Die Lebertransplantat-Spontantoleranz ist eines der immunologisch beeindruckensten Phänomene. Die Fähigkeit, Toleranz für sich selbst und auch für mittransplantierte Organe über die MHC Barriere hinweg zu induzieren, ist einzigartig. Dies steht im Gegensatz zum zentralen Dogma der Transplantationsimmunologie, wonach MHC differente Organe nach Transplantation ohne Immunsuppression irreversibel zerstört werden. Ziel der Arbeit war, die verschiedenen intrahepatischen CD4+ T-Lymphozyten durchflusszytometrisch zu charakterisieren und eine in vitro Aktivierung naiver Leukozyten mit der in vivo zu vergleichen. Dies wurde mit der Fragestellung verknüpft, ob es einen möglichen Phänotyp regulatorischer intrahepatischer T-Lymphozyten gibt, der für die Induktion von Spontantoleranz verantwortlich ist. Auch wurde das Zytokinmuster dieser intrahepatischen T-Lymphozyten bestimmt, um eine mögliche funktionelle Aussage treffen zu können. Für die Versuche wurden Lebertransplantationen von Lewis-Spendertieren (LEW) auf Dark Agouti (DA) Empfänger durchgeführt. Die Transplantate wurden dauerhaft (> 300 Tage p.op.) spontan, d.h. ohne Immunsuppression toleriert. Andere vaskularisierte Organe, wie z.B. Herzen, wurden von DA-Empfängern abgestoßen. Die intrahepatischen und Milz T-Lymphoyzten wurden an den Tagen 3, 30 und 100 nach Transplantation untersucht. Unmittelbar nach Lebertransplantation kam es zu einem massiven Anstieg der intrahepatischen Leukozyten. Das Leberinfiltrat wurde dabei von aktivierten CD4+ T-Lymphozyten dominiert (ca. 23 Prozent CD4+CD45RCneg T-Lymphozyten), von denen ca. 50 Prozent den IL-2 Rezeptor exprimierten. Als Zeichen einer intrahepatischen Immunantwort wurde gewertet, dass das Verhältnis aktivierter CD4+CD45RCneg zu ruhenden CD4+CD45RCpos T-Lymphozyten dynamisch war. Zur funktionellen Charakterisierung der verschiedenen Populationen an CD4+ T-Lymphozyten wurden die Zytokinmuster dieser Zellen mit zwei verschiedenen Verfahren bestimmt. Zum einen wurden die sezernierten Zytokine INF-gamma, TNF-alpha, IL-10 und IL-13, zum anderen deren spezifische mRNAs semiquantitativ nachgewiesen. Für die sezernierten Zytokine waren keine wesentlichen Unterschiede zwischen den beiden Subpopulationen nachzuweisen. Bei der Analyse der mRNA-Transkription zeigte sich jedoch, dass die CD4+CD45RCneg T-Lymphozyten stärker die Th2-Zytokine IL-10 und IL-13 exprimierten, während die CD4+CD45RCpos T-Lymphozyten stärker die Th1-Zytokine INF-gamma, TNF-alpha exprimierten. In dieser Arbeit wurde zudem gezeigt, dass die CD4+CD45RCpos und CD4+CD45RCneg T-Lymphozyten jeweils heterogene Zellpopulationen sind, die noch in CD4posCD45RCpos, CD4hochposCD45RChochpos, CD4posCD45RCneg und CD4hochposCD45RCneg T-Lymphozyten differenziert werden können. Die CD4hochposCD45RChochpos repräsentieren dabei mit großer Wahrscheinlichkeit naive T-Lymphozyten, wohingegen die CD4hochposCD45RCneg aktivierte T-Lymphozyten darstellen. Wir gehen davon aus, dass die CD4posCD45RCpos T-Lymphozyten auch ruhende Gedächtniszellen und die CD4posCD45RCneg T-Lymphozyten auch aktivierte Gedächtniszellen enthalten. Der Nachweis intrahepatischer Gedächtnis T-Lymphoyzten zeigt, dass die Leber immunologisch ein überaus interessantes Organ repräsentiert. Ihre Fähigkeit zur Induktion von Spontantoleranz ist einzigartig, so dass von diesem Organ weitere grundlegende Erkenntnisse zum Phänomen der Toleranz erwartet werden. / Summary of Paul Schmitz’s thesis with the title “Mechanisms of immunological tolerance after liver transplantation: Analysis of the cytokine pattern of intrahepatic CD4+ CD45pos and CD4+ CD45RCneg T lymphocytes” Without the