Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

CD4 T cells and B cells are cells associated with the adaptive immune system. The adaptive immune system is designed to mount a rapid antigen-specific response to pathogens by way of clonal expansions of T and B cells bearing discrete antigen-specific receptors. During viral infection, interactions between CD4 T cells and B cells occur in a dynamic process, where B cells that bind to the virus internalize and degrade virus particles. The B cells then present viral antigens to virus-specific CD4 T cells that activate the B cells and cause them to proliferate and differentiate into virus-specific antibody-secreting cells. Yet, non-specific hypergammaglobulinemia and the production of self-reactive antibodies occur during many viral infections, and studies have suggested that viral antigen-presenting B cells may become polyclonally activated by CD4 T cells in vivo in the absence of viral engagement of the B cell receptor. This presumed polyclonal B cell activation associated with virus infection is of great medical interest because it may be involved in the initiation of autoimmunity or contribute to the long-term maintenance of B cell memory. In order to directly examine the interactions that occur between T cells and B cells, I asked what would happen to a polyclonal population of B cells that are presenting viral antigens, if they were transferred into virus-infected hosts. I performed these studies in mice using the well-characterized lymphocytic choriomeningitis virus (LCMV) model of infection. I found that the transferred population of antigen-presenting B cells had two fates. Some antigen-expressing B cells were killed in vivo by CD4 T cells in the first day after transfer into LCMV-infected hosts. However, B cells that survived the cytotoxicity underwent a dynamic polyclonal activation manifested by proliferation, changes in phenotype, and antibody production. The specific elimination of antigen-presenting B cells following adoptive transfer into LCMV-infected hosts is the first evidence that MHC class II-restricted killing can occur in vivo during viral infection. This killing was specific, because only cells expressing specific viral peptides were eliminated, and they were only eliminated in LCMV-infected mice. In addition to peptide specificity, killing was restricted to MHC class II high cells that expressed the B cell markers B220 and CD19. Mice depleted of CD4 T cells prior to adoptive transfer did not eliminate virus-specific targets, suggesting that CD4 T cells are required for this killing. I found that CD4 T cell-dependent cytotoxicity cannot be solely explained by one mechanism, but Fas-FasL interactions and perforin are mechanisms used to induce lysis. Polyclonal B cell activation, hypothesized to be the cause of virus-induced hypergammaglobulinemia, has never been formally described in vivo. Based on previous studies of virus-induced hypergammaglobulinemia, which showed that CD4 T cells were required and that hypergammaglobulinemia was more likely to occur when virus grows to high titer in vivo, it was proposed that the B cells responsible for hypergammaglobulinemia may be expressing viral antigens to virus-specific CD4 T cells in vivo. CD4 T cells would then activate the B cells. However, because the antibodies produced during hypergammaglobulinemia are predominantly not virus-specific, nonvirus-specific B cells must be presenting viral antigens in vivo. In my studies, the adoptively transferred B cells that survived the MHC class II-restricted cytotoxicity became polyclonally activated in LCMV-infected mice. Most of the surviving naïve B cells presenting class II MHC peptides underwent an extensive differentiation process involving both proliferation and secretion of antibodies. Both events required CD4 cells and CD40/CD40L interactions to occur but B cell division did not require MyD88-dependent signaling, type I interferon signaling, or interferon γ signaling within B cells. No division or activation of B cells was detected at all in virus-infected hosts in the absence of cognate CD4 T cells and class II antigen. B cells taken from immunologically tolerant donor LCMV carrier mice with high LCMV antigen load became activated following adoptive transfer into LCMV-infected hosts, suggesting that B cells can present sufficient antigen for this process during a viral infection. A transgenic population of B cells presenting viral antigens was also stimulated to undergo polyclonal activation in LCMV-infected mice. Due to the high proportion of B cells stimulated by virus infection and the fact that transgenic B cells can be activated in this manner, I conclude that virus-induced polyclonal B cell activation is independent of B cell receptor specificity. This approach, therefore, formally demonstrates and quantifies a virus-induced polyclonal proliferation and differentiation of B cells which can occur in a B cell receptor-independent manner. By examining the fate of antigen-presenting B cells following adoptive transfer into LCMV-infected mice, I have been able to observe dynamic interactions between virus-specific CD4 T cells and B cells during viral infection. Adoptive transfer of antigen-presenting B cells results in CD4 T cell-mediated killing and polyclonal activation of B cells during LCMV infection. Studies showing requirements for CD4 T cells or MHC class II to control viral infections must now take MHC class II-restricted cytotoxicity into account. Polyclonal B cell activation after viral infection has the potential to enhance the maintenance of B cell memory or lead to the onset of autoimmune disease.

