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Background: Life-prolonging tumour removal surgery is associated with increased metastasis and disease recurrence. Natural Killer (NK) cells are critical for the anti-tumour immune response. Postoperatively, NK cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed and this dysfunction has been linked to increased metastases/poor patient outcomes. NK cell activity depends on the integration of signals through receptors and can be modulated by soluble factors, including transforming growth factor- beta (TGFβ). The postoperative period is characterized by the expansion of myeloid-derived suppressor cells (sxMDSCs), which inhibit NK cell effector functions. I hypothesize that impaired NK cell IFNγ production is due to altered signaling pathways caused by sxMDSC-derived TGFβ. Methods: Postoperative changes in NK cell receptor expression, receptor-dependent phosphorylation of downstream targets, and rIL-2/12-stimulated IFNγ production were assessed using newly developed whole blood assays utilizing peripheral blood samples from cancer surgery patients. Isolated healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma or isolated sxMDSCs and NK cell phenotype and function were assessed. NK cells were also cultured with plasma in the presence/absence of a TGFβ blocking monoclonal antibody (mAb) or a TGFβ RI small molecule inhibitor (smi). Single-cell RNA-sequencing was performed on six colorectal cancer surgery patients at baseline and on POD1. S6 phosphorylation was used as a proxy for mammalian target of rapamycin complex (mTORC) 1 activity to investigate the mechanism of TGFβ-mediated NK cell dysfunction. Results: Intracellular NK cell IFNγ, activating receptors CD132 (IL-2R), CD212 (IL-12R), NKG2D, and DNAM-1, and the phosphorylation of downstream targets STAT5, STAT4, p38 MAPK, and S6 were significantly reduced on POD1. TGFβ was increased in patient plasma on POD1. The dysfunctional phenotype could be phenocopied in healthy NK cells through the addition of rTGFβ1 or by incubation with POD1 plasma. This dysfunctional phenotype could be prevented with the addition of an anti-TGFβ mAb or a TGFβ RI smi in culture. RNA-sequencing revealed a reduction in transcripts associated with mTOR effector functions, suggesting an impairment in mTOR. S6 phosphorylation was maintained with the addition of TGFβ-specific therapies. The hyporesponsive NK cell phenotype was reproduced upon culture of healthy NK cells with sxMDSCs and sxMDSCs were shown to produce soluble TGFβ in culture. Conclusion: Surgically stressed NK cells display a dysfunctional phenotype, which could be prevented in vitro through the addition of TGFβ-specific blocking therapies. sxMDSCs produced TGFβ and co- incubation induced dysfunction in healthy NK cells. The recovery of impaired S6 phosphorylation with TGFβ-specific therapies suggests that TGFβ is inducing NK cell dysfunction via inhibition of mTORC1 activity. The perioperative period of immunosuppression presents a window of opportunity for novel therapeutics to prevent metastases and cancer recurrence among cancer surgery patients.

Evidence suggests that the neurotropic parasite Toxoplasma gondii may play a role in the development of cognitive impairments. My hypothesis was that congenital exposure to T. gondii would lead to detectable age and sex related differences in behavior and neurotransmitter levels in mice. The neurotransmitter dopamine and commonly used anti-schizophrenic agents were evaluated against T. gondii in human fibroblast cells. Dopamine caused a significant increase in tachyzoite numbers at 250 nM but not 100 nM and the drugs valproic acid, fluphenazine, thioridazine and trifluoperazine inhibited T. gondii development. The effects T. gondii infection had on behavior were examined using a congenital mouse model. Previous work demonstrated maternal immune stimulation (MIS) with interferon gamma (INF-g) resulted in decreased fetal mortality from congenital T. gondii infections; therefore I examined the effects of INF- g treatment of mothers to determine if protection from the behavioral effects of T. gondii occurred in their offspring. No differences in concentrations of neurotransmitters in the brains of congenitally infected mice were observed. I found that mice infected with T. gondii developed adult onset behavior impairments with decreased rate of learning, increased activity and decreased memory, indicating cognitive impairment for male mice and not female mice. My findings support the evidence T. gondii is a factor in the development of cognitive impairments. My results for T. gondii exposed male mice are consistent with the convention that males have more cognitive impairments in the prodromal stage of schizophrenia. MIS with IFN-g had a minimal effect on behavior post sexual maturity but had a greater effect on pre sexual maturity female mice which exhibited difficulties with spatial memory, coordination and the ability to process stimuli. The results indicate the behavior alterations from IFN- g are transient. When MIS is given prior to congenital infection with T. gondii, we detected no behavior deficits in any group of mice, including male mice post sexual maturity. Based on the results of my study, I must reject the hypothesis that neurotransmitter levels are influenced by congenital toxoplasmosis and accept the hypothesis that congenital T. gondii infection caused cognitive impairments in male mice post sexual maturity. / Ph. D.

