5. T/B Immune Marker Panel

Immunophenotyping of CDR3 clonotypes with T/B Immune Marker Assay

In addition to AIR RNA profiling, sorted immune cell subfractions can be phenotypically characterized by an additional targeted multiplex RT-PCR DriverMap T/B Immune Marker assay. To develop the T/B Immune Marker assay, we combined a list of more than 3,000 T and B-specific cell subtyping and activation markers from more than 100 different public databases, commercial assays, and scientific publications. We also designed several sets of redundant primers and validated the performance of these primers against a panel of 40 different immune cell types and activated PBMC and whole blood samples. As a result of these validation studies, we arrived at approximately 500 highly expressed T/B subtyping and activation marker genes that confirm the quality of cell sorting and could phenotypically characterize different immune cell subtypes.

In addition to this, we also offer the DriverMap EXP assay, a genome-wide expression profiling assay to measure the expression levels of 19,000 human protein-coding genes. If the research goal is the unbiased discovery of novel immunity biomarkers, we recommend using a genome-wide expression profiling panel.

Combining AIR profiling with the T/B Immune Marker assay in sorted immune cells will link specific CDR3 sequences with the comprehensive phenotype of different immune subfractions (e.g., CD4. CD8, naïve, memory, cytotoxic, effector, etc.) as illustrated in Fig. 18. When performing a combined AIR assay and T/B Immune Marker assay, we recommend splitting each sorted cell fraction in a 5:1 ratio (e.g., 25,000 and 5,000 cells) and mixing amplified samples at a 1:1 ratio at the NGS step to account for the differences in the complexity of CDR3 sequences and genes in the T/B Immune Marker Panel.

Fig. 18. Characterization of different T and B cell types (T, CD8, CD8 naive, CD8 memory, CD4, CD4 naive, CD4 memory, Treg, NK, B, B naive, B memory) with DriverMap T/B Immune Marker Panel