Made available in DSpace on 2014-06-11T19:25:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-18Bitstream added on 2014-06-13T19:12:32Z : No. of bitstreams: 1 caetano_mc_me_araca.pdf: 1024075 bytes, checksum: aa12fb1ffe3cd0c2176481b1472ff87e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As células-tronco (stem cells) têm um papel fundamental nos processos reparativos e regenerativos da córnea. A exata localização destas é bem conhecida no olho do homem, bem como as suas funções. Entretanto, as mesmas ainda não foram descritas em medicina veterinária, sequer a sua localização em olhos de animais de companhia, tratando-se assim, de assunto que merece novas e originais investigações. Anticorpos monoclonais que reagem superficialmente com as citoqueratinas são empregados para determinar este tipo celular presente nos tecidos, no entanto, existem outros marcadores capazes de reagir antigenicamente com núcleos celulares. Entre eles, o PCNA (antígeno nuclear de proliferação celular) e o Ki-67 (antígeno de proliferação Ki-67) são capazes de marcar células em proliferação. Uma vez que isso nunca fora descrito anteriormente, e considerando-se a necessidade e importância científica em se determinar a localização de células em proliferação na superfície ocular de cães, o presente estudo teve por objetivos: 1) determinar a expressão de células em proliferação pela marcação imunoistoquímica com anticorpos monoclonais anti-PCNA e anti-MIB-1 (anticorpo monoclonal anti-Ki-67), bem como, sua localização na superfície ocular; 2) comparar se há diferença estatística significante na marcação dessas células em diferentes idades. Para tanto, foram utilizados 24 cães jovens e adultos, sadios, dos quais o bulbo ocular esquerdo foi removido, fixado e corado pela H.E. (hematoxilina e eosina), além da imunoistoquímica empregando-se os anticorpos monoclonais anti-PCNA e anti-MIB-1. Concluiu-se que a presença de células em proliferação imunomarcadas pelo PCNA e pelo MIB-1 foi observada em diferentes tecidos da superfície ocular de cães, sendo os tecidos mais marcados: o limbo e o epitélio da córnea... / The stem-cells has a fundamental paper in process of cornea s tissue repair and regeneration. In man, the well-know localization and functions had been descriebed a long time ago, however the same ones had not yet been descriebed in veterinary medicine, at least your localization in eyes of company animals, what deserve new and original inquiries. Monoclonais antibodies that react superficially with the cytokeratins are used to determine this cell type presence in ocular surface. However, exist another markers capable to react with cell nucleoli, for example, PCNA and MIB-1 are capable to mark proliferation cells. Considering the necessity and scientific importance to determining the localization of proliferation cells on dog s ocular surface, a time that, never was descriebed previously, the present study had for objectives: 1) determineted the expression of proliferation cells immunohistochemically by PCNA and MIB-1 and its localization in ocular surface; 2) compare if has statistically significant difference in immunoexpression of proliferation cells in different ages. In the study was used 24 healthy dogs, young and adults, witch had the left eyes removed, fixed in buffered formalin and stained with H.E. (hematoxilin and eosin) and with monoclonal antibodies anti-PCNA and anti-MIB-1. The presence of immunostained cells was most observed in corneal ephithelium and limbo. Probably the immunostained cells are stem-cells in dog s ocular surface, however for such evidence it is necessary ...(Complete abstract click electronic address below)

Orientador: Alexandre Lima de Andrade / Banca: Adriana Morales / Banca: Renée Laufer Amorim / Resumo: As células-tronco (stem cells) têm um papel fundamental nos processos reparativos e regenerativos da córnea. A exata localização destas é bem conhecida no olho do homem, bem como as suas funções. Entretanto, as mesmas ainda não foram descritas em medicina veterinária, sequer a sua localização em olhos de animais de companhia, tratando-se assim, de assunto que merece novas e originais investigações. Anticorpos monoclonais que reagem superficialmente com as citoqueratinas são empregados para determinar este tipo celular presente nos tecidos, no entanto, existem outros marcadores capazes de reagir antigenicamente com núcleos celulares. Entre eles, o PCNA (antígeno nuclear de proliferação celular) e o Ki-67 (antígeno de proliferação Ki-67) são capazes de marcar células em proliferação. Uma