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Breakthrough Technology Characterizes Immune Response

By LabMedica International staff writers
Posted on 16 Nov 2015
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Image: Colored scanning electron micrograph of T-lymphocytes (pink) that recognize antigens on a tumor cell (yellow) through T-cell receptors (Photo courtesy of Steve Gschmeissner).
Image: Colored scanning electron micrograph of T-lymphocytes (pink) that recognize antigens on a tumor cell (yellow) through T-cell receptors (Photo courtesy of Steve Gschmeissner).
Assays enabling the identification and enumeration of antigen-specific T cells are critical tools in characterizing immune responses and harnessing T cell function for treatment of numerous diseases including cancer.

A novel multiplex assay has been developed that combines conventional immune monitoring techniques and immune receptor repertoire sequencing to enable identification of T cells specific to large numbers of antigens simultaneously.

Scientists at Adaptive Biotechnologies (South San Francisco, CA, USA) multiplexed 30 different antigens and identified 427 antigen-specific clonotypes from five individuals with frequencies as low as one per million T cells. The clonotypes identified were validated several ways including repeatability, concordance with published clonotypes, and high correlation with Enzyme-Linked ImmunoSpot (ELISPOT).

Antigen-specific T cells were identified using one of two approaches: either by dextramer binding or by CD137 upregulation following overnight incubation with mixtures of peptides. Dextramer-specific T cells were identified by incubating peripheral blood mononuclear cells (PBMCs) with pools of eight dextramers. The new assay was named MIRA for Multiplexed Identification of T cell Receptor Antigen specificity.

The ELISPOT results for four antigens were independently generated for each donor. ELISPOT measures the total number of antigen-specific T cells secreting a particular cytokine. If all antigen-specific T cells secrete the cytokine measured by ELISPOT then results would be analogous to the sum frequency of antigen-specific clonotypes identified by MIRA. The scientists compared IFN-γ ELISPOT results with the sum frequency of antigen-specific clonotypes from each donor. There was a high correlation between results from both assays and MIRA readily detected antigen-specific clonotypes below 1 in 100,000 PBMCs, below estimates of the limit of detection for ELISPOT of around 4 spots per 100,000 PBMCs.

Harlan Robins, PhD, Chief Scientific Officer and Co-Founder at Adaptive Biotechnologies, said, “With this new multiplex technology we now have the ability to assign antigen-specificity to T cell receptors (TCR) sequences at a massive scale. Combined with our first-in-class technology for pairing TCR alpha and beta chain sequences at high throughput, we now have the tools needed for efficient identification of functional immune receptors, which may lead to tremendous advancements in biomarker discovery and therapeutic development.” The study was published on October 28, 2015, in the journal Public Library of Science ONE.

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