Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against epitopes at the N-terminus and reverse transcriptase domain (rtTERT) of TERT. Photon emission from mice boosted with TERT/TERT-HA+Luc DNA was 100 times lower than from vector+Luc DNA-boosted controls. Bioluminescence loss correlated with percent of IFN-γ/IL-2/TNF-α producing CD8+ and CD4+ T-cells specific to rtTERT, indicating immune clearance of TERT/Luc-coexpressing cells. We made murine adenocarcinoma 4T1luc2 cells to express rtTERT by lentiviral transduction. Expression of rtTERT significantly reduced the capacity of 4T1luc2 to form tumors and metastasize in mice, while not affecting in vitro growth. Mice which rejected the tumors developed T-cell response against rtTERT and low/no response to the autoepitope of TERT. This advances rtTERT as key component of TERT-based therapeutic vaccines against cancer.

Chemokines and their receptors direct migration and infiltration of immune cells. CCR1 and CCR2 maintain sequence similarity and respond to a number of the same chemokines secreted in lymphoid organs. Expression of CD38 on leukemic cells has been associated with poor clinical outcomes in patients with chronic lymphocytic leukemia (CLL) and is considered as the negative predictor of progression. In our study of newly diagnosed CLL patients, which included 39 CD38-positive and 22 CD38-negative patients, CCR1 and/or CCR2 were always detected, using flow cytometry, on the peripheral blood (PB) CD19+CD5+ lymphocytes in patients with >30% of the CD38+ CD19+CD5+ lymphocytes (n = 16). Spearman’s rank correlation analysis determined correlations between the frequency of the CCR1- and CCR2-expressing PB CD19+CD5+ lymphocytes and the frequency of the CD38-positive CD19+CD5+ lymphocytes (rs = 0.50 and rs = 0.38, respectively). No significant correlations were observed between ZAP70 mRNA expression levels in PB mononuclear cells and the frequency of the circulating CCR1+ or CCR2+ CD19+CD5+ lymphocytes. Further association studies are needed to verify prognostic relevance of the CCR1/CCR2 expression on leukemic cells in CLL patients at diagnosis. We suggest that CCR1/CCR2 signaling pathways could represent attractive targets for development of CLL anti-progression therapeutics.

The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.