Made available in DSpace on 2015-04-11T13:54:58Z (GMT). No. of bitstreams: 1 eduardo bentes.pdf: 1682417 bytes, checksum: be32237b0a1e23a7f3225f1419caf484 (MD5) Previous issue date: 2012-05-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis complex species, which infect the exposed individuals, initiate an immune response with production of several cytokines, among them interferon-gamma (IFN-gamma), which is crucial for activation of cellular immune response against TB bacillus. This cytokine has been extensively studied as a possible biomarker for diagnosis of TB. In this work, we analyzed the levels of IFN-gamma in response to in vitro stimulation with ESAT-6, CFP10 and TB7.7, by QuantiFERON ®-TB Gold (In-tube method) (QTF-IT) in 155 participants: 47 patients confirmed pulmonary TB (patient group), 49 direct contacts (group contact), and 59 individuals without history or contact with TB (control group). The levels of IFN-gamma were different among the groups (p=0.0001); the highest level was observed in the patients group (median=1.43 IU/mL). In contacts group, 20/49 (40.8%) subjects were positive for the QTF-IT (median = 0.26 IU / mL), and among these positive contacts 14/20 (70.0%) exhibited high levels of IFN-gamma, (≥ 1.05 IU/mL). The performance testing of TB patients versus controls, resulted in 80.9% sensitivity (95% CI = 69.6% to 92.1%) and 93.2% specificity (95% CI = 86.8 % to 99.6%), but when the results of contacts and control group were combined, the specificity decreased to 77.8% (95% CI = 69.9% to 85.6%). Thus, according to our data, there is a distinctive profile of IFN-gamma, with higher levels in the patients group. The QTF-IT positive in contacts, especially those with high levels of IFN-gamma, should be monitored, especially those with high levels of IFN-gamma. This result may be a risk factor for developing TB disease. In addition, the QTF-IT, properly executed, can be useful in endemic areas for the diagnosis of latent and active TB (infection) when evaluated with other routine tests. / A Tuberculose (TB) é uma doença infecciosa causada por espécies do complexo Mycobacterium tuberculosis, que ao infectar os indivíduos expostos, iniciam uma resposta imunológica com produção de várias citocinas, dentre elas o Interferon-gama (IFN-gama), que é crucial para ativação da resposta imune celular contra o bacilo da TB. Esta citocina tem sido amplamente estudada como um possível biomarcador para auxiliar no diagnóstico da TB. Neste trabalho foram analisados as concentrações de IFN-gama em resposta a estímulos in vitro com ESAT-6, CFP10 e TB7.7, utilizando o QuantiFERON®-TB Gold (In-tube method) (QTF-IT), em 155 participantes, sendo: 47 pacientes com TB pulmonar confirmada (grupo paciente), 49 contatos de pacientes com TB pulmonar (grupo contato), e 59 indivíduos sem história e/ou contato com TB (grupo controle). As concentrações de IFN-gama foram diferentes entre os grupos avaliados (p=0,0001), sendo mais elevado nos pacientes com TB (Mediana = 1.43 UI/mL). Dos contatos diretos, 20/49 (40,8%) foram positivos para o QTF-IT (Mediana=0,26 UI/mL), e destes 14/20 (70,0%) apresentaram níveis elevados de IFN-gama, superior à 1,05 UI/mL. O desempenho do teste relacionando os pacientes de TB como os doentes e os controles como sadios, resultou em 80,9% de sensibilidade (IC 95% = 69,6% a 92,1%) e 93,2% de especificidade (IC 95% = 86,8% a 99,6%), mas quando os resultados dos contatos diretos foram adicionados aos controles, a especificidade reduziu para 77,8% (IC 95% = 69,9% a 85,6%). Concluiu-se que há um perfil diferenciado na produção de IFN-gama, com níveis mais elevados nos pacientes de TB ativa, e que os contatos positivos para o QTF-IT, principalmente, aqueles com altas concentrações de IFN-gama, devem ser monitorados, considerando que o perfil ou concentração elevado desta citocina, pode ser um fator de risco para o desenvolvimento da TB doença. Além disso, o QTF-IT, executado corretamente, pode ser útil em áreas endêmicas para auxiliar no diagnóstico da TB latente (infecção) e ativa, quando avaliados com outros exames de rotina.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative incurable, qui touche les motoneurones de la moelle épinière et du cerveau. Elle se manifeste par une faiblesse musculaire qui évolue rapidement vers une paralysie générale, entrainant la mort du patient. Les principes moléculaires conduisant à la dégénérescence sélective des motoneurones demeurent encore mal connus, entravant le développement de nouvelles thérapies. Mon travail de thèse a permis l'identification d'une nouvelle voie de mort spécifique aux motoneurones, qui dépend du récepteur LT-βR et de son ligand LIGHT. De plus, cette voie de mort peut être déclenchée par une cytokine pro-inflammatoire, qui est l'interféron gamma (IFNγ). Nous avons pu montrer des signes d'activation de cette voie de mort chez des souris modèles de la SLA ainsi que dans les tissus de patients atteints de la maladie. En effet, on observe au cours de la maladie une augmentation des niveaux d'IFNγ dans les astrocytes et les motoneurones. Une approche génétique a par la suite permis de démontrer l'implication fonctionnelle de cette voie de mort dans le processus pathologique. / Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease affecting primarily motoneurons in the brain and spinal cord. Symptoms of the disease include general muscle weakness, rapidly evolving in an overall paralysis, leading to the death of the patient. The precise mechanisms responsible for the selective vulnerability of motoneurons remain largely unknown, impeding therefore the development of effective therapies. My thesis work led to the discovery of a novel motoneuron selective death pathway dependent on the activation of LT-βR by LIGHT. This death pathway might also be triggered by the pro-inflammatory cytokine interferon gamma (IFNγ). Interestingly, we have documented signs of activation of this pathway in ALS mice and sporadic ALS patients, with IFNγ being upregulated in astrocytes and motoneurons. Furthermore, a genetic approach has provided evidence of the functional involvement of this death pathway in the pathogenic process.

