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Clinical Chemistry 51: 768-775, 2005. First published February 3, 2005; 10.1373/clinchem.2004.047266
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(Clinical Chemistry. 2005;51:768-775.)
© 2005 American Association for Clinical Chemistry, Inc.

Clinical Immunology

Analysis of T-Cell Receptor Vß Gene Repertoires after Immune Stimulation and in Malignancy by Use of Padlock Probes and Microarrays

Johan Banér1, Per Marits2,3, Mats Nilsson, Ola Winqvist2,3 and Ulf Landegren1,a

1 Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala Universitet, Uppsala, Sweden.
2 Department of Medical Sciences, Uppsala University Hospital, Uppsala University, Uppsala, Sweden.
3 Department of Medicine, Unit of Clinical Allergy Research L2:04, Karolinska Hospital, Stockholm, Sweden.

aAddress correspondence to this author at: Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala Universitet, SE-751 85 Uppsala, Sweden. Fax 46-18-4714808; e-mail ulf.landegren{at}genpat.uu.se.

Background: Detection of expanded T-cell clones, identified by their receptor (TCR) repertoires, can assist diagnosis and guide therapy in infectious, inflammatory, and autoimmune conditions as well as in tumor immunotherapy. Analysis of tumor-infiltrating lymphocytes often reveals preferential use of one or a few TCR Vß genes, compared with peripheral blood, indicative of a clonal response against tumor antigens.

Methods: To simultaneously measure the relative expression of all Vß gene families, we combined highly specific and sensitive oligonucleotide reagents, called padlock probes, with a microarray read-out format. T-Cell cDNA was combined with a pool of Vß subfamily-specific padlock probes. Reacted probes were selectively amplified and the products hybridized to a microarray, from which the Vß subfamily distribution in each sample could be determined relative to a control sample.

Results: In lymphocytes stimulated with the superantigen staphylococcal enterotoxin B, we detected expansions at the mRNA level of TCR subfamilies previously shown to respond to staphylococcal enterotoxin B. Expansions of the same Vß families could also be detected by flow cytometry. In samples from two bladder cancer patients, we detected predominant representations of specific Vß subfamilies in both tumor-infiltrating lymphocytes and in the draining lymph nodes, but not in non-tumor-draining lymph nodes or peripheral blood. Several expression profiles from draining lymph nodes in patients with malignant melanoma were divergent from profiles seen in non-tumor-draining lymph nodes.

Conclusion: Padlock probe-based parallel analysis of TCR Vß gene distributions provides an efficient method for screening multiple samples for T-cell clonal expansions with reduced labor and time of analysis compared with traditional methods.




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