Browsing by Author "Sirc, Neja"

Large granular lymphocytic (LGL) leukemia is a rare form of chronic lymphocytic leukemia, that is characterized by clonal expansion of mature cytotoxic T- or natural killer (NK)- cells. As the white cell count in patients is predominantly not distinguishably altered, it often goes underdiagnosed or is diagnosed accidentally. T lymphocytic LGL leukemia (T-LGLL), that makes up 85% of all LGL leukemia (LGLL) cases is characterized by a prolonged expansion of peripheral blood T-lymphocytes, mostly CD8+ lymphocytes. 40 % of T-LGLL patients harbor mutations in the Signal Transducer and Activator of Transcription 3 (STAT3) gene. Y640F mutation of STAT3 (STAT3 Y640F) is the most commonly occurring alteration, present in approximately 17% of all T-LGLL patients, and 42% of patients that bear a mutation in STAT3. Furthermore, a higher prevalence of rheumatoid arthritis (RA) can be observed in patients with mutated STAT3 (26% vs 6%, p=0.02). As T-LGLL patients with the Y640F mutation have a higher incidence of co-occurring RA, we aimed to understand the possible role CD8+ T-cell clones carrying somatic mutation of STAT3 may play in the autoimmune process. We applied lentiviral vectors to express STAT3 wild type (wt) and STAT3 Y640F in murine and human CD8+ T cells. We were able to show their successful integration into the host genome using droplet digital PCR (ddPCR). ddPCR showed high selectivity in its ability to differentiate between the hosts’ gDNA and virally inserted cDNA. The custom-designed probes showed high specificity for either STAT3 wt or STAT3 Y640F, proving the functionality of the assay. Sensitivity studies provided us with accurate quantification even with the presence of STAT3 wt or STAT3 Y640F cDNA under 1%, displaying successful detection of rare variants in low concentration samples. In our expression studies, using Flow cytometry and Western Blotting (WB), we detected a modest rise in STAT3 expression in the virally transduced CD8+ cells. We hypothesized that the CD8+ cells were successfully transduced, but unable to accommodate sufficient STAT3 expression.To determine the role of Y640F mutation in the migration of CD8+ lymphocytes in different tissues in vivo, we injected lentivirally transduced cells, mixed in a 1:1 ratio (wt:mut), into the mice. Unfortunately, our ddPCR method was not sensitive enough to reliably quantitate the transduced cells in the diverse tissue samples. Consequently, we decided that any further mouse experiments cannot be justified. In conclusion, we present successful integration of lentivirally expressed wt and mutant STAT3 in both human and mouse primary CD8+ T lymphocytes and human peripheral blood mononuclear cells. The successfully constructed and optimized ddPCR assay was not, however sensitive enough for in vivo quantification of the transduced cells. As the lentivirally mediated expression of STAT3 variants was low, new approaches and tools are needed to study the role of STAT3 mutated T cells in the pathogenesis of RA.