requirement for immunosuppression liver allografts of some rat strain combinations are spontaneously accepted despite a complete mismatch of molecules of the major histoincompatibility complex (MHC). Furthermore, liver grafts may induce immunological tolerance to a subsequent heart, kidney or skin graft from the same donor. Without liver grafts these organs are destroyed by the recipient’s immune system. The survival of liver grafts is, however, not related to a failure of the recipient to mount an immunological response against donor MHC molecules. An immune-mediated damage of the transplanted livers with an accompanying infiltrate and up-regulation of pro-inflammatory cytokines is temporarily observed. This is in contrast to the well-established maxim of transplantation immunology that rejection will occur when donor and recipient transplantation antigens are mismatched, especially those encoded by MHC. The liver contains a large population of resident and migratory T lymphocytes. Since T lymphocytes mediate both rejection and tolerance, the aim of the study was to phenotype intrahepatic CD4+ T lymphocytes by flow cytometry. In addition, secreted cytokines as well as cytokine-specific mRNA were analysed. With these investigations we wanted to identify an intrahepatic T cell subpopulation with regulatory properties. For the orthotopic liver transplantation we used rats of the inbred strains Lewis, as donors, and Dark Agouti, as recipients. The liver grafts in this strain combination survived permanently (>300 days), although heart grafts from the same donor were rejected. T lymphocytes isolated from liver grafts and spleens were analysed on days 3, 30, and 100 after transplantation. The amount of immune cells from the recipient increased dramatically in liver allografts within 3 days after transplantation. With approximately 23% activated CD4+ CD45RCneg dominated this infiltrate and the ratio of activated CD4+CD45RCneg and rested CD4+CD45RCpos increased. This observation was interpreted as an intrahepatic immune response mediated by infiltrated immune cells. The presence of the cytokines INF-gamma, TNF-alpha, IL-10 und IL-13 was analysed in the supernatant of isolated intrahepatic T incubated for 3 days in vitro. Cytokine-specific mRNA was semiquantified by ELISA-PCR. The supernatants did not demonstrate any differences between both subpopulations. In contrast, the results of the ELISA-PCR revealed increased mRNA levels of the Th2 cytokines IL-10 and IL-13 for CD4+CD45RCneg T lymphocytes and increased levels of the Th1 cytokines INF-gamma and TNF-alpha for CD4+CD45RCpos T lymphocytes. In the present study it was also shown that both populations of CD4+CD45RCpos (rested) and CD4+CD45RCneg (activated) T lymphocytes were heterogeneous and divisible into CD4posCD45RCpos, CD4strong pos CD45RCstrong pos and CD4pos CD45RCneg, CD4strong posCD45RCneg T cells by flow cytometry. The CD4strong pos CD45RCstrong pos T cells may also represent naïve T cells, whereas the CD4strong pos CD45RCneg may be a certain type of activated T cells. We have evidence that rested memory T lymphocytes are located within the population of CD4pos CD45RCpos T lymphocytes and activated memory T lymphocytes are present within the population of CD4pos CD45RCneg T lymphocytes. The proof of intrahepatic memory T cells indicates that the liver represents an immunologically important organ. Its ability to induce spontaneous tolerance in many fully mismatched donor recipient combinations is unique and additional studies are necessary to further explore the true potential of intrahepatic T cells for tolerance induction.

