Refine
Has Fulltext
- yes (29)
Is part of the Bibliography
- yes (29)
Year of publication
Document Type
- Journal article (24)
- Doctoral Thesis (5)
Keywords
- B cells (29) (remove)
Institute
- Medizinische Klinik und Poliklinik II (8)
- Institut für Anatomie und Zellbiologie (7)
- Neurologische Klinik und Poliklinik (6)
- Pathologisches Institut (5)
- Abteilung für Molekulare Innere Medizin (in der Medizinischen Klinik und Poliklinik II) (2)
- Graduate School of Life Sciences (2)
- Institut für Virologie und Immunbiologie (2)
- Kinderklinik und Poliklinik (2)
- Klinik und Poliklinik für Mund-, Kiefer- und Plastische Gesichtschirurgie (2)
- Lehrstuhl für Tissue Engineering und Regenerative Medizin (2)
Sonstige beteiligte Institutionen
Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospot\(^{®}\) assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)-\({\gamma}\) and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN-\({\gamma}\) and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.
There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.
The first description of neuromyelitis optica by Eugène Devic and Fernand Gault dates back to the 19th century, but only the discovery of aquaporin-4 autoantibodies in a major subset of affected patients in 2004 led to a fundamentally revised disease concept: Neuromyelits optica spectrum disorders (NMOSD) are now considered autoantibody-mediated autoimmune diseases, bringing the pivotal pathogenetic role of B cells and plasma cells into focus. Not long ago, there was no approved medication for this deleterious disease and off-label therapies were the only treatment options for affected patients. Within the last years, there has been a tremendous development of novel therapies with diverse treatment strategies: immunosuppression, B cell depletion, complement factor antagonism and interleukin-6 receptor blockage were shown to be effective and promising therapeutic interventions. This has led to the long-expected official approval of eculizumab in 2019 and inebilizumab in 2020. In this article, we review current pathogenetic concepts in NMOSD with a focus on the role of B cells and autoantibodies as major contributors to the propagation of these diseases. Lastly, by highlighting promising experimental and future treatment options, we aim to round up the current state of knowledge on the therapeutic arsenal in NMOSD.
B cell-derived interleukin-10 (IL-10) production has been described as a hallmark for regulatory function in B lymphocytes. However, there is an ongoing debate on the origin of IL-10-secreting B cells and lack of specific surface markers has turned into an important obstacle for studying human B regulatory cells. In this study, we propose that tumor necrosis factor receptor 2 (TNFR2) expression can be used for enrichment of IL-10-secreting B cells. Our data confirm that IL-10 production can be induced by TLR9 stimulation with CpG ODN and that IL-10 secretion accompanies differentiation of peripheral blood B cells into plasma blasts. We further show that CpG ODN stimulation induces TNFR2 expression, which correlates with IL-10 secretion and terminal differentiation. Indeed, flow cytometric sorting of TNFR2+ B cells revealed that TNFR2+ and TNFR2− fractions correspond to IL-10+ and IL-10− fractions, respectively. Furthermore, CpG-induced TNFR2+ B cells were predominantly found in the IgM+ CD27+ B cell subset and spontaneously released immunoglobulin. Finally, our data corroborate the functional impact of TNFR2 by demonstrating that stimulation with a TNFR2 agonist significantly augments IL-10 and IL-6 production in B cells. Altogether, our data highlight a new role for TNFR2 in IL-10-secreting human B lymphocytes along with the potential to exploit this finding for sorting and isolation of this currently ill-defined B cell subset.
