Lack of acid sphingomyelinase (ASM) activity, either through genetic deficiency or through pharmacological inhibition, is linked with increased activity and frequency of Foxp3+ regulatory T cells (Treg) among cluster of differentiation (CD) 4+ T cells in mice in vivo and in vitro1. Thus, pharmacological blockade of ASM activity, which catalyzes the cleavage of sphingomyelin to ceramide and phosphocholine, might be used as a new therapeutic mechanism to correct numeric and/ or functional Treg de-ficiencies in diseases like multiple sclerosis or major depression. In the present study, the effect of pharmacological inhibition of ASM in humans, in vitro and in vivo, was analyzed. In the in vitro experiments, peripheral blood mono-nuclear cells (PBMC) of healthy human blood donors were treated with two widely prescribed antidepressants with high (sertraline, Ser) or low (citalopram, Cit) capaci-ty to inhibit ASM activity. Similar to the findings in mice an increase in the frequency of Treg among human CD4+ T cells upon inhibition of ASM activity was observed. For the analysis in vivo, a prospective study of the composition of the CD4+ T cell com-partment of patients treated for major depression was done. The data show that pharmacological inhibition of ASM activity was superior to antidepressants with little or no ASM-inhibitory activity in increasing CD45RA- CD25high effector Treg (efTreg) frequencies among CD4+ T cells to normal levels. Independently of ASM inhibition, correlating the data with the clinical response, i.e. improvement of the Hamilton rat-ing scale for depression (HAMD) by at least 50 per cent (%) after four weeks of treatment, it was found that an increase in efTreg frequencies among CD4+ cells dur-ing the first week of treatment identified patients with a clinical response. Regarding the underlying mechanism, it could be found that the positive effect of ASM inhibition on Treg required CD28 co-stimulation suggesting that enhanced CD28 co-stimulation was the driver of the observed increase in the frequency of Treg among human CD4+ T cells. Inhibition of ASM activity was further associated with changes in the expression and shuttling of CTLA-4, a key inhibitory molecule ex-pressed by Treg, between cellular compartments but the suppressive activity of CTLA-4 through its transendocytosis activity was unaffected by the inhibition of ASM activity. In summary, the frequency of (effector) Treg among CD4+ T cells in mice and in hu-mans is increased after inhibition of ASM activity suggesting that ASM blockade might beneficially modulate autoimmune diseases and depression-promoting in-flammation. / Ein Mangel an Aktivität der sauren Sphingomyelinase (ASM), entweder durch ge-netisches Defizit oder durch pharmakologische Hemmung, ist mit einer erhöhten Aktivität und Häufigkeit von Foxp3+ regulatorischen T-Zellen (Treg) innerhalb der CD4+ (cluster of differentation 4) T-Zellen in Mäusen in vivo und in vitro verbun-den1. Daher könnte die pharmakologische Blockade der ASM-Aktivität, die die Spaltung von Sphingomyelin in Ceramid und Phosphocholin katalysiert, als neuer therapeutischer Mechanismus zur Korrektur von numerischen und/oder funktionel-len Treg-Defiziten bei Erkrankungen wie Multipler Sklerose oder schwerer Depres-sion eingesetzt werden. In der vorliegenden Studie wurde die Wirkung der pharmakologischen Hemmung von ASM beim Menschen, in vitro und in vivo analysiert. In den In-vitro-Experimenten wurden die peripheren mononukleären Blutzellen (PBMC) gesunder menschlicher Blutspender mit zwei weithin verschriebenen Antidepressiva mit ho-her (Sertralin, Ser) oder niedriger (Citalopram, Cit) Fähigkeit zur Hemmung der ASM-Aktivität untersucht. Ähnlich wie bei Mäusen wurde bei Hemmung der ASM-Aktivität ein Anstieg der Häufigkeit von Treg innerhalb der menschlichen CD4+ T-Zellen festgestellt. Für die Analyse in vivo wurde eine prospektive Studie über die Zusammensetzung des CD4+ T-Zellkomplexes bei Patienten, die wegen einer De-pression im Krankenhaus behandelt wurden, durchgeführt. Die Daten zeigen, dass die pharmakologische Hemmung der ASM-Aktivität Antidepressiva mit ge-ringer oder keiner ASM-hemmenden