Effizientes in vitro priming von tumor- und virusspezifischen CD8+ T-Zellen mit kalibrierten künstlichen antigenpräsentierenden Zellen

T cells are important effectors in the defense of human pathogens entering the organism. CD8+ T cells recognize peptides which are presented by MHC class I molecules and lyse cells which are infected by viruses or intracellular pathogens. One aim of this thesis was the development of a fast and reproducible method to prime and expand functional antigen-specific CD8+ effector T cells. We used the modified HLA-A*0201 restricted epitope ELAGIGILTV as a model system. Priming experiments were performed using artificial antigen presenting cells with defined MHC densities. Different settings for coating aAPCs had to be tested until we came up with an optimized ratio of 3:1 for costimuatory antibodies anti-4-1BB and anti-CD28, which enabled in vitro priming of high- or low-avidity effector T cells. To further characterize the T cell responses, we used fluorescently labeled MHC I tetramers as well as intracellular cytokine staining and 51Cr release assay. Induced CTLs were of an effector memory phenotype and therefore fully functional. Repeated stimulation of CD8-enriched T cells of different donors pointed out that on average a fivefold higher percentage of tetramer-specific T cells could be obtained by costimulation with anti-4-1BB/anti-CD28 in a 3:1 ratio if, compared to anti-CD28 alone. The optimal ratio between anti-CD28 and anti-4-1BB, values of up to 72% of antigen-specific T cells were monitored. Similar results could be obtained for the HLA-A*0201 restricted epitope SLAPPVHNV referred to tetramer staining. T cells specific for this altered peptide ligand recognize the cognate peptide SLAPPVHNV derived from the tumor antigen MUC1. HCMV infection is a major cause of death and disease in immunocompromised patients, especially organ transplant recipients, haemodialysis patients, cancer patients, people receiving immunosuppressive drugs and HIV-patients. Therefore, it is of utmost remaining interest to research for innovative approaches to generate antigen-specific T cells. The developed artificial priming method was highly effective in priming and expanding HCMV-specific CD8+ T cells from freshly isolated CD8+ T cells from HCVM inexperienced healthy donors. As expected the induced effector T cells were capable of lysing target cells efficiently and secreted INF-gamma. The efficiency of such a highly controlled T cell stimulation system holds great promisefor future therapeutic settings.T-Zellen sind wichtige Effektoren bei der Abwehr von Krankheitserregern im menschlichen Organismus. CD8+ T-Zellen erkennen Peptide, die von MHC-Klasse I-Molekülen präsentiert werden und zerstören Zellen, die durch Viren oder intrazelluläre Erreger infiziert sind. Die vorgelegte Arbeit hatte zum Ziel, eine Methode zu entwickeln, mit der es möglich ist schnell und reproduzierbar CD8+ T-Zellen gegen tumorassozierte Antigene zu induzieren und diese zu expandieren. Als Modellsystem wurde das veränderte HLA-A*0201 restringierte Epitop ELAGIGILTV, das vom Tumorantigen MelanA/Mart1 abstammt, genutzt. Priming-Experimente wurden mit Hilfe von Antigen-präsentierende Zellen mit kontrollierter MHC-Beladung durchgeführt. Es mussten verschiedene Beladungsstrategien für die künstlichen Antigen-präsentierenden Zellen ausgetestet werden, bis schließlich das optimale Verhältnis an kostimulatorischen Antikörpern von 3:1 (anti-4-1BB zu anti-CD28) ermittelt werden konnte. Mit dieser Kombination war es möglich in vitro gezielt hoch- oder niedrig-avide Effektor-T-Zellen zu generieren. Um die Funktionalität der T-Zellantworten näher zu beschreiben wurden Tetramerfärbungen, intrazelluläre Zytokinfärbungen und 51Chrom-Freisetzungstests durchgeführt. Die erzeugten T-Zellen wiesen einen Effektor-Phänotyp auf und waren voll funktionstüchtig. Durch wiederholte Stimulationsversuche mit T-Zellen verschiedener Spender konnte gezeigt werden, dass 5-fach größere Tetramer-spezifsche T-Zellpopulationen mit einer 3:1-Kombination der kostimulatorischen Antikörper, im Vergleich zu anti-CD28 alleine, erzielt werden konnten. Außerdem konnten nach Stimulation T-Zellpopulationen mit bis zu 72% Tetramer-spezifischer T-Zellen damit erhalten werden. Ähnliche Ergebnisse konnten auch mit dem HLA-A*0201 restringierten Epitop SLAPPVHNV beobachtet werden, auch wenn hier noch die funktionalen Tests ausstehen. Die Infektion mit dem Humanen Cytomegalievirus (HCMV) ist eine der Hauptursachen für den Tod bzw. schwere Krankheitsverläufe, bei immunsupprimierten Patienten. Daher ist es immer noch von dringender Notwendigkeit, innovative Ansätze zur Generierung von Antigenspezifischen T-Zellen in großer Zahl zu entwickeln. Die zuvor beschriebene Priming Methode war auch bei der Induktion und Expansion von HCMV-spezifischen CD8+ T-Zellen, die zuvor aus Virus-unerfahrenen gesunden Spender isoliert wurden, sehr effektiv. Die induzierten Zellen wiesen wie erwartet eine Effektorfunktion auf. Das hohe Maß an Effizienz und Kontrolle dieses Systems könnte sich bei zukünftigen immuntherpeutischen Anwendungen auszahlen

