability of antibodies to TCR repertoire is incomplete.
      Complete
      Further information about TCR sequence repertoireCells can be sorted and then subjected to HTSNo, cells are destroyed through the mass cytometry processCan identify and quantify the V(D)J genes and the nucleotide and amino acid sequences
      Abbreviations: HTS, high-throughput sequencing; TCR, T-cell receptor; V(D)J, variable, diversity, joining.
      1 Possible only when starting with sorted single cells or cloned cells.
      2 Availability of antibodies to TCR repertoire is incomplete.

      Terms to Describe TCR Repertoire Diversity

      The lexicon of terms applied to describe the diversity of T-cell clones in biological samples includes metric terms such as clonality, richness, and entropy.
      Clonality is a metric of relative abundance that allows evaluation of clonal expansion based on the probability of finding the same sequence in two different replicates. A higher clonality score reflects greater clonal expansion and can be attributed to expansion within the memory compartment, an uneven homeostatic proliferation of the naive T-cell repertoire, or may be indicative of a T-cell tumor. For example, cutaneous T-cell lymphoma (CTCL) samples commonly have a high clonality value, because there is a single pathogenic T-cell clone largely predominating in skin lesions (
      • Chitgopeker P.
      • Sahni D.
      T-cell receptor gene rearrangement detection in suspected cases of cutaneous T-cell lymphoma.
      ).
      Richness measures how many distinct T-cell clones (unique TCRs) are present in a sample. The diversity richness of the repertoire is strongly linked to a healthy immune system, ensuring continuous surveillance and response to unlimited foreign antigens, controlling for acute and chronic infections, and preventing mutant cells from unrestricted proliferation.
      Entropy provides a theoretic measurement of the probability of a certain clone being present within the total T-cell repertoire, combining information on abundance and richness. Entropy is the degree of uncertainty associated with identifying clones in the total repertoire based on the total number of clones, their identity, and their relative abundances.

      Applications of High-Throughput TCR Sequencing in Dermatology

      High-throughput TCR sequencing is increasingly becoming an important tool in the field of investigative dermatology, including monitoring the immune response to infectious diseases or vaccines, studying pathogenesis of T-cell–associated diseases, facilitating diagnosis and early recurrence detection, and assessing T-cell responses during therapies.

      Monitoring the immune response to infectious diseases or vaccines

      The exposure to pathogens or vaccines induces a specific adaptive immune reaction, which can be monitored by HTS. The study of the TCRs of all present T cells allows analyzing the efficacy of inducing persistent T-cell memory by tracking unique sequences that were initially present in the naive T-cell population but appear in the T-cell population with a memory phenotype after an immune response. Moreover, it allows tracking the memory T cells over time, unveiling whether the vaccine or infection provides long-lasting protection. This cumulative knowledge may allow researchers to identify T-cell clones that exclusively react to an antigen of interest, which might eventually be used as a biomarker of exposure or for early detection and prevention of disease spread.
      • Gaide O.
      • Emerson R.O.
      • Jiang X.
      • Gulati N.
      • Nizza S.
      • Desmarais C.
      • et al.
      Common clonal origin of central and resident memory T cells following skin immunization.
      used HTS of the TCR to clarify the clonal origin of both central memory T cells in lymph nodes and resident memory T (TRM) cells in peripheral tissues after skin vaccination. They noted that after skin immunization, a mutual native T-cell precursor gives rise to both antigen-reactive skin TRM cells and lymph node central memory T cells with overlapping TCR repertoires, hence creating memory T cells with different effector properties but with identical antigen specificity in distinct tissue compartments. In addition, TRM cells could rapidly generate a contact hypersensitivity response, whereas central memory T cells were responsible for a moderate and delayed response. These data indicate that TRM cells mediate allergic contact dermatitis, which explains the site-specificity, recurrence, and refractory characteristics of this disease. Similar approaches may help in the study of human diseases for which the clinical features resemble TRM-mediated diseases, including psoriasis, vitiligo, and fixed drug eruption.

