Email updates

Keep up to date with the latest news and content from BMC Genomics and BioMed Central.

Open Access Methodology article

Quantitative measurement of pathogen specific human memory T cell repertoire diversity using a CDR3β-specific microarray

Xujing Wang12, Shuang Jia1, Lisa Meyer1, Maryam B Yassai3, Yuri N Naumov4, Jack Gorski3 and Martin J Hessner12*

Author Affiliations

1 The Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics, The Medical College of Wisconsin and The Children's Hospital Research Institute of Children's Hospital of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA

2 The Human and Molecular Genetics Center, The Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA

3 Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA

4 The Blood Research Institute, BloodCenter of Wisconsin, 8727 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA

For all author emails, please log on.

BMC Genomics 2007, 8:329  doi:10.1186/1471-2164-8-329

Published: 19 September 2007

Abstract

Background

Providing quantitative microarray data that is sensitive to very small differences in target sequence would be a useful tool in any number of venues where a sample can consist of a multiple related sequences present in various abundances. Examples of such applications would include measurement of pseudo species in viral infections and the measurement of species of antibodies or T cell receptors that constitute immune repertoires. Difficulties that must be overcome in such a method would be to account for cross-hybridization and for differences in hybridization efficiencies between the arrayed probes and their corresponding targets. We have used the memory T cell repertoire to an influenza-derived peptide as a test case for developing such a method.

Results

The arrayed probes were corresponded to a 17 nucleotide TCR-specific region that distinguished sequences differing by as little as a single nucleotide. Hybridization efficiency between highly related Cy5-labeled subject sequences was normalized by including an equimolar mixture of Cy3-labeled synthetic targets representing all 108 arrayed probes. The same synthetic targets were used to measure the degree of cross hybridization between probes. Reconstitution studies found the system sensitive to input ratios as low as 0.5% and accurate in measuring known input percentages (R2 = 0.81, R = 0.90, p < 0.0001). A data handling protocol was developed to incorporate the differences in hybridization efficiency. To validate the array in T cell repertoire analysis, it was used to analyze human recall responses to influenza in three human subjects and compared to traditional cloning and sequencing. When evaluating the rank order of clonotype abundance determined by each method, the approaches were not found significantly different (Wilcoxon rank-sum test, p > 0.05).

Conclusion

This novel strategy appears to be robust and can be adapted to any situation where complex mixtures of highly similar sequences need to be quantitatively resolved.