Office of Technology Development (OTD)

Martin Hessner, PhD

During the initiation of immune responses, individual T cell clones recognize peptides presented by major histocompatibility complex (MHC) molecules through T cell receptors (TCR).  Within the TCR, the amino acid sequence of the third complimentarity-determining region (CDR3) conveys fine antigen specificity. The DNA sequence at CDR3 defines a clonotype and serves as a fingerprint for the T cell lineage bearing it. The frequency with which a particular TCR clonotype is encountered can be taken to be a measure of clonal expansion. Often recognition of a particular antigen-MHC complex is mediated by TCR using identical V chains with very similar CDR amino acid sequences. Because of the degeneracy of the genetic code this can lead to responses characterized by multiple clonotypes that encode identical CDR3 amino acid sequences. Since antigen specific T cell responses are central to human immunity, and there is significant interest in the relationship between lymphocyte diversity and immunocompetence, the analysis of T cell repertoires is highly relevant. T cell repertoire analysis for the past decade has relied on spectratyping to characterize T cell receptor (TCR) families by size, followed by repetitive cloning/sequencing to resolve clonal diversity. As an alternative, researchers at the Medical College of Wisconsin have developed a novel, array assay that accurately identifies and quantifies T cell repertoires.

Among HLA-A2 individuals, response to influenza A M1 peptide is recognized by T cells expressing ß variable 17 (VB17) chains possessing the amino acid sequence IRSS. As a high-throughput alternative to cloning and sequencing, Dr. Hessner developed an array-based assay for quantitative detection of 108 IRSS encoding TCR. The assay design is illustrated below. The array is comprised of 108 63-mer probes. The probes were designed with a 23 nucleotide 5’-region specific for the Arabidopsis thaliana LTP4 gene; a 23 nucleotide polyA spacer, and a 17 nucleotide IRSS-specific region (sense strand) placed at the 3’-end of the probe. The A. thaliana specific region offers advantages in terms of quality control. Hybridization efficiency between highly related Cy5-labeled subject sequences was normalized by including a equimolar mix of Cy3-labeled synthetic targets representing all 108 arrayed probes.

Analysis of T cell cultures determined that array-based clonotyping is a reliable, efficient, and cost effective approach that is at least as informative as traditional cloning and sequencing. This novel strategy is directly relevant to any researcher investigating T cell receptor usage. It is also applicable for quantitative genotyping of complex mixtures or highly polymorphic systems where the constituent sequences can differ by only one or a few nucleotides, such as human leukocyte antigen (HLA) system and human immunodeficiency virus (HIV) genotyping.

• Accurately identifies clonotypes
• Provides a high-throughput tool for determining T cell repertoire
• Eliminates cloning and sequencing 
• Identifies and quantifies genotypes within a complex mixture
• Differentiates genotypes with as little as one base difference

Working prototypes