Lung cancer is one of the main causes of death worldwide, with most patients suffering from an advanced unresectable or metastatic non-small cell lung cancer. The mortality trends are mostly related to patterns of tobacco use, specifically from cigarettes. Tobacco is the basic etiological agent found as a consequence of the inhalation of tobacco smoke. Published data show the use of interferons (IFNs) in the treatment of lung tumours due to their potential in displaying antiproliferative, anti-angiogenic, immunoregulatory, and proapoptotic effects. Type1 IFNs have been employed as treatments for many types of cancer, both for haematological cancers and solid tumours. The IFN-γ (naked) functions as an anticancer agent against various forms of cancer. Hence, this study aimed to investigate the genoprotective and genotoxic effects of IFN-γ liposome (nano) on 42 blood samples from lung cancer patients, compared to the same sample size from healthy individuals. The effectiveness of IFN- γ liposome against oxidative stress was also evaluated in this study. A concentration of 100U/ml of IFN-γ liposome was used to treat the lymphocytes in: Comet and micronucleus assays, Comet repair, Western blotting and real-time polymerase chain reaction (qPCR) were based on a preliminary test for the optimal dose. The lymphocytes from lung cancer patients presented with higher DNA damage levels than those of healthy individuals. IFN-γ liposome was not found to induce any DNA damage in the lymphocytes. Also, it caused a significant reduction in DNA damage in the lymphocytes from lung cancer patients in; Comet, Comet repair and micronucleus assays. Furthermore, the 100U/ml of IFN-γ liposome significantly reduced the oxidative stress caused by H2O2 and appeared to be effective in both groups using the Comet and micronucleus assays. Results from; Comet, Comet repair and micronucleus assays were consistent. The data obtained indicated that IFN-γ in both forms (naked INF-γ and INF-γ nano-liposome) may potentially be effective for the treatment of lung cancer and showed the ability of IFN-γ liposome to reduce the DNA damage more than the naked form. The IFN-γ in both forms has also shown anti-cancer potential in the lymphocytes from lung cancer patients by regulating the expression of p53, p21, Bcl-2 at mRNA and protein levels by up-regulating the p53 and p21 to mediate cell cycle arrest and DNA repair in lung cancer patients. The findings of this study are consistent with the view that the naked IFN-γ and liposome could have a significant role in cancer treatment, including lung cancer. / Mutah University in Jordan

Das 26S Proteasom ist ein großer Proteinase-Komplex, der aus 32 unterschiedlichen Untereinheiten aufgebaut ist. Das 26S Proteasom ist involviert in die ATP-abhängige De-gradation von ubiquitinierten Proteinen, die eine Vielfalt an zellulären Prozessen wie Signaltransduktion, Stressantwort, transkriptionelle Regulation, Chromosomen-Segregation, DNA-Reparatur, Zellzyklus-Steuerung und die Prozessierung von Peptiden für die MHC I Antigen Präsentation regulieren. Die Prozessierung von Peptiden wird ver-stärkt durch eine Interferon ? stimulierbare Variante des Proteasoms übernommen, dem so genannten Immunoproteasom. Die Biogenese dieses großen Komplexes ist ein komplizierter Mechanismus, welcher Expression und Assemblierung der proteasomalen Untereinheiten beinhaltet. In Eukaryonten sind für die Assemblierung und Maturierungsprozesse Helferproteine notwendig. In Mammalia übernimmt diese Funktion das Proteasom maturation Protein POMP. POMP ist wahrscheinlich auch bei der Biogenese des Immunoproteasoms von Bedeutung, da die mRNA von POMP durch Interferon ? induziert wird. Um die Regulation dieser Induktion zu untersuchen wurde der Promotor von POMP für die erste Fragestel-lung dieser Arbeit charakterisiert und seine Induzierbarkeit durch Interferon ? untersucht. Es konnte nachgewiesen werden, dass die erhöhte mRNA-Menge durch Interferon ?