2.2. RNA vs. DNA Template

Both genomic DNA (gDNA) and mRNA are commonly used as starting material for immune receptor profiling, and each has advantages and disadvantages. Cellecta offers both an RNA-based and a DNA-based AIR assay. Based on our experience, the AIR RNA assay has a significant advantage over the AIR DNA assay for the following reasons:

• Sensitivity: The copy number of mRNA per cell is at least 10 to 100-fold greater than gDNA, significantly increasing sensitivity due to the higher number of starting RNA template copies [4]. The increased AIR sensitivity from using RNA templates is an essential advantage for reliable profiling of TCR and BCR clonotypes in small biological samples (e.g., sorted cells, tumors biopsy, FFPE, etc.) (Fig. 10).

• Functionality: Using RNA as a template limits amplification to only the receptor chains which are functional and productively expressed. AIR profiling from gDNA amplifies many additional sequences, including non-rearranged and rearranged but non-functional (usually expressed at low level) receptor genes, which increases undesirable background in the NGS data, especially in tissue samples with low immune cell content.

• Coverage: BCR repertoire analysis using gDNA as a template does not allow identification of the Ig isotype, as V(D)J and C regions are separated by an intron and cannot be effectively amplified by a multiplex-PCR reaction.

The drawbacks of using RNA include requiring reasonable RNA template integrity (RIN > 5). Furthermore, if your research needs to accurately quantify the T and B cell number for each clonotype, a gDNA-based assay based on UMI (Cellecta AIR DNA assay) or in combination with calibration standards is a better choice. Please note that precise quantitation of cell number for each CDR3 clonotype (based on receptor gene copy number) is not critical if the goal is the detection of “up-regulated” disease-associated, activated clonotypes (similar to differential gene-expression analysis), e.g., in serial / time course studies in blood samples.

Identification of disease-activated CDR3 clonotypes

The most common strategy for discovering antigen-induced clonotypes is AIR repertoire profiling in multiple samples collected in serial/time course studies of infection, immunization, immune therapy treatment, etc. [1-5]. Antigen-activated clonotypes are detected by an increase in the copy number of RNA or DNA CDR3-specific sequences compared to control non-activated samples.
In another application, the identification of overlapping, transcriptionally activated CDR3 clonotypes in the tumor, blood, or tumor-draining lymph nodes can be used for identifying tumor-reactive T cells [24]. Unfortunately, for many human diseases, only a single sample (e.g., a heterogeneous tumor biopsy) is available for AIR profiling.

Studies demonstrate that adaptive immunity activation induces significant up-regulation of both BCR and TCR transcription in antigen-specific clonotypes (e.g., up to 1,000-fold for plasma B cells [13]) as illustrated in Fig.10. Furthermore, the transcriptional up-regulation of TCR genes in large CD8+ effector memory clonotypes in peripheral blood samples was associated with durable responses to immune checkpoint blockade in patients with metastatic melanoma [23].

As a result of transcriptional activation, an RNA-based immune receptor repertoire is usually dominated by a low number of abundant clonotypes, some of which are antigen-induced or disease-specific. Up-regulation of TCR and BCR receptor gene expression in activated effector/memory T and B cells can identify and differentiate antigen-induced CDR3 sequences from non-activated, bystander CDR3 sequences commonly present in tumor samples [20].

Fig 10. Expression of TCR and BCR genes in naive and activated T and B cells. Normalization of RNA to DNA copy number allows effective discrimination of antigen-activated vs naive cells.

Combining mRNA and gDNA-based assay technology

A simple and efficient strategy to differentiate antigen-induced clonotypes from abundant naive or bystander clonotypes commonly present in clinical samples is to normalize RNA-based CDR3 profiling to similar data from a DNA-based assay. To facilitate this approach, we developed UMI-based AIR RNA and AIR DNA assays, which can be combined to identify antigen-activated clonotypes.
As a practical guideline, we suggest that AIR profiling be performed using both RNA and DNA isolated from the same biological sample (e.g., PBMC with high content of immune cells). In another approach, RNA-based and DNA-based AIR profiling could be performed directly in T and/or B cells sorted from tissue or blood samples (see below). Furthermore, to reveal disease-associated, activated CDR3 clonotypes, RNA-based data can be normalized to DNA-based AIR profiling data for overlapping clonotypes, as illustrated in Fig.11.

Fig 11. Comparison of RNA-based to DNA-based immune receptor repertoire profiling data from RNA and DNA purified from the T and B cells sorted from metastatic brain tumor samples [20]. Results show the presence of 5-10% tumor-activated immune receptor clones (marked by brackets) that can be activated by up to 1000-fold (B cells) and to 50-fold (T cells) by transcriptional activation of immune receptor genes.