Pathologic Clonal CTLResponses - Non Random Nature of the TCR Restriction in LGL Leukemia.

T-LGL leukemia, a clonal lymphoproliferation of CTL associated with cytopenias, seems to be triggered/sustained by antigenic drive. It is likely that hematopoietic progenitors are the targets in this process. We hypothesized that CTL clones evolve not randomly but in the context of an autoimmune response. We studied 75 patients with suspected LGL leukemia. The detection strategy for signature clonotypes included VB flow cytometric typing and VB specific PCR or multiplex PCR on cDNA from sorted CD8+ cells, followed by colony sequencing of the VB CDR3 regions (clonotypes). In 60 patients, we were able to isolate and characterize 86 pathologically expanded clonotypes (in 22 patients >1 immunodominant clone was detected); in most of the cases, the over-represented VB families nominally were >20% of all CD8+ cells. Clonal frequency was 65%± 30% of a given VB family or 35.4%± 31% of the total CD8+ population. By comparison, the most expanded clones in controls constitute to only 0.7% of the CD8+ repertoire.

Two patients showed an identical immunodominant clonotype also shared with a third patient in whom it was not expanded. We also found sequence identity between an immunodominant and a minor clonotype and between 2 minor clonotypes in 2 LGL patient pairs. In 2 other patient pairs, a major clonotype showed a striking homology to a minor clonotype. Not only were identical clonotypes shared between the patients, a high degree of similarity was found between a large number of minor clonotypes. Likely, these minor clonotypes are the remnants of the initial polyclonal response. When up to 172 clones were sequenced per VB family in 24 healthy donors, only one shared clonotype was found in 2 donors and only a total of 11 sequences were detected to show the level of homology as detected in LGL. Immunodominant clonotypes can be used as tumor markers or to study expansion of LGL clones in correlation with disease activity. Identification of pathologic clonotypes allowed for design of a clonotype-specific real time PCR that allowed for monitoring of the clonal behavior over the time. For 7 patients followed serially either by sequencing or clonotype-specific Taq-Man PCR, a marked decrease in the frequency of the immunodominant clone was observed with successful therapy. This process resulted in increased clonal diversity and decreased redundancy. Some clonotypes that appeared post therapy showed sequence similarity to the original immunodominant clones.

Given the normally immense variability of the TCR repertoire and the very low frequency of shared clonotypes identified in healthy donors, our findings suggest a non-random nature of LGL transformation. This possibly occurred in the context of initially polyclonal immune response directed against common antigenic target. Clonotypic sequences may be used to monitor the frequency of malignant T cell clones, but such an approach may also be applied for polyclonal responses in which clonotypes derived from most significantly expanded T cell clones may serve as marker for specific autoimmune processes. Finally, the availability of the clonotypic sequences may allow for measurements of anti-idiotypic responses and study of the applicability of clonotype-based anti-idiotypic vaccination as a potential therapeutic modality for T cell malignancies.