What is TCR Sequencing

T cell receptor sequencing profiles the T cell repertoire. It reveals which T cells are present in a sample, how diverse the T cell response is and which clones have expanded in response to antigen.

What TCR sequencing is

TCR sequencing reads the T cell receptor genes from T cells. Every T cell expresses a unique receptor on its surface. By sequencing these genes across thousands or millions of T cells, you build a catalog of the T cell receptors present in a sample.

This catalog is called the T cell repertoire. It is a snapshot of the adaptive cellular immune system at a specific moment. The repertoire changes over time in response to infection, vaccination, cancer and autoimmune disease.

TCR sequencing is also called:

• TCR-seq
• Rep-seq (repertoire sequencing, shared term with BCR sequencing)
• Immune repertoire sequencing (when combined with BCR sequencing)

Why it matters

T cells are the adaptive immune system’s main weapon against intracellular threats. They recognise and kill virus-infected cells, control intracellular bacteria and eliminate cancer cells. While antibodies bind free-floating pathogens, T cells survey the surfaces of other cells for signs of infection or malignancy.

Traditional assays provide limited views of T cell immunity. ELISPOT measures T cells responding to a handful of known peptides. Flow cytometry counts T cell populations by surface markers. Neither reveals the full sequence-level diversity of the T cell repertoire.

TCR sequencing fills this gap. It provides single-sequence resolution of the entire T cell receptor pool in a sample.

TCR structure

The T cell receptor is a heterodimer. Most T cells in the blood express an alpha chain and a beta chain. This is the alpha-beta TCR. A smaller population of T cells expresses a gamma chain and a delta chain. This is the gamma-delta TCR.

Each chain has a variable region and a constant region. The variable region determines what the TCR binds. It contains three loops called complementarity-determining regions (CDR1, CDR2, CDR3). Among these, CDR3 of the beta chain is the most variable and the primary determinant of antigen specificity.

Unlike antibodies, the TCR does not exist in a secreted form. It is always membrane-bound. The TCR also has only one binding site, while antibodies have two.

Chain pair	Frequency	Primary location
Alpha-beta	~95% of blood T cells	Blood, lymph nodes, tissues
Gamma-delta	~5% of blood T cells	Epithelial surfaces, gut, skin

Most TCR sequencing studies focus on the alpha-beta TCR. The beta chain is sequenced most often because it has higher diversity than the alpha chain and is technically simpler to sequence.

V(D)J recombination

Like the BCR, TCR diversity comes from V(D)J recombination. This process assembles the variable region gene from smaller gene segments during T cell development in the thymus.

The beta chain is assembled from three types of gene segments:

• V (variable): ~48 functional genes in humans (TRBV)
• D (diversity): 2 functional genes (TRBD)
• J (joining): 13 functional genes (TRBJ)

One V, one D and one J segment are randomly selected and joined together. The alpha chain uses only V and J segments, similar to the antibody light chain.

Junctional diversity adds variation at each junction. The recombination machinery deletes a few nucleotides and inserts random N-nucleotides at the V-D and D-J junctions. This makes the CDR3 region essentially random in sequence.

The theoretical diversity of the TCR repertoire exceeds 10^15. This is even greater than the antibody repertoire because TCR diversity relies almost entirely on V(D)J recombination rather than somatic hypermutation.

No somatic hypermutation

This is a key difference between BCR and TCR biology. T cell receptors do not undergo somatic hypermutation. Once a TCR is assembled in the thymus, its sequence is fixed for the lifetime of the T cell.

Antibodies improve their binding over time through rounds of mutation and selection in germinal centres. T cells cannot do this. The TCR you sequence is the same TCR the cell has always carried.

This has practical consequences for analysis. In BCR sequencing, mutation frequency is a key metric that reveals affinity maturation. In TCR sequencing, mutation analysis is not relevant. If you see many mutations in a TCR sequence relative to the germline, it is likely a sequencing error or a misassignment, not a biological process.

Thymic selection

Because TCRs cannot improve after assembly, the thymus performs strict quality control during T cell development.

