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In this report we present a confocal Raman system to identify the unique spectral features of two proteins, Interleukin-10 and Angiotensin Converting Enzyme. Characteristic Raman spectra were successfully acquired and identified for the first time to our knowledge, showing the potential of Raman spectroscopy as a non-invasive investigation tool for biomedical applications.

In this report we present a confocal Raman system to identify the unique spectral features of two proteins, Interleukin-10 and Angiotensin Converting Enzyme. Characteristic Raman spectra were successfully acquired and identified for the first time to our knowledge, showing the potential of Raman spectroscopy as a non-invasive investigation tool for biomedical applications.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replicate during acute infection in lymphocytes of the gastrointestinal tract, before disseminating systemically. Localized replication and associated loss of gut-resident CD4+ T cells occur regardless of the portal of entry of the virus (e.g., intravenous vs. rectal). Thus, HIV and SIV are tropic for gut tissue, and their pathogenesis requires the special environment of the intestine. T helper 17 (Th17) cells are important contributors to microbial defense in the gut that are vulnerable to HIV infection and whose loss is associated with translocation of microbial products to the systemic circulation, leading to chronic immune activation and disease progression. Interleukin (IL)-21 promotes differentiation and survival of Th17 cells and stimulates CD8+ T cell function. By promoting Th17 cell survival, IL-21 could limit bacterial translocation and immune activation in the setting of acute or rebounding HIV/SIV disease. In this study, we tested the effect of recombinant IL-21-IgFc treatment, given at the time of infection, on SIVmac251 infection. We found that rIL-21-IgFc decreases immune activation and maintains effective antiviral responses by CD8+ T cells in blood, but this maintenance is not associated with lower viral loads. rIL-21-IgFc treatment also did not generally support Th17 cell populations, but Th17 cells remained strongly and independently associated with control of plasma viremia. For example, the single animal exhibiting greatest control over viremia in our study also manifested the highest levels of IL-21 in plasma, Th17 cell maintenance in blood, and Th17 cells in intestinal tissue. These findings provide rationale for further exploration of IL-21 treatment as a support for host CD8+ T cell responses in HIV cure strategies.

Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n = 18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL, AuthorCount:5

T cell help to B cells is a fundamental property of adaptive immunity, yet only recently have many of the cellular and molecular mechanisms of T cell help emerged. T follicular helper (Tfh) cells are the CD4+ T helper cells that provide cognate help to B cells for high affinity antibody production in germinal centres (GC). This study has reveals a critical role of IL-21 in the upregulation of Tfh signature molecules. Expression of ICOS was found to be necessary for optimal production of IL-21; indicative of interplay between two Tfh expressed molecules. We also demonstrate that IL-21's costimulatory capacity for T helper differentiation operates at the level of the TCR through Vav1 signalling that controls T cell helper function and survival. Tfh cells express uniquely high levels of the IL-21 receptor relative to other T cell subsets, which reflects an IL-21-driven autocrine loop that is important for the generation and function of Tfh cells, which in turn were critical for supporting primary and secondary T dependent antibody responses. This study reveals a previously unappreciated role for Tfh cells in the formation of the GC through a CD4+ T cell intrinsic requirement for responsiveness to IL-21. IL-2 and IL-21 are crucial growth factors for distinct T helper subsets with opposing regulatory and effector functions, respectively. Mice made genetically deficient in IL-2 or its high affinity receptor chain (CD25) suffer from a fatal autoimmune disease characterized by ulcerative colitis and haemolytic anaemia. The observed autoimmunity and associated splenomegally are thought to be caused, in part, by a loss of regulation of effector T cells due to a deficit in IL-2-dependent Foxp3 regulatory T cells. Since IL-21 is known to facilitate the development of a number of autoimmune diseases, the possible contribution of IL-21 to the autoimmune pathology observed in Il2-/- mice was investigated in this study. Our findings demonstrate that IL-21:IL-21R signalling contribu