vez que isso nunca fora descrito anteriormente, e considerando-se a necessidade e importância científica em se determinar a localização de células em proliferação na superfície ocular de cães, o presente estudo teve por objetivos: 1) determinar a expressão de células em proliferação pela marcação imunoistoquímica com anticorpos monoclonais anti-PCNA e anti-MIB-1 (anticorpo monoclonal anti-Ki-67), bem como, sua localização na superfície ocular; 2) comparar se há diferença estatística significante na marcação dessas células em diferentes idades. Para tanto, foram utilizados 24 cães jovens e adultos, sadios, dos quais o bulbo ocular esquerdo foi removido, fixado e corado pela H.E. (hematoxilina e eosina), além da imunoistoquímica empregando-se os anticorpos monoclonais anti-PCNA e anti-MIB-1. Concluiu-se que a presença de células em proliferação imunomarcadas pelo PCNA e pelo MIB-1 foi observada em diferentes tecidos da superfície ocular de cães, sendo os tecidos mais marcados: o limbo e o epitélio da córnea...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The stem-cells has a fundamental paper in process of cornea’s tissue repair and regeneration. In man, the well-know localization and functions had been descriebed a long time ago, however the same ones had not yet been descriebed in veterinary medicine, at least your localization in eyes of company animals, what deserve new and original inquiries. Monoclonais antibodies that react superficially with the cytokeratins are used to determine this cell type presence in ocular surface. However, exist another markers capable to react with cell nucleoli, for example, PCNA and MIB-1 are capable to mark proliferation cells. Considering the necessity and scientific importance to determining the localization of proliferation cells on dog’s ocular surface, a time that, never was descriebed previously, the present study had for objectives: 1) determineted the expression of proliferation cells immunohistochemically by PCNA and MIB-1 and its localization in ocular surface; 2) compare if has statistically significant difference in immunoexpression of proliferation cells in different ages. In the study was used 24 healthy dogs, young and adults, witch had the left eyes removed, fixed in buffered formalin and stained with H.E. (hematoxilin and eosin) and with monoclonal antibodies anti-PCNA and anti-MIB-1. The presence of immunostained cells was most observed in corneal ephithelium and limbo. Probably the immunostained cells are stem-cells in dog’s ocular surface, however for such evidence it is necessary ...(Complete abstract click electronic address below) / Mestre

Background Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that play an important role in the peripheral tolerance mechanisms of the immune system. Their suppressive function on autoreactive T cells can prevent autoimmunity. In type 1 diabetes (T1D), Treg have been inconsistently reported to be impaired in their capability to suppress autoreactive T cells (Tan, et al., 2014; Zhang, et al., 2012). Treg can be thymus derived (tTreg) or generated from naïve CD4+ CD25- T cells in the periphery (pTreg), which exhibit similar suppressive qualities as tTreg. They have also been reported to be actively induced (iTreg) under tolerogenic conditions (Kleijwegt, et al., 2010; Yuan and Malek, 2012). Although several Treg subpopulations have been described, the archetypical Treg express the major markers CD4, CD25 and FOXP3, while CD127 is heavily downregulated. However, activated conventional T cells (Tconv) show a similar phenotype, at least transiently (Miyara, et al., 2009). Since Treg and Tconv have opposing functions and therapeutic indications, it is important to obtain markers that confidently identify bona fide Treg. Scientific aim The aim of my thesis is to define the heterogeneity of human T cells with a specific emphasis to identify bona fide Treg. I examined heterogeneity of this population in healthy controls and T1D patients, as my model disease, and examined how T cells that are exposed to antigen can be defined as Treg or Tconv. Material and Methods For marker phenotyping I used samples from new onset T1D patients (age 7-11 years), autoantibody positive (Aab+) patients and age-matched healthy controls, which were tested by flow cytometry with an array of Treg-associated markers. Separately, freshly isolated CD4+CD25+CD127lo Treg and CD+CD25- Tconv were used for transcriptomic analysis, which was done by RNAseq on isolated whole RNA. For functional analysis of antigen specific gene