In dieser Arbeit wurde die Zytolyse, die durch HIV-spezifische zytotoxische T-Lymphozyten (CTL) bewirkt wird, untersucht. Grundsätzlich erscheinen in der Abwehr viraler Infekte sowohl CD4+ wie CD8+ CTL. In chronisch HIV-Infizierten sind in der Regel lediglich CD8+ CTL zu finden. HIV-spezifische CD4+ CTL können aber zum Beispiel in Impfstudien aus Probandenblut isoliert werden. Sie erwiesen sich als zytolytisch aktiv und waren außerdem, wie in dieser Arbeit erstmals gezeigt werden konnte, auch an einer Fusions-vermittelten Lyse beteiligt. Diese ist Kalzium-unabhängig, auch mit heterologen Zellen möglich und die Expression des HIV-gp120 an der Zelloberfläche der Zielzellen ist essentiell, während die Präsentation von gp120 Epitopen nicht ausreicht. Die Konsequenz dieser Lyseform ist die apoptotische Zerstörung aller beteiligten Zellen. CD4+ HIV-spezifische CTL können über diesen Lysemechanismus bereits in der akuten Phase der Infektion einer negativen Selektion unterliegen, was ein Grund für das Fehlen der HIV-spezifischen CD4+ CTL in chronisch Infizierten darstellen kann. HIV-spezifische CD8+ CTL konnten aus einem HIV-positiven Patienten isoliert werden. Insgesamt wurden fünf Klone näher charakterisiert. Zwei erkannten jeweils ein Epitop aus unterschiedlichen Bereichen des extrazellulären Anteils des Glykoproteins, einer einen Bereich des p17-Anteils und zwei dasselbe Epitop im p24-Anteil des gag-Proteins. Dieses Epitop, plaziert innerhalb der Aminosäuren 71 bis 85, konnte hier zum erstenmal für die Präsentation mit HLA B51 beschrieben werden. In der spezifisch induzierten Lyse wiesen alle CD8+ CTL-Klone zwei Lysemechanismen auf, einen Perforin-vermittelten und einen CD95-vermittelten Anteil. Die CD95-vermittelte Lyse war stets prozentual geringer, und zusätzlich, sowohl Klon- wie Zielzell-spezifisch, unterschiedlich stark ausgeprägt . In HIV-infizierten primären Zellen ließ sich kein CD95-Anteil signifikant nachweisen, was darauf hinweist, daß dieser Lysemechanismus wahrscheinlich keine Rolle bei der Zerstörung infizierter Zellen im peripheren Blut spielt. In anderen Geweben kann sich die Situation unterschiedlich darstellen. Desweiteren konnte in dieser Arbeit nachgewiesen werden, daß das Vakzinia-Virus B13R-Genprodukt die CD95-vermittelte Apoptose hemmt. Daher muß bei der Untersuchung apoptotischer Vorgänge, die Wahl der eingesetzten Vektoren genauestens bedacht werden. / In this work the HIV-specific cytotoxic T-lymphocyte (CTL) mediated cytolysis was investigated. Normally CD4+ and CD8+ CTL are generated to defend viral infections. In chronically infected HIV-patients generally only CD8+ CTL are detectable. But HIV-specific CD4+ CTL can be isolated fram blood of HIV-vaccine candidates, for example. These CTL were cytolytic active and, as shown in this work for the first time, were also involved in fusion-mediated lyses. This lyses is independent of extracellular calcium, works with heterologous cells and the expression of HIV-gp 120 at the cell surface is essential. A presentation of gp 120 epitopes by HLA is not sufficient. In consequence this form of lysis is destructive in an apoptotic manner for all participating cells. CD4+ HIV-specific CTL could be negative selected by this mechanism in the early stage of infection. This could be a reason for the absence of HIV-specific CD4+ CTL in chronically infected patients. HIV-specific CD8+ CTL were isolated from one patient. Five were characterized in more detail. Two of them recognized an epitope in different parts of the extracellular region of the glycoprotein, one discerned the p17-protein and two the same epitope of the p24-part of the gag-protein. This epitope, localized between amino acids 71 to 85, is here reported for the fist time for presentation with HLA B51. In specific induced lyses all CD8+ CTL-clones presented two lyses-mechanisms, a perforin-mediated and a CD95-mediated portion. The CD95-mediated lyses always showed lower percentages and differed dependant on the CTL-clone and the target-cells in its markedness. For HIV-infected primary cells no significant CD95-mediated lyses was detectable, leading to that there is no role for this lyses mechanism in destruction of infected cells in peripheral blood. In other tissues the situation can differ. In this work, furthermore the inhibition of CD95-mediated apoptosis by the Vaccinia Virus B13R gene product was proven. Therefore, the vectors used in investigations of apoptosis has to be chosen carefully.

