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Dynamics of T cell distribution

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Immunotherapy of cancer with CD3-targeting bispecific antibodies (CD3 bsAb) is a fast developing field, and multiple tumor-associated antigens (TAA) are evaluated for hematologic and solid malignancies. The efficacy of these CD3 bsAb is usually examined in xenograft mouse tumor models with human T cells or in genetically engineered mouse models, where human TAA are introduced. These models often fail to fully recapitulate the natural tumor environment, especially for solid cancers, because of interspecies differences. Here, we investigated the systemic and intratumoral effects of a mouse CD3 bsAb in a fully immune-competent mouse melanoma model. Systemic administration of 0.5 mg/kg antibody induced a brief overall T-cell activation that was selectively sustained in the tumor microenvironment for several days. A fast subsequent influx of inflammatory macrophages into the tumor microenvironment was observed, followed by an increase in the number of CD4+ and CD8+ T cells. Although the capacity to directly kill melanoma cells in vitro was very modest, optimal tumor elimination was observed in vivo, even in the absence of CD8+ T cells, implying a redundancy in T-cell subsets for therapeutic efficacy. Finally, we took advantage of the full immune competence of our mouse model and tested immune memory induction. Despite a strong initial immunity against melanoma, treatment with the CD3 bsAb did not install protective memory responses. The observed mechanisms of action revealed in this immune-competent mouse model might form a rational basis for combinatorial approaches.

Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined. Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (FcγR) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely FcγR dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity. These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development.

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fc gamma receptors (Fc gamma R) I, III, and IV. Whereas LDV infection decreases Fc gamma R III expression, it enhances Fc gamma R I and IV expression in wild-type animals. The LDV-associated increase in the expression of Fc gamma R I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of Fc gamma R I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.

The diverse roles of monocytes in SLE and RA, AbstractMonocytes are evolutionally conserved innate immune cells that play essential roles for the protection of the host against pathogens and also produce several inflammatory cytokines. Thus, the aberrant functioning of monocytes may affect not only host defense but also the development of inflammatory diseases. Monocytes are a heterogeneous population with phenotypical and functional differences. Most recent studies have shown that monocytes are divided into three subsets, namely classical, intermediate and non-classical subsets, both in humans and mice. Accumulating evidence showed that monocyte activation is associated with the disease progression in autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). However, it remains to be determined how monocytes contribute to the disease process and which subset is involved. In this review, we discuss the pathogenic role of monocyte subsets in SLE and RA on the basis of current studies by ourselves and others to shed light on the suitability of monocyte-targeted therapies in these diseases.

Therapy with tumor-specific Abs is common in the clinic but has limited success against solid malignancies. We aimed at improving the efficacy of this therapy by combining a tumor-specific Ab with immune-activating compounds. In this study, we demonstrate in the aggressive B16F10 mouse melanoma model that concomitant application of the anti-TRP1 Ab (clone TA99) with TLR3-7/8 or -9 ligands, and IL-2 strongly enhanced tumor control in a therapeutic setting. Depletion of NK cells, macrophages, or CD8+ T cells all mitigated the therapeutic response, showing a coordinated immune rejection by innate and adaptive immune cells. FcγRs were essential for the therapeutic effect, with a dominant role for FcγRI and a minor role for FcγRIII and FcγRIV. FcγR expression on NK cells and granulocytes was dispensable, indicating that other tumoricidal functions of NK cells were involved and implicating that FcγRI, -III, and -IV exerted their activity on macrophages. Indeed, F4/80+Ly-6C+ inflammatory macrophages in the tumor microenvironment displayed high levels of these receptors. Whereas administration of the anti-TRP1 Ab alone reduced the frequency of these macrophages, the combination with a TLR agonist retained these cells in the tumor microenvironment. Thus, the addition of innate stimulatory compounds, such as TLR ligands, to tumor-specific Ab therapy could greatly enhance its efficacy in solid cancers via optimal exploitation of FcγRs.

