Publications by authors named "Thomas H Winkler"

58 Publications

SARS-CoV-2 in severe COVID-19 induces a TGF-β-dominated chronic immune response that does not target itself.

Nat Commun 2021 03 30;12(1):1961. Epub 2021 Mar 30.

Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany.

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-β, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-β. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-β, and is distracted from itself.
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http://dx.doi.org/10.1038/s41467-021-22210-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010106PMC
March 2021

Upregulation of CCR4 in activated CD8 T cells indicates enhanced lung homing in patients with severe acute SARS-CoV-2 infection.

Eur J Immunol 2021 Mar 30. Epub 2021 Mar 30.

Department of Internal Medicine 5, Hematology, and Oncology, University Hospital Erlangen and Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.

COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK-/B-cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T-follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T cells in COVID-19 patients exhibit a stronger activation profile with differentiation toward effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted.
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http://dx.doi.org/10.1002/eji.202049135DOI Listing
March 2021

Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2.

Eur J Clin Microbiol Infect Dis 2021 Apr 20;40(4):751-759. Epub 2020 Oct 20.

Institute of Clinical and Molecular Virology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.
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http://dx.doi.org/10.1007/s10096-020-04072-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572153PMC
April 2021

Siglec-15 on Osteoclasts Is Crucial for Bone Erosion in Serum-Transfer Arthritis.

J Immunol 2020 11 5;205(10):2595-2605. Epub 2020 Oct 5.

Division of Genetics, Department of Biology, University of Erlangen, 91058 Erlangen, Germany;

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15 mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.
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http://dx.doi.org/10.4049/jimmunol.2000472DOI Listing
November 2020

TFG is required for autophagy flux and to prevent endoplasmic reticulum stress in CH12 B lymphoma cells.

Autophagy 2020 Sep 22:1-19. Epub 2020 Sep 22.

Division of Molecular Immunology, Department of Internal Medicine 3, Nikolaus-Fiebiger-Zentrum, Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Erlangen, Germany.

Plasma cells depend on quality control of newly synthesized antibodies in the endoplasmic reticulum (ER) via macroautophagy/autophagy and proteasomal degradation. The cytosolic adaptor protein TFG (Trk-fused gene) regulates ER-Golgi transport, the secretory pathway and proteasome activity in non-immune cells. We show here that TFG is upregulated during lipopolysaccharide- and CpG-induced differentiation of B1 and B2 B cells into plasmablasts, with the highest expression of TFG in mature plasma cells. CRISPR-CAS9-mediated gene disruption of in the B lymphoma cell line CH12 revealed increased apoptosis, which was reverted by BCL2 but even more by ectopic TFG expression. Loss of TFG disrupted ER structure, leading to an expanded ER and increased expression of ER stress genes. When compared to wild-type CH12 cells, KO CH12 cells were more sensitive toward ER stress induced by tunicamycin, monensin and proteasome inhibition or by expression of an ER-bound immunoglobulin (Ig) μ heavy (µH) chain. CH12 KO B cells displayed more total LC3, lower LC3-II turnover and increased numbers and size of autophagosomes. Tandem-fluorescent-LC3 revealed less accumulation of GFP-LC3 in starved and chloroquine-treated CH12 KO B cells. The GFP:RFP ratio of tandem-fluorescent-LC3 was higher in tunicamycin-treated CH12 KO B cells, suggesting less autophagy flux during induced ER stress. Based on these data, we suggest that TFG controls autophagy flux in CH12 B cells and propose that TFG is a survival factor that alleviates ER stress through the support of autophagy flux in activated B cells and mature plasma cells.: Ab, antibody; Ag, antigen; ASC, antibody-secreting cells; ATG, autophagy-related; BCR, B cell receptor; COPII, coat protein complex II; CpG, non-methylated CpG oligonucleotide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FO, follicular; GFP, green fluorescent protein; HC, heavy chain; Ig, immunoglobulin; IRES, internal ribosomal entry site; LC, light chain; MZ, marginal zone; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; TLR, toll-like receptor; UPR, unfolded protein response.
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http://dx.doi.org/10.1080/15548627.2020.1821546DOI Listing
September 2020

Cell Fusion Induced by a Fusion-Active Form of Human Cytomegalovirus Glycoprotein B (gB) Is Inhibited by Antibodies Directed at Antigenic Domain 5 in the Ectodomain of gB.

