Abstract
A patient with large granular lymphocyte (LGL) expansion (T-gamma lymphocytosis), neutropenia and thrombocytopenia was studied longitudinally. Analysis of peripheral blood mononuclear cells (PBMC) demonstrated an unusual large proportion of CD3+ T-lymphocytes expressing a gamma delta T-cell receptor (TcR-gamma delta). Immunofluorescence (IF) stainings with subset-specific monoclonal antibodies showed a fluctuating expansion of TcR-gamma delta+ T-cells expressing V gamma 9 and V delta 2 variable (V) gene segments. Biochemical characterization of PBMC showed the presence of a disulphide-linked TcR-gamma delta. TcR gene rearrangement studies on sorted TcR-gamma delta+ T-cells showed rearrangements of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 V and joining (J) gene segments, thereby confirming the IF staining results. These data alone did not allow us to determine whether the TcR-gamma delta+ LGL expansion represented a polyclonal or monoclonal proliferation, because the combinatorial repertoire of TcR-gamma delta receptors is limited due to the availability of only a few V and J segments within the TcR-gamma and TcR-delta genes and because of the preferential usage of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 rearrangements by TcR-gamma delta+ T-cells in blood of healthy individuals. We therefore used polymerase chain reaction (PCR)-mediated amplification of the TcR-gamma delta rearrangements, using specific V gamma 9, J gamma 1.2, V delta 2, and J delta 1 oligonucleotides to determine the junctional diversity of the TcR-gamma delta+ T-cell population. Sequence analysis of the PCR products obtained revealed a mixture of different junctional region sequences compatible with a polyclonal expansion. This is in contrast to the few reported TcR-gamma delta+ LGL and the majority of TcR-alpha beta+ LGL expansions, which appeared to consist of monoclonal proliferations.