Analysis of T-cell receptor Vβ gene repertoires after immune stimulation and in malignancy by use of padlock probes and microarrays

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. (c) 2005 American Association for Clinical Chemistry.