      Studying pathogenesis of T-cell diseases

      Besides elucidating the pathogenesis of allergic contact dermatitis, HTS also allowed demonstration that allopurinol-induced severe cutaneous adverse reactions are driven by clonotype-specific T cells in a dose-dependent response to oxypurinol (
      • Chung W.H.
      • Pan R.Y.
      • Chu M.T.
      • Chin S.W.
      • Huang Y.L.
      • Wang W.C.
      • et al.
      Oxypurinol-specific T cells possess preferential TCR clonotypes and express granulysin in allopurinol-induced severe cutaneous adverse reactions.
      ). Studies of the TCR γ gene by HTS showed that CTCL is caused by mature memory T cells after undergoing normal thymic maturation and not by lymphoid progenitor cells or immature T cells (
      • Kirsch I.R.
      • Watanabe R.
      • O’Malley J.T.
      • Williamson D.W.
      • Scott L.L.
      • Elco C.P.
      • et al.
      TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.
      ). By means of a modified HTS technique,
      • Ruggiero E.
      • Nicolay J.P.
      • Fronza R.
      • Arens A.
      • Paruzynski A.
      • Nowrouzi A.
      • et al.
      High-resolution analysis of the human T-cell receptor repertoire.
      assessed the TCR α- and β-chain diversity in Sézary syndrome, and correlations between the restriction of the repertoire and clinical severity of CTCL skin involvement were found.

      Facilitating diagnosis and early recurrence detection

      High-throughput TCR sequencing has been shown to facilitate the diagnosis of CTCL. TCRγ PCR is currently the common diagnostic method of CTCL; however, it detects only the malignant T-cell clone in a subgroup of patients.
      • Kirsch I.R.
      • Watanabe R.
      • O’Malley J.T.
      • Williamson D.W.
      • Scott L.L.
      • Elco C.P.
      • et al.
      TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.
      showed that HTS of the TCR β and γ alleles is more sensitive and specific than TCRγ PCR in detecting the pathogenic expanded T-cell clone. HTS can function as an early definitive diagnostic tool for CTCL, and importantly, distinguish early stages of CTCL from benign inflammatory skin diseases (Figure 4).
      Figure 4
      Figure 4High-throughput TCRβ CDR3 region sequencing identifies expanded T-cell clones and discriminates CTCL from benign inflammatory skin disorders. (a) Clonality of lesional skin T cells increased with advanced stage of CTCL. (b, c) TCR sequencing identified expanded populations of clonal malignant T cells in CTCL skin lesions. (b) The V versus J gene usages of T cells from a lesional skin sample are shown. The green peak includes the clonal malignant T-cell population. (c) The individual T-cell clone sequence is shown with detailed information on the CDR3 amino acid sequence and V and J gene usage. The nine most frequent TCR sequences of benign infiltrating T cells are also shown. In this patient, the malignant T-cell clone made up 10.3% of the total T-cell population in lesional skin. (d, e) The most frequent T-cell clone expressed as the fraction of total nucleated cells successfully discriminates CTCL from benign inflammatory skin diseases. The most frequent TCR sequence expressed as a fraction of total nucleated cells is shown for (d) individual samples and (e) aggregate data. This analysis allowed discrimination of CTCL from benign inflammatory skin diseases and healthy skin. Reprinted from
      • Kirsch I.R.
      • Watanabe R.
      • O’Malley J.T.
      • Williamson D.W.
      • Scott L.L.
      • Elco C.P.
      • et al.
      TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.
      , with permission from The American Association for the Advancement of Science. ACD, allergic contact dermatitis; CDR3, complementarity determining region 3; CTCL, cutaneous T-cell lymphoma; ED, eczematous dermatitis; J, joining; MF, mycosis fungoides; Nml, normal; TCR, T-cell receptor; V, variable.

      Assessing immune response to therapy

      In a clinical trial testing topical resiquimod gel (an agonist for toll-like receptors 7 and 8) as a treatment for CTCL, it was noted that HTS of the TCR was more specific in assessing malignant T-cell clone clearance than clinical score evaluation (
      • Rook A.H.
      • Gelfand J.C.
      • Wysocka M.
      • Troxel A.B.
      • Benoit B.
      • Surber C.
      • et al.
      Topical resiquimod can induce disease regression and enhance T-cell effector functions in cutaneous T-cell lymphoma.
      ). HTS showed that the malignant T-cell clone was reduced in 90% of the treated patients and that malignant T-cell eradication was correlated with the recruitment and expansion of new benign T cells.