J Virol 2020 08 31;94(18). Epub 2020 Aug 31.

Virologisches Institut, Klinische und Molekulare Virologie, Friedrich Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected Virus-neutralizing antibodies defined have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection. HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.
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http://dx.doi.org/10.1128/JVI.01276-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459561PMC
August 2020

Measuring the cellular memory B cell response after vaccination in patients after allogeneic stem cell transplantation.

Ann Hematol 2020 Aug 9;99(8):1895-1906. Epub 2020 Jun 9.

Department of Biology, Division of Genetics, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.

After allogeneic hematopoietic stem cell transplantation (HSCT), patients are repetitively vaccinated to reduce the risk of infection caused by the immune deficiency following allogeneic HSCT. By the vaccination of transplanted patients, the humoral memory function can be restored in the majority of cases. It is unknown, however, to what extent memory B cells derived from the donor contribute to the mobilization of antibody-secreting cells and long-term humoral memory in patients after allogeneic HSCT. We therefore analyzed patients after allogeneic HSCT for memory B cell responses 7 days after single vaccination against tetanus toxoid (TT), diphtheria toxoid (DT), pertussis toxoid (PT), Haemophilus influenzae type b (Hib), and poliovirus. Patients showed an insufficient mobilization of plasmablasts (PB) after vaccination, whereas healthy subjects (HD, n = 13) exhibited a significant increase of PB in the peripheral blood. Regarding vaccine-specific antibody-secreting PB, all HD responded against all vaccine antigens, as expected. However, only 65% of the patients responded with a measurable increase in IgG-secreting PB against TT, 65% against DT, 33% against PT, and 53% against poliovirus. Correspondingly, the antibody titers on day 7 after vaccination did not increase in patients. A significant increase of serum titers for the vaccine antigens was detectable in the majority of patients only after repetitive vaccinations. In contrast to the low mobilization of vaccine-specific PB after vaccination, a high number of PB before vaccination was detectable in patients following allogeneic HSCT. High frequencies of circulating PB correlated with the incidence of moderate/severe chronic GVHD. In summary, patients showed a weak mobilization of antigen-specific PB and an inadequate increase in antibody titers 7 days after the first vaccination. Patients with moderate or severe chronic GVHD in their history had a significantly higher percentage of IgG-secreting PB prior to vaccination. The antigen specificity of these IgG-secreting PB is currently unknown.
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http://dx.doi.org/10.1007/s00277-020-04072-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340644PMC
August 2020

Resolving the mystery-How TCR transgenic mouse models shed light on the elusive case of gamma delta T cells.

J Leukoc Biol 2020 06 18;107(6):993-1007. Epub 2020 Feb 18.

Department of Biology, Division of Genetics, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg (FAU), Erlangen, Germany.

Cutting-edge questions in αβ T cell biology were addressed by investigating a range of different genetically modified mouse models. In comparison, the γδ T cell field lacks behind on the availability of such models. Nevertheless, transgenic mouse models proved useful for the investigation of γδ T cell biology and their stepwise development in the thymus. In general, animal models and especially mouse models give access to a wide range of opportunities of modulating γδ T cells, which is unachievable in human beings. Because of their complex biology and specific tissue tropism, it is especially challenging to investigate γδ T cells in in vitro experiments since they might not reliably reflect their behavior and phenotype under physiologic conditions. This review aims to provide a comprehensive historical overview about how different transgenic mouse models contributed in regards of the understanding of γδ T cell biology, whereby a special focus is set on studies including the elusive role of the γδTCR. Furthermore, evolutionary and translational remarks are discussed under the aspect of future implications for the field. The ultimate full understanding of γδ T cells will pave the way for their usage as a powerful new tool in immunotherapy.
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http://dx.doi.org/10.1002/JLB.1MR0120-237RDOI Listing
June 2020

Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis.