Aktivität überlegen war, was die Vermehrung der CD45RA- CD25hoch-Effektor-Treg (efTreg)-Frequenzen innerhalb der CD4+ T-Zellen betraf. Unabhängig von der Untersuchung zur ASM-Aktivität beobachteten wir, dass die klinische Reaktion (d.h. der Verbesserung der Hamilton-Bewertungsskala für Depressionen (HAMD) um mindestens 50 Prozent (%) nach vierwöchiger Behandlung) mit einem frühen Anstieg der efTreg-Frequenzen unter CD4+-Zellen während der ersten Behandlungswoche positiv korrelierte. Hinsichtlich des zugrunde liegenden Mechanismus konnte festgestellt werden, dass die positive Wirkung der ASM-Hemmung auf Treg eine CD28-Kostimulation erforderte, was darauf hindeutet, dass eine verstärkte CD28-Kostimulation die Ur-sache für den beobachteten Anstieg der Frequenz von Treg innerhalb menschli-cher CD4+ T-Zellen war. Die Hemmung der ASM-Aktivität war darüber hinaus mit Veränderungen in der Expression und im zellulären Umsatz von CTLA-4, einem von Treg exprimierten inhibitorischen Schlüsselmolekül, verbunden. Die suppressi-ve Aktivität von CTLA-4 durch seine Transendozytose-Aktivität wurde jedoch durch die Hemmung der ASM-Aktivität nicht beeinflusst. Zusammenfassend lässt sich sagen, dass die Häufigkeit von (Effektor-)Treg unter-halb der CD4+ T-Zellen in Mäusen und beim Menschen nach Hemmung der ASM-Aktivität erhöht ist, was darauf hindeutet, dass eine ASM-Blockade Autoimmuner-krankungen und depressionsfördernde Entzündungen vorteilhaft modulieren könn-te.

The adaptive immune system is known to provide highly specific and effective immunity against a broad variety of pathogens due to different effector cells. The most prominent are CD4+ T-cells which differentiate after activation into distinct subsets of effector and memory cells, amongst others T helper 1 (Th1) cells. We have recently shown that mouse as well as human Th1 cells depend on T cell receptor (TCR) signals concomitant with CD28 costimulation in order to secrete interferon  (IFN) which is considered as their main effector function. Moreover, there is a class of anti-CD28 monoclonal antibodies that is able to induce T cell (re-)activation without concomitant TCR ligation. These so-called CD28-superagonists (CD28-SA) have been shown to preferentially activate and expand CD4+ Foxp3+ regulatory T (Treg) cells and thereby efficaciously conferring protection e.g. against autoimmune responses in rodents and non-human primates. Considering this beneficial effect, CD28-SA were thought to be of great impact for immunotherapeutic approaches and a humanized CD28-SA was subjected to clinical testing starting with a first-in-man trial in London in 2006. Unexpectedly, the volunteers experienced life-threatening side effects due to a cytokine release syndrome (CRS) that was unpredicted by the preclinical studies prior to the trial. Retrospectively, CD4+ memory T cells within the tissues were identified as source of pro-inflammatory cytokines released upon CD28-SA administration. This was not predicted by the preclinical testing indicating a need for more reliable and predictive animal models. Whether mouse CD4+ T cells are generally irresponsive to CD28-SA stimulation or rather the lack of a bona fide memory T cell compartment in cleanly housed specific-pathogen-free (SPF) mice is the reason why the rodent models failed to predict the risk for a CRS remained unclear. To provide SPF mice with a true pool of memory/effector T cells, we transferred in vitro differentiated TCR-transgenic OT-II Th1 cells into untreated recipient mice. Given that Treg cells suppress T cell activation after CD28- SA injection in vivo, recipients were either Treg-competent or Treg-deficient, wild type or DEREG mice, respectively. Subsequent CD28-SA administration resulted in induction of systemic pro-inflammatory cytokine release, dominated by IFN, that was observed to be much more pronounced and robust in Treg-deficient recipients. Employing a newly established in vitro system mirroring the in vivo responses to CD28-SA stimulation of Th1 cells revealed that antigen-presenting cells (APCs) amplify CD28-SAinduced IFN release by Th1 cells due to CD40/CD40L-interactions. Thus, these data are the