Das Hauptprojekt dieser Dissertation war der Aufklärung der Rolle des Orphan-Nuclear-Rezeptors NR2F6 bei der Reaktion auf das Keimzentrum (GC) gewidmet. Frühere Arbeiten aus unserem Labor haben gezeigt, dass NR2F6 eine Rolle bei der Unterdrückung von Immunantworten spielt, insbesondere bei der Kontrolle der Cytokinexpression. In diesen früheren Studien haben wir in einem Mausmodell für Multiple Sklerose eine erhöhte Clearance von Krebs bei Mäusen mit Nr2f6-Mangel sowie schwerwiegendere Autoimmunreaktionen bei Mäusen mit Nr2f6-Mangel gezeigt. Wir haben auch zuvor gezeigt, dass Mäuse mit Nr2f6-Mangel beim Altern eine systemische Lupus erythematodes (SLE) -ähnliche Krankheit entwickeln, die durch selbstreaktive Antikörper gegen dsDNA und nukleares Antigen gekennzeichnet ist. Dies lässt vermuten, dass Nr2f6 eine wichtige Rolle bei den Reaktionen des Keimzentrums spielt. Literaturrecherchen deuteten auch auf eine mögliche Rolle von Nr2f6 bei menschlichem SLE hin, da mindestens eine Gruppe eine verringerte Nr2f6-Expression in B-Zellen von SLE-Patienten nachgewiesen hat. Wir zeigen, dass Nr2f6-defiziente Mäuse größere Keimzentren entwickeln, einschließlich erhöhter Populationen von T-follikulären Helferzellen (Tfh), Keimzentrum-B-Zellen und frühen Plasmazellen. Interessanterweise führte der Verlust von Nr2f6 zu einer erhöhten Überlebensrate und Akkumulation, insbesondere bei Tfh-Zellen. Der Nr2f6-Mangel innerhalb des CD4 + -Zellkompartiments war der Haupttreiber der veränderten GC-Reaktion und war nicht abhängig von dem Nr2f6-Mangel in B-Zellen. Wir zeigen ferner, dass der Verlust von Nr2f6 zu einer erhöhten Il21-Expression und Proteinproduktion durch CD4-Zellen führte und dass NR2F6-Protein den Il21-Promotor direkt an drei getrennten Stellen und einer konservierten nicht-codierenden Sequenz band. Schließlich zeigen wir, dass eine Unterbrechung der IL-21R-Signalübertragung durch blockierende Antikörper in Mäusen mit Nr2f6-Mangel eine übermäßige Akkumulation von Tfh-Zellen, The main project found within this thesis was devoted to elucidating the role of the orphan nuclear receptor NR2F6 in germinal center (GC) responses. Previous work from our lab has indicated a role for NR2F6 in the suppression of immune responses, particularly in control of cytokine expression. In these earlier studies, we demonstrate increased clearance of cancer in mice deficient for Nr2f6 as well as more severe autoimmune responses by Nr2f6-deficient mice in a mouse model of multiple sclerosis. We have also previously demonstrated that Nr2f6-deficient mice when allowed to age, develop a systemic lupus erythematosus (SLE)-like disease characterized by self-reactive antibodies to dsDNA and nuclear antigen. Suggesting, that Nr2f6 may play an important role in germinal center responses. Literature searches also suggested a potential role for Nr2f6 in human SLE as at least one group has demonstrated reduced Nr2f6 expression in B cells from SLE patients. We demonstrate that Nr2f6-deficient mice develop larger germinal centers, including increased populations of T follicular helper cells (Tfh), germinal center B cells and early-plasma cells. Interestingly, Nr2f6-loss resulted in increased survival and accumulation particularly for Tfh cells. Nr2f6-deficiency within the CD4+ cell compartment was the primary driver of the altered GC response and was not dependent on Nr2f6-deficiency in B cells. We further show that loss of Nr2f6 resulted in increased Il21 expression and protein production by CD4 cells and that NR2F6 protein directly bound the Il21 promotor at three separate sites and one conserved non-coding sequence. Finally, we show that interruption of IL-21R signaling via blocking antibody in Nr2f6-deficient mice prevented excessive accumulation of Tfh cells. With this work we have now linked the nuclear receptor NR2F6 to control of Tfh accumulation through regulation of Il21 expression. This finding is a significant step towards a better understanding of Il21 control, submitted by William Joseph Olson, MSc, Kumulative Dissertation aus drei Artikeln, Dissertation Medical University of Innsbruck 2019