Nat Immunol 2020 03 17;21(3):331-342. Epub 2020 Feb 17.

Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA.

Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells, as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification rate but consumed oxygen in response to FAs. [C]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into tricarboxylic acid (TCA) cycle intermediates. Conversely, [C]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation was functionally important, as drug-mediated and genetic dampening of FA oxidation resulted in a selective reduction of GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.
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http://dx.doi.org/10.1038/s41590-020-0598-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112716PMC
March 2020

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

Eur J Immunol 2019 Oct;49(10):1457-1973

Flow Cytometry Laboratory, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, München, Germany.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
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http://dx.doi.org/10.1002/eji.201970107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350392PMC
October 2019

IFN-γ drives inflammatory bowel disease pathogenesis through VE-cadherin-directed vascular barrier disruption.

J Clin Invest 2019 11;129(11):4691-4707

Division of Molecular and Experimental Surgery, Translational Research Center, Department of Surgery, University Medical Center Erlangen.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with rising incidence. Diseased tissues are heavily vascularized. Surprisingly, the pathogenic impact of the vasculature in IBD and the underlying regulatory mechanisms remain largely unknown. IFN-γ is a major cytokine in IBD pathogenesis, but in the context of the disease, it is almost exclusively its immune-modulatory and epithelial cell-directed functions that have been considered. Recent studies by our group demonstrated that IFN-γ also exerts potent effects on blood vessels. Based on these considerations, we analyzed the vessel-directed pathogenic functions of IFN-γ and found that it drives IBD pathogenesis through vascular barrier disruption. Specifically, we show that inhibition of the IFN-γ response in vessels by endothelial-specific knockout of IFN-γ receptor 2 ameliorates experimentally induced colitis in mice. IFN-γ acts pathogenic by causing a breakdown of the vascular barrier through disruption of the adherens junction protein VE-cadherin. Notably, intestinal vascular barrier dysfunction was also confirmed in human IBD patients, supporting the clinical relevance of our findings. Treatment with imatinib restored VE-cadherin/adherens junctions, inhibited vascular permeability, and significantly reduced colonic inflammation in experimental colitis. Our findings inaugurate the pathogenic impact of IFN-γ-mediated intestinal vessel activation in IBD and open new avenues for vascular-directed treatment of this disease.
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http://dx.doi.org/10.1172/JCI124884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819119PMC
November 2019

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

Nat Microbiol 2019 12 23;4(12):2273-2284. Epub 2019 Sep 23.

Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.
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http://dx.doi.org/10.1038/s41564-019-0529-zDOI Listing
December 2019

CD83 orchestrates immunity toward self and non-self in dendritic cells.

JCI Insight 2019 10 17;4(20). Epub 2019 Oct 17.

Department of Immune Modulation and.

Dendritic cells (DCs) are crucial to balance protective immunity and autoimmune inflammatory processes. Expression of CD83 is a well-established marker for mature DCs, although its physiological role is still not completely understood. Using a DC-specific CD83-conditional KO (CD83ΔDC) mouse, we provide new insights into the function of CD83 within this cell type. Interestingly, CD83-deficient DCs produced drastically increased IL-2 levels and displayed higher expression of the costimulatory molecules CD25 and OX40L, which causes superior induction of antigen-specific T cell responses and compromises Treg suppressive functions. This also directly translates into accelerated immune responses in vivo. Upon Salmonella typhimurium and Listeria monocytogenes infection, CD83ΔDC mice cleared both pathogens more efficiently, and CD83-deficient DCs expressed increased IL-12 levels after bacterial encounter. Using the experimental autoimmune encephalomyelitis model, autoimmune inflammation was dramatically aggravated in CD83ΔDC mice while resolution of inflammation was strongly reduced. This phenotype was associated with increased cell influx into the CNS accompanied by elevated Th17 cell numbers. Concomitantly, CD83ΔDC mice had reduced Treg numbers in peripheral lymphoid organs. In summary, we show that CD83 ablation on DCs results in enhanced immune responses by dysregulating tolerance mechanisms and thereby impairing resolution of inflammation, which also demonstrates high clinical relevance.
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http://dx.doi.org/10.1172/jci.insight.126246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824307PMC
October 2019