The evolutionary distance of lampreys from humans in conjunction with their distinct antibody architecture is profound. Thus, lampreys may provide antibodies with specificity for antigens unrecognized by conventional mammalian antibodies. This study investigates lamprey based monoclonal variable lymphocyte receptor antibodies (VLRs) for purifying and identifying an antigen by tandem mass spectrometry. VLRs specific for clinically relevant cell populations were isolated. Subsequently, utilizing intrinsic VLR affinity, with or without covalent cross-linking molecules, for immunoprecipitating VLR protein antigens was tested. In one case, CD5 glycoprotein from Jurkat T cells was purified by a VLR; the antigen was identified by tandem mass spectrometry. Antibody specificity was validated by western blotting and flow cytometry. Furthermore, VLR binding to CD5 required multimerization of the antibody, indicating the individual VLR units likely bind antigen with low affinity. The study provides ‘proof of concept’ for human biomarker identification using novel lamprey monoclonal antibodies.

The evolutionary distance of lampreys from humans in conjunction with their distinct antibody architecture is profound. Thus, lampreys may provide antibodies with specificity for antigens unrecognized by conventional mammalian antibodies. This study investigates lamprey based monoclonal variable lymphocyte receptor antibodies (VLRs) for purifying and identifying an antigen by tandem mass spectrometry. VLRs specific for clinically relevant cell populations were isolated. Subsequently, utilizing intrinsic VLR affinity, with or without covalent cross-linking molecules, for immunoprecipitating VLR protein antigens was tested. In one case, CD5 glycoprotein from Jurkat T cells was purified by a VLR; the antigen was identified by tandem mass spectrometry. Antibody specificity was validated by western blotting and flow cytometry. Furthermore, VLR binding to CD5 required multimerization of the antibody, indicating the individual VLR units likely bind antigen with low affinity. The study provides ‘proof of concept’ for human biomarker identification using novel lamprey monoclonal antibodies.

Cancerassocierade antigen (CA) används vid diagnostisering, prognosbestämning och för att följa behandling vid cancer. Exempel på CA är CA 15–3, CA 19–9 och CA 125. Syftet med arbetet var att undersöka hållbarheten för blodprover förvarade i primärrör som skulle analyseras för CA 15–3, CA 19–9 och CA 125 och jämföra med nuvarande metod på klinisk kemi, Oskarshamn, Region Kalmar län. Enligt nuvarande metod överförs plasman till nya provrör innan analys. Metoden som användes var att dubbelprov togs och det ena förvarades i primärrör och analyserades på Roche Cobas 411® tre, fem och tio dagar efter provtagning. Det andra provet överfördes till nytt provrör inför analys. Den procentuella skillnaden mellan analys av CA i primärrör och överfört prov beräknades. För alla tre analyserna var den procentuella skillnaden störst hos de lägsta koncentrationerna av CA. Medelvärde för analyserna av CA, standardavvikelse (SD) och variationskoefficient beräknades. SD för CA jämfördes mot kontrollerna MAS Omni Immune Pro 1 och MAS Omni Immune Pro 3s SD. Provernas SD var mindre för kontrollerna förutom för CA 19–9 >1100 kIE/L. I den gruppen analyserades dock för få analyser för att utföra statistisk analys och gav därmed ingen slutsats hur CA 19–9 påverkades av de olika förvaringarna. För övriga analyser och för CA 19–9 i lägre koncentrationer kunde slutsatsen dras att prover kunde förvaras i primärrör i tio dagar utan att analysresultatet påverkades. / Cancer-associated antigens (CA) are used for diagnosis, establish prognosis and to monitor treatment of cancer. Three CA are CA 15-3, CA 19-9 and CA 125. The aim of this study was to evaluate the stability of CA 15-3, CA 19-9 and CA 125 blood samples in primary tubes coated with Litium-heparin gel and compare the results with the method used at Klinisk Kemi Oskarshamn, Region Kalmar where the samples today are transferred to new tubes before analysis. In this study two samples were collected from each patient, one was stored and analyzed according to the current method. The other sample was stored in the primary tube and analyzed three, five and ten days after