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DriverMap Adaptive Immune Receptor (AIR) TCR-BCR Profiling

Cellecta's DriverMap™ Adaptive Immune Receptor (AIR) Profiling Assay is the only assay technology on the market for bulk and single-cell T-cell receptor (TCR) and B-cell receptor (BCR) using multiplex RT-PCR and next-generation sequencing (NGS) protocols without the need for any additional specialized equipment.

Cellecta offers bulk DriverMap AIR assays to profile human or mouse RNA, DNA, or both, looking at either full-length receptor regions or only the CDR3 region of all TCR chains--TRA, TRB, TRG, and TRD--and BCR chains--IGH, IGK, and IGL. Additionally, the simultaneous profiling of DNA and RNA from the same sample enables the identification of antigen-activated clonotypes to provide even greater insight into the immune response.

The DriverMap single-cell AIR (scAIR) assays use primers designed to enable full-length receptor sequencing to obtain paired-chain information from individually flow-sorted or diluted T or B cells. This easy-to-use, 96-well plate-based RT-PCR workflow is available for human, mouse, or hybrid human-mouse samples.

How does the DriverMap AIR assay work?

  • Start from bulk human or mouse RNA or DNA from a variety of immune sample types, such as whole blood, PBMCs, cancer biopsies, tissue samples, FFPE or dried blood microsamples to obtain information on clonotype repertoire
  • Start by introducing flow-sorted individual T or B cells or diluted (pipetted) T or B cells into a 96-well plate to run the single-cell AIR assay
  • Detect and accurately quantify AIR clonotypes with the multiplex PCR-based assay, using primer-incorporated, proprietary validator barcodes (VBCs).
  • Obtain more sensitive and reproducibleprofiling data.

    • BULK AIR Repertoire Profiling

      Since T- and B-cells work synergistically in the adaptive immune response, we have designed an assay that profiles both T-cell receptor (TCR) and B-cell receptor (BCR) repertoires in a single convenient reaction. Separate assays specific for T- or B-cell chains are also available.

      The DriverMap AIR-RNA assay (bulk) quantifies T-cell and B-cell receptor transcripts. Assaying expression of the immune receptors from either the CDR3 OR the full-length receptor region enables highly sensitive detection of low-frequency, rare TCR and BCR clonotypes, and more comprehensive profiling when working with small samples and limited numbers of cells. Fig. 1 shows that the DriverMap AIR-RNA assay can detect all seven TCR and BCR chains from a single sample of RNA.

      The DriverMap AIR-DNA assay (bulk) amplifies receptor genes directly from genomic DNA. The AIR-DNA assay provides a more quantitative measurement of the genetic copies for each CDR3-specific clonotype which correlates to the number of cells with that clonotype in the sample. This data enables the measurement of clonal expansion in T and B cells.

      Combining data obtained from both the AIR-DNA and AIR-RNA assays enables the assessment of both transcriptional activation and number of cells with a particular clonotype. The ability to differentiate these two effects provides a quantitative basis for detecting antigen-activated clonotypes. For example, it is evident from the data in Fig. 2, that particular TRB clonotypes, even when present in only a small portion of the cells in a population, can be highly up-regulated.

      SINGLE-CELL TCR / BCR Profiling

      The DriverMap scAIR assayfills a critical gap in immune profiling by providing the full-length, chain-pair sequences for T-cell or B-cell receptors. This easy-to-use, multiplex PCR-based workflow does not require RNA extraction, a microfluidics platform, or complex barcoding protocols. The researcher simply sorts individual cells or introduces diluted cells into a 96-well plate, performs the hybridization step, and then pools all the wells into a single reaction tube for the subsequent NGS library preparation steps.

      All 7 TCR and BCR Chains Identified from a Single Sample of RNA DriverMap™ AIR TCR-BCR Profiling Kit (Human RNA)g - Kits

      Fig 1: Number of clonotypes for 7 TCR/BCR chains identified in 50 ng of normal PBMC or whole blood RNA (10x10^6 reads per sample, triplicates).

      Detection of Cancer-Activated CDR3 Clones Cellecta_DriverMap_Assay

      Fig. 2: The red intensity of each line in the paired heat maps shows the detection level of each TCR or BCR clonotype in each sample with the DriverMap AIR-RNA (R) and a competitor DNA (D) assay. By comparing the normalized level of clonotype expression as measured by the AIR-RNA assay with the frequency of each clonotype in the population as measured by the DNA-based assay, it is evident that 5-10% of the clones (indicated by green brackets) are strongly up-regulated even when they are only weakly detected at the DNA level, indicating they are not highly represented in the population.

       

    • How is the DriverMap AIR Assay Different from other Adaptive Immune Receptor Repertoire (AIRR) Assays?

      • DriverMap™ Multiplex PCR technology uses gene-specific primers which significantly reduce the level of non-specific binding and primer-dimer amplification products, and are designed to target only TCR/BCR isoforms.
      • Validator barcodes (VBCs) facilitate accurate quantitation of the copy number of cDNA or DNA molecules in amplification steps, as well as detection of low abundance clonotypes and correction of amplification biases and sequencing errors.
      • Dual-index amplicon labeling strategy minimizes index hopping during NGS allowing for comprehensive readouts.
      • Full profiles of the antigen-recognition CDR3 region enable assessment of CDR3 length distribution, V(D)J segment usage, isotype composition for BCRs, somatic mutations, and similar characteristics with immune receptor profiling software such as MiXCR (MiLaboratories Inc.).
      • The Single-cell AIR assay workflow and analysis are economical, starting with costs starting at US$ 0.10 per cell, not requiring use of a microfluidics platform, complex barcoding protocols, or expensive reagents.

       

      DriverMap™ Adaptive Immune Receptor (AIR) TCR-BCR Profiling - Manual

    • Applications of BCR sequencing (BCR-Seq)

      • Identify broadly neutralizing antibodies (BNAbs) and map Ig-seq datasets to known antibody structures for antibody and vaccine development.
      • Track B-cell migration and development patterns
      • Find markers of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and cancers (e.g. B-cell lymphoma)
      • Contrast naïve and antigenically challenged datasets to understand antibody maturation

      Applications of TCR sequencing (TCR-Seq)

      • Track T-cell clonality and diversity for insights into mechanisms of action of immune checkpoint inhibitors for immunotherapies
      • Assess TCR overlap between repertoires to define spatial and temporal heterogeneity of the anti-tumoral immune response
      • Analyze TCR sequence and structure to annotate antigenic specificity for developing personalized cellular immunotherapies

       

      Applications of single-cell TCR or BCR sequencing (scTCR-Seq or scBCR-Seq)

      • Characterizing T-cell or B-cell chain pairs and immunophenotypes will aid in the discovery of neo-antigen biomarkers and novel epitopes
      • Identifying antigen-specific clonotypesand their functional profiles can guide the design and evaluation of vaccines and immunotherapies, i.e., checkpoint inhibitors and CAR-T cells
      • High-throughput screening will be useful in the development of humanized antibodies and for TCR validation studies

       

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