Amplification of TCRβ gene rearrangements from micromanipulated single cells: T cells rosetting around Hodgkin and Reed-Sternberg cells in Hodgkin's disease are polyclonal

Abstract

Despite accounting for only a minor fraction of all cells in Hodgkin's lymphoma tissue, the Hodgkin and Reed-Sternberg (HRS) cells represent the malignant tumor cell clone in Hodgkin's disease (HD). By far the most abundant cell type in the tumor tissue are CD4+ T cells. Some of them intimately associate with HRS cells forming rosettes around them. This study addresses the question whether the rosetting phenomenon reflects a specific interaction between T and HRS cells by asking whether the rosettes are composed of T cells expressing a restricted TCR repertoire. Single rosetting T cells were micromanipulated from frozen sections of tumor tissue in two cases of nodular sclerosing HD and one case of lymphocyte predominant HD. TCR Vbeta gene rearrangements were amplified from these single cells by PCR. Of 83 potentially functional Vbeta gene rearrangements obtained altogether from the three cases, 81 were found to be clonally unrelated. Furthermore, they did not show signs of selection of the receptor chains for recognition of common epitopes: The usage of Vbeta and Jbeta gene segments as well as the distribution of complementarity-determining region (CDR) 3 lengths was similar to what was seen in a collection of 60 Vbeta gene rearrangements from blood of healthy donors and no recurrent CDR3 amino acid motifs were found. These data suggest that the HRS cells attract CD4+ T cells nonspecifically.

PCR Analysis of Immunoglobulin Heavy Chain (IgH) and TcR-γ Chain Gene Rearrangements in the Diagnosis of Lymphoproliferative Disorders: Results of a Study of 525 Cases

“…The detection of such a rearrangement may simply reflect the presence of oligoclonal expansion of tumor-infiltrating T-lymphocytes rather than a dual rearrangement involving neoplastic B-cells (49 -51). In such circumstances, only single-cell gene rearrangement study of tumor-infiltrating T-lymphocytes in non-Hodgkin's B-cell lymphoma-affected tissues could address this issue (52). Clonal populations of nonneoplastic T-cells have also been described in patients with B-cell chronic lymphocytic leukemia and multiple myeloma (53,54) and even in healthy elderly humans (55).…”

Molecular Single-Cell Analysis of Hodgkin- and Reed-Sternberg Cells Harboring Unmutated Immunoglobulin Variable Region Genes

“…The same cells were subjected to semi-nested amplification of TCR␤ VDJ and DJ rearrangements and fragments specific for germline configuration of the C␤1 and C␤2 loci, as previously described (Müschen et al, 2000;Roers et al, 1998).…”

Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis

“…Overview of expanded and persisting TCR BV sequences in muscle and blood of all investigated patients conjunction with single-cell PCR (39). The single-cell PCR protocol used in our study (19,23) allows the amplification of TCR BV chain rearrangements from single molecules of rearranged genomic DNA, which is more stable than RNA (23). A drawback of this approach is that the initial spectratype screening may miss expanded clones among muscle-infiltrating T cells.…”

“…Rearranged TCR BV chains were amplified from DNA of single T cells essentially as described (19,23), with minor modifications. Positive products were sequenced as described above.…”