collection. Roche Cobas 411 instrument was used for analysis. The mean value for the samples in primary tubes was compared to the current method and were calculated in percent. The difference was more distinct when the concentration of the analyte was low. The mean value, standard deviation (SD) and variation coefficient were calculated for each analyte. The SD of the samples were compared to the SD of the controls IM 1 and IM3. All the obtained SD from the samples were lower than the control SD, except for CA 19-9 > 1100 kIE/L. Unfortunately, there were only three test results in the concentration interval and therefore no statistic conclusion could be made. The conclusion for the other analytes CA 15-3, CA 125 and CA 19-9 in lower concentrations was that the storage in primary tubed up to ten days did not affect the test results.

The aim of my project was to examine the extent to which endogenous expression of a largely renal-specific antigen influences the repertoire in adulthood of autoreactive T cells specific to that antigen. The renal-specific antigen, human α3(IV)NC1, is the target of autoimmune attack in Goodpasture’s disease. This protein was expressed and purified in recombinant (using bacterial and mammalian cell expression systems) and purified in native (extracted from human tissue) forms. Transgenic mice were generated that express HLA-DR15 (associated with Goodpasture’s disease) as their sole MHC class II molecule, and for which α3(IV)NC1 can be endogenous or exogenous. The CD4 T cell responses of these mice were then tested following immunisation with α3(IV)NC1. In mice with endogenous expression of α3(IV)NC1 there were no consistent detectable proliferative T cell responses to any α3(IV)NC1 peptides in a set of overlapping peptides representative of the entire sequence. In the mice lacking endogenous α3(IV)NC1 there were consistent responses to the peptide α3(IV)NC1 136-150. This contains part of the peptide recognised by the most abundant autoreactive T cells in patients with acute Goodpasture’s disease. Therefore, the T cell responses seen in man to an endogenous (auto)antigen have similar fine specificity to those seen in mice responding to the same protein as a foreign antigen. This is surprising as one might expect self-tolerance in man to be most secure to such dominantly presented and immunogenic (in HLA DR15 mice) self peptides. However, recent work suggests that the peptide most commonly presented in humans is normally destroyed during antigen processing, giving a possible explanation for the lack of tolerance. Future work should study why tolerance is ineffective to this particular peptide, whether tolerance can be reinforced, these questions could be addressed using a transgenic mouse model that develops Goodpasture-like pathology. In addition, how processing is defective in Goodpasture’s disease could be explored by making antigen presenting hybridomas from patient samples or from the transgenic mouse line described within this thesis.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Vaccines play a vital role in public health by preventing infectious disease across the globe. Vaccine formulations represent a weakened form of a microbe or toxin that is injected into the human body to elicit an immune response, generating antibodies to protect against a future infection. To this day, it is a challenge to identify and engineer important antigens and epitopes to focus this immune response in a safe and effective manner. The example of Bordetella pertussis is used to highlight the problems and lessons learned in designing a vaccine for this global epidemic. In particular, this review will focus on the advantages and disadvantages of chemical versus genetic detoxification and whole cell versus acellular vaccines in the context of pertussis. The latter part of this review will provide a summary of general strategies, such as epitope mapping and manipulation, synthesis of truncated variants, reverse vaccinology, and structural vaccinology, that have been successful in addressing increasingly complex diseases. Collectively, these techniques provide an invaluable set of tools to focus the immune response by finding and engineering specific antigens and epitopes. / text

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