Die zentrale Rolle von B- Zellen in der Pathogenese und Therapie von Autoimmunerkrankungen hat in den letzten Jahren zu unterschiedlichen therapeutischen Ansätzen geführt, B- Zellen direkt oder indirekt zu targetieren. Der bisher effektivste Ansatz stellt der monoklonale anti- CD20 Antikörper Rituximab dar. Nach Gabe von Rituximab kommt es zu einer passageren, in der Regel sechs bis neun Monate anhaltenden peripheren B- Zelldepletion. Die anti- CD20 vermittelte B- Zelldepletion stellt zwar ein vielversprechendes Therapieverfahren in der Behandlung der Rheumatoiden Arthritis dar, derzeit ist noch wenig über das Regenerationsverhalten von B- Zellen nach Therapie mit Rituximab bekannt. Daher wurde in dieser Arbeit die B-Zellrepopulation insbesondere hinsichtlich der Modulation des Mutationsmusters des B- Zellrezeptors untersucht. Dazu wurde die VH4- Familie des Immunglobulinrezeptors- prospektiv vor und nach anti- CD20 vermittelter B- Zelldepletion analysiert. Bei drei Patienten (A-C) wurden die Ig-VH4 Gene aus genomischer DNA peripherer B- Zellen amplifiziert, subkloniert und sequenziert. Die Analyse erfolgte zu drei verschiedenen Zeitpunkten: Vor Therapie, in der frühen Regenerationsphase mit einem B- Zellanteil von 1% bis 1,3% im peripheren Blut und in der späten Regenerationsphase, zwei bis drei Monate nach der frühen Regenerationsphase Mini Gene wie VH4-34 und VH4-39, die in Verbindung mit Autoimmunität stehen, waren vor Einleitung der Therapie relativ überexprimiert. Die Behandlung mit Rituximab führte bei allen drei Patienten zu einer Veränderung des Repertoires der regenerierenden B- Zellen mit einer reduzierten Benutzung der VH4-39 Gene, außerdem bei Patient A zusätzlich der VH4-34 Gene. Tief greifende Veränderungen fanden sich während der frühen Regenerationsphase durch den Nachweis einer rezirkulierenden Population hochmutierter B- Zellen, die in einer durchgeführten Immunophänotypisierung mit spezifischen Oberflächenmarkdern als Plasmazellen identifiziert wurden. Da Plasmazellen kein CD20 Molekül exprimieren, werden sie durch eine anti-CD20 vermittelte Therapie nicht direkt eliminiert. Sie zirkulieren während der Phase der B- Zelldepletion aber auch nicht im peripheren Blut. Interessanterweise sind sie in der frühen Regenerationsphase in der Peripherie als erste mit hohem relativem Anteil nachweisbar. Daher wurde untersucht, ob auch Plasmazellen durch die Therapie moduliert werden, obwohl sie durch Rituximab nicht direkt targetiert werden. Hierfür wurden die Sequenzen mit hochmutiertem Ig- Rezeptor (>9 Mutationen/Sequenz) im Verlauf einer detaillierten Analyse zugeführt. Dabei wurde insbesondere das Mutationsmuster in RGYW/WRCY Hotspot Motiven und in den CDR- Regionen untersucht. Die Analyse der Mutationshäufigkeit in RGYW/WRCY Hotspot Motiven erlaubt eine Abschätzung, in wieweit die somatische Hypermutation der B- Zellen durch T- Zell abhängige Differenzierung erfolgte. Die als Plasmazellen identifizierten hochmutierten Sequenzen zeigten vor der Therapie Charakteristika einer aktiven Erkrankung mit einem verminderten Targeting der RGYW/WRCY Motive. Dagegen zeigte sich in den rezirkulierenden Plasmazellen während der frühen Regenerationsphase ein zunehmendes Targeting der RGYW/WRCY Motive. Dies spricht für einen Repertoire Shift zu mehr T- Zell abhängigen B- Zell Mutationen. Ein Zustand, wie er bei Gesunden beobachtet wird. Um die Hypothese einer Rituximab- induzierten Modulation des Plasmazellkompartimentes weiter zu untermauern, wurde der R/S Quotient, d.h. das Verhältnis von Silent zu Replacement Mutationen in den hypervariablen Regionen (CDRs) der hochmutierten Plasmazell-Ig Sequenzen bestimmt. Interessanterweise fanden sich in der Regenerationsphase signifikant erhöhte R/S Ratios in den rezirkulierenden Plasmazellen.. Die signifikante Zunahme an Replacement Mutationen in den CDR- Regionen, welche sich in einer Zunahme des R/S Verhältnisses wiederspiegelt, kann als Entwicklung des Ig- Repertoires durch positive Antigenselektion interpretiert werden und weist somit eine Rituximab- induzierte Veränderung auf, wie man sie sonst bei gesunden Individuen findet. Zusammenfassend zeigt unsere Studie, dass die transiente anti- CD20 vermittelte B- Zelldepletion auch zu einer indirekten Modulation des Plasmazellkompartimentes führt. Insbesondere werden postrekombinatorische Imprints des B- Zell Rezeptors, wie somatische Hypermutation und Antigen Selektion, verändert, die mit hoher Wahrscheinlichkeit für die Entstehung von Autoimmunität bei der Rheumatoiden Arthritis eine Rolle spielen. Zusätzlich kann die Modulation des genetischen Imprints der Ig Rezeptoren bei der Rheumatoiden Arthritis eventuell als möglicher Biomarker entwickelt werden, um ein Ansprechen auf die Therapie vorherzusagen. Dies bedarf weiterer Untersuchungen, um tiefer greifende Einblicke in Prozesse zu erlangen, die durch zukünftige Therapien beeinflussbar werden.
