In Search for the Specificity of Clonal CTL in T-LGL Leukemia - Generation of Soluble LGL-Derived T Cell Receptor.

Large granular lymphocyte leukemia (LGL) is a clonal lymphoproliferation of CTL associated with immune cytopenias. Hematopoietic progenitor cells may be targets for the immune attack by clonal CTL. The rearranged unique portion of the T cell receptor (TCR), the complementarity determining region 3 (CDR3) of the variable beta chain can serve as molecular marker of the clonal process. Previously, we applied molecular analysis of TCR repertoire to the analysis of CDR3 regions of the variable beta-chain in LGL leukemia and obtained 70 individually specific CDR3 clonotypes. Moreover, using clonotype-specific PCR clonal size was determined. Shared or highly homologous clonotypic CDR3 sequences in some patients provide evidence for the non-random nature of the clonal transformation in LGL and the presence of a common antigenic target. Such an antigen may constitute a drive for the expansion of LGL and its distribution may explain lineage-specificity of the LGL CTL. Identification of the target antigen is essential for understanding of immune-mediated hematologic disorders, including autoimmune cytopenias such as those observed in LGL. T-LGL has inherent advantage of the availability of clonal CTL for experimentation. We hypothesized here that generation of a soluble TCR (sTCR) derived from T-LGL clone could facilitate identification of target cells and analysis of the cellular distribution of peptide presentation. For cloning of sTCR we selected 4 patients with extreme clonal CTL expansion and profound neutropenia. Flow cytometry with VB specific antibodies was applied to determine VB family utilization by the clonal TCR. B-chains were amplified with specific primers so that C region is truncated and the transmembrane and intracellular portions are deleted. Due to the lack of VA specific antibodies, a 5′RACE RT-PCR with a constant A primer was applied. Expanded LGL-specific A chain was determined and cloned in its truncated form as described for B-chain. For example, clonal TCR for patient #1 had a structure VB1/JB1.6/CB1 and VA9-2/JA41*01/CA. To construct a sTCR truncated VA and VB products were subcloned into a dual promoter baculovirus expression vector containing the k and H-chain cassettes (pAC-k-CH3 or alternatively pAC-Fc). This vector allows for expression of a bivalent TCR:Ig protein that can be produced in a baculovirus system. A flow cytometry on fluorochrome conjugated sTCR after co-culture with HLA matched normal bone marrow cells allows for the detection of cells expressing target peptides. Therapeutically, malignant T cell clone-specific sTCR may be used as a potential idiotypic vaccine for T cell lymphomas/leukemias expressing TCR.

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