Application to the Analysis of Germinal Center Reactions In Vivo

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method

In vivo analysis of B cell receptor signaling in germinal centers : new insights into affinity maturation via intravital microscopy

Unser Immunsystem stellt uns eine Vielzahl von Effektormechanismen zur Verfügung, die für den lebenslangen Schutz vor Krankheitserregern sorgen. Ein Aspekt dieser Mechanismen ist die Produktion von Immunglobulin-Antikörpern durch langlebige Plasmazellen, die aus B-Zellen in Keimzentren der sekundären lymphatischen Organe stammen. Innerhalb der Keimzentren bilden B-Zellen, T-Zellen und follikuläre dendritische Zellen stabile Kontakte, die für einen Prozess, der Affinitätsreifung genannt wird, unerlässlich sind. Diese Kontakte führen zum Erwerb des Antigens aus follikulären dendritischen Zellen durch B-Zellen, zu kognitiven T-Zell-B-Zell-Interaktionen, die das Überleben von affinen B-Zell-Klonen sichern, und schließlich zur Rezirkulation von B-Zellen innerhalb bestimmter Zonen des Keimzentrums. Während der Rezirkulation nimmt die Antigen-Affinität der membrangebundenen Form des Immunglobulins, des B-Zell-Rezeptors, durch akkumulierte Mutationen in den Immunglobulin-Genen stetig zu. In der Keimzentrumsreaktion kommen ihm zwei Rollen zu: Er ist verantwortlich für die Aufnahme des Antigens und überträgt Signale ins Innere der Zelle. So könnte die Affinität des B-Zell-Rezeptors neben der Erleichterung der Antigenaufnahme auch eine entscheidende Rolle bei der Signaltransduktion spielen, eine Rolle des Keimzentrums-B-Zell-Rezeptors, die schon lange in der Diskussion steht. Wir zeigen hier, dass durch die Stimulation des B-Zellrezeptors der Calciumeinstrom in B-Zellen initiiert wird und die Calciumkonzentration mit der Zeit und mit der Affinität des Rezeptors zum Antigen ansteigt. Zur Überwachung der Signaltransduktion durch B-Zell-Rezeptoren in vivo haben wir einen neuartigen transgenen Reporter-Mausstamm namens YellowCaB entwickelt, der das calciumempfindliche Förster-Resonanzenergietransfer-Sensorprotein TN-XXL in CD19+ B-Zellen exprimiert. Mithilfe der intravitalen Zweiphotonenmikroskopie sind wir erstmals in der Lage, das Signaling von B-Zell-Rezeptoren zeitlich und räumlich im lebenden Organismus zu analysieren und gleichzeitig Zell-zu-Zell-Interaktionen und Migration zu untersuchen. Unser System erhält dabei die naturgegebenen Strukturen des Gewebes, was so in Kultur nicht machbar wäre. Darüber hinaus bietet uns die Fluoreszenzlebensdauer-Bildgebung die Möglichkeit, Calciumkonzentrationen mit Entscheidungen über das Zellschicksal in Beziehung zu setzen. Mit dieser Methode konnten wir zeigen, dass naive und antigenspezifische B-Zellen innerhalb und außerhalb von Keimzentren eine differentielle Faltung von TN-XXL (und damit Calciumkonzentration) aufweisen. Die Beobachtung von kolokalisierten B-Zellen, die mit dem Calciumzufluss in einem der Reaktionspartner zusammenfiel, führte zu der Hypothese, dass neben follikulären dendritischen Zellen auch B-Zellen selbst eine Quelle der Stimulation von B-Zell-Rezeptoren sind. Zusammen mit CD40L-Signalen von T-follikulären Helferzellen und Signalen, die von Toll-ähnlichen Rezeptoren stammen, wird der B-Zell-Calciumspiegel streng reguliert, um das Ergebnis der Keimzentrumsreaktion zu kontrollieren. Die terminale Differenzierung könnte eine direkte Folge der Aufsummierung dieser Signale sein. Um dies zu unterstützen, konnten wir zeigen, dass B-Zellen in der Lage sind, auf äußere Reize mehrmals durch Calciumzufuhr ohne Erschöpfung zu reagieren. Zusammengefasst stellen wir hier einen neuen Wirkmechanismus für B-Zellen vor, die an der Keimzentrumsreaktion teilnehmen und ihr eigenes Schicksal rückkoppeln. Daher ist das Signaling von B-Zell-Rezeptoren ein wesentlicher Bestandteil der B-Zell-, Plasmazell- und Gedächtnis-B-Zell-Entwicklung.Our immune system provides us with a variety of effectors that account for life-long protection against pathogens. One aspect of this mechanism is the production of antibodies by long-lived plasma cells that derive from B cells of germinal centers within secondary lymphoid organs. Within germinal centers, B cells, T cells and follicular dendritic cells form stable contacts that are essential for a process called affinity maturation. These contacts lead to acquisition of antigen from follicular dendritic cells by B cells, cognate T cell-B cell interactions that ensure survival of B cell clones and finally to the recirculation of B cells within distinct zones of the germinal center. During recirculation, the membrane-bound form of immunoglobulin, the B cell receptor, becomes increasingly affine to the antigen thanks to accumulating mutations in the immunoglobulin genes. The B cell receptor is not only responsible for antigen uptake but is also able to transduce signals to inner parts of the cell. Thus, apart from its facilitating antigen uptake, affinity of the B cell receptor might play a pivotal role in signaling as well, a role of the germinal center B cell receptor that has long been up for discussion. We here show that B cell receptor signaling-related Calcium influx is active in a subset of germinal center B cells and that calcium concentration is increasing over time and with affinity of the receptor to the antigen. To monitor B cell receptor signaling in vivo we developed a novel transgenic reporter mouse strain termed YellowCaB that expresses the calcium sensitive Förster Resonance Energy Transfer sensor protein TN-XXL in CD19+ B cells. Employing intravital two-photon microscopy, we are the first group to be able to spatiotemporally resolve B cell receptor signaling in living organisms, while simultaneously monitoring cell-to-cell interactions and migration. This allows us to preserve the natural architecture of the tissue, what otherwise would not have been possible in culture systems. Furthermore, fluorescence lifetime imaging is offering us the possibility to relate calcium concentrations to cell fate decisions, as we could show that naive and antigenspecific B cells inside and outside germinal centers show distinct quenching of TN-XXL. The observation of colocalized B cells that coincided with calcium influx in one reaction partner led us to hypothesize that apart from follicular dendritic cells, B cells itself are a source of B cell receptor stimulation via complement receptors. Together with CD40L-signals from T follicular helper cells and signals stemming from Toll like receptor engagement, the B-cellular calcium level is tightly regulated to control the outcome of the germinal center reaction. Terminal differentiation might be a direct downstream result of these signals adding up. To support this, we could show that B cells are able to react to external stimuli several times via calcium influx without exhaustion. Taken together, we here present a new mode of action for B cells taking part in the germinal center reaction and feedbacking their own fate. Thus, B cell receptor signaling is an essential part of B-cell-, plasma cell- and memory B cell development

