Faculty Collaboration Database

Quantitative measurement of pathogen-specific human memory T cell repertoire diversity using a CDR3 beta-specific microarray. BMC Genomics 2007 Sep 19;8:329

Date

09/21/2007

Pubmed ID

17880719

Pubmed Central ID

PMC2039751

DOI

10.1186/1471-2164-8-329

Scopus ID

2-s2.0-35448956566 (requires institutional sign-in at Scopus site)   5 Citations

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.

Author List

Wang X, Jia S, Meyer L, Yassai MB, Naumov YN, Gorski J, Hessner MJ

Authors

Martin J. Hessner PhD Professor in the Pediatrics department at Medical College of Wisconsin
Shuang Jia Biostatistician III in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Motifs
Antigens, Viral
Cells, Cultured
Complementarity Determining Regions
Gene Rearrangement, beta-Chain T-Cell Antigen Receptor
Genes, T-Cell Receptor beta
HLA-A2 Antigen
Humans
Immunologic Memory
Influenza A virus
Oligonucleotide Array Sequence Analysis
Oligonucleotide Probes
Peptide Fragments
Sequence Homology, Nucleic Acid
T-Cell Antigen Receptor Specificity
T-Lymphocyte Subsets
T-Lymphocytes, Cytotoxic
Viral Matrix Proteins