Immune monitoring technology primer: immunosequencing
© Kirsch. 2015
Received: 6 June 2015
Accepted: 11 June 2015
Published: 25 June 2015
Profiling of the immune receptor repertoire is becoming increasingly relevant to the understanding and clinical management of cancer, autoimmunity, aging, and infectious disease.
A platform technology is described that provides comprehensive immune receptor profiling.
Immunosequencing is a platform technology that allows the enumeration, specification and quantification of each and every B-and/or T-cell in any biologic sample of interest. It is based on bias-controlled multiplex PCR and high throughput sequencing and is highly accurate, standardized, and sensitive.
Description of the technology
The adaptive immune system generates a remarkable breadth of diversity in antigen-specific TCRs and Igs by combinatorial recombination of gene segments in lymphocytes. The TCR is composed of two peptide chains, encoded by the TCRA and TCRB or TCRG and TCRD genes, respectively. There are thus two types of T-cell receptors, αβ and γδ, that differ by the TCR heterodimer type and immune function. The antigenic specificity of T-cells is in large part determined by the amino acid sequence in the hypervariable complementarity-determining region 3 (CDR3) regions of the T-cell receptors. The existence of multiple V and J gene segments at these T-cell loci permits a large combinatorial diversity in receptor composition; and the non-templated insertion or deletion of nucleotides at the V-J, V-D, and D-J junctions further adds to the potential diversity of receptors that can be encoded. Because of the potential diversity of receptors it is highly improbable to randomly converge on the same TCRB nucleotide CDR3 sequence, effectively making each CDR3 sequence a unique tag for a T-cell clone.
Type of data obtained/readout
For each sample, DNA is extracted and the relevant immune receptor CDR3 regions amplified and sequenced. Bias-controlled V and J gene primers are used to amplify rearranged V(D)J segments for high throughput sequencing at ~10 × coverage. Raw sequence data is uploaded and after correcting sequencing errors via a clustering algorithm, primary nucleotide sequence of the amplified regions from the immune receptors’ unique CDR3 segments are identified, quantified, and annotated according to the International ImMunoGeneTics collaboration, identifying which V, D, and J genes contributed to each rearrangement.
Limitations of the approach
In isolation, immunosequencing only provides information relevant to the enumeration, specification, and quantification of each and every B-and/or T-cell in a given sample of interest. It can be combined with other technologies focused on immunophenotypic characterization or single cell isolation to provide more comprehensive information on the physiologic and developmental status of the individual cells or populations.
Types of samples needed and special issues pertaining to samples
The assay is capable of providing information from fresh, frozen, or FFPE samples and has been tested on blood, bone marrow, lymph node, spleen, kidney, liver, lung, brain, ovary, skin, cell-free DNA, primary and metastatic tumor and other tissues.
Level of evidence
Approximately 100 manuscripts and poster presentations have been published using this platform. The analytic validity, clinical validity, and clinical utility is presented in many of these publications (see for example, refs [1–7]). The process is conducted in CLIA and CAP certified laboratories. An RUO kit is also available.
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