C57BL/6 (B6).FcγRIIb −/− . Yaa mice spontaneously develop lethal lupus nephritis. To define the cell type–specific role of FcγRIIb in Yaa -associated lupus, we established B cell– (CD19 Cre . Yaa ), myeloid cell– (C/EBPα Cre . Yaa ), and dendritic cell– (DC) (CD11c Cre . Yaa ) specific FcγRIIb-deficient B6. Yaa mouse strains. CD19 Cre . Yaa mice developed milder lupus than B6.FcγRIIb −/− . Yaa mice, indicating that FcγRIIb deficiency on B cells is not sufficient for the development of severe disease. Surprisingly, C/EBPα Cre . Yaa mice also showed autoantibody production and mild lupus similar to that in CD19 Cre . Yaa mice, whereas CD11c Cre . Yaa mice stayed disease free. These observations indicate that FcγRIIb deficiency in B cells and myeloid cells, but not DCs, contributes to the severe disease in B6.FcγRIIb −/− . Yaa mice. Flow cytometric analysis showed that the frequency of peripheral Gr-1 − but not Gr-1 + monocyte was increased in B6.FcγRIIb −/− . Yaa and C/EBPα Cre . Yaa but not CD19 Cre . Yaa mice, suggesting a link between FcγRIIb deficiency on myeloid cells and the high frequency of Gr-1 − monocytes. RNA sequencing revealed that compared with Gr-1 + monocytes, Gr-1 − monocytes expressed higher levels of the B cell–stimulating cytokines BSF-3, IL-10, and IL-1β, the DC markers CD11c, CD83, and Adamdec1, and the antiapoptotic factors Bcl2 and Bcl6. In conclusion, in Yaa -associated lupus nephritis, FcγRIIb on B cells and myeloid cells modulates B cell activation via different but synergistic pathways. Gr-1 − monocytes are the most likely candidate myeloid cells involved.

The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2+ breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu+ tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours.

SummaryThe clinical application of monoclonal antibodies (mAbs) has revolutionized the field of cancer therapy, as it has enabled the successful treatment of previously untreatable types of cancer. Different mechanisms play a role in the anti-tumour effect of mAbs. These include blocking of tumour-specific growth factor receptors or of immune modulatory molecules as well as complement and cell-mediated tumour cell lysis. Thus, for many mAbs, Fc-mediated effector functions critically contribute to the efficacy of treatment. As immunoglobulin (Ig) isotypes differ in their ability to bind to Fc receptors on immune cells as well as in their ability to activate complement, they differ in the immune responses they activate. Therefore, the choice of antibody isotype for therapeutic mAbs is dictated by its intended mechanism of action. Considering that clinical efficacy of many mAbs is currently achieved only in subsets of patients, optimal isotype selection and Fc optimization during antibody development may represent an important step towards improved patient outcome. Here, we discuss the current knowledge of the therapeutic effector functions of different isotypes and Fc-engineering strategies to improve mAbs application.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.

Dendritic cells (DCs) are specialized in Ag engulfment via a wide variety of uptake receptors on their cell surface. In the present study we investigated Ag uptake and presentation of in vivo–formed Ag–Ab complexes by i.v. injecting mice with Ag-specific Abs followed by the cognate Ag. We show by this natural Ab-mediated Ag targeting system that uptake by splenic APC subsets is severely hampered in mice lacking complement factor C1q (C1qa−/−). Moreover, no detectable Ag cross-presentation by CD8α+ DCs from C1qa−/− mice was found. On the contrary, Ag uptake was not hampered by APCs in FcγRI/II/III/IV-deficient (FcγR quadruple−/−) mice, and the cross-presentation ability of CD8α+ DCs was not affected. In conclusion, we show that C1q rather than FcγRs controls the Ab-mediated Ag uptake and its presentation by spleen APC subsets to T cells.

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8(+) T cells independently of CD4(+) T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4(+) T cells during acute LCMV Arm infection generated high levels of virus-specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV-specific IgG after transient CD4(+) T cell depletion was dependent on Fc gamma receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV-specific IgG, the generation of LCMV-specific isotype-switched memory B cells after transient CD4(+) T cell depletion was considerably reduced. Moreover, mice depleted of CD4(+) T cells during acute infection were strongly impaired in generating a secondary LCMV-specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus-specific IgG. They failed, however, to induce robust virus-specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen.

Summary Vaccines can serve as essential tools to prevent bacterial diseases via the induction of long‐lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc‐gamma receptors (FcγRs) can potentially play a crucial role in IgG‐mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene‐targeted mice lacking either Fcγ RI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody‐mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O‐antigen in gene‐targeted mice, we demonstrate that the complement system is essential for the IgG‐mediated reduction of bacterial numbers in the tissues.