Dann bilden wir mal asynchron eine Gruppe...! - Wie bitte? Was machen wir?

Im vergangenen Jahr startete das Evangelische Erwachsenenbildungswerk Nordrhein ein Pilotprojekt: Im sechswöchigen Onlinekurs "OLiWe - OnLine in der Weiterbildung" ging es darum, herauszufinden, wie sich asynchrone Kommunikation und kooperatives Onlinearbeiten anfühlt, um schließlich selbst kleine Onlinemodule zu konzipieren. Ein Moderator, fünf Tutor/innen und zwanzig Teilnehmende nutzten den Kurs, um digitale Tools zu erproben, didaktisch zu experimentieren und ihren Arbeitsalltag zu bereichern. (DIPF/Orig.

Intravital FRET: Probing Cellular and Tissue Function in Vivo

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice

The chronically inflamed central nervous system provides niches for long-lived plasma cells

Abstract Although oligoclonal bands in the cerebrospinal fluid have been a hallmark of multiple sclerosis diagnosis for over three decades, the role of antibody-secreting cells in multiple sclerosis remains unclear. T and B cells are critical for multiple sclerosis pathogenesis, but increasing evidence suggests that plasma cells also contribute, through secretion of autoantibodies. Long-lived plasma cells are known to drive various chronic inflammatory conditions as e.g. systemic lupus erythematosus, however, to what extent they are present in autoimmune central nervous system inflammation has not yet been investigated. In brain biopsies from multiple sclerosis patients and other neurological diseases, we could detect non-proliferating plasma cells (CD138+Ki67−) in the parenchyma. Based on this finding, we hypothesized that long-lived plasma cells can persist in the central nervous system (CNS). In order to test this hypothesis, we adapted the multiple sclerosis mouse model experimental autoimmune encephalomyelitis to generate a B cell memory response. Plasma cells were found in the meninges and the parenchyma of the inflamed spinal cord, surrounded by tissue areas resembling survival niches for these cells, characterized by an up-regulation of chemokines (CXCL12), adhesion molecules (VCAM-1) and survival factors (APRIL and BAFF). In order to determine the lifetime of plasma cells in the chronically inflamed CNS, we labeled the DNA of proliferating cells with 5-ethynyl-2′-deoxyuridine (EdU). Up to five weeks later, we could detect EdU+ long-lived plasma cells in the murine CNS. To our knowledge, this is the first study describing non-proliferating plasma cells directly in the target tissue of a chronic inflammation in humans, as well as the first evidence demonstrating the ability of plasma cells to persist in the CNS, and the ability of the chronically inflamed CNS tissue to promote this persistence. Hence, our results suggest that the CNS provides survival niches for long-lived plasma cells, similar to the niches found in other organs. Targeting these cells in the CNS offers new perspectives for treatment of chronic autoimmune neuroinflammatory diseases, especially in patients who do not respond to conventional therapies

Lymphocyte access to lymphoma is impaired by high endothelial venule regression

International audienceBlood endothelial cells display remarkable plasticity depending on the demands of a malignant microenvironment. While studies in solid tumors focus on their role in metabolic adaptations, formation of high endothelial venules (HEVs) in lymph nodes extends their role to the organization of immune cell interactions. As a response to lymphoma growth, blood vessel density increases; however, the fate of HEVs remains elusive. Here, we report that lymphoma causes severe HEV regression in mouse models that phenocopies aggressive human B cell lymphomas. HEV dedifferentiation occurrs as a consequence of a disrupted lymph-carrying conduit system. Mechanosensitive fibroblastic reticular cells then deregulate CCL21 migration paths, followed by deterioration of dendritic cell proximity to HEVs. Loss of this crosstalk deprives HEVs of lymphotoxin-β-receptor (LTβR) signaling, which is indispensable for their differentiation and lymphocyte transmigration. Collectively, this study reveals a remodeling cascade of the lymph node microenvironment that is detrimental for immune cell trafficking in lymphoma

T-bet and RORα control lymph node formation by regulating embryonic innate lymphoid cell differentiation.

The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZFhi ILCP expressing central LTi molecules in a RORα-dependent fashion. Our data unveil an unexpected role for T-bet and RORα during embryonic ILC function and highlight that RORγt is crucial in counteracting the suppressive effects of T-bet