Proteomics of diphtheria toxoid vaccines reveals multiple proteins that are immunogenic and may contribute to protection of humans against Corynebacterium diphtheriae.

Vaccine 2019 05 26;37(23):3061-3070. Epub 2019 Apr 26.

Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. Electronic address:

Introduced for mass immunization in the 1920s, vaccines against diphtheria are among the oldest and safest vaccines known. The basic principle of their production is the inactivation of purified diphtheria toxin by formaldehyde cross-linking, which converts the potentially fatal toxin in a completely harmless protein aggregate, which is still immunogenic. Since in addition to diphtheria toxin also other proteins may be secreted by Corynebacterium diphtheriae during cultivation, we assumed that diphtheria toxoid might not be the only component present in the vaccine. To address this question, we established a protocol to reverse formaldehyde cross-linking and carried out mass spectrometric analyses. Different secreted, membrane-associated and cytoplasmic proteins of C. diphtheriae were detected in several vaccine preparations from across the world. Based on these results, bioinformatics and Western blot analyses were applied to characterize if these proteins are immunogenic and may therefore support protection against C. diphtheriae. In frame of this study, we could show that the C. diphtheriae toxoid vaccines induce antibodies against different C. diphtheriae proteins and against diphtheria toxin secreted by Corynebacterium ulcerans, an emerging pathogen which is outnumbering C. diphtheriae as cause of diphtheria-like illness in Western Europe.
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http://dx.doi.org/10.1016/j.vaccine.2019.04.059DOI Listing
May 2019

The Role of the Pre-B Cell Receptor in B Cell Development, Repertoire Selection, and Tolerance.

Front Immunol 2018 15;9:2423. Epub 2018 Nov 15.

Department of Rheumatology and Inflammation Research, Institute of Medicine, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Around four decades ago, it had been observed that there were cell lines as well as cells in the fetal liver that expressed antibody μ heavy (μH) chains in the apparent absence of light chains. It was thus possible that these cells expressed another molecule(s), that assembled with μH chains. The ensuing studies led to the discovery of the pre-B cell receptor (pre-BCR), which is assembled from Ig μH and surrogate light (SL) chains, together with the signaling molecules Igα and β. It is expressed on a fraction of pro-B (pre-BI) cells and most large pre-B(II) cells, and has been implicated in IgH chain allelic exclusion and down-regulation of the recombination machinery, assessment of the expressed μH chains and shaping the IgH repertoire, transition from the pro-B to pre-B stage, pre-B cell expansion, and cessation.
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http://dx.doi.org/10.3389/fimmu.2018.02423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249383PMC
October 2019

Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA.

Front Immunol 2018 5;9:1551. Epub 2018 Jul 5.

Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.

