Role of regulatory CD4+ T lymphocytes in developing autoimmune diseases.

Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Apoptosis of tumor cells : role of TRAIL and caspase 10

One of the key features of cancer cells is the ability to escape programmed cell death (apoptosis). As a mechanism of apoptosis inactivation in cancer cells, somatic mutations of pro-apoptotic genes have been reported in many cancers. Caspase 10 is an initiator caspase whose physiological function remains poorly understood. Also the ability of caspase 10 to substitute for caspase 8 in the death receptors apoptotic pathway is still controversial. However, the fact that some of the mutations found in CASP10 gene was associated with apoptosis defects (79, 81) suggest that caspase 10 could be also important in apoptosis initiation. In our lab, there was found a heterozygous mutation in CASP10 gene of Jurkat (human T-acute lymphoblastic leukemia) clone resistant to TRAIL (J-TR1). This mutation influence the amino acid composition close to the active site of the enzyme. The aim of this thesis was to confirm the mutation by ARMS-PCR and to determine if an overexpression of normal (unmutated) or mutated caspase 10 D in TRAIL sensitive and/or TRAIL resistant Jurkat cells (J-WT and/or J-TR1) will influence TRAIL induced apoptosis. Mutation was confirmed. We created J-WT and J-TR1 stable clones transfected by vector with unmutated or mutated CASP10 D (CASP10 D WT or CASP10 D MUT). CASP10 D MUT overexpression in J-WT..

Immunogenic cell death and it's relevance for biology and therapy of malignant diseases

Immunostimulatory potential of tumor cells depends on various factors, including primarily tumor antigen repertoire and the capacity to emit molecules associated with cellular stress or injury, so called DAMPs, during immunogenic forms of cell death. These molecules mainly act on dendritic cells (DCs), thus activating the antitumor immune response. Several immunogenic cell death (ICD) inducers have been described in the past years. The contribution of my PhD thesis into this topic was the characterization of the apoptotic pathways activated by high hydrostatic pressure (HHP). HHP induces rapid tumor cell death accompanied by DAMP release (mainly calreticulin (CRT), HSP70, HSP90, HMGB1 and ATP) that is characterized by the overproduction of reactive oxygen species (ROS) causing the establishment of integrated stress response. ROS-PERK-eIF2α-caspase-2-caspase-8 signaling pathway plays an essential role in CRT translocation to the tumor cell surface upon HHP treatment, thus influencing the immunogenic potential of these cells. Moreover, the importance of ICD concept was also confirmed in vivo. The results point out that the presence of CRT on the surface of malignant blasts from AML patients correlates with the activation of specific antitumor immune response and improved clinical outcome. Another..

Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-gamma and multiple toll-like receptor agonists

Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFN gamma and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFN gamma, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFN gamma, R848, and poly I:C had the ability to activate IFN gamma production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFN gamma and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines