Assessment of clonality of rosetting T lymphocytes in Hodgkin's disease by single-cell polymerase chain reaction: detection of clonality in a polyclonal background in a case of lymphocyte predominance Hodgkin's disease

Rosetting of CD4+ T cells around the neoplastic Hodgkin and Reed-Sternberg (H&RS) cells is a characteristic feature of Hodgkin's disease (HD). To answer the question whether this phenomenon is solely due to chemokine-mediated attraction of T cells or whether the rosetting T cells in addition recognize antigens presented by the H&RS cells, we examined the T cells adherent to H&RS cells. Cells from five cases of HD [four classic HD and one lymphocyte-predominant (LP) HD] were examined by single-cell analysis for the T-cell receptor (TCR) γ gene. Between 5 and 17 rosettes containing one to ten rosetting lymphocytes and the corresponding H&RS cells were amplified in separate plastic tubes. Of the resulting 119 TCRγ polymerase chain reaction (PCR) products, 87 were sequenced. While no evidence of a clonal expansion was obtained in the lymph nodes from four of five patients with classic HD, clonal TCRγ sequences were found in the lymph node from the patient within LPHD in two independent experiments analyzing seven and ten different rosetting complexes, respectively. Of 13 products, 11 showed identical Vγ9 sequences. Unrelated products were found in all other TCRγ family subgroups in this case. Single H&RS cells picked as controls were negative for TCRγ rearrangements. Our results demonstrate that clonal proliferations on a polyclonal background can occur among the T cells forming rosettes with Hodgkin cells and lend support to the view that Hodgkin cells may also function as cells presenting antigens to the adhering T cells.