-Stimulation nicht auf eine Promotor-Induktion, sondern auf post-transkriptionelle Ereig-nisse zurückzuführen ist. In der zweiten Fragestellung dieser Arbeit sollte die Genregulation des Proteasoms unter Stressbedingungen untersucht werden. Der Stress wurde durch Inhibition der proteolyti-schen Aktivität des Proteasoms ausgelöst. Wie seit längerem bekannt ist, werden in Bakterien und Hefe die ATP-abhängigen Pro-teasekomplexe über ein kompliziertes regulatorisches Netzwerk gesteuert. Über die transkriptionelle Regulation des Mammalia Proteasoms war bisher wenig bekannt. Im Rahmen der hier vorliegenden Dissertation konnte gezeigt werden, dass die Reduktion der proteolytischen Aktivität des Proteasoms durch Behandlung von Mammalia-Zellen mit Proteasom-Inhibitoren durch eine gesteigerte Genexpression der proteasomalen Unter-einheiten kompensiert wird. Alle proteasomalen Untereinheiten werden konzertiert hoch-reguliert. Exemplarisch an der proteasomalen Untereinheit Rpt1(S7) und an dem Matu-rierungsfaktor POMP konnte eine posttranskriptionelle Regulation unter Proteasom-Inhibitor Einfluss ausgeschlossen werden. Die vom Inhibitor induzierte Genaktivierung resultiert in einer de novo Protein-Synthese und führt daher zu einer gesteigerten de no-vo Biogenese des Proteasoms. Dieses Phänomen ist begleitet durch eine vermehrte Ex-pression vom POMP. Damit konnte erstmals gezeigt werden, dass die Menge an Protea-som in Mammalia auf transkriptioneller Ebene reguliert wird und dass vermutlich ein au-toregulatorischer feedback-Mechanismus eine verminderte proteolytische Aktivität kom-pensieren kann. Diese Daten werden durch Ergebnisse der CAT-Reportergen-Assays des ?1(?)-Promotors gestützt. Exemplarisch konnte gezeigt werden, dass die Aktivität dieses Promotors in Anwesenheit von Proteasom-Inhibitoren ansteigt. Die induzierbare Promotorregion konnte bis auf 130 bp eingegrenzt werden. Innerhalb dieser Promotorse-quenz konnte die Bindung eines Transkriptionsfaktors (Nrf2) durch EMSA-Technik nach-gewiesen werden. / The 26 S proteasome is a high molecular mass proteinase complex that is built by of least 32 different protein subunits. The 26S proteasome is involved in the ATP-dependent degradation of ubiquitinated proteins that regulate a variety of cellular proc-esses including signal transduction, stress response, transcriptional control, chromosome segregation, DNA repair, cell cycle progression and processing of antigenic Peptides for the MHC I pathway. Biogenesis of this large complex is a complicated process compris-ing expression, assembly and maturation of all subunits. This crucial step is supported by POMP (proteasome maturation protein). POMP mRNA is induced by Interferon gamma (IFN ?). We investigated this phenomenon via Reportergen assays with the Promoter re-gion of POMP. POMP mRNA seems not to be regulated on a trancriptional level, but on posttranscriptional events. ATP-dependent protease complexes in bacteria and yeast are systems that undergo a highly sophisticated network of gene expression regulation. However, regulation of mammalian proteasome gene expression has been neglected so far as a possible control mechanism for the amount of proteasomes in the cell. We showed that treatment of cells with proteasome inhibitors and the concomitant impairment of proteasomal enzyme activ-ity induce a transient and concerted up-regulation of all mammalian 26S proteasome subunit mRNAs. Proteasome inhibition in combination with inhibition of transcription re-vealed that the observed up-regulation is mediated by coordinated transcriptional activa-tion of the proteasome genes and not by post-transcriptional events. Our experiments also demonstrate that inhibitor-induced proteasome gene activation results in enhanced de novo protein synthesis of all subunits and in increased de novo formation of the pro-teasome. This phenomenon is accompanied by enhanced expression of the proteasome maturation factor POMP. Thus, our experiments present first evidence that the amount of proteasomes in mammalia is regulated at the transcriptional level and that an auto regu-latory feedback mechanism exists that allows the compensation of reduced proteasome activity. These data are also supported by CAT reportergene assays with the protea-somal subunit ?1(?)-promoter. Exemplary we show the increase of CAT activities in re-sponse to proteasome inhibition. We can restrict the region of the promoter to 130 bp and identify Nrf2 as a possible candidate for a transcription factor via EMSA.

