@phdthesis{Pusch2015, author = {Pusch, Tobias}, title = {The transcription factor NFATc1 mediates cytotoxic T cell function in vitro and in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123690}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment. Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells. Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade. We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation. Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse.}, subject = {Transkriptionsfaktor}, language = {en} } @article{KarulinKaracsonyZhangetal.2015, author = {Karulin, Alexey Y. and Karacsony, Kinga and Zhang, Wenji and Targoni, Oleg S. and Moldova, Ioana and Dittrich, Marcus and Sundararaman, Srividya and Lehmann, Paul V.}, title = {ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting}, series = {Cells}, volume = {4}, journal = {Cells}, number = {1}, doi = {10.3390/cells4010056}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149648}, pages = {56-70}, year = {2015}, abstract = {Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7\% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.}, language = {en} }