first to show that mouse Th1 cells are indeed sensitive to CD28-SA stimulation in vivo and in vitro responding with strong IFN release accompanied by secretion of further pro-inflammatory cytokines, which is compatible with a CRS. In conclusion, this study will facilitate preclinical testing of immunomodulatory agents providing a mouse model constituting more “human-like” conditions allowing a higher degree of reliability and translationability. / Das adaptive Immunsystem ermöglicht mittels hocheffektiver, antigen-spezifischer Mechanismen und unterschiedlicher Effektorzellen den Schutz vor einer nahezu unbegrenzten Vielfalt von Pathogenen. Die Hauptakteure stellen hierbei CD4+ T-Zellen dar, welche nach Aktivierung distinkte Effektorpopulationen, unter anderem Th1 Zellen, bilden. Wir zeigten kürzlich, dass sowohl für Maus- als auch humane Th1-Zellen CD28-Kostimulation mit zeitgleicher T-Zellrezeptor (TZR)-Aktivierung essentiell für die Sekretion von Interferon  (IFN), deren Haupteffektorfunktion, ist. Allerdings sind monoklonale anti-CD28 Anti-körper bekannt, die auch ohne TZR-Signal T-Zellen aktivieren können. Diese sogenannten CD28 Supera-gonisten (CD28-SA) aktivieren und expandieren vorrangig CD4+ Foxp3+ regulatorische T-Zellen (Treg) und vermitteln wirksamen Schutz vor z.B. Autoimmunreaktionen in Nagern und Primaten. Um diesen erfolgversprechenden Effekt für immuntherapeutische Ansätze nutzen zu können, wurde 2006 in Lon-don eine erste klinische Erprobung eines humanisierten CD28-SA begonnen. Unerwarteterweise zeigten sich bei den Probanden lebensbedrohliche Nebenwirkungen, die Ausdruck eines Zytokin-Ausschüttungs-Syndroms (Cytokine Release Syndrome, CRS) waren, welches durch die vorangegangenen präklinischen Studien nicht vorhersagbar war. Rückblickend konnte die Sekretion pro-inflammatorischer Zytokine auf CD4+ Gedächtnis-T-Zellen im Gewebe zurückgeführt werden, die so auf die Gabe des CD28-SA reagier-ten. Die unvorhersehbare Reaktion im Menschen zeigt deutlich, dass verlässlichere und prädiktivere Tiermodelle unverzichtbar sind. Ob Maus CD4+-T-Zellen möglicherweise nicht durch CD28-SA stimulier-bar sind oder dieser fehlgeleiteten Einschätzung über das mögliche Risiko eines CRS eher das Fehlen eines echten CD4+ Gedächtnis-T-Zellen-Kompartiments in sauber gehaltenen spezifischen-Pathogen-freien (SPF) Mäusen zugrunde liegt, ist bisher ungeklärt. Um in SPF-Mäusen ein Gedächtnis-T-Zell-Kompartiment zu etablieren, wurden in vitro-differenzierte Th1 Zellen, die TZR-transgenen OT-II-Mäusen entstammen, in unbehandelte Empfängermäuse transferiert. Da bekannt ist, dass Treg-Zellen die Aktivierung von T-Zellen nach Anwendung von CD28-SA in vivo supprimieren, wurden Treg-kompetente (wildtypische) oder -defiziente (DEREG) Empfänger verwendet. Die anschließend erfolgte Injektion von CD28-SA löste die systemische Sekretion pro-inflammatorischer Zytokine aus, wobei eine stark erhöhter IFN-Konzentration im Serum zu beobachten war, welche deutlich ausgeprägter und robuster bei den Treg-defizienten Empfängern ausfiel. Ein neu etabliertes in vitro-System, welches die in vivo Antwort der Th1-Zellen auf CD28-SA-Stimulation widerspiegelt, identifizierte Antigen-präsentierende Zellen (APZs) als essentiellen Faktor für die erhöhte IFN-Sekretion der Th1-Zellen nach CD28-SA-Stimulation in Abhängigkeit von CD40/CD40L-Interaktionen. Zusammenfassend zeigt diese Thesis zum ersten Mal, dass Maus Th1 Zellen sowohl in vivo als auch in vitro durch CD28 SA stimulierbar sind, wodurch eine starke IFN-Sekretion induziert wird, die von der gesteigerten Ausschüttung anderer pro-inflammatorischer Zytokine begleitet wird und in Abwesenheit von Treg einem CRS gleicht. Folglich kann diese Erkenntnis die präklinische Forschung bei der Erprobung neuer immuntherapeutischer Ansät-ze durch ein neues Mausmodell voranbringen, das dem menschlichen erfahreneren Immunsystem mehr als bisherige Modelle entspricht und somit verlässlichere Vorhersagen erlaubt und eine verbesserte Übertragbarkeit von Maus zu Mensch ermöglicht.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, June 2001. / Includes bibliographical references. Also available on the Internet.