Here, we assessed whether 36 single nucleotide polymorphisms (SNPs) within the TNFSF4 and MAPKAPK2 loci influence the risk of developing invasive aspergillosis (IA). We conducted a two-stage case control study including 911 high-risk patients diagnosed with hematological malignancies that were ascertained through the aspBIOmics consortium. The meta-analysis of the discovery and replication populations revealed that carriers of the TNFSF4\(_{rs7526628T/T}\) genotype had a significantly increased risk of developing IA (p = 0.00022). We also found that carriers of the TNFSF4\(_{rs7526628T}\) allele showed decreased serum levels of TNFSF14 protein (p = 0.0027), and that their macrophages had a decreased fungicidal activity (p = 0.048). In addition, we observed that each copy of the MAPKAPK2\(_{rs12137965G}\) allele increased the risk of IA by 60% (p = 0.0017), whereas each copy of the MAPKAPK2\(_{rs17013271T}\) allele was estimated to decrease the risk of developing the disease (p = 0.0029). Mechanistically, we found that carriers of the risk MAPKAPK2\(_{rs12137965G}\) allele showed increased numbers of CD38+IgM-IgD- plasmablasts in blood (p = 0.00086), whereas those harboring two copies of the allele had decreased serum concentrations of thymic stromal lymphopoietin (p = 0.00097). Finally, we also found that carriers of the protective MAPKAPK2\(_{rs17013271T}\) allele had decreased numbers of CD27-IgM-IgD- B cells (p = 0.00087) and significantly lower numbers of CD14+ and CD14+CD16- cells (p = 0.00018 and 0.00023). Altogether, these results suggest a role of the TNFSF4 and MAPKAPK2 genes in determining IA risk.
Background:
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable.
Methods:
We used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 μg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA.
Results:
Treatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment.
Conclusion:
Anti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.
Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor β (TGFβ) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies.
Background
Lymphocytes have been shown to play an important role in the pathophysiology of acute ischemic stroke, but the properties of B cells remain controversial. The aim of this study was to unravel the role of B cells during acute cerebral ischemia using pharmacologic B cell depletion, B cell transgenic mice, and adoptive B cell transfer experiments.
Methods
Transient middle cerebral artery occlusion (60 min) was induced in wild-type mice treated with an anti-CD20 antibody 24 h before stroke onset, JHD\(^{−/−}\) mice and Rag1\(^{−/−}\) mice 24 h after adoptive B cell transfer. Stroke outcome was assessed at days 1 and 3. Infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain sections, and neurological scores were evaluated. The local inflammatory response was determined by real-time PCR and immunohistochemistry. Apoptosis was analyzed by TUNEL staining, and astrocyte activation was revealed using immunohistochemistry and Western blot.
Results
Pharmacologic depletion of B cells did not influence infarct volumes and functional outcome at day 1 after stroke. Additionally, lack of circulating B cells in JHD\(^{−/−}\) mice also failed to influence stroke outcome at days 1 and 3. Furthermore, reconstitution of Rag1\(^{−/−}\) mice with B cells had no influence on infarct volumes.
Conclusion
Targeting B cells in experimental stroke did not influence lesion volume and functional outcome during the acute phase. Our findings argue against a major pathophysiologic role of B cells during acute ischemic stroke.
Primary prevention strategies, such as vaccinations at the age extremes, in neonates and elderly individuals, demonstrate a challenge to health professionals and public health specialists. The aspects of the differentiation and maturation of the adaptive immune system, the functional implications of immunological immaturity or immunosenescence and its impact on vaccine immunogenicity and efficacy will be highlighted in this review. Several approaches have been undertaken to promote Th1 responses in neonates and to enhance immune functions in elderly, such as conjugation to carrier proteins, addition of adjuvants, concomitant vaccination with other vaccines, change in antigen concentrations or dose intervals or use of different administration routes. Also, early protection by maternal vaccination seems to be beneficial in neonates. However, it also appears necessary to think of other end points than antibody concentrations to assess vaccine efficacy in neonates or elderly, as also the cellular immune response may be impaired by the mechanisms of immaturity, underlying health conditions, immunosuppressive treatments or immunosenescence. Thus, lifespan vaccine programs should be implemented to all individuals on a population level not only to improve herd protection and to maintain protective antibody levels and immune memory, but also to cover all age groups, to protect unvaccinated elderly persons and to provide indirect protection for neonates and small infants.