Positive selection ensures the TCR can bind MHC molecules at all. T cells whose TCR cannot recognise MHC die by neglect. This guarantees that every mature T cell can survey peptide-MHC complexes.

Negative selection eliminates T cells whose TCR binds self-peptides too strongly. These would become autoreactive and attack the body’s own tissues. T cells that fail negative selection are deleted.

Only about 2% of developing T cells survive both positive and negative selection. The mature repertoire is shaped by this bottleneck.

What TCRs recognise

TCRs do not bind free antigen. They recognise short peptide fragments displayed on the surface of cells by major histocompatibility complex (MHC) molecules.

The process works like this:

1. A cell degrades intracellular proteins into short peptides.
2. MHC molecules bind these peptides and display them on the cell surface.
3. The TCR scans the peptide-MHC complex. If it recognises the peptide as foreign, the T cell activates.

There are two classes of MHC molecules:

MHC class	Presents peptides from	Recognised by
MHC class I (HLA-A, B, C)	Intracellular proteins	CD8+ cytotoxic T cells
MHC class II (HLA-DR, DP, DQ)	Extracellular proteins	CD4+ helper T cells

This means TCR specificity depends on both the peptide and the MHC molecule presenting it. The same peptide presented by different MHC alleles may be recognised by completely different TCRs. This is called MHC restriction.

Clonotypes

T cells descended from the same V(D)J recombination event share the same TCR sequence. This group is called a clonotype. In practice, a clonotype is defined by its CDR3 amino acid or nucleotide sequence along with the V and J gene assignments.

Clonal expansion occurs when a T cell recognises its antigen and proliferates. An expanded clone can grow from a single cell to millions of cells. In sequencing data, expanded clones appear as many sequences with identical or near-identical CDR3 regions.

Key clonal metrics include:

• Clonality: the fraction of the repertoire occupied by the top clones. High clonality means a few clones dominate.
• Diversity: Shannon or Simpson indices applied to clone sizes. Low diversity can indicate an antigen-driven response, chronic infection or malignancy.
• Public clonotypes: identical CDR3 sequences found across unrelated individuals. These arise from convergent V(D)J recombination and often target common pathogens like CMV, EBV and influenza.

Applications

TCR sequencing is used across immunology, oncology and clinical diagnostics.

Cancer immunology. Characterise tumour-infiltrating lymphocytes (TILs). Expanded TIL clonotypes suggest active anti-tumour immunity. Track clonal dynamics during immunotherapy with checkpoint inhibitors. Identify neoantigen-reactive T cells.

Infectious disease. Profile the T cell response to SARS-CoV-2, HIV, CMV, EBV and tuberculosis. Identify public TCR sequences shared across patients with the same infection. Monitor T cell memory after vaccination.

Autoimmune disease. Identify autoreactive T cell clones in type 1 diabetes, multiple sclerosis, rheumatoid arthritis and coeliac disease. Track pathogenic clones across disease flares.

Transplant monitoring. Detect alloreactive T cell clones that mediate graft rejection or graft-versus-host disease. Monitor donor-reactive repertoire changes after transplantation.

Minimal residual disease. In T cell lymphomas and leukaemias, track the malignant clone by its TCR sequence. TCR sequencing can detect one malignant cell among a million normal cells, far more sensitive than flow cytometry or morphology.

Antigen specificity prediction. Computational tools can predict what antigen a TCR recognises based on its CDR3 sequence. This is unique to TCR analysis and is covered in detail on the Repertoire Analysis page.

Summary

• TCR sequencing catalogues the T cell receptors present in a sample at single-sequence resolution.
• V(D)J recombination generates receptor diversity. Unlike antibodies, TCRs do not undergo somatic hypermutation.
• The CDR3 region of the beta chain is the most variable part and the primary determinant of antigen specificity.
• TCRs recognise peptide-MHC complexes, not free antigen. Specificity depends on both the peptide and the MHC allele.
• Clonal expansion reveals which T cells are actively responding to antigen.
• Applications span cancer immunology, infectious disease, autoimmunity, transplant monitoring and antigen specificity prediction.