2.1. Combined TCR and BCR Analysis

In most clinical responses, T and B cells work synergistically in the adaptive immune response [13,18]. Therefore, for comprehensive analysis of the adaptive immune response and unbiased discovery of antigen-induced immunity receptors, we recommend profiling the repertoire of both TCR (TRA, TRB, TRD, TRG) and BCR (IGH, IGK, and IGL) genes simultaneously.

Hence, the AIR RNA assay is designed to profile all TCR and BCR isoforms in a single reaction without compromising sensitivity for robust repertoire detection. It is important to note that TRA and TRB clonotypes have similar complexity and representation at the RNA level in blood samples, which allows them to be profiled together, as illustrated in Fig.8. TRD and TRG clonotypes have significantly lower complexity but can easily be detected together with other TCR and BCR chains. In studies designed for immune receptor repertoire analysis of only T or B cells or in the samples with significant (e.g., 5-10-fold) differences in T/B content, Cellecta also offers primer sets to amplify only the TCR or BCR chains in separate reactions.

Fig 8. Number of clonotypes for 7 TCR/BCR chains identified in 50 ng of normal PBMC or whole blood RNA using the AIR RNA assay. 10x10^6 reads per sample, each sample run in triplicates.

Similarly, in the AIR DNA assay, we recommend amplifying the CDR3 regions separately for TCR and BCR, considering the significant differences (e.g., 4-5-fold in the blood) in the abundance of T and B cells in many biological samples. Furthermore, the number of clonotypes identified for TRA, IGK, and TRG are usually higher than for other matched chains, as illustrated in Fig.9. One of the reasons for these differences is that a significant fraction of lymphocytes have two rearranged TRA, IGK, and TRG genes wherein one copy is non-productive and silenced or expressed at a very low level [20-22]. An extreme case is seen with TRG rearranged genes which are present not only in gamma-delta T cells but also present but silenced in alpha-beta T cells [22].

Fig 9A. AIR DNA repertoire analysis in all three BCR chains (IGH, IGK and IGL). PBMC gDNA in triplicates, 5x10^6 reads per sample.

Fig 9B. . AIR DNA repertoire analysis of four TCR chains (TRB, TRA, TRD and TRG) .PBMC gDNA in triplicates, 5x10^6 reads per sample.