expression patterns I developed a multi-dye proliferation assay. Treg (CD4+CD25+CD127lo) and Tconv (CD4+CD25-CD127+/lo) were sorted from isolated peripheral blood mononuclear cells (PBMC). I recombined the sorted and proliferation dye stained subsets with CD4- cells to simulate whole PBMC assays and stimulated them with tetanus-, influenza- or auto-antigens (GAD65, proinsulin). Cells were incubated for 5 days and responding proliferating cells as well as non-responding cells were single cell sorted and analyzed by multiplex qPCR. In investigating therapeutic approaches to expand or generate Treg, I examined in vitro approaches for de novo induction of Tregs with tolerogenic dendritic cells (tDCs). The tDCs were differentiated from monocytes either in the presence of 1α,25-OH(2)Vitamin D3 and/or Dexamethasone and matured with lipopolysaccharide. In a multistep assay, naïve T cells were incubated with DCs for two rounds and functional suppression assays were performed. The resultant T cells were analyzed at the DNA, protein, and functional level. Results Substantial phenotypic heterogeneity of peripheral blood CD4+ T cells was observed and documented for three major populations: resting Tconv (CD25-CD127+/lo), activated Tconv (CD25+CD127+) and Treg (CD25+CD127lo) in healthy controls. Despite this, I observed no differences between the Treg subpopulations from new onset T1D patients, Aab+ patients and healthy controls. In addition, there were no differences in the Treg transcriptome of T1D patients and healthy controls by RNAseq. I was, however, able to identify a small set of differentially expressed genes was discovered in Tconv suggesting a role of neutrophils in the onset of T1D. Heterogeneity of antigen-responsive Tconv and Treg was identified by gene expression profiling. I was able to define Treg specific as well as activation specific profiles, and found different expression profiles if T cells are foreign antigen or autoantigen activated and if the responding cells are Treg or Tconv. Genes that define the specific profiles include FOXP3, CD127, several cytokines, transcription factors and activation markers. The manipulation of naïve CD4+CD25- T cells by tDCs led to an unstable CD25+CD127loFOXP3+ phenotype of the generated cells. However, none of the subsequently performed functional assays could confirm that the resultant cells were iTreg or exhausted activated Tconv. In particular, methylation status of the Treg-specific demethylated region (TSDR) was inconsistent with stable Treg, suggesting that so-called tolerogenic protocols may not lead to a long-lived Treg phenotype. Conclusion CD4+CD25+ T cells are heterogeneous. I defined marker combinations that will help distinguish Treg from ex vivo and in vitro activated Tconv cells. With these tools, I was able to show that healthy controls and patients with type 1 diabetes cannot be distinguished by Treg phenotype. Comprehensive single cell analysis of antigen activated T cells provided the most promising avenue for identifying antigen-specific Treg and opens new possibilities to analyze immune therapeutic approaches, particularly when Treg expansion is the therapeutic objective. The findings will be used for monitoring children participating in antigen-based prevention studies in children at risk for T1D. / Hintergrund Regulatorische T Zellen (Treg) sind eine Subpopulation der CD4+ T Zellen, welche eine wichtige Rolle in den peripheren Toleranzmechanismen des Immunsystems spielen. Ihre suppressive Funktion auf autoreaktive T Zellen kann Autoimmunität verhindern. Verschiedene Studien berichteten widersprüchlich, dass Treg in Typ 1 Diabetes (T1D) in ihrer Fähigkeit beeinträchtigt sind autoreaktive T Zellen zu supprimieren (Tan et al., 2014; Zhang et al., 2012). Treg können im Thymus differenzieren (tTreg) oder aus peripheren naïven CD4+CD25- T Zellen generiert werden (pTreg), welche ähnliche suppressive Eigenschaften wie tTreg besitzen. Es wurde außerdem berichtet, dass Treg aktiv unter tolerisierenden Konditionen induziert werden können (iTreg) (Kleijwegt et al., 2010; Yuan and Malek, 2012). Obwohl verschiedene Treg Subpopulationen beschrieben wurden, exprimieren die archetypischen humanen Treg die Hauptmarker CD4, CD25 und FOXP3 exprimieren, während CD127 herunterreguliert ist. Jedoch zeigen auch aktivierte konventionelle T Zellen (Tconv) diesen Phänotyp (Miyara et al., 2009). Da Treg und Tconv gegensätzliche Funktionen und therapeutische Indikationen aufweisen, ist es wichtig Marker zu erhalten, die sicher bona fide Treg identifizieren. Fragestellung