Autoantibodies against double-stranded DNA (anti-dsDNA) are a hallmark of systemic lupus erythematosus (SLE). It is well documented that anti-dsDNA reactive B lymphocytes are normally controlled by immune self-tolerance mechanisms operating at several levels. The evolution of high levels of IgG anti-dsDNA in SLE is dependent on somatic hypermutation and clonal selection, presumably in germinal centers from non-autoreactive B cells. Twin studies as well as genetic studies in mice indicate a very strong genetic contribution for the development of anti-dsDNA as well as SLE. Only few single gene defects with a monogenic Mendelian inheritance have been described so far that are directly responsible for the development of anti-dsDNA and SLE. Recently, among other mutations, rare null-alleles for the deoxyribonuclease 1 like 3 () and the Fc gamma receptor IIB () have been described in SLE patients and genetic mouse models. Here, we demonstrate that double Dnase1l3- and FcgR2b-deficient mice in the C57BL/6 background exhibit a very early and massive IgG anti-dsDNA production. Already at 10 weeks of age, autoantibody production in double-deficient mice exceeds autoantibody levels of diseased 9-month-old NZB/W mice, a long established multigenic SLE mouse model. In single gene-deficient mice, autoantibody levels were moderately elevated at early age of the mice. Premature autoantibody production was accompanied by a spontaneous hyperactivation of germinal centers, early expansions of T follicular helper cells, and elevated plasmablasts in the spleen. Anti-dsDNA hybridomas generated from double-deficient mice show significantly elevated numbers of arginines in the CDR3 regions of the heavy-chain as well as clonal expansions and diversification of B cell clones with moderate numbers of somatic mutations. Our findings show a strong epistatic interaction of two SLE-alleles which prevent early and high-level anti-dsDNA autoantibody production. Both genes apparently synergize to keep in check excessive germinal center reactions evolving into IgG anti-dsDNA antibody producing B cells.
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http://dx.doi.org/10.3389/fimmu.2018.01551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041390PMC
July 2018

Lupus nephritis progression in FcγRIIB-/-yaa mice is associated with early development of glomerular electron dense deposits and loss of renal DNase I in severe disease.

PLoS One 2017 30;12(11):e0188863. Epub 2017 Nov 30.

RNA and Molecular Pathology Research Group, Department of Medical Biology, Faculty of Health Sciences, UIT-The Arctic University of Norway, Tromsø, Norway.

FcγRIIB-/-yaa mice develop severe lupus glomerulonephritis due to lack of an inhibitory immune cell receptor combined with a Y-chromosome linked autoimmune accelerator mutation. In the present study, we have investigated nephritis development and progression in FcγRIIB-/-yaa mice to find shared features with NZB/NZW F1 lupus prone mice and human disease. We sacrificed 25 male FcγRIIB-/-yaa mice at various disease stages, and grouped them according to activity and chronicity indices for lupus nephritis. Glomerular morphology and localization of electron dense deposits containing IgG were further determined by immune electron microscopy. Renal DNase I and pro-inflammatory cytokine mRNA levels were measured by real-time quantitative PCR. DNase I protein levels was assessed by immunohistochemistry and zymography. Our results demonstrate early development of electron dense deposits containing IgG in FcγRIIB-/-yaa mice, before detectable levels of serum anti-dsDNA antibodies. Similar to NZB/NZW F1, electron dense deposits in FcγRIIB-/-yaa progressed from being confined to the mesangium in the early stage of lupus nephritis to be present also in capillary glomerular basement membranes. In the advanced stage of lupus nephritis, renal DNase I was lost on both transcriptional and protein levels, which has previously been shown in NZB/NZW F1 mice and in human disease. Although lupus nephritis appears on different genetic backgrounds, our findings suggest similar processes when comparing different murine models and human lupus nephritis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188863PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708736PMC
December 2017

GMP-production of purified human B lymphocytes for the adoptive transfer in patients after allogeneic hematopoietic stem cell transplantation.

J Transl Med 2017 11 7;15(1):228. Epub 2017 Nov 7.

Department of Internal Medicine 5, Hematology/Oncology, University Hospital Erlangen, Erlangen, Germany.

Background: We have recently shown that memory B cells from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of immunodeficiency after allogeneic hematopoietic stem cell transplantation. As adoptive transfer of B cells has not been used before in a clinical setting it was necessary to establish a technology for the generation of good manufacturing practice (GMP)-grade B cell products.

Methods: Starting from the leukapheresis product of healthy blood donors, B cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS system. A one-step protocol was used for positive enrichment of B lymphocytes with anti-CD19 microbeads. In a two-step enrichment protocol, first T lymphocytes were depleted by anti-CD3 microbeads and the remaining fraction was positively selected by anti-CD19 microbeads.

