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)
- •Recognize the newest techniques in biomedical research.
- •Describe how these techniques can be utilized and their limitations.
- •Describe the potential impact of these techniques.
Overview of Methodology
Variants of PCR and Other Techniques to Study the TCR
|Type of Information||Flow Cytometry||Mass Cytometry||HTS of the TCR|
|Tissue sample preparation||Obligatory to prepare single-cell suspension from tissue||Obligatory to prepare single-cell suspension from tissue||Direct extraction of mRNA/DNA from tissue|
|Cellular throughput (approximate maximum)||25,000 events/second||2,000 events/second||Not applicable|
|Combined analysis of other markers||Up to ±20||Up to ±40||Not possible|
|Dependent on availability of specific antibodies||Yes||Yes||No|
|Antigen specificity possible||Up to ±30 (combination of tetramers)||Up to ±100 (combination of tetramers)||No|
|TCR repertoire information||Limited|
|Further information about TCR sequence repertoire||Cells can be sorted and then subjected to HTS||No, cells are destroyed through the mass cytometry process||Can identify and quantify the V(D)J genes and the nucleotide and amino acid sequences|
Terms to Describe TCR Repertoire Diversity
Applications of High-Throughput TCR Sequencing in Dermatology
Monitoring the immune response to infectious diseases or vaccines
Studying pathogenesis of T-cell diseases
Facilitating diagnosis and early recurrence detection
Assessing immune response to therapy
Summary and Future Directions
Conflict of Interest
What HTS of the TCR Does
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
- Quiz and brief explanation of correct answers
- Teaching Slides
- T-cell receptor gene rearrangement detection in suspected cases of cutaneous T-cell lymphoma.J Invest Dermatol. 2014; 134: e19
- Oxypurinol-specific T cells possess preferential TCR clonotypes and express granulysin in allopurinol-induced severe cutaneous adverse reactions.J Invest Dermatol. 2015; 135: 2237-2248
- Comprehensive genomic access to vector integration in clinical gene therapy.Nat Med. 2009; 15: 1431-1436
- Common clonal origin of central and resident memory T cells following skin immunization.Nat Med. 2015; 21: 647-653
- High-throughput pairing of T cell receptor α and β sequences.Sci Transl Med. 2015; 7: 301ra131
- TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.Sci Transl Med. 2015; 7: 308ra158
- IMGT, the international ImMunoGeneTics information system.Nucleic Acid Res. 2009; 37: D1006-D1012
- Janeway’s immunobiology, 8th ed.Science, New York: Garland2011
- Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells.Blood. 2009; 114: 4099-4107
- Topical resiquimod can induce disease regression and enhance T-cell effector functions in cutaneous T-cell lymphoma.Blood. 2015; 126: 1452-1461
- High-resolution analysis of the human T-cell receptor repertoire.Nat Commun. 2015; 6: 8081
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