B cells play diverse roles in the immunopathogensis of autoimmune diseases several approaches targeting B cell directly or indirectly are in clinical practice in the treatment of autoimmunity. In this regard, temporal B cell depletion by rituximab (anti CD20 antibody) is being appreciated and gaining more importance in recent years. To date, little is known about the regeneration profile of B cells following B cell depletion. We wanted to investigate the early replenishing B cells and examine the dynamic changes in the repertoire. we studied the immunoglobulin receptor (IgR) modulation of Ig-VH4 genes as representative of the heavy chain family. Five patients were included in the study and therapy induced alterations were assessed. Three time points namely before therapy, early regeneration phase (ERP- the early time point during regeneration where just above 1% B cells were found in the peripheral lymphocyte pool) and later regeneration phase (LRP- which commenced 2-3 months following ERP) were chosen. In three patients (A-C), Ig-VH4 genes were amplified from total genomic DNA during the above-mentioned all time points and in another two patients (D and E), Ig genes during ERP were studied by single cell amplification technique. Firstly, B cell regeneration followed the characteristic regeneration pattern as reported by several groups, with a predominant circulation of CD38hi expressing plasma cells and immature B cells in the ERP. During LRP, the proportion of these cells reduced relatively and the levels of naïve B cells rose gradually. On a molecular level, Ig-VH4 variable gene usage prior and post B cell depletion was determined and it was noticed that a diverse set of Ig-VH4 genes were employed in the repertoire before and after therapy. Mini gene segments such as VH4-34 and VH-4-39, which were reported to be connected with autoimmunity, were over expressed in the B cell repertoire before therapy. Profound changes were noticed in the early reemerging repertoire with a relatively increased population of intensely mutated B cells. These B cells acquired >=9 mutations in the Ig genes. Immunophenotyping with specific surface markers revealed that these highly mutated B cells evolve from the isotype-switched memory compartment especially the plasma cells. To support the hypothesis that the highly mutated B cells observed during ERP were plasma cells we carried out single cell amplification of individual plasma cells in another two patients during ERP and compared the mutational load, which remained similar. Actually plasma cells do not express CD20 on their surface and are not eliminated by rituximab therapy. However they were not observed in the peripheral blood following B cell depletion. The earliest time point when plasma cells are found again in peripheral circulation is the early recovery period (ERP). Therefore, it was intriguing to ascertain if the plasma cells were also modulated by rituximab therapy although they were not directly targeted by the therapy. We investigated if there is a therapy mediated mutational modulation of the plasma cells though these are not directly targeted by the therapy. We examined the confinement of mutations to the pre-defined RGYW/WRCY hotspot motifs (R=purine, Y=pyrimidine, W=A/T) in the plasma cells, which provides information on the involvement of T cells in B cell somatic hypermutation (SHM). Plasma cells before rituximab manifested the characteristics of active disease, which was revealed by restricted mutational targeting to the RGYW/WRCY motifs. The reemerging plasma cells during ERP had an increased targeting of the RGYW/WRCY motifs which indicated for a more pronounced T cell mediated B cell mutations which is the scenario observed in the healthy subjects. To further support the hypothesis of rituximab-mediated plasma cell modulation, we delineated the replacement to silent mutations ratio (R/S) in the hypervariable regions (CDRs) of the plasma cell Ig sequences. Within our study, the mean R/S ratio in the plasma cell CDRs of the patient group was relatively low (1.87) before rituximab treatment and interestingly this ratio increased significantly in the recirculating plasma cells to values of 2.67 and 3.60 in ERP and LRP status respectively. The increase in R/S ratios in reemerging plasma cells can be interpreted as a shaping of the Ig-repertoire by positive antigen selection as seen in healthy individuals. To conclude, our study demonstrates temporal B cell depletion by rituximab therapy seems to modulate also the plasma cell compartment, which is not directly targeted by the therapy. Modulation of plasma cells in RA could be also used as a potential biomarker in studying the effective response in RA treatment. This needs to be further explored to gain deeper insights into the underlying processes, which may be influenced by future therapies.
In lymphocytes, immune receptor signals induce the rapid nuclear translocation of preformed cytosolic NFAT proteins. Along with co-stimulatory signals, persistent immune receptor signals lead to high levels of NFATc1/αA, a short NFATc1 isoform, in effector lymphocytes. Whereas NFATc1 is not expressed in plasma cells, in germinal centers numerous centrocytic B cells express nuclear NFATc1/αA. When overexpressed in chicken DT40 B cells or murine WEHI 231 B cells, NFATc1/αA suppressed their cell death induced by B cell receptor signals and affected the expression of genes controlling the germinal center reaction and plasma cell formation. Among those is the Prdm1 gene encoding Blimp-1, a key factor of plasma cell formation. By binding to a regulatory DNA element within exon 1 of the Prdm1 gene, NFATc1/αA suppresses Blimp-1 expression. Since expression of a constitutive active version of NFATc1/αA interfered with Prdm1 RNA expression, LPS-mediated differentiation of splenic B cells to plasmablasts in vitro and reduced immunoglobulin production in vivo, one may conclude that NFATc1/αA plays an important role in controlling plasmablast/plasma cell formation.
Memory B cells have known to play an important role in the pathogenesis of rheumatoid arthritis (RA). With the emergence of B cell-targeted therapies, the modulation of memory B cells appears to be a key therapeutic target. Human peripheral memory B cells can be distinguished based on the phenotypic expression of CD27 and IgD, characterizing the three major B cell subpopulations: CD27+IgD+ pre-switch, CD27+IgD- post-switch, and CD27-IgD- double-negative memory B cells. We evaluated different memory cell populations for activation markers (CD95 and Ki-67) and chemokine receptors (CXCR3 and 4) expressing B cells in active RA, as well as under IL6-R blockade by tocilizumab (TCZ) and TNF-α blockade by adalimumab (ADA). Memory B cells were phenotypically analyzed from RA patients at baseline, week 12, and week 24 under TCZ or ADA treatment, respectively. Using flow cytometry, surface expression of CD95, intracellular Ki-67, and surface expressions of CXCR3 and CXCR4 were determined. Compared with healthy donors (n = 40), the phenotypic analysis of RA patients (n = 80) demonstrated that all three types of memory B cells were activated in RA patients. Surface and intracellular staining of B cells showed a significantly higher percentage of CD95+ (p < 0.0001) and Ki-67+ (p < 0.0001) cells, with numerically altered CXCR3+ and CXCR4+ cells in RA. CD95 and Ki-67 expressions were highest in post-switch memory B cells, whereas CD19+CXCR3+ and CD19+CXCR4+ expressing cells were substantially higher in the pre-switch compartment. In all subsets of the memory B cells, in vivo IL-6R, and TNF-α blockade significantly reduced the enhanced expressions of CD95 and Ki-67. Based on our findings, we conclude that the three major peripheral memory B cell populations, pre-, post-switch, and double-negative B cells, are activated in RA, demonstrating enhanced CD95 and Ki-67 expressions, and varied expression of CXCR3 and CXCR4 chemokine receptors when compared with healthy individuals. This activation can be efficaciously modulated under cytokine inhibition in vivo.
Multiple myeloma (MM), a malignancy of the bone marrow, is characterized by a pathological increase in antibody-producing plasma cells and an increase in immunoglobulins (plasmacytosis). In recent years, bone morphogenetic proteins (BMPs) have been reported to be activators of apoptotic cell death in neoplastic B cells in MM. Here, we use bone morphogenetic protein 2 (BMP2) to show that the "apoptotic" effect of BMPs on human neoplastic B cells is dominated by anti-proliferative activities and cell cycle arrest and is apoptosis-independent. The anti-proliferative effect of BMP2 was analysed in the human cell lines KMS12-BM and L363 using WST-1 and a Coulter counter and was confirmed using CytoTox assays with established inhibitors of programmed cell death (zVAD-fmk and necrostatin-1). Furthermore, apoptotic activity was compared in both cell lines employing western blot analysis for caspase 3 and 8 in cells treated with BMP2 and FasL. Additionally, expression profiles of marker genes of different cell death pathways were analysed in both cell lines after stimulation with BMP2 for 48h using an RT-PCR-based array. In our experiments we observed that there was rather no reduction in absolute cell number, but cells stopped proliferating following treatment with BMP2 instead. The time frame (48–72 h) after BMP2 treatment at which a reduction in cell number is detectable is too long to indicate a directly BMP2-triggered apoptosis. Moreover, in comparison to robust apoptosis induced by the approved apoptotic factor FasL, BMP2 only marginally induced cell death. Consistently, neither the known inhibitor of apoptotic cell death zVAD-fmk nor the necroptosis inhibitor necrostatin-1 was able to rescue myeloma cell growth in the presence of BMP2.
Nach Aktivierung differenzieren B-Zellen entweder direkt zu IgM sezernierenden Plasmazellen oder treten in den Differenzierungsweg zur Gedächtniszelle ein, der sowohl durch die Affinitätsreifung als auch den Klassensprung zu sekundären Immunglobulin-Isotypen gekennzeichnet ist. Welchen Weg die B-Zelle durchläuft, ist abhängig von der Intensität und der Dauer des BZR-Signals, von der Verfügbarkeit und der Art der T-Zell-Hilfe und von weiteren Signalen in der speziellen Mikroumgebung des Keimzentrums. Der Transkriptionsfaktor Blimp-1 ("B lymphocyte induced maturation protein 1") wird als ein „Mastergen“ der terminalen B-Zell-Differenzierung betrachtet und ist in der Lage, die komplexen Differenzierungsprozesse zu Ig-sezernierenden Plasmazellen auszulösen und voranzutreiben. Die Ergebnisse der vorliegenden Arbeit identifizieren Blimp-1 als wichtigen Regulator, der bestimmt, ob eine B-Zelle zur Plasmazelle oder zur Gedächtniszelle differenziert. Unter Verwendung ruhender, primärer B-Zellen der Maus, die in vitro mit Interleukin-4 (IL-4), anti-mF(ab´)2 oder anti-CD40 in verschiedenen Kombinationen sowohl in An- als auch in Abwesenheit von LPS stimuliert wurden, konnte in der vorliegenden Arbeit gezeigt werden, dass die IgM-Sekretion und die Expression von Blimp-1 durch Signalgebung über den BZR oder CD40 und durch IL-4 entweder nicht induziert oder sogar unterdrückt wird. Die Zugabe von IL-2 und IL-5 induziert die Expression von Blimp-1 und erleichtert die Sekretion von IgM und IgG1 in diesem System. Gleiches kann durch direkte Transduktion der B-Zellen mit rekombinanten Retroviren erreicht werden, die für Blimp-1 codieren. Auf der anderen Seite wird der durch IL-4 induzierte Klassensprung nach IgG1 durch Blimp-1 gehemmt. Blimp-1 bewirkt daher ein Umschalten des B-Zell-Differenzierungsweges von der Gedächtniszelle zur Plasmazelle. Die Unterdrückung der Expression von Blimp-1 sowohl durch Antigen-BZR-Wechselwirkungen als auch durch die von T-Helferzellen abhängige Signalgebung über CD40 und IL-4 unterdrückt die terminale Differenzierung zur Plasmazelle und ist für den Eintritt und das Durchlaufen des Gedächtniszell-Differenzierungsweges notwendig. Zur Identifikation von Genen, deren Expression durch Blimp-1 direkt oder indirekt