Results: The purity and recovery after enrichment of B lymphocytes from the leukapheresis material in both separations strategies was not statistically different. However, contamination of the B-cell product with T cells was significantly lower after the two-step protocol (0.16%, range 0.01-0.43% after two-step separation and 0.55%, range 0.28-0.85% after one-step separation, p < 0.05). Therefore, a combined CD3 depletion and CD19 enrichment was used for the production of GMP-conform B-cell products from the leukapheresis material of 17 healthy stem cell donors. The absolute B-cell numbers obtained in the final product was 4.70 ± 3.64 × 10 with a purity of 95.98 ± 3.31% B lymphocytes and a recovery of 18.9 ± 10.6%. Importantly, the contamination with CD3 T cells was extremely low in the final B- cell products (0.10 ± 0.20%). Purified B cells exhibited normal antibody production after in vitro stimulation and showed excellent viability after cryopreservation.

Conclusions: A GMP-grade B-cell product can be obtained with high purity and very low T-cell contamination using the two-step enrichment protocol based on CliniMACS® technology.
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http://dx.doi.org/10.1186/s12967-017-1330-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678567PMC
November 2017

Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus.

PLoS Pathog 2017 Aug 30;13(8):e1006601. Epub 2017 Aug 30.

Virologisches Institut, Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.

Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.
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http://dx.doi.org/10.1371/journal.ppat.1006601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595347PMC
August 2017

Synergistic Strategy for Multicolor Two-photon Microscopy: Application to the Analysis of Germinal Center Reactions In Vivo.

Sci Rep 2017 08 2;7(1):7101. Epub 2017 Aug 2.

Deutsches Rheumaforschungszentrum, a Leibniz Institute, Berlin, Germany.

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.
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http://dx.doi.org/10.1038/s41598-017-07165-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540998PMC
August 2017

A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1.

Cell Death Differ 2017 07 19;24(7):1239-1252. Epub 2017 May 19.

Department of Internal Medicine 3, Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.

B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1;33.C9 μ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared to pro B cells from Rag1 mice. We were interested in the step regulating this metabolic switch from pro to pre B cells and uncovered that Swiprosin-2/EFhd1, a Ca-binding protein of the inner mitochondrial membrane involved in Ca-induced mitoflashes, is expressed in pro B cells, but downregulated by surface pre B-cell receptor expression. Knockdown and knockout of EFhd1 in 38B9 pro B cells decreased the oxidative phosphorylation/glycolysis (OCR/ECAR) ratio by increasing glycolysis, glycolytic capacity and reserve. Prolonged expression of EFhd1 in EFhd1 transgenic mice beyond the pro B cell stage increased expression of the mitochondrial co-activator PGC-1α in primary pre B cells, but reduced mitochondrial ATP production at the pro to pre B cell transition in IL-7 cultures. Transgenic EFhd1 expression caused a B-cell intrinsic developmental disadvantage for pro and pre B cells. Hence, coordinated expression of EFhd1 in pro B cells and by the pre BCR regulates metabolic changes and pro/pre B-cell development.
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http://dx.doi.org/10.1038/cdd.2017.52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520169PMC
July 2017

Systems Analysis Reveals High Genetic and Antigen-Driven Predetermination of Antibody Repertoires throughout B Cell Development.