Mit durchflusszytometrischen und molekularbiologischen Verfahren wurden verschiedene Subpopulationen peripherer T-Lymphozyten bei gesunden Spendern und Nierentransplantatempfängern phänotypisch und funktionell untersucht. Wir fanden, dass eine T-Zell-Population, welche unter anderem die Oberflächenmarker LFA-1 und CD57 exprimierte, nicht aber CD28, terminale Effektorzellen enthielt. Wegen der bekannten Assoziation der Expansion CD57-positiver CD8-positiver T-Zellen mit einer Infektion mit dem Zytomegalievirus (CMV), wollten wir untersuchen, ob auch CMV-spezifische Effektorzellen darin enthalten waren. Um diesen möglichen Zusammenhang zwischen Phänotyp und Spezifität zu untersuchen, wurde ein bekanntes durchflusszytometrisches Verfahren zur Erfassung antigen-spezifischer CD4-T-Zellen so modifiziert, dass damit auch antigen-spezifische CD8-T-Zellen erfasst werden konnten. Dazu wurden frisch isolierte periphere mononukleäre Zellen in vitro mit löslichen Peptiden oder Peptidgemischen inkubiert (anstelle von Erregerlysaten oder Proteinantigenen wie im bekannten Verfahren), so dass durch direkte externe Beladung von MHC-I- und MHC-II- Molekülen nicht nur CD4- sondern auch CD8-T-Zellen stimuliert wurden. Anschließend konnten CD4 - und/oder CD8-positive T-Zellen nachgewiesen werden, in welchen es zur Synthese von Interferon-gamma gekommen war (intrazelluläre Färbung), wodurch diese Zellen als antigen-spezifisch identifiziert wurden. Diese Zellen konnten dann weiter phänotypisch analysiert werden. Unter Verwendung bekannter CD8-T-Zellen-stimulierender CMV-Peptide konnte der Phänotyp CMV-spezifischer CD8-T-Zellen untersucht werden, wobei sich zeigte, dass das CD57-positive Subset tatsächlich den gößeren Teil der CMV-spezifischen CD8 T-Zellen enthielt. Andererseits konnte das neue Verfahren verwendet werden, um weitere Peptide zu identifizieren, welche eine T-Zellstimulation bewirkten (Epitopkartierung). In zwei von uns untersuchten Proteinen des CMV (pp65 und IE-1) wurden so mehrere neue CD4- und CD8-T-Zellepitope beschrieben. Die Vewendung komplexer Peptidgemische erlaubte darüber hinaus die Untersuchung der T-Zellantwort gegen ganze Proteine (repräsentiert durch die Gesamtheit aller denkbaren Epitope), was insbesondere für die Untersuchung von CD8-T-Zellen eine große Bereicherung darstellte und vom MHC-Typ unabhängig war. Wir verwendeten dieses neue Verfahren bisher zur Analyse und zum Monitoring der T-Zellantwort gegen bestimmte Erregerproteine oder -peptide (z.B. aus CMV oder HIV). Es eignet sich darüber hinaus auch zur Untersuchung der Immunantwort gegen Impfstoffe, welche zur Induktion von T-Zellen führen sollen. / Using flow-cytometric and molecular-biology methods subpopulations of peripheral blood T-lymphocytes were examined in healthy donors and renal transplant recipients with respect to phenotype and function. We found that a T-cell population that expressed the surface markers LFA-1 and CD57 (among others), but not CD28, contained terminal effector cells. Because of the known association of an expansion of CD57-positive CD8-positive T-cells with an infection with Cytomegalovirus (CMV), we wanted to examine if CMV-specific effector cells were also contained in this subset. In order to investigate this association between phenotype and specificity, we modified a known flow-cytometric method for the detection of antigen-specific CD4 T-cells in such a way that antigen-specific CD8 T-cells could also be detected. For this purpose freshly isolated mononuclear cells were incubated in vitro with soluble peptides or peptide mixes (instead of pathogen lysates or protein antigens as used in the original method) so that following direct external loading of MHC-I and MHC-II molecules not only CD4 T-cells but also CD8-T-cells were stimulated. Subsequently, CD4 and/or CD8 T-cells that had synthesized Interferon-gamma (intracellular staining), which identified them as being antigen-specific, could be detected. These cells could then be analyzed with regard to phenotype. Using known CD8-T-cell stimulating CMV-peptides, the phenotype of CMV-specific CD8 T-cells could be analyzed. Thus it was demonstrated that the majority of CMV-specific CD8 T-cells was indeed contained in the CD57-positive subset. On the other hand, this new approach allowed the identification of additional peptides that stimulated T-cells (epitope mapping). In two CMV-proteins that we examined (pp65 and IE-1) several new CD4 and CD8-T-cell epitopes were described. The use of complex peptide mixes in this approach allowed the analysis of T-cell responses to complete proteins (represented by the entirety of all possible epitopes), which was a great benefit to the analysis of CD8 T-cells and independent of MHC-type. Until now, we have used this new method for the analysis and the monitoring of the T-cell response to specific pathogen proteins or peptides (e.g. from CMV or HIV). It is suitable, moreover, for the analysis of the immune response to vaccinations that aim at the induction of T-cells.