Azimuthal anisotropy of charged jet production in root s(NN)=2.76 TeV Pb-Pb collisions

We present measurements of the azimuthal dependence of charged jet production in central and semi-central root s(NN) = 2.76 TeV Pb-Pb collisions with respect to the second harmonic event plane, quantified as nu(ch)(2) (jet). Jet finding is performed employing the anti-k(T) algorithm with a resolution parameter R = 0.2 using charged tracks from the ALICE tracking system. The contribution of the azimuthal anisotropy of the underlying event is taken into account event-by-event. The remaining (statistical) region-to-region fluctuations are removed on an ensemble basis by unfolding the jet spectra for different event plane orientations independently. Significant non-zero nu(ch)(2) (jet) is observed in semi-central collisions (30-50% centrality) for 20 <p(T)(ch) (jet) <90 GeV/c. The azimuthal dependence of the charged jet production is similar to the dependence observed for jets comprising both charged and neutral fragments, and compatible with measurements of the nu(2) of single charged particles at high p(T). Good agreement between the data and predictions from JEWEL, an event generator simulating parton shower evolution in the presence of a dense QCD medium, is found in semi-central collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Forward-central two-particle correlations in p-Pb collisions at root s(NN)=5.02 TeV

Two-particle angular correlations between trigger particles in the forward pseudorapidity range (2.5 2GeV/c. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B. V.Peer reviewe

Event-shape engineering for inclusive spectra and elliptic flow in Pb-Pb collisions at root(NN)-N-S=2.76 TeV

Peer reviewe

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Pseudorapidity and transverse-momentum distributions of charged particles in proton-proton collisions at root s=13 TeV

The pseudorapidity (eta) and transverse-momentum (p(T)) distributions of charged particles produced in proton-proton collisions are measured at the centre-of-mass energy root s = 13 TeV. The pseudorapidity distribution in vertical bar eta vertical bar <1.8 is reported for inelastic events and for events with at least one charged particle in vertical bar eta vertical bar <1. The pseudorapidity density of charged particles produced in the pseudorapidity region vertical bar eta vertical bar <0.5 is 5.31 +/- 0.18 and 6.46 +/- 0.19 for the two event classes, respectively. The transverse-momentum distribution of charged particles is measured in the range 0.15 <p(T) <20 GeV/c and vertical bar eta vertical bar <0.8 for events with at least one charged particle in vertical bar eta vertical bar <1. The evolution of the transverse momentum spectra of charged particles is also investigated as a function of event multiplicity. The results are compared with calculations from PYTHIA and EPOS Monte Carlo generators. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Elliptic flow of muons from heavy-flavour hadron decays at forward rapidity in Pb-Pb collisions at root s(NN)=2.76TeV