Cell Rep 2017 05;19(7):1467-1478

Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland. Electronic address:

Antibody repertoire diversity and plasticity is crucial for broad protective immunity. Repertoires change in size and diversity across multiple B cell developmental stages and in response to antigen exposure. However, we still lack fundamental quantitative understanding of the extent to which repertoire diversity is predetermined. Therefore, we implemented a systems immunology framework for quantifying repertoire predetermination on three distinct levels: (1) B cell development (pre-B cell, naive B cell, plasma cell), (2) antigen exposure (three structurally different proteins), and (3) four antibody repertoire components (V-gene usage, clonal expansion, clonal diversity, repertoire size) extracted from antibody repertoire sequencing data (400 million reads). Across all three levels, we detected a dynamic balance of high genetic (e.g., >90% for V-gene usage and clonal expansion in naive B cells) and antigen-driven (e.g., 40% for clonal diversity in plasma cells) predetermination and stochastic variation. Our study has implications for the prediction and manipulation of humoral immunity.
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http://dx.doi.org/10.1016/j.celrep.2017.04.054DOI Listing
May 2017

The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer.

Nat Cell Biol 2017 05 17;19(5):518-529. Epub 2017 Apr 17.

Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany.

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.
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http://dx.doi.org/10.1038/ncb3513DOI Listing
May 2017

Regulation of autoantibody activity by the IL-23-T17 axis determines the onset of autoimmune disease.

Nat Immunol 2017 01 7;18(1):104-113. Epub 2016 Nov 7.

Department of Internal Medicine 3 and Institute for Clinical Immunology, University Hospital Erlangen, Erlangen, Germany.

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the T17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, T17 cells regulated the expression of β-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23-T17 cell-dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.
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http://dx.doi.org/10.1038/ni.3579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5164937PMC
January 2017

Siglec-H protects from virus-triggered severe systemic autoimmunity.

J Exp Med 2016 07 4;213(8):1627-44. Epub 2016 Jul 4.

Division of Genetics, Department of Biology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany

It is controversial whether virus infections can contribute to the development of autoimmune diseases. Type I interferons (IFNs) are critical antiviral cytokines during virus infections and have also been implicated in the pathogenesis of systemic lupus erythematosus. Type I IFN is mainly produced by plasmacytoid dendritic cells (pDCs). The secretion of type I IFN of pDCs is modulated by Siglec-H, a DAP12-associated receptor on pDCs. In this study, we show that Siglec-H-deficient pDCs produce more of the type I IFN, IFN-α, in vitro and that Siglec-H knockout (KO) mice produce more IFN-α after murine cytomegalovirus (mCMV) infection in vivo. This did not impact control of viral replication. Remarkably, several weeks after a single mCMV infection, Siglec-H KO mice developed a severe form of systemic lupus-like autoimmune disease with strong kidney nephritis. In contrast, uninfected aging Siglec-H KO mice developed a mild form of systemic autoimmunity. The induction of systemic autoimmune disease after virus infection in Siglec-H KO mice was accompanied by a type I IFN signature and fully dependent on type I IFN signaling. These results show that Siglec-H normally serves as a modulator of type I IFN responses after infection with a persistent virus and thereby prevents induction of autoimmune disease.
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http://dx.doi.org/10.1084/jem.20160189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986536PMC
July 2016

CD83 Modulates B Cell Activation and Germinal Center Responses.

J Immunol 2016 05 16;196(9):3581-94. Epub 2016 Mar 16.

Division of Genetics, Department of Biology, University of Erlangen, 91058 Erlangen, Germany;

CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on light zone B cells during the germinal center (GC) reaction. The function of CD83 during GC responses is unclear. CD83(-/-) mice have a strong reduction of CD4(+) T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell-specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo.
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http://dx.doi.org/10.4049/jimmunol.1502163DOI Listing
May 2016

Reactivation of IgG-switched memory B cells by BCR-intrinsic signal amplification promotes IgG antibody production.

Nat Commun 2015 Oct 13;6:8575. Epub 2015 Oct 13.

Institute of Cellular and Molecular Immunology, Georg-August-University of Göttingen, Medical Faculty, Humboldtallee 34, 37073 Göttingen, Germany.