beeinflusst wird, wurde Blimp-1 in WEHI 231 B-Lymphomzellen unter Verwendung rekombinanter Retroviren überexprimiert. Messika et al. zeigten, dass die Überexpression von Blimp-1 in B-Lymphomzellen in Abhängigkeit vom Reifungsstadium der Zelle entweder einen Wachstumsnachteil, gefolgt vom Zelltod, induziert oder zur terminalen Differenzierung führt. Obwohl WEHI 231 Zellen unreife, d.h. sIgM+ B-Zellen repräsentieren, exprimieren Blimp-1 transduzierte WEHI 231 Zellen die J-Kette, zeigen eine erhöhte Konzentration der für die sekretorische Form der my-Kette codierenden mRNA, exprimieren den Plasmazellmarker Syndecan-1 auf ihrer Oberfläche und sezernieren für kurze Zeit IgM. Diese Differenzierungsprozesse gehen allerdings mit einem Wachstumsnachteil und Zellzyklus-Arrest, gefolgt vom Zelltod, einher. Blimp-1 exprimierende WEHI 231 Zellen zeigen somit den Phänotyp kurzlebiger Plasmazellen. Eine Langzeitkultur Blimp-1+ WEHI 231 Zellen führt zum Verlust des differenzierten Phänotyps, d.h der Erhalt IgM-sezernierender WEHI 231 Zellen ist nicht ohne weitere Maßnahmen möglich. Auf molekularer Ebene hemmt Blimp-1 die Expression von c-myc und diejenige des antiapoptotischen Bcl-2 Familienmitgliedes A1, stimuliert aber die Expression von mad4. Die Verschiebung des Verhältnisses von Myc/Max- zu Mad/Max-Heterodimeren zugunsten von Mad/Max-Komplexen und die daraus resultierende Inhibition der Transkription von Myc-abhängigen, proiliferationsfördernden Genen ist in vielen Systemen als von zentraler Bedeutung für die Initiation von Differenzierungsprozessen beschrieben und wurde auch bereits für B-Zellen diskutiert. Auch in primären B-Zellen führt Blimp-1 zu einer verstärkten Expression von mad4. Wird der durch Blimp-1 bewirkte Verlust der Expression von A1 durch dessen Überexpression in Blimp-1+ WEHI 231 Zellen kompensiert, so überleben diese Zellen wieder erheblich länger, bleiben aber weiterhin im Zellzyklus arretiert. Der differenzierte Phänotyp, charakterisiert durch die verstärkte Expression von mad4 und der Sekretion von IgM, wird dabei aufrechterhalten. Wachstumsnachteil und Zelltod können in diesem System daher entkoppelt werden. In primären Zellen führt Blimp-1 ebenfalls zur Erniedrigung der A1 Expression. Da diese Zellen aber nicht in dem Maße wie WEHI 231 Zellen absterben, kann der Verlust von A1 offensichtlich besser kompensiert werden. Die Lebensdauer einer Blimp-1+ Plasmazelle ist somit durch Manipulation der Expression von antiapoptotischen Molekülen wie A1 verlängerbar.
Atopic dermatitis (AD) is one of the most prevalent inflammatory disease among non-fatal skin diseases, affecting up to one fifth of the population in developed countries. AD is characterized by recurrent pruritic and localized eczema with seasonal fluctuations. AD initializes the phenomenon of atopic march, during which infant AD patients are predisposed to progressive secondary allergies such as allergic rhinitis, asthma, and food allergies. The pathophysiology of AD is complex; onset of the disease is caused by several factors, including strong genetic predisposition, disrupted epidermal barrier, and immune dysregulation. AD was initially characterized by defects in the innate immune system and a vigorous skewed adaptive Th2 response to environmental agents; there are compelling evidences that the disorder involves multiple immune pathways. Symptomatic palliative treatment is the only strategy to manage the disease and restore skin integrity. Researchers are trying to more precisely define the contribution of different AD genotypes and elucidate the role of various immune axes. In this review, we have summarized the current knowledge about the roles of innate and adaptive immune responsive cells in AD. In addition, current and novel treatment strategies for the management of AD are comprehensively described, including some ongoing clinical trials and promising therapeutic agents. This information will provide an asset towards identifying personalized targets for better therapeutic outcomes.
