Work

Digital Multiplexed Quantification of Both T-Cell Receptor β-Chain and α-Chain Diversity in T Cells Using the Nanostring nCounter Assay System

Year: 2011

Type: article

Abstract: Measurement and skewing of T-cell receptor (TCR) α and β diversity in the tumor-bearing host (i) engrafted with allogeneic hematopoietic stem cells and/or (ii) after vaccination or adoptive T-cell transfer, are used for monitoring the effect of immunotherapies. TCR diversity is mostly concentrated within the third complementarity-determining region (CDR3) of α and β chains. Currently available methods for measuring diversity are primarily limited to TCRVβ usage and are (i) flow-cytometric detection by using antibody panel against TCRVβ, (ii) CDR3 length spectratyping by semi-quantitative RT-PCR, and (iii) DNA sequencing of the rearranged CDR3 regions. Although these methods can detect oligoclonal expansion of T cells they are costly, labor-intensive, and require substantial amount of sample. Here, we adapted the newly-developed Nanostring nCounter Analysis System to quantify gene expression of 45 TCRVβ and 44 TCRVα gene families in a single non-enzymatic reaction from a small number of T cells. Two sequence-specific gene probes, one mRNA target sequence-specific capture probe, and a second mRNA target sequence-specific fluorescent-labeled color-coded probe were produced for each of the TCR genes. These were used to determine TCRVβ and TCRVα usage based on profiling the 99 V region families of T cells from healthy volunteers by nCounter System and a subset of these data were validated by flow cytometry with TCRVβ-specific mAb. Subsequently, we analyzed clinical samples obtained from 10 patients with advanced melanoma treated with ex vivo propagated tumor infiltrating lymphocyte (TIL) therapy. To validate this data set, the TCRVβ panel analysis obtained from nCounter assay of these samples agreed with CDR3 DNA sequencing data. This is the first time that digital mRNA profiling for TCRVβ and TCRVα has been undertaken without requiring cDNA synthesis, enzymatic reactions, or mAb usage. Our approach requires just a small amount of total RNA and at $1.40/gene, this assay is cost-effective. It is anticipated that this approach will be of significant interest to immunologists seeking to manipulate the human immune system.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2010.12.329