<p>In affinity protein biotechnology the selective bindingbetween a chosen protein and an interacting biomolecule isutilized for a variety of applications including bioseparation,detection and therapy. Traditionally, affinity proteinsrecruited for such applications have been derived from naturalproteins or immunoglobulins generated via immunization routes.More recently, advances in the construction and handling oflarge collections of proteins(denoted libraries) generated invitro have opened up for new routes for the development ofaffinity proteins with desired properties.</p><p>In this study, phage display selection technology was usedfor the isolation of novel human CD28 (hCD28)-specific affinityproteins from a protein library constructed by combinatorialprotein engineering of a 58 aa protein domain (Z) derived fromstaphylococcal protein A (SPA). From selections using hCD28 asa target molecule, several hCD28-specific affinity proteins(denoted affibodies) could be identified and analysis of theisolated affibody variants revealed a high degree of sequencehomology between the different clones. The biosensor analysisshowed that all variants bound to hCD28 with micromolardissociation constants (KD) and no significant cross-reactivitytowards the structurally related T-cell receptor hCTLA-4 couldbe observed. The apparent binding affinity for hCD28 of one ofthe isolated affibodies was further improved through fusion toa human Fc fragment fusion partner, resulting in a homodimericversion of the affibody ligand showing avidity effects uponhCD28 binding. Further, a co-culture experiment involvingJurkat T-cells and CHO cell lines tranfected to express eitherhuman CD80 or LFA-3 on the cell surface showed that apreincubation of Jurkat cells with one of the affibody variantsresulted in a specific concentration-dependent inhibition ofthe CD80 induced IL-2 production. This indicates that thisaffibody binds to hCD28 and specifically interferes with theco-stimulation signal mediated via hCD28 and hCD80. ACD28-specific binding protein could have potential as an agentfor various immunotherapy applications. In a second study, anaffinity protein-based strategy was investigated forsite-specific anchoring of proteins onto cellulose for woodfiber engineering purposes. Here, affinity proteins derivedfrom different sources were used for the assembly of acellulosome-like complex for specific and reversible anchoringof affinity domain-tagged reporter proteins to acellulose-anchored fusion protein. A fusion protein between acellulose binding module (Cel6A CBM1) derived from the fungalTrichoderma reesei and a five-domain staphylococcal protein A(SPA) moiety was constructed to serve as a platform for thedocking of reporter proteins produced as fusion to two copiesof a SPA-binding affibody affinity protein (denoted ZSPA-1),selected by phage display technology from a Z domain basedprotein library. In a series of experiments, involving repeatedwashing and low pH elutions, affinity tagged Enhanced GreenFluorescent Protein (EGFP) and Fusarium solani pisi lipasecutinase reporter proteins were both found to be specificallydirected from solution to a region of a cellulose-based filterpaper where the SPA-CBM fusion protein previously had beenpositioned. This showed that the cellulose-anchored SPA-Cel6ACBM1 fusion protein had been stably anchored to the surfacewith retained binding activity and that the interaction betweenSPA and the ZSPA-1 affibody domain was selective.</p><p>phage display, combinatorial, selection, CD28, cellulosome,cellulose, affibody</p>

Die Kostimulation über den CD28 Oberflächenrezeptor spielt eine entscheidende Rolle sowohl in der Proliferation von T-Zellen, in deren Differenzierung zu Effektorzellen, als auch in der Entwicklung und Kontrolle von regulatorischen T-Zellen. Diese Schlüsselrolle macht CD28 zu einer interessanten Zielstruktur, um den Einfluss monoklonaler Antikörper auf die Entwicklung und Homöostase von T-Zellen zu analysieren. Die hier verwendeten Antikörper lassen sich funktionell in zwei verschiedene Kategorien einteilen: zum Einen in konventionelle Antikörper (E18 mAk), welche die physiologische Rezeptor-Liganden Interaktion blockieren, und zum Anderen in superagonistische Antikörper (D665 mAk), die in der Lage sind ruhende T-Zellen ohne eine Ligation des T-Zell Rezeptors voll zu aktivieren. In der vorliegenden Arbeit konnte in verschiedenen in-vitro und in-vivo Experimenten gezeigt werden, dass eine Behandlung mit dem CD28 Superagonisten keinen Einfluss auf die Differenzierung von T-Zellen im Thymus nimmt, und die Zusammensetzung der einzelnen Zellpopulationen in diesem Organ unbeeinflusst bleibt. Bei Verwendung blockierender anti-Maus CD28 mAk zeigte sich in sämtlichen Untersuchungen eine beeinträchtigte Entwicklung von regulatorischen T-Zellen innerhalb des Thymus, während die anderen Zellpopulationen unbeeinflusst blieben. Auch in Analysen peripherer Lymphknoten lag der Anteil regulatorischer T-Zellen nach