      Summary and Future Directions

      HTS of the TCR is a highly sensitive and precise technique that allows quantification of the relative frequency of each clone within the full T-cell repertoire. It enables concomitant study of each unique T cell and all clonal populations over time and comparison of different biologic tissues of the same individual or among different individuals who may be either healthy or suffering from a certain disease.
      HTS technology is based on the specific detection of the CDR3 region on just one of the two chains of the TCR heterodimer. An important next step forward would be establishment of technology that is able to simultaneously define both chains of the TCR. Currently, the most common way to define both peptide chains of the same TCR is through single-cell analysis. However, a recently developed method called pairSEQ seems to be able to accurately pair TCRα and TCRβ sequences out of hundreds of thousands of lymphocytes without the need for single-cell technologies (
      • Howie B.
      • Sherwood A.M.
      • Berkebile A.D.
      • Berka J.
      • Emerson R.O.
      • Williamson D.W.
      • et al.
      High-throughput pairing of T cell receptor α and β sequences.
      ). Once established, this development may significantly contribute to another highly anticipated goal: being able to link TCR sequences to their specific target antigenic peptides. These new methods will augment TCR datasets with complementary information on the target epitope recognized by each TCR and link these TCR sequences to lymphocytic phenotypic markers. In addition, these new methods build on the current applications of HTS as a monitoring tool for lymphoid malignancies and hematopoietic transplants and potentiate the identification and development of lymphocytes specific for tumor antigens or self-antigens for anticancer or autoimmune therapeutics, respectively.
      HTS is meaningfully expanding our understanding of the complex and exquisite role of T cells in the immune system and may lead us in groundbreaking personalized medicine by accurately monitoring and improving therapeutic interventions and possibly uncovering diagnostic and prognostic biomarkers.

      Conflict of Interest

      The authors state no conflict of interest.

      Summary Points

      What HTS of the TCR Does

      HTS of the TCR accurately identifies and quantifies each and every T cell present in a certain biological sample, allowing study of each unique clone and the full T-cell repertoire, including over time and among different tissues and/or individuals.

      Advantages of HTS of TCR

      • High clone detection sensitivity. Approximately 100-fold greater than other current technologies (e.g., flow cytometry).
      • Extremely accurate, with lower rate of false negatives and false positives than analysis of the TCR by PCR.
      • Capable of successfully studying any type of tissue and small samples, including standard-size punch biopsy or shave biopsy samples, because it is a cDNA- or DNA-based technology, and the amplification step enables the detection of scarce cells.
      • Technique does not require radioactive agents as in the previously commonly used Southern blotting.
      • Can be used for a wide range of applications, from expanding our knowledge of the adaptive immune system to monitoring responses to therapeutic interventions and studying new diagnostic and prognostic biomarkers.

      Limitations of HTS of TCR

      • Technology’s sensitivity is limited only by the amount of DNA probed. For example, if the DNA of a million cells is analyzed, then the clone detection sensitivity is about 1:1,000,000.
      • Variations in tissue processing can lead to DNA degradation. Section thickness and sample cell size may affect accurate amplification and representation of all gene segments. A certain gene may also be lost as a cell undergoes malignant transformation.
      • Pairing the α with β or γ with δ chains of a specific TCR is generally possible only when analyzing single cells.
      • It is generally still not possible to match the studied TCR sequences to their specific epitope.

      Multiple Choice Questions

      • 1.
        What does HTS of the TCR identify?
        • A.
          Identifies the DNA sequence of the entire TCR
        • B.
          Identifies the specific epitope of each TCR
        • C.
          Identifies the variable and constant region of each TCR
        • D.
          Identifies and quantifies each and every T cell present in a sample
      • 2.
        All of the following are advantages of HTS of the TCR, except the following:
        • A.
          Uses highly accurate radioactive agents
        • B.
          High clone detection sensitivity
        • C.
          Can be applied to any biologic tissue and small samples
        • D.
          Low rate of false positives and false negatives
      • 3.
        All of the following steps are part of the HTS of the TCR methodology, except the following:
        • A.
          Bias-controlled multiplexed PCR
        • B.
          Western blot
        • C.
          Advanced bioinformatics
        • D.
          HTS of the CDR3 of the TCR
      • 4.
        What does the clonality score measure?
        • A.
          The probability of a clone being present within the total cell repertoire
        • B.
          How much a sample is dominated by clonal expansion
        • C.
          How many distinct clones are present in a sample
        • D.
          How many T cells belong to each clonal population
      • 5.
        HTS can be used to study the following:
        • A.
          The immune response to infectious diseases or vaccine
        • B.
          Pathogenesis of T-cell–associated diseases
        • C.
          Diagnosis biomarkers and/or immune response to therapies.
        • D.
          All of the above

      Acknowledgments

      We would like to thank Dr. Jodi L. Johnson for helpful comments, critical reading of the manuscript, and editorial assistance, and Dr. Rachael A. Clark for the valuable scientific advice and guidance. This work was supported by Fondation René Touraine.

      Supplementary Material

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