The elliptic flow, v(2), of muons from heavy-flavour hadron decays at forward rapidity (2.5 <y <4) is measured in Pb-Pb collisions at root s(NN)= 2.76TeVwith the ALICE detector at the LHC. The scalar product, two- and four-particle Q cumulants and Lee-Yang zeros methods are used. The dependence of the v(2) of muons from heavy-flavour hadron decays on the collision centrality, in the range 0-40%, and on transverse momentum, p(T), is studied in the interval 3 <p(T)<10 GeV/c. A positive v(2) is observed with the scalar product and two-particle Q cumulants in semi-central collisions (10-20% and 20-40% centrality classes) for the p(T) interval from 3 to about 5GeV/c with a significance larger than 3 sigma, based on the combination of statistical and systematic uncertainties. The v(2) magnitude tends to decrease towards more central collisions and with increasing pT. It becomes compatible with zero in the interval 6 <p(T)<10 GeV/c. The results are compared to models describing the interaction of heavy quarks and open heavy-flavour hadrons with the high-density medium formed in high-energy heavy-ion collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V.Peer reviewe

Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at root s(NN)=2.76TeV

Peer reviewe

Thiazide-Associated Hyponatremia, Report of the Hyponatremia Registry: An Observational Multicenter International Study

Background: Hyponatremia is a frequent and potentially life-threatening adverse side effect of thiazide diuretics. This sub-analysis of the Hyponatremia Registry database focuses on current management practices of thiazide-associated hyponatremia (TAH) and compares differences between TAH and syndrome of inappropriate antidiuretic hormone secretion (SIADH). Methods: We analyzed 477 patients from 225 US and EU sites with euvolemic hyponatremia ([Na+] <= 130 mEq/L) who were receiving a thiazide diuretic. Of these, 118 met criteria for true thiazide-induced hyponatremia (TIH). Results: Thiazide was withdrawn immediately after hyponatremia was diagnosed only in 57% of TAH; in these patients, the median rate of [Na+] change (. daily [Na+]) was significantly higher than those with continued thiazide treatment (3.8 [interquartile range: 4.0] vs. 1.7 [3.8] mEq/L/day). The most frequently employed therapies were isotonic saline (29.6%), fluid restriction (19.9%), the combination of these two (8.2%), and hypertonic saline (5.2%). Hypertonic saline produced the greatest. daily [Na+] (8.0[6.4] mEq/L/day) followed by a combination of fluid restriction and normal saline (4.5 [3.8] mEq/L/day) and normal saline alone (3.6 [3.5] mEq/L/day). Fluid restriction was markedly less effective (2.7 [2.7] mEq/L/day). Overly rapid correction of hyponatremia occurred in 3.1% overall, but in up to 21.4% given hypertonic saline. Although there are highly significant differences in the biochemical profiles between TIH and SIADH, no predictive diagnostic test could be derived. Conclusions: Despite its high incidence and potential risks, the management of TAH is often poor. Immediate withdrawal of the thiazide is crucial for treatment success. Hypertonic saline is most effective in correcting hyponatremia but associated with a high rate of overly rapid correction. We could not establish a diagnostic laboratory-based test to differentiate TIH from SIADH. (C) 2017 The Author(s) Published by S. Karger AG, Base

Euvolemic hyponatremia in cancer patients. Report of the Hyponatremia Registry: an observational multicenter international study

Hyponatremia secondary to SIADH is frequent in cancer patients and potentially deleterious. The aim of this sub-analysis of the Hyponatremia Registry database is to analyze current diagnostic and therapeutic management practices in cancer patients with SIADH. We analyzed 358 cancer patients who had serum sodium concentration ([Na+]) = 130 mEq/L. Overly rapid correction of hyponatremia occurred in 11.7%. Although essential for successful hyponatremia management, appropriate diagnostic testing is not routinely performed in current practice. The most frequently employed monotherapies were often ineffective and sometimes even aggravated hyponatremia. Tolvaptan was used less often but showed significantly greater effectiveness. Despite clear evidence that hyponatremia is associated with poor outcome in oncology patients, most patients were discharged still hyponatremic. Further studies are needed to assess the beneficial impact of hyponatremia correction with effective therapies