Behandlung mit E18 mAk unter den Kontrollwerten. Durch eine chronische Blockade von CD28 mittels E18 mAk ließ sich die Gesamtanzahl an Tregs in peripheren Lymphknoten, Milz und Thymus drastisch senken, ohne dass es zu einem vollständigen Verschwinden dieser Zellpopulation kam. Auch die absolute Zahl an CD4 positiven T-Zellen innerhalb der peripheren Lymphknoten, Milz und Thymus der behandelten Tiere lag deutlich unter der Kontrolle. Diese Beobachtungen waren 13 Wochen nach Beendigung der Antikörper-Applikationen vollständig reversibel und zu keinem Zeitpunkt des Experimentes ergaben sich klinische oder serologische Hinweise auf die Entwicklung von Autoimmunität trotz niedriger Treg- Zahlen über mehrere Wochen. / Costimulation via the CD28 receptor plays an important role not only in the proliferation of T cells and the differentiation to effector T cells, but also in the development and control of regulatory T cells (Treg). This key role makes CD28 an attractive target for analysing the influence of monoclonal antibodies on the development und homeostasis of T cells. The antibodies used in this work could be devided into two types: conventional antibodies (E18 mAb) on the one hand that are per se not stimulatory in vitro and block the interaction of CD28 with its ligands, and superagonistic antibodies (D665 mAb) on the other hand which can fully activate resting T cells even without ligation of the T-cell receptor. Different in vitro and in vivo experiments in this work could show that application of CD28 superagonists has no influence on differentiation of T cells in thymus. The composition of the single cell subsets in this organ stays completely unaffected. Treatment with conventional E18 mAb leads to an impaired development of Treg cells in thymus, while other populations stay unaffected. Similar results were obtained by analysing peripheral lymphnodes. Chronic application of E18 mAb reduces the representation of Treg cells within the CD4 T-cell compartment in peripheral lymphnodes, spleen and thymus, along with a strong reduction of the CD4 compartment at large. Importanty, however, no signs of autoimmunity were detected during or after release from anti-CD28 treatment, during which Treg numbers swiftly recover.

Regulatorische T-Zellen (Tregs) spielen eine ntscheidende Rolle beim Erhalt der Immunhomöostase und bei der Kontrolle überschießender Immunantworten. Sie können anhand ihres Entstehungsortes in im Thymus generierte natürliche Tregs (nTregs) und in der Peripherie generierte induzierte Tregs (iTregs) unterteilt werden. Ihr Phänotyp wie auch ihre Funktion werden zu einem großen Teil durch den transkriptionellen Masterregulator Foxp3 kontrolliert. Das kostimulatorische Molekül CD28 wird von nTregs für die Differenzierung benötigt und von Tregs und konventionellen T-Zellen (Tkons) für ihre Aktivierung. Superagonistische CD28 spezifische monoklonale Antikörper (CD28SA) aktivieren T-Zellen im Gegensatz zu konventionellen anti-CD28 Antikörpern ohne zusätzliche Ligation des T-Zellrezeptors. Die in vivo Applikation des CD28SA bewirkt eine starke Aktivierung der Tregs und eine präferentielle Expansion der Tregs gegenüber Tkons. Dies erklärt die präventive und therapeutische Wirkung der CD28SA Behandlung in verschiedenen Krankheitsmodellen bei Nagern. Die erste Anwendung des humanisierten CD28SA TGN1412 führte in den Testpersonen jedoch zu einem unerwarteten „Cytokine-Release Syndrom“. Daher wurde hier am Mausmodell der Zusammenhang zwischen Treg Aktivierung und systemischer Zytokinausschüttung näher untersucht. Es konnte gezeigt werden, dass die CD28SA vermittelte Proliferation der T-Zellen abhängig vom CD28 Signal und von parakrinem Interleukin (IL)-2 ist. Durch die in vivo Depletion der Tregs vor der CD28SA Injektion wurde deutlich, dass es auch in Mäusen nach CD28SA Stimulation zu einer systemischen Ausschüttung pro-inflammatorischer Zytokine kommt, die jedoch, im Gegensatz zum humanen System, von Tregs effektiv kontrolliert werden kann. Um die usschüttung pro-inflammatorischer Zytokine zu verhindern, wäre eine zusätzliche prophylaktische Behandlung mit Corticosteroiden möglich, da diese auch in hohen Dosen die CD28SA vermittelte Aktivierung und Expansion der Tregs nicht beeinflussen. Neben der Expansion wird durch die Stimulation mit CD28SA auch die Produktion des anti-inflammatorischen Zytokins IL-10 in Tregs induziert und so eine genauere Untersuchung des Ursprungs und des Schicksals IL-10 produzierender Tregs ermöglicht. Diese Tregs exprimieren im Vergleich zu IL-10 negativen Tregs ein höheres Niveau an Molekülen, die mit einer supprimierenden Aktivität verbunden sind. Zudem werden IL-10 Produzenten aufgrund der Veränderung im Expressionsmuster der Migrationsrezeptoren nach der Stimulation von einem lymphknotensuchenden CCR7+CCR5-CCR6- zu einem entzündungssuchenden CCR7-CCR5+CCR6+ Phänotyp verstärkt in Bereiche mit stattfindender Immunantwort rekrutiert. Schließlich sind IL-10 produzierende Tregs von CD28SA