Adaptive immunity relies on B and T cells that recognize foreign antigens via hypervariable B cell and T cell receptors (BCRs and TCRs). Diversity among B cell and T cell receptors is primarily produced by V(D)J recombination, which involves the shuffling and joining of the variable (V), diversity (D), joining (J), and constant region (C) gene segments. This results in a diverse repertoire called the adaptive immune repertoire (AIR) that comprises multiple individual clonotypes (sequence) for particular receptor chains.
Adaptive Immune Repertoire (AIR) Profiling is essential for designing effective therapeutic strategies for immune-mediated diseases. For example, tracking TCR/BCR diversity in patients across time points can reveal clonal dynamics that correlate with treatment response or other clinically relevant features. Additionally, pairing the clonal information with cellular phenotype can identify disease-relevant cell types that can be insightful for disease prognosis and to develop targeted immunotherapy strategies.
Although both genomic DNA and RNA can be used for AIR Profiling, the copy number of mRNA per cell is at least 10- to 100-fold more than gDNA. The higher number of starting RNA template copies has been shown to significantly increase the detection level of T-cell receptors . Also, the clonotypes express receptor mRNA at different levels, and activation of adaptive immunity induces significant up-regulation of TCR and BCR transcription in antigen-specific clonotypes (e.g., up to 1,000-fold for plasma B cells) . As a result, RNA-based immune receptor repertoire is usually dominated by a low number of abundant clonotypes and many of these abundant clonotypes could be antigen-induced or disease-specific.
Another benefit of using mRNA over genomic DNA starting material is that using RNA as a template limits amplification to only “functional” expressed receptor chains rather than non-functional genes–such as pseudogenes and ORFs–which reduces background in the NGS data. Moreover, repertoire analysis using genomic DNA as a template doesn’t identify the Ig isotype since the V(D)J and C regions on this isotype are separated from each other by an intron that prevents them from being amplified together by PCR.
To assess the difference between Immune Repertoire profiling of RNA vs. genomic DNA samples, we compared profiles from the same sorted T or B cells (1K-50K) from tumor metastatic biopsy samples generated using our DriverMap AIR assay, which employs mRNA as the template, with Adaptive Technology’s genomic DNA-based assay. After NGS analysis, we found that the DriverMap AIR assay from mRNA detects 1.5-2x more TCR/BCR Clonotypes than gDNA from Adaptive Technology. (Figure 1)
Figure 1: Number of clonotypes detected from gDNA vs mRNA from 27 patient tumor samples
Do you have immune cell samples that you are interested in profiling? Cellecta is actively seeking collaborators to test our DriverMap Adaptive Immune Repertoire Profiling (AIR) assay. Find out more here.
1. Pai, J.A., and Satpathy, A.T. High-throughput and single-cell T cell receptor sequencing technologies. Nat. Methods (2021) 18: 881-892.
2. Minervina, A., Pogorely, M., and Mamedov, I. T-cell receptor and B-cell receptor repertoire profiling in adaptive immunity. Transplant Intern. (2019) 32: 1111-1123.