Introduction
B cells are attracting increasing attention in the pathogenesis of multiple sclerosis (MS). B cell-targeted therapies with monoclonal antibodies or plasmapheresis have been shown to be successful in a subset of patients. Here, patients with either relapsing-remitting (n = 24) or secondary progressive (n = 6) MS presenting with an acute clinical relapse were screened for their B cell reactivity to brain antigens and were re-tested three to nine months later. Enzyme-linked immunospot technique (ELISPOT) was used to identify brain-reactive B cells in peripheral blood mononuclear cells (PBMC) directly ex vivo and after 96 h of polyclonal stimulation. Clinical severity of symptoms was determined using the Expanded Disability Status Scale (EDSS).
Results
Nine patients displayed B cells in the blood producing brain-specific antibodies directly ex vivo. Six patients were classified as B cell positive donors only after polyclonal B cell stimulation. In 15 patients a B cell response to brain antigens was absent. Based on the autoreactive B cell response we categorized MS relapses into three different patterns. Patients who displayed brain-reactive B cell responses both directly ex vivo and after polyclonal stimulation (pattern I) were significantly younger than patients in whom only memory B cell responses were detectable or entirely absent (patterns II and III; p = 0.003). In one patient a conversion to a positive B cell response as measured directly ex vivo and subsequently also after polyclonal stimulation was associated with the development of a clinical relapse. The evaluation of the predictive value of a brain antigen-specific B cell response showed that seven of eight patients (87.5%) with a pattern I response encountered a clinical relapse during the observation period of 10 months, compared to two of five patients (40%) with a pattern II and three of 14 patients (21.4%) with a pattern III response (p = 0.0005; hazard ratio 6.08 (95% confidence interval 1.87-19.77).
Conclusions
Our data indicate actively ongoing B cell-mediated immunity against brain antigens in a subset of MS patients that may be causative of clinical relapses and provide new diagnostic and therapeutic options for a subset of patients.
INTRODUCTION:
B cells are attracting increasing attention in the pathogenesis of multiple sclerosis (MS). B cell-targeted therapies with monoclonal antibodies or plasmapheresis have been shown to be successful in a subset of patients. Here, patients with either relapsing-remitting (n = 24) or secondary progressive (n = 6) MS presenting with an acute clinical relapse were screened for their B cell reactivity to brain antigens and were re-tested three to nine months later. Enzyme-linked immunospot technique (ELISPOT) was used to identify brain-reactive B cells in peripheral blood mononuclear cells (PBMC) directly ex vivo and after 96 h of polyclonal stimulation. Clinical severity of symptoms was determined using the Expanded Disability Status Scale (EDSS).
RESULTS:
Nine patients displayed B cells in the blood producing brain-specific antibodies directly ex vivo. Six patients were classified as B cell positive donors only after polyclonal B cell stimulation. In 15 patients a B cell response to brain antigens was absent. Based on the autoreactive B cell response we categorized MS relapses into three different patterns. Patients who displayed brain-reactive B cell responses both directly ex vivo and after polyclonal stimulation (pattern I) were significantly younger than patients in whom only memory B cell responses were detectable or entirely absent (patterns II and III; p = 0.003). In one patient a conversion to a positive B cell response as measured directly ex vivo and subsequently also after polyclonal stimulation was associated with the development of a clinical relapse. The evaluation of the predictive value of a brain antigen-specific B cell response showed that seven of eight patients (87.5%) with a pattern I response encountered a clinical relapse during the observation period of 10 months, compared to two of five patients (40%) with a pattern II and three of 14 patients (21.4%) with a pattern III response (p = 0.0005; hazard ratio 6.08 (95% confidence interval 1.87-19.77).
CONCLUSIONS:
Our data indicate actively ongoing B cell-mediated immunity against brain antigens in a subset of MS patients that may be causative of clinical relapses and provide new diagnostic and therapeutic options for a subset of patients.
