What is a T-cell receptors (TCRs)?

T-cell receptors (TCRs) are heterodimeric proteins expressed on the surface of all T cells. In humans, the majority (95%) of TCRs consist of an alpha and beta chain (αβ TCRs), while the remaining 5% are composed of a gamma and delta chain (γδ TCRs).

T-cell receptors recognize antigens presented by major histocompatibility complex (MHC) molecules, which are essential for T-cell activation and the subsequent immune response. The sequence diversity of TCRs, generated through somatic recombination, enables the recognition of a wide array of antigens, increasing the likelihood of mounting an effective immune response against pathogens. TCRs specifically recognize short peptides bound within the binding groove of MHC molecules. In TCR-T cell therapies for cancer, TCRs are engineered to recognize tumor-specific antigens. These therapies introduce TCR-coding genes into ordinary T cells to endow them with enhanced tumor-specific killing capabilities.

Why sequence TCRs?

TCR sequencing is essential for understanding the biology of T-cell receptors, enabling the characterization of TCR repertoire diversity and clonality. TCR sequencing is critical in immunotherapy by identifying optimal target antigens and tumor antigen-specific TCRs. Additionally, TCR sequencing is used to assess various immunotherapies’ efficacy and predict patient outcomes and responses.

The role of TCR sequencing in immunotherapy

Sequencing TCRs from tumor-infiltrating lymphocytes allows for identifying those recognizing tumor-specific neoantigens, mutated proteins expressed by cancer cells. Genetically modified T cells can be engineered to express novel TCRs selected for their ability to elicit strong tumor-specific immune responses against targeted neoantigens.
Tracking TCR clones over time is crucial to understanding the persistence and expansion of T-cell populations post-administration. This monitoring helps identify which TCRs contribute to tumor regression and assess the efficacy of immunotherapy. A diverse TCR repertoire is often associated with better therapeutic outcomes.
TCR sequencing also enables monitoring changes in T-cell clonality, which can reflect tumor evolution and immune escape mechanisms. A decrease in T-cell clonality may indicate tumor progression and signal the need for therapeutic adjustments.
Finally, TCR repertoire metrics derived from sequencing data can predict patient outcomes and responses to immunotherapy, guiding the development of personalized treatment plans.

TCR profiling

RNA TCR profiling
We utilize Cellecta’s DriverMap™ Adaptive Immune Receptor (AIR) TCR-BCR Profiling Kit, which combines multiplexed reverse transcription polymerase chain reaction (RT-PCR) for RNA amplification with next-generation sequencing (NGS) to analyze the full-length variable regions of TCR chains, including complementarity-determining regions 1, 2, and 3 (CDR1, CDR2, and CDR3).

For RNA profiling, the process focuses on expressed TCR transcripts. Total RNA is isolated from whole blood, PBMCs, or tissues. Gene-specific primers with unique molecular identifiers (UMIs) target all TCR and BCR constant region isoforms to synthesize cDNA.
The cDNA undergoes two rounds of PCR amplification:

• The first round amplifies the CDR regions.
• The second round incorporates indexed primers to prepare the library for sequencing.

The amplified, indexed libraries are sequenced on an Illumina platform with sufficient depth to detect clonal populations. Sequencing data is processed to identify and quantify TCR clonotypes.

DNA TCR profiling
Cellecta’s DriverMap™ Adaptive Immune Receptor DNA (AIR-DNA) TCR and BCR Kits are designed to profile the genomic diversity of T-cell receptors (TCRs) and B-cell receptors (BCRs) from various sample types, including whole blood, tissue biopsies, and formalin-fixed paraffin-embedded (FFPE) samples. These kits use a multiplex PCR-based approach followed by NGS to analyze the full-length variable regions of TCR and BCR chains, providing comprehensive insights into the immune receptor repertoire.
DNA is extracted and amplified using gene-specific primers targeting the variable regions of the TCR and BCR chains. UMIs are introduced to enhance accuracy. Sequencing adapters and sample-specific indexes are then added. The sequencing is performed on an Illumina platform with sufficient coverage to assess the diversity of the immune receptor repertoire.

Conclusion
Sequencing data enables tracking changes in TCR repertoires in response to infections, vaccinations, immunotherapies, and graft acceptance or rejection after transplantation. It also enhances understanding of anti-tumor immune responses and aids in identifying potential biomarkers through the analysis of tumor-infiltrating lymphocytes. TCR sequencing is a vital tool in drug development, particularly for immunotherapies.