Secondary antibody responses are marked by faster kinetics, improved antibody affinity and a switch from IgM to other immunoglobulin isotypes, most notably IgG, compared with primary responses. These changes protect from reinfection and represent the principle of most vaccination strategies. Yet, the molecular mechanisms that underlie B-cell memory responses are unclear. Here we show, by inactivating the immunoglobulin tail tyrosine (ITT) signalling motif of membrane-bound IgG1 in the mouse, that the ITT facilitates maintenance and reactivation of IgG-switched memory B cells in vivo. The ITT motif equips IgG-switched cells with enhanced BCR signalling capacity, which supports their competitiveness in secondary immune reactions and drives the formation of IgG-secreting plasma cells even in the absence of T-cell help. Our results demonstrate that ITT signalling promotes the vigorous production of IgG antibodies and thus provide a molecular basis for humoral immunological memory.
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http://dx.doi.org/10.1038/ncomms9575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633962PMC
October 2015

Control of murine cytomegalovirus infection by γδ T cells.

PLoS Pathog 2015 Feb 6;11(2):e1004481. Epub 2015 Feb 6.

Nikolaus-Fiebiger-Zentrum für Molekulare Medizin, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1(-/-) mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1(-/-) mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.
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http://dx.doi.org/10.1371/journal.ppat.1004481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450058PMC
February 2015

The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module.

Nat Commun 2014 Nov 21;5:5456. Epub 2014 Nov 21.

Institute of Cellular and Molecular Immunology, Georg-August-University of Göttingen, Medical Faculty, Humboldtallee 34, 37073 Göttingen, Germany.

The vigorous response of IgG-switched memory B cells to recurring pathogens involves enhanced signalling from their B-cell antigen receptors (BCRs). However, the molecular signal amplification mechanisms of memory-type BCRs remained unclear. Here, we identify the immunoglobulin tail tyrosine (ITT) motif in the cytoplasmic segments of membrane-bound IgGs (mIgGs) as the principle signal amplification device of memory-type BCRs in higher vertebrates and decipher its signalling microanatomy. We show that different families of protein tyrosine kinases act upstream and downstream of the ITT. Spleen tyrosine kinase (Syk) activity is required for ITT phosphorylation followed by recruitment of the adaptor protein Grb2 into the mIgG-BCR signalosome. Grb2 in turn recruits Bruton's tyrosine kinase (Btk) to amplify BCR-induced Ca(2+) mobilization. This molecular interplay of kinases and adaptors increases the antigen sensitivity of memory-type BCRs, which provides a cell-intrinsic trigger mechanism for the rapid reactivation of IgG-switched memory B cells on antigen recall.
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http://dx.doi.org/10.1038/ncomms6456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263166PMC
November 2014

Identification of a neutralizing epitope within antigenic domain 5 of glycoprotein B of human cytomegalovirus.

J Virol 2015 Jan 15;89(1):361-72. Epub 2014 Oct 15.

Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

Unlabelled: Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus-neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus-neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a target site on gB, despite originating from different, healthy, HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND mutations in AD-5 were resistant to virus-neutralizing MAbs. Competition enzyme-linked immunosorbent assays (ELISAs) with human HCMV-convalescent-phase sera from unselected donors confirmed the conserved antibody response for the YNND epitope in HCMV-infected individuals and, because a significant fraction of the gB AD-5 response was directed against the YNND epitope, further argued that this epitope is a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed reactivity to AD-5 and neutralization activity toward gB mutant viruses that were similar to those of AD-5-specific MAbs. Taken together, our data indicate that the YNND epitope represents an important target for anti-gB antibody responses as well as for anti-AD-5 virus-neutralizing antibodies.

Importance: HCMV is a major global health concern, and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing antibodies and hence an interesting molecule for intervention strategies, e.g., vaccination. Mapping the target structures of neutralizing antibodies induced by naturally occurring HCMV infection can aid in defining the properties required for a protective capacity of vaccine antigens. The data presented here extend our knowledge of neutralizing epitopes within gB to include AD-5. Collectively, our data will contribute to optimal vaccine design and development of antibody-based therapies.
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http://dx.doi.org/10.1128/JVI.02393-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301166PMC
January 2015