Das Hauptprojekt dieser Dissertation war der Aufklärung der Rolle des Orphan-Nuclear-Rezeptors NR2F6 bei der Reaktion auf das Keimzentrum (GC) gewidmet. Frühere Arbeiten aus unserem Labor haben gezeigt, dass NR2F6 eine Rolle bei der Unterdrückung von Immunantworten spielt, insbesondere bei der Kontrolle der Cytokinexpression. In diesen früheren Studien haben wir in einem Mausmodell für Multiple Sklerose eine erhöhte Clearance von Krebs bei Mäusen mit Nr2f6-Mangel sowie schwerwiegendere Autoimmunreaktionen bei Mäusen mit Nr2f6-Mangel gezeigt. Wir haben auch zuvor gezeigt, dass Mäuse mit Nr2f6-Mangel beim Altern eine systemische Lupus erythematodes (SLE) -ähnliche Krankheit entwickeln, die durch selbstreaktive Antikörper gegen dsDNA und nukleares Antigen gekennzeichnet ist. Dies lässt vermuten, dass Nr2f6 eine wichtige Rolle bei den Reaktionen des Keimzentrums spielt. Literaturrecherchen deuteten auch auf eine mögliche Rolle von Nr2f6 bei menschlichem SLE hin, da mindestens eine Gruppe eine verringerte Nr2f6-Expression in B-Zellen von SLE-Patienten nachgewiesen hat. Wir zeigen, dass Nr2f6-defiziente Mäuse größere Keimzentren entwickeln, einschließlich erhöhter Populationen von T-follikulären Helferzellen (Tfh), Keimzentrum-B-Zellen und frühen Plasmazellen. Interessanterweise führte der Verlust von Nr2f6 zu einer erhöhten Überlebensrate und Akkumulation, insbesondere bei Tfh-Zellen. Der Nr2f6-Mangel innerhalb des CD4 + -Zellkompartiments war der Haupttreiber der veränderten GC-Reaktion und war nicht abhängig von dem Nr2f6-Mangel in B-Zellen. Wir zeigen ferner, dass der Verlust von Nr2f6 zu einer erhöhten Il21-Expression und Proteinproduktion durch CD4-Zellen führte und dass NR2F6-Protein den Il21-Promotor direkt an drei getrennten Stellen und einer konservierten nicht-codierenden Sequenz band. Schließlich zeigen wir, dass eine Unterbrechung der IL-21R-Signalübertragung durch blockierende Antikörper in Mäusen mit Nr2f6-Mangel eine übermäßige Akkumulation von Tfh-Zellen, The main project found within this thesis was devoted to elucidating the role of the orphan nuclear receptor NR2F6 in germinal center (GC) responses. Previous work from our lab has indicated a role for NR2F6 in the suppression of immune responses, particularly in control of cytokine expression. In these earlier studies, we demonstrate increased clearance of cancer in mice deficient for Nr2f6 as well as more severe autoimmune responses by Nr2f6-deficient mice in a mouse model of multiple sclerosis. We have also previously demonstrated that Nr2f6-deficient mice when allowed to age, develop a systemic lupus erythematosus (SLE)-like disease characterized by self-reactive antibodies to dsDNA and nuclear antigen. Suggesting, that Nr2f6 may play an important role in germinal center responses. Literature searches also suggested a potential role for Nr2f6 in human SLE as at least one group has demonstrated reduced Nr2f6 expression in B cells from SLE patients. We demonstrate that Nr2f6-deficient mice develop larger germinal centers, including increased populations of T follicular helper cells (Tfh), germinal center B cells and early-plasma cells. Interestingly, Nr2f6-loss resulted in increased survival and accumulation particularly for Tfh cells. Nr2f6-deficiency within the CD4+ cell compartment was the primary driver of the altered GC response and was not dependent on Nr2f6-deficiency in B cells. We further show that loss of Nr2f6 resulted in increased Il21 expression and protein production by CD4 cells and that NR2F6 protein directly bound the Il21 promotor at three separate sites and one conserved non-coding sequence. Finally, we show that interruption of IL-21R signaling via blocking antibody in Nr2f6-deficient mice prevented excessive accumulation of Tfh cells. With this work we have now linked the nuclear receptor NR2F6 to control of Tfh accumulation through regulation of Il21 expression. This finding is a significant step towards a better understanding of Il21 control, submitted by William Joseph Olson, MSc, Kumulative Dissertation aus drei Artikeln, Dissertation Medical University of Innsbruck 2019