stimu2 lierten Mäusen in vitro stärker apoptoseanfällig als die IL-10 negativen Tregs. Die Aktivierung der Tregs scheint somit die terminale Differenzierung zu einem IL-10 produzierenden Effektorphänotyp mit begrenzter Lebensdauer zu induzieren. Dies führt auch zur Beendigung der Immunsuppression. Die Kombination aus schwachem TZR und starkem CD28 Signal, die die CD28SA Stimulation in naiven T-Zellen auslöst, induziert zumindest in vitro abhängig von IL-2 und TGFβ effizient die Expression von Foxp3. Die so generierten iTregs haben, ähnlich wie konventionell in vitro erzeugte iTregs, in Bezug auf die Expression von Oberflächenmolekülen und den Methylierungsstatus bestimmter Regionen des Foxp3 Gens einen Phänotyp, der zwischen dem von Tkons und Tregs liegt. Da auch die supprimierende Aktivität der iTregs geringer ist als die der ex vivo Tregs bedarf es einer weiteren Optimierung des Stimulationsprotokolls, um diese Zellen für therapeutische Zwecke verwenden zu können. Zusammenfassend zeigt diese Arbeit, dass die superagonistische Stimulation des CD28 Moleküls ein vielseitig einsetzbares Instrument ist. Einerseits können durch die CD28SA Stimulation Tregs polyklonal aktiviert und für therapeutische Zwecke mobilisiert werden und andererseits kann die besondere Art der T-Zellstimulation auch dazu genutzt werden, neue Aspekte von nTregs und iTregs zu untersuchen. / Regulatory T-cells (Tregs) are important to maintain immune homeostasis and to control overshooting immune responses. Depending on the place where they are generated Tregs can be divided into thymus-derived natural Tregs (nTregs) and peripheral-derived induced Tregs (iTregs). Their phenotype and function is controlled by the transcriptional masterregulator Foxp3. The costimulatory molecule CD28 is required for nTreg differentiation and for the activation of Tregs and conventional T-cells (Tcons). Superagonistic CD28 specific monoclonal antibodies (CD28SA) activate T-cells without additional T-cell receptor ligation in contrast to conventional anti-CD28 antibodies. In vivo CD28SA treatment induces strong Treg activation and preferential expansion of Tregs over Tcons. This explains the preventive and therapeutic effects of CD28SA in various rodent disease models. In contrast, a first-in-man trail of the human CD28SA TGN1412 resulted in an unexpected cytokine release syndrome. Thus, using the mouse system the relationship between Treg activation and systemic cytokine release was reinvestigated. It could be shown that CD28SA induced T-cell proliferation is dependent on the CD28 signal and on paracrine interleukin (IL)-2. In vivo depletion of Tregs prior CD28SA injection showed that in mice, too, CD28SA stimulation induces a systemic release of pro-inflammatory cytokines, but in contrast to humans, this can be effectively controlled by Tregs. To prevent the pro-inflammatory cytokine release an additional prophylactic treatment with corticosteroids might be feasible since even high doses of corticosteroids have no effect on CD28SA driven Treg activation and expansion. Beside expansion, CD28SA stimulation also induces the production of anti-inflammatory IL-10 in Tregs. This allows for examination of the origin and fate of IL-10 producing Tregs. In comparison to IL-10 negative Tregs, these cells express higher levels of molecules associated with suppressive activity. In addition IL-10 producers are preferentially recruited to sites of ongoing immune responses, as they shift their expression pattern of migration receptors after stimulation from a CCR7+CCR5-CCR6- lymph node-seeking to a CCR7-CCR5+CCR6+ inflammation-seeking phenotype. Finally, IL-10 producing Tregs of CD28SA stimulated mice are more apoptosis-prone in vitro than their IL-10 negative counterparts. Thus, activation of Tregs induces terminal differentiation towards an IL-10 producing effector phenotype with a limited life span. This also terminates the immune suppression. The special combination of weak TCR and strong CD28 signals following CD28SA stimulation of naïve T-cells can, at least in vitro and dependent on IL-2 and TGFβ, efficiently induce Foxp3 expression. The phenotype regarding expression of certain surface molecules and the methylation level of particular regulatory regions of the Foxp3 gene is similar in CD28SA and anti-CD3/ anti-CD28 (+ IL-2/TGFβ) induced iTregs, and shows properties of both Tcons and Tregs. Since the suppressive activity of these iTregs is also lower than that of ex vivo Tregs, further improvement of the stimulation protocol is needed before these cells could be used for therapeutic purposes. In conclusion, this study shows that superagonistic stimulation of CD28 molecules is a versatile tool. On the one hand CD28SA stimulation polyclonally activates Tregs and can be used as a therapeutic, on the other hand the special way of T-cell stimulation can also be utilized to investigate new aspects of nTregs and iTregs.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [121]-138).