Peripheral neuropathies can severely affect patients. Causes for the disease are diverse but can be classified into two main groups, acquired and hereditary. Examples for these two types are chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and Charcot-Marie-Tooth disease type 1A (CMT1A). CIDP has an estimated prevalence of about 1-9:100 000. In this pathogenetically hetereo- geneous patient group about 5-10% show auto-antibodies against the node of Ranvier and present with distinct symptoms. Treatment with rituximab - a monoclonal antibody that deletes CD20 + B cells - has been shown to be effective in a majority of auto-antibody as- sociated CIDP cases. This suggests that B cells and the produced auto-antibodies might be pathogenic. Previous studies delivered evidence that auto-antibodies alone can induce nerve damage. In this study, the aim was to investigate the pathomechanism of auto-antibodies in vivo and their exact origin: For the analysis of the pathogenicity of auto-antibodies, passive transfer experiments on Lewis rats were performed with whole IgG from a patient with anti-contactin-1 (CNTN1) IgG4 auto-antibodies. IgG was infused through an intrathe- cal catheter targeting the thoracic/lumbar region of the spine over a long-term, 3-week period. In a previous study of our group, the IgG from the same patient has been re- ported to have mild pathogenic effects when applied intraneurally into the sciatic nerve of Lewis rats. In this study however, binding of auto-antibodies to nerve roots could not be detected. Neither evaluation of electrophysiological properties after the injection period nor motor and sensory skills tested throughout the injection period showed differences when compared to animals infused with control IgG. This suggests that in the chronic intrathecal protocol anti-CNTN1 auto-antibodies did not have a pathogenic effect. In peripheral blood, four B cell subsets capable to produce antibodies were previously described: memory B cells, plasmablasts (PBs), B1 cells and CD20 + CD38 hi cells. For the identification of the B cell subsets that produce auto-antibodies, purification and sort protocols as well as an enzyme-linked immuno spot (ELISpot) assay for IgG and IgM were established successfully. Since unstimulated B cell subsets produced very small amounts of IgG and IgM, peripheral blood mononuclear cells (PBMCs) were stimulated with IL-2 and R848 for 72 h prior to sorting. While the memory B cell frequency decreased after stimulation, the frequency of CD20 + CD38 hi cells increased and the overall number of antibody-secreting cells was increased. When stimulating patient PBMCs for 10 days though, detection of anti-neurofascin-155 (NF155) auto-antibodies in supernatants by enzyme-linked immunosorbent assay (ELISA) was possible in two out of three patient samples. Even though cell sorting was feasible after 10 days of stimulation, detection of auto-antibodies could not be accomplished using antigen-specific ELISpot. Although the implementation of the cell sorting and purification protocol was successful, further adjustments of the antigen-specific ELISpot need to be performed. However, we could show that after 10 days of stimulation auto-antibody detection is possible by ELISA which helps to pre-screen if patient PBMC contain auto-reactive B cells. CMT1A has an estimated prevalence of 1:5000 and is caused by a duplication of the peripheral myelin protein 22 kDa (PMP22) gene. Patients suffer from distal weakness and muscle wasting leading even to wheelchair-dependency in some cases. Although different treatment options for CMT1A have been tested in previous clinical trials, none of them have been successful. In this study, the aim was to identify objective and reproducible outcome measures that assess the actual nerve damage in a large cohort of CMT1A patients by analyzing a series of parameters. Glabrous skin samples were collected from 48 CMT1A, 7 CIDP and 16 small fiber neuropathy patients and 45 healthy controls. 40-µm cryosections from the lateral part of the index finger were double-labeled using immunoflu- orescence to investigate cutaneous innervation. The disease severity which was assessed using the Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2) and ranged between mild to severe (3-27) correlated with age in CMT1A patients. Furthermore, the intraepidermal nerve fiber density (IENFD) was reduced in CMT1A patients in comparison to controls and correlated negatively with the disease severity. In controls however, the IENFD correlated inversely with age. Meissner corpuscle density tended to be reduced and correlated inversely with age in CMT1A patients. This was not observed in healthy controls though. Compared to controls, Merkel cell density was also reduced in CMT1A, while the fraction of denervated Merkel cell was increased and correlated with age. Further differences were revealed concerning the node of Ranvier. Paranodes were shortened and the fraction of long nodes was decreased in CMT1A patients compared to controls. These data suggest that the IENFD, the Meissner corpuscle and Merkel cell densities are possible candidates for outcome measures as they are associated with disease severity or age of patients. However, a reliable statement about the suitability as a marker for disease progression can not be made in this study since only six CMT1A patients agreed to give a follow-up biopsy two years later.
Background
Autologous hematopoietic stem cell transplantation (aHSCT) is performed in patients with aggressive forms of systemic sclerosis (SSc). The profile of B cell reconstitution after aHSCT is not fully understood. The aim of this study was to investigate changes of B cell subsets and cytokine production of B cells in patients with SSc after aHSCT.
Methods
Peripheral blood of six patients with SSc was collected at defined intervals up to 16 months after aHSCT. Immunophenotyping was performed, and B cell function was determined by measuring cytokine secretion in supernatants of stimulated B cell cultures.
Results
Within 1 month after aHSCT, a peak in the percentage of CD38\(^{++}\)/CD10\(^+\)/IgD\(^+\) transitional B cells and CD38\(^{++}\)/CD27\(^{++}\)/IgD\(^−\) plasmablasts was detected. Long-term changes persisted up to 14 months after aHSCT and showed an increased percentage of total B cells; the absolute B cell number did not change significantly. Within the B cell compartment, an increased CD27/IgD\(^+\) naïve B cell percentage was found whereas decreased percentages of CD27\(^+\)/IgD\(^+\) pre-switched memory, CD27\(^+\)/IgD\(^−\) post-switched memory, and CD27\(^−\) /IgD\(^−\) double-negative B cells were seen after aHSCT. Cytokine secretion in B cell cultures showed significantly increased IL-10 concentrations 13 to 16 months after aHSCT.
Conclusion
A changed composition of the B cell compartment is present for up to 14 months after aHSCT indicating positive persisting effects of aHSCT on B cell homeostasis. The cytokine secretion profile of B cells changes in the long term and shows an increased production of the immune regulatory cytokine IL-10 after aHSCT. These findings might promote the clinical improvements after aHSCT in SSc patients.