Hintergrund: Um akute Rejektionen nach Nierentransplantation früh erkennen und behandeln zu können, gleichzeitig die Nebenwirkungen einer immunsuppressiven Therapie zu minimieren, wäre die Etablierung eines “Immunmonitorings”, welches zu jedem Zeitpunkt Hinweise auf die Aktivierung des Immunsystems des Empfängers gegen das Transplantat gibt, wünschenswert. Methodik: In dieser Studie wurden die Anzahl der spenderreaktiven, IFNgamma-produzierenden T-Zellen von 52 nierentransplantierten Patienten zu verschiedenen Zeitpunkten vor (prä-TX) und nach Transplantation (post-TX) im ELISpot-Assay gemessen und in Zusammenhang mit der klinischen Entwicklung gebracht. Außerdem wurde das Assay auf Reproduzierbarkeit untersucht und versucht zu optimieren. Ergebnisse: Eine stark erhöhte Anzahl spenderreaktiver Zellen prä-TX (>200 IFNgamma-spots/3*100000 PBMZ, n = 5) war immer mit einer akuten Rejektion des Transplantats assoziiert. Post-TX korrelierte die Anzahl der spenderreaktiven, IFNgamma-produzierenden Zellen mit der Nierenfunktion ein Jahr nach Transplantation. Diese Korrelation wurde in den Wochen 2 und 3 post-TX und bei Patienten ohne akute Rejektion, besonders deutlich. Hinsichtlich der methodischen Optimierung hat sich die magnetische Depletion CD2pos-Zellen als effektiv gezeigt, die IFNgamma-Sekretion von Stimulatorzellen zu unterbinden. Um die Reproduzierbarkeit des Assays zu verbessern sollten Stimulatorzellen im Überschuss und Empfänger-T-Zellen in einer konstanten Anzahl eingesetzt werden. Dabei sollte die Gesamtzellzahl über 1000000 Zellen/ml betragen. Conclusion: Das ELISpot-Assay ist zur Erkennung klinisch relevanter T-Zellsensibilisierungen vor und nach Transplantation geeignet. Vor einem Einsatz in der klinischen Routine sollten jedoch einige methodische Verbesserungen vorgenommen werden. / Background: In order to perform early diagnosis and treatment of acute rejections after renal transplantation while minimizing side effects of immunosuppression, an immune monitoring tool is needed, which gives information on the activation state of the immune system of the transplant recipient against the allograft at any given time. Methods: In this study, frequencies of donor-reactive, IFNgamma-producing T cells where measured in 52 renal transplant recipients at different time points before (pre-TX) and after transplantation (post-TX) using the ELISPOT-assay. The frequencies were correlated with clinical outcome. Also, the reproducibility of the assay and possibilities of optimization were tested. Results: Highly elevated frequencies of donor-reactive cells pre-TX (>200 IFNgamma-spots/3*100000 PBMC´s, n = 5) were always associated with acute rejection episodes after transplantation. Post-TX frequencies of donor-reactive, IFNgamma-producing cells correlated significantly with graft function one year post-TX. This correlation was strongest for frequencies in week 2 and 3 post-TX and in patients without acute rejection. Regarding the methodical optimization, magnetic CD2pos-cell depletion of donor leucocytes proved useful to inhibit IFNgamma secretion of stimulating cells. To improve reproducibility of the assay stimulating cells should be used as a surplus, a constant number of responding T cells should be chosen, and overall cell concentration should exceed 1000000 cells/ml. Conclusion: The ELISPOT-assay is a useful tool to detect clinically relevant T cell sensibilisation pre- and post-TX. Before it is routinely used some methodical alterations must be performed.