Würzburg, Univ., Diss., 2004. / Erscheinungsjahr an der Haupttitelstelle: 2004

Made available in DSpace on 2016-08-17T18:39:44Z (GMT). No. of bitstreams: 1 4594.pdf: 3287424 bytes, checksum: 45ec60b0a38c4b296ddba2d66d8cd69c (MD5) Previous issue date: 2011-10-27 / Universidade Federal de Minas Gerais / Schistosomiasis mansoni is a major problem affecting public health, according to World Health Organization (WHO), 210 million individuals worldwide. In the infected host, the disease is characterized by the presence of granuloma, immunopathological outcome of the cellular infiltrate and in many cases a consequence of tissue fibrosis. Thus, the host-parasite relationship may lead to morbidity from the disease result in hepatosplenomegaly, hepatic fibrosis and ascites. The granulomatous process in schistosomiasis is dependent on CD4 + and requires recruitment and accumulation of inflammatory cells at the site of deposition of eggs. Schistosomal fibrosis is the result of granulomatous reaction that develops in response to antigens released by eggs of Schistosoma mansoni retained in the portal veins of smaller caliber. A host of disease caused by S. mansoni is mediated by the immune response by T cells through the dissemination of eggs that are housed in the liver and intestine. Manipulation of the interaction between B7 molecules of antigen presenting cells (APC) and T cell receptors CD28/CTLA4 modulate, and in some circumstances block the immunological response in vivo. The CTLA4 is structurally homologous to CD28 but is expressed in CD4 + and CD8 + newly activated, and its function is to inhibit T cell activation by inhibiting the signals released by CD28. Thus, CTLA4 is involved in the completion of T cell responses The way in which the two receptors release opposite signs and recognize the same molecule B7 of APC is an intriguing question and a matter of research. Coestimulatory these signals may have different roles in various types of immune response. Thus the treatment strategy of using monoclonal antibodies (mAb) anti-CTLA4, anti-CD28 in murine schistosomiasis was to evaluate the modulation of inflammatory response and parasite-induced S. mansoni as CTLA4 and CD28 were blocked. Our results showed that treatment with (mAb) anti CD28 and CTLA4 molecules coestimulatory favored changes in the number of leukocytes, modulations in the levels of circulating antibodies, cytokines, and the response profile of T helper (Th) and change in the parasitic activity. Our data showed that the reduction of parasite burden was 21.3% in the group treated with anti-CTLA4 mAb and 46.2% in the group treated with anti-CD28. Suggesting that these treatments should be considered interesting candidates for immunotherapy and for further investigation, since it is necessary to better understand the response of these molecules in schistosomiasis mansoni that when blocked or did not show the hypothetical anti-parasitic and anti-inflammatory activity in this model. / A esquistossomose mansônica representa um dos grandes problemas de Saúde Pública acometendo, segundo a Organização Mundial de Saúde (OMS), 210 milhões de indivíduos no mundo todo. No hospedeiro infectado, a doença é caracterizada pela presença de granuloma, resultado imunopatológico do infiltrado celular e em muitos casos consequência de fibrose tecidual. Dessa forma, a relação parasito-hospedeiro pode levar a morbidade devido à doença resultar em hepatoesplenomegalia, fibrose hepática e ascite. O processo granulomatoso na esquistossomose é dependente de linfócitos T CD4+ e requer recrutamento e acumulo de células inflamatórias no sítio de deposição dos ovos. A fibrose esquistossomótica é resultado da reação granulomatosa que se desenvolve em resposta a antígenos liberados pelos ovos do Schistosoma mansoni retidos nas veias portais de menor calibre. A patologia causada em hospedeiros com S. mansoni é mediada pela resposta imune, por células T através da disseminação dos ovos que são alojados no fígado e intestino. Manipulações para a interação entre moléculas B7 de células apresentadoras de antígenos (APC) e receptores CD28/CTLA4 de células T modulam, e em algumas circunstâncias, bloqueiam a resposta imunopatológica in vivo. O CTLA4 é estruturalmente homólogo ao CD28, mas é expresso nas células T CD4+ e CD8+ recém ativadas, e sua função é inibir a ativação de células T pela inibição dos sinais liberados pelo CD28. Assim, o CTLA4 está envolvido na finalização das respostas das células T. A maneira pelos quais os dois receptores liberam sinais opostos e reconhecem a mesma molécula B7 das APC é uma intrigante questão e motivo de pesquisa. Esses sinais coestimulatórios podem