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Clinical Chemistry 0: clinchem.2004.047266v1, 2005; 10.1373/clinchem.2004.047266
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Received on December 30, 2004
Accepted on January 19, 2005

Clinical Immunology

Analysis of T-Cell Receptor V{beta} Gene Repertoires after Immune Stimulation and in Malignancy by Use of Padlock Probes and Microarrays

Johan Banér 1, Per Marits 2, Mats Nilsson 1, Ola Winqvist 2, Ulf Landegren 1*

1 Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala Universitet, Uppsala, Sweden
2 Department of Medical Sciences, Uppsala University Hospital, Uppsala University, Uppsala, Sweden

* To whom correspondence should be addressed. 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 V{beta} genes, compared with peripheral blood, indicative of a clonal response against tumor antigens.

Methods: To simultaneously measure the relative expression of all V{beta} 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 V{beta} subfamily-specific padlock probes. Reacted probes were selectively amplified and the products hybridized to a microarray, from which the V{beta} 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 V{beta} families could also be detected by flow cytometry. In samples from two bladder cancer patients, we detected predominant representations of specific V{beta} 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 V{beta} 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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