Das Ziel meiner Arbeit ist es, die Heterogenität von humanen T Zellen zu definieren mit einen spezifischen Fokus bona fide Treg zu identifizieren. Dafür untersuchte ich die Heterogenität dieser Zellpopulation in gesunden Individuen und T1D Patienten, als Krankheitsmodell, und wie T Zellen als Treg oder Tconv definiert werden können wenn sie einem Antigen ausgesetzt sind. Material und Methoden Für das Phänotypisieren habe ich Proben von Patienten mit beginnendem T1D (Alter 7-11 Jahre), Autoantikörper positiven Patienten (Aab+) und gesunden Individuen mittels Durchflusszytometrie auf eine Reihe von Treg-assoziierten Markern getestet. Des Weiteren wurden frisch isolierte CD4+CD25+CD127lo Treg und CD+CD25- Tconv für die Transkriptomanalyse (RNAseq) genutzt, welche mit der Gesamt-RNA durchgeführt wurden. Für die funktionelle Analyse von Antigen-spezifischen Genexpressionsmustern habe ich ein Multifarbenproliferationstest entwickelt. Treg (CD4+CD25+CD127lo) und Tconv (CD4+CD25-CD127+/lo) wurden aus isolierten mononukleären Zellen des peripheren Blutes (PBMC) sortiert. Ich habe die sortierten und gefärbten Zellen mit CD4- Zellen zusammengefügt, um einen Gesamt-PBMC-Test zu simulieren und habe die Zellen mit Tetanus-, Influenza- oder Auto-antigen (GAD65, Proinsulin) stimuliert. Die Zellen wurden für 5 Tage inkubiert und die Antigen-reagierenden und -proliferierenden Zellen sowie die nicht-reagierenden Zellen Einzelzell sortiert und mittels Multiplex qPCR analysiert. Um therapeutische Ansätze zum Expandieren oder Generieren von Treg zu untersuchen, habe ich in vitro Ansätze für die de novo Induktion von Treg durch die Nutzung von tolerisierenden dendritischen Zellen (tDCs) untersucht. Die tDCs wurden von Monozyten in Anwesenheit von 1α,25-OH(2)Vitamin D3 und/oder Dexamethason differenziert und mit Lipoploysaccharid maturiert. Naïve T Zellen wurden in einem Mehrschrittverfahren mit DCs inkubiert. Die resultierenden T Zellen wurden auf DNA, Protein und funktioneller Ebene analysiert. Ergebnisse Substantielle phänotypische Heterogenität von peripheren Blut CD4+ T Zellen wurde in drei Hauptpopulationen in gesunden Individuen beobachtet und dokumentiert: ruhende Tconv (CD25-CD127+/lo), aktivierte Tconv (CD25+CD127+) und Treg (CD25+CD127lo). Weiterführend ergab der phänotypische Vergleich von Patienten mit beginnender T1D, Aab+ Patienten und gesunden Individuen keine Unterschiede in den Treg Subpopulationen. Außerdem zeigten sich keine Unterschiede in den durch RNAseq gemessenen Treg Transkriptomen von T1D Patienten und gesunden Individuen. Jedoch wurde ein kleine Gruppe von differentiell exprimierten Genen in Tconv entdeckt, welche eine mögliche Rolle von Neutrophilen in T1D andeuten. Heterogenität von Antigen-spezifischen Tconv und Treg Antworten wurde durch Genexpressionsanalysen identifiziert. Ich konnte Treg- sowie Aktivierungs-spezifische Muster definieren und verschiedene Expressionsprofile finden, wenn T Zellen durch Fremd- oder Autoantigen aktiviert wurden und ob sie die reagierenden Zellen Treg oder Tconv sind. Folgende Gene waren hauptsächlich in die Profilbildung involviert: FOXP3, CD127, mehrere Zytokine, Transkriptionsfaktoren und Aktivierungsmarker. Die Manipulation von naïven CD4+CD25- T Zellen durch tDCs führte zu einem instabilen CD25+CD127loFOXP3+ Phänotyp der generierten Zellen. Jedoch konnte keiner der weiterführenden funktionellen Analysen unterscheiden, ob die resultierenden Zellen iTreg oder aktivierte erschöpfte T Zellen waren. Insbesondere war der Methylierungsstatus der Treg-spezifisch demethylierten Region (TSDR) nicht konsistent mit einen stabilen Treg Phänotyp, was darauf hinweist, dass sogenannte tolerisiernde Protokolle nicht zu einem langlebigen Treg Phänotyp führen. Schlussfolgerungen CD4+CD25+ T Zellen sind heterogen. Ich habe Markerkombinationen definiert die helfen werden Treg von ex vivo und in vitro aktivierten Tconv Zellen zu unterscheiden. Mit diesen Mitteln war ich in der Lage zu zeigen, dass gesunde Individuen und Patienten mit Typ 1 Diabetes nicht anhand ihres Treg Phänotyps unterschieden werden können. Umfassende Einzelzell-Analysen von Antigen aktivierten T Zellen lieferten den vielversprechendsten Ansatz für die Identifizierung von Antigen-spezifischen Treg und eröffnen neue Möglichkeiten um immuntherapeutische Ansätze zu analysieren, insbesondere wenn Treg Expansion das therapeutische Ziel ist. Diese Erkenntnisse werden zukünftig für das Monitoring von Kindern, mit einem hohen T1D Risiko, genutzt die an Antigen-basierten Präventionsstudien teilnehmen.

Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...

Les lymphocytes T (LT) CD4+ auxiliaires soutiennent l‘action des LT CD8+ cytotoxiques (LTC) au cours des réponses immunitaires anti-tumorales. Des protocoles d‘immunothérapie cellulaire adoptive (ICA) basés sur l‘injection d‘effecteurs T CD4+ ont donc été développés pour traiter les cancers, et ils ont montré une efficacité thérapeutique. Cependant, la difficulté de disposer de cellules présentatrices d‘antigène (CPA) autologues permettant de générer un nombre suffisant de LT CD4+ spécifiques fonctionnels in vitro dans un court délai représente un obstacle majeur au développement de telles approches. Pour contourner cette difficulté, notre groupe a précédemment développé des cellules présentatrices d‘antigène artificielles (CPAA) dérivant de fibroblastes murins NIH/3T3 et exprimant les molécules nécessaires pour activer des LT CD4+ humains : une molécule HLA (Human Leucocyte Antigen) de classe II (ici HLA-DR1), la molécule de costimulation CD80 et les molécules d‘adhérence CD54 et CD58. Dans ce travail, nous avons cherché à optimiser nos CPAA DR1+ (CPAADR1) en permettant une expression endogène et constitutive de l‘antigène d‘intérêt (ici l‘hémagglutinine, HA), sous forme de peptide ou de protéine, au niveau des compartiments cellulaires impliqués dans la présentation des antigènes par les molécules HLA-II. Nous avons montré que les CPAADR1 « endogènes » exprimant le peptide HA au niveau du réticulum endoplasmique (RE) ou la protéine HA à la membrane plasmique, possédaient les meilleures capacités de présentation de l‘antigène. En stimulation primaire, ces deux lignées de CPAADR1 activaient des LT CD4+ spécifiques de HA, mais avec une capacité moindre que des CPA autologues. En revanche, en restimulation, les CPAADR1 exprimant le peptide HA dans le RE étaient même plus efficaces pour amplifier des LT CD4+ spécifiques fonctionnels que des CPAADR1 ou des CPA autologues chargées avec le peptide d‘intérêt. Les LT obtenus étaient des cellules Th1 mémoires exprimant du granzyme B et produisant de l‘IFN-γ et du TNF-α. C‘est la première fois à notre connaissance qu‘un antigène exprimé de façon endogène dans une lignée cellulaire peut-être présenté de façon efficace sur une molécule HLA de classe II. Nos CPAA « endogènes » constituent donc un nouvel outil unique pour générer de façon reproductible et standardisable des réponses T CD4+ spécifiques, et pourraient déboucher sur le développement de nouvelles approches d‘ICA / CD4+ helper T lymphocytes (TLs) sustain CD8+ cytotoxic TL (CTL) activity during anti-tumor immune responses. Adoptive cell immunotherapy (ACI) protocols based on the injection of CD4+ T-effectors have therefore been developed to treat cancers, and they have proven therapeutic efficacy. However, the difficulty of obtaining autologous antigen presenting cells (APCs) for generating a sufficient number of functional specific CD4+ TLs in a short time in vitro is a major obstacle to the development of such approaches. To bypass this difficulty, our group has previously developed artificial antigen presenting cells (AAPCs) derived from NIH/3T3 murine fibroblasts expressing molecules necessary to activate human CD4+ TLs: an HLA (Human Leucocyte Antigen) class II molecule (in this study HLA-DR1), CD80 costimulatory molecule, and CD54 and CD58 adhesion molecules. In this work, we sought to optimize our DR1+ AAPCs (AAPCDR1) by allowing an endogenous and constitutive expression of the antigen of interest (in this study hemagglutinin, HA), as a peptide or a whole protein, in different cell compartments involved in antigen presentation by HLA-II molecules. We have shown that ―endogenous‖ AAPCDR1 expressing HA peptide in the endoplasmic reticulum (ER) or HA protein at the plasma membrane had the best antigen presentation abilities. In a first stimulation, both AAPCDR1cell lines activated HA-specific CD4+ TLs, but to a lower extent than autologous APCs. However, in a second stimulation, AAPCDR1 expressing HA peptide in the ER were even more effective for amplifying functional specific CD4+ TLs than AAPCDR1 or autologous APCs loaded with the peptide of interest. Obtained TLs were memory Th1 cells expressing granzyme B and producing IFN-γ and TNF-α. This is the first time to our knowledge that an antigen expressed endogenously in a cell line can be efficiently presented on an HLA class II molecule. Our ―endogenous‖ AAPCs represent therefore a new and unique tool for reproducible and standardizable generation of specific CD4+ T responses, and could lead to the development of new ACI approaches.

Trotz der längst etablierten und weit verbreiten Impfprophylaxe mit potenten Toxoidimpfstoffen sind dramatisch verlaufende Tetanusinfektionen noch immer im Alltag des Pferdepraktikers präsent. Dies gab Anlass, verschiedene Impfprotokolle und die daraus resultierende humorale Immunitätslage zu überprüfen. Kenntnis über die durch Vakzination erwirkte Tetanusimmunität ist im Falle der Versorgung von Verletzungen oder vor elektiven Eingriffen hinsichtlich der Entscheidung für oder gegen eine neuerliche aktive und/oder passive Immunisierung erforderlich. Weiterhin ermöglicht die Kontrolle auf Persistenz homologer maternaler Antikörper vor Durchführung der Erstvakzination eine optimale Impfprophylaxe. Für diese beiden Indikationen wurde der Fassisi® TetaCheck als direkt am Patienten anwendbarer Schnelltest entwickelt. Dieser Streifentest wurde mit besonderem Augenmerk auf der zuverlässigen Identifizierung nicht ausreichend geschützter Individuen und der Unempfindlichkeit gegenüber der Testdurchführung und Interpretation durch ungeschulte Personen evaluiert. Zunächst wurden 91 Serumproben von Klinikpatienten mit glaubhafter Impfanamnese mittels Doppel-Antigen-ELISA (DAE) untersucht. Neben der Bestimmung der Seroprävalenz protektiver Tetanustoxoid-Antikörperkonzentrationen (TTAK) von > 0,1 IE/ml in dieser Population wurden mögliche Einflussgrößen auf die Höhe der TTAK zum Zeitpunkt der Blutentnahme analysiert. Zu diesen zählten das Alter der Tiere, die Impfintervalle, der Zeitabstand zur letzten Vakzination und das gleichzeitige Verimpfen weiterer Komponenten. Der Tetanus-Streifentest (TST) wurde evaluiert, indem die durch zwei unabhängige Untersucher ermittelten qualitativen Resultate des Schnelltestes mit den mittels DAE quantifizierten Antitoxinkonzentrationen in 99 Serumproben retrospektiv verglichen wurden. Ergänzend erfolgte die objektive Quantifizierung der Farbreaktion im Testfeld des TST durch Fotografieren und anschließender Analyse mittels einer Bildbearbeitungssoftware. Die Seroprävalenz protektiver TTAK betrug 92,3 %. 89 % der untersuchten Pferde waren ihrem jeweiligen Alter entsprechend gemäß der ‚Leitlinie zur Impfung von Pferden‘, herausgegeben von der Ständigen Impfkommission Vet. des Bundesverbandes Praktizierender Tierärzte immunisiert. Fünf dieser Pferde waren jedoch nicht ausreichend geschützt. Hierzu zählten ein fünf Monate altes Fohlen, bei welchem die maternalen Antikörper bereits unter die Schutzgrenze abgefallen waren, zwei juvenile Pferde ohne abgeschlossene Grundimmunisierung und zwei adulte Pferde. Abweichungen von der Impfempfehlung bestanden ausschließlich in Form verlängerter Abstände der Wiederholungsimpfungen von drei bis zu acht Jahren. Trotzdem wiesen diese Tiere protektive TTAK auf. Unter