ter diferentes papéis nos vários tipos de resposta imune. Dessa forma a estratégia de utilizar o tratamento com anticorpos monoclonais (mAb) anti-CTLA4 e anti-CD28 na esquistossomose mansônica murina foi de avaliar a modulação da resposta inflamatória e parasitária induzida pelo S. mansoni quando CTLA4 e CD28 fossem bloqueados. Nossos resultados mostraram que o tratamento com (mAb) anti moléculas coestimulatórias CTLA4 e CD28 favoreceram alterações no número de leucócitos, modulações nos níveis de anticorpos circulantes, citocinas, e do perfil da resposta T helper (Th) e alteração na atividade parasitária. Nossos dados demonstraram que, a redução da carga parasitária foi de 21,3% no grupo tratado com mAb anti- CTLA4 e 46,2% no grupo tratado com anti-CD28. Sugerindo que, estes tratamentos devem ser considerados interessantes candidatos para imunoterapia e para maiores investigações, uma vez que se faz necessário conhecer melhor a resposta destas moléculas na esquistossomose mansônica que quando bloqueadas ou não apresentaram a hipotética atividade antiparasitária e antiinflamatória nesse modelo.

In affinity protein biotechnology the selective bindingbetween a chosen protein and an interacting biomolecule isutilized for a variety of applications including bioseparation,detection and therapy. Traditionally, affinity proteinsrecruited for such applications have been derived from naturalproteins or immunoglobulins generated via immunization routes.More recently, advances in the construction and handling oflarge collections of proteins(denoted libraries) generated invitro have opened up for new routes for the development ofaffinity proteins with desired properties. In this study, phage display selection technology was usedfor the isolation of novel human CD28 (hCD28)-specific affinityproteins from a protein library constructed by combinatorialprotein engineering of a 58 aa protein domain (Z) derived fromstaphylococcal protein A (SPA). From selections using hCD28 asa target molecule, several hCD28-specific affinity proteins(denoted affibodies) could be identified and analysis of theisolated affibody variants revealed a high degree of sequencehomology between the different clones. The biosensor analysisshowed that all variants bound to hCD28 with micromolardissociation constants (KD) and no significant cross-reactivitytowards the structurally related T-cell receptor hCTLA-4 couldbe observed. The apparent binding affinity for hCD28 of one ofthe isolated affibodies was further improved through fusion toa human Fc fragment fusion partner, resulting in a homodimericversion of the affibody ligand showing avidity effects uponhCD28 binding. Further, a co-culture experiment involvingJurkat T-cells and CHO cell lines tranfected to express eitherhuman CD80 or LFA-3 on the cell surface showed that apreincubation of Jurkat cells with one of the affibody variantsresulted in a specific concentration-dependent inhibition ofthe CD80 induced IL-2 production. This indicates that thisaffibody binds to hCD28 and specifically interferes with theco-stimulation signal mediated via hCD28 and hCD80. ACD28-specific binding protein could have potential as an agentfor various immunotherapy applications. In a second study, anaffinity protein-based strategy was investigated forsite-specific anchoring of proteins onto cellulose for woodfiber engineering purposes. Here, affinity proteins derivedfrom different sources were used for the assembly of acellulosome-like complex for specific and reversible anchoringof affinity domain-tagged reporter proteins to acellulose-anchored fusion protein. A fusion protein between acellulose binding module (Cel6A CBM1) derived from the fungalTrichoderma reesei and a five-domain staphylococcal protein A(SPA) moiety was constructed to serve as a platform for thedocking of reporter proteins produced as fusion to two copiesof a SPA-binding affibody affinity protein (denoted ZSPA-1),selected by phage display technology from a Z domain basedprotein library. In a series of experiments, involving repeatedwashing and low pH elutions, affinity tagged Enhanced GreenFluorescent Protein (EGFP) and Fusarium solani pisi lipasecutinase reporter proteins were both found to be specificallydirected from solution to a region of a cellulose-based filterpaper where the SPA-CBM fusion protein previously had beenpositioned. This showed that the cellulose-anchored SPA-Cel6ACBM1 fusion protein had been stably anchored to the surfacewith retained binding activity and that the interaction betweenSPA and the ZSPA-1 affibody domain was selective. phage display, combinatorial, selection, CD28, cellulosome,cellulose, affibody / NR 20140805