alleiniger Betrachtung des Patientenalters wiesen alle geriatrischen Patienten (n = 12) TTAK weit oberhalb der Schutzgrenze auf. Hinsichtlich der Einhaltung unterschiedlicher Boosterintervalle unterschieden sich die TTAK nicht signifikant (p = 0,117). Der zeitliche Abstand zur letzten Tetanusimpfung ließ keine Prognose über die zu erwartenden TTAK zu. TTAK nach Impfung mit monovalenten Vakzinen unterschieden sich nicht signifikant von denen nach Durchführung einer Kombinationsimpfung (p = 0,63). Für den TST ergaben sich eine Sensitivität von 83,6 % und eine Spezifität von 100 %. Die Übereinstimmung der Untersucher hinsichtlich eines binären Resultats war fast vollkommen (K = 0,88). Die Durchführung des TST durch den jeweils anderen Untersucher hatte keinen maßgeblichen Einfluss auf die Auswertung des Teststreifens (K = 0,80 und K = 0,84). Durch Erweiterung des vom Hersteller vorgegebenen Bewertungsmaßstabes „negativ“, „schwach positiv“ und „positiv“ auf fünf unterschiedliche Farbintensitäten konnte eine bessere Differenzierung ungeschützter Individuen von Tieren mit belastbarer Immunität ermöglicht werden. Zwischen der objektiv gemessenen Farbintensität und der TTAK bestand ein positiver linearer Zusammenhang (r² = 0,74). Auf diesen Ergebnissen basierend sollte zur Vermeidung ineffektiver Immunisierungen vor der Erstvakzination eine Bestimmung der TTAK mit Vollendung des fünften Lebensmonats erfolgen. Hierzu erwies sich der Fassisi® TetaCheck aufgrund seiner Zuverlässigkeit und Unempfindlichkeit als überaus geeignet. Da auch das strikte Einhalten der Impfempfehlung keine ausreichende Seroprotektion garantiert und die Eintragungen im Pferdepass fehlerhaft sein können, kann nur über eine Bestimmung der TTAK Gewissheit über den tatsächlichen Immunstatus erlangt werden. Die routinemäßige Implementierung des TST in die Pferdepraxis kann dazu beitragen, die Notwendigkeit einer Immuntherapie zu diagnostizieren und damit unnötige und nebenwirkungsbehaftete TT- oder Antiserumgaben zu minimieren. Im zweijährlichen Abstand vorgenommene Wiederholungsimpfungen führen zu keiner besseren Immunitätslage gegenüber wesentlich längeren Impfintervallen. Die Impfempfehlung könnte daher ein acht- bis zehnjähriges Boosterintervall ausweisen. Die humorale Tetanusimmunität betreffend ergeben sich keine Nachteile bei gleichzeitiger Impfung weiterer Komponenten.

Background: To compare the clinical value of the measurement of complex and total PSA in the discrimination between benign prostatic hyperplasia (BPH) and prostate cancer. Methods: In serum samples collected from 166 men with histopathologically proven clinically localized prostate cancer and of 97 men with BPH, total prostate-specific antigen (PSA), complexed PSA and the free to total PSA ratio were determined. The statistical analysis was done by the comparison of the receiver operator characteristic (ROC) curves. Results: The areas under the ROC curves were 0.776 for total PSA, 0.799 for complexed PSA (total PSA vs. cPSA: p < 0.0001) and 0.812 for the free to total PSA ratio. With a cut-off of 3.0 ng/ml for complexed PSA, the sensitivity was 90%, the specificity 58%, the positive and the negative predictive values 79 and 78%, respectively. With a cut-off of 4.0 ng/ml for total PSA, the sensitivity was 87%, the specificity 59%, the positive and the negative predictive values were 78 and 72%, respectively. Conclusions: There was a statistically significant advantage for complexed PSA compared to total PSA in the discrimination between BPH and prostate cancer. The difference was, however, small and its clinical relevance is questionable. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...