Simple SummaryCancer treatments are increasingly based on therapeutic antibodies to clear tumors. While in vivo mouse models are useful to predict effectiveness of human antibodies it is not completely clear how useful these models are to test antibodies engineered with enhanced effector functions designed for humans. One of the changes considered for many new antibody-based drugs is the removal of fucose (resulting in afucosylated IgG) which enhances IgG-Fc receptor (Fc gamma R) mediated effector functions in humans through Fc gamma RIIIa. Here we show that afucosylated human IgG1 also have enhanced effector functions against peritoneal metastasis of melanoma cells in mice through the evolutionary related mouse Fc gamma RIV. This shows that afucosylated human IgG is functionally recognized across species and shows that mouse tumor models can be used to assess the therapeutic potential of afucosylated IgG1.Promising strategies for maximizing IgG effector functions rely on the introduction of natural and non-immunogenic modifications. The Fc domain of IgG antibodies contains an N-linked oligosaccharide at position 297. Human IgG antibodies lacking the core fucose in this glycan have enhanced binding to human (Fc gamma R) IIIa/b, resulting in enhanced antibody dependent cell cytotoxicity and phagocytosis through these receptors. However, it is not yet clear if glycan-enhancing modifications of human IgG translate into more effective treatment in mouse models. We generated humanized hIgG1-TA99 antibodies with and without core-fucose. C57Bl/6 mice that were injected intraperitoneally with B16F10-gp75 mouse melanoma developed significantly less metastasis outgrowth after treatment with afucosylated hIgG1-TA99 compared to mice treated with wildtype hhIgG1-TA99. Afucosylated human IgG1 showed stronger interaction with the murine Fc gamma RIV, the mouse orthologue of human Fc gamma RIIIa, indicating that this glycan change is functionally conserved between the species. In agreement with this, no significant differences were observed in tumor outgrowth in Fc gamma RIV-/- mice treated with human hIgG1-TA99 with or without the core fucose. These results confirm the potential of using afucosylated therapeutic IgG to increase their efficacy. Moreover, we show that afucosylated human IgG1 antibodies act across species, supporting that mouse models can be suitable to test afucosylated antibodies.

Summary Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcγR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer., Graphical Abstract, Highlights • Antagonistic anti-CD40 mAbs can be converted into agonists by isotype switching to hIgG2 • Transformation is based upon the hIgG2 hinge • Transforms an antagonist to an agonist four times more potent than existing anti-CD40 mAbs • This converted antagonist exhibits antitumor synergy with cell therapy and vaccination, Yu et al. show that isotype switching can convert clinically relevant anti-CD40 antagonistic antibodies to potent FcγR-independent agonists. The converted antibodies can elicit strong antitumor responses in mouse models.

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8

Immunotherapy of cancer with CD3-targeting bispecific antibodies (CD3 bsAb) is a fast developing field, and multiple tumor-associated antigens (TAA) are evaluated for hematologic and solid malignancies. The efficacy of these CD3 bsAb is usually examined in xenograft mouse tumor models with human T cells or in genetically engineered mouse models, where human TAA are introduced. These models often fail to fully recapitulate the natural tumor environment, especially for solid cancers, because of interspecies differences. Here, we investigated the systemic and intratumoral effects of a mouse CD3 bsAb in a fully immune-competent mouse melanoma model. Systemic administration of 0.5 mg/kg antibody induced a brief overall T-cell activation that was selectively sustained in the tumor microenvironment for several days. A fast subsequent influx of inflammatory macrophages into the tumor microenvironment was observed, followed by an increase in the number of CD4+ and CD8+ T cells. Although the capacity to directly kill melanoma cells in vitro was very modest, optimal tumor elimination was observed in vivo, even in the absence of CD8+ T cells, implying a redundancy in T-cell subsets for therapeutic efficacy. Finally, we took advantage of the full immune competence of our mouse model and tested immune memory induction. Despite a strong initial immunity against melanoma, treatment with the CD3 bsAb did not install protective memory responses. The observed mechanisms of action revealed in this immune-competent mouse model might form a rational basis for combinatorial approaches.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV−/− mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV−/− mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

Background Osteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA). Methods AIA was induced in knee joints of wild-type (WT), FcγRI,II,III−/−, and FcγRI,II,III,IV−/− mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR. Results FcγRI,II,III,IV−/− mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV−/− mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV−/− and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV−/− mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV−/− mice, AIA induction in knee joints of FcγRI,II,III−/− mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity. Conclusions FcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint. Electronic supplementary material The online version of this article (10.1186/s13075-018-1584-1) contains supplementary material, which is available to authorized users.

Objective Pathogenic immunoglobulins are produced during the development of obesity and contribute to the development of insulin resistance (IR). However, the mechanisms by which these antibodies affect IR are largely unknown. This study investigated whether Fc-receptors contribute to the development of diet-induced obesity and IR by studying FcRγ−/− mice that lack the γ-subunit necessary for signaling and cell surface expression of FcγR and FceRI. Methods FcRγ−/− and wild-type (WT) mice were fed a high-fat diet (HFD) to induce obesity. At 4 and 11 weeks, body weight and insulin sensitivity were measured, and adipose tissue (AT) inflammation was determined. Furthermore, intestinal triglyceride (TG) uptake and plasma TG clearance were determined, and gut microbiota composition was analyzed. Results FcRγ−/− mice gained less weight after 11 weeks of HFD. They had reduced adiposity, adipose tissue inflammation, and IR. Interestingly, FcRγ−/− mice had higher lean mass compared to WT mice, which was associated with increased energy expenditure. Intestinal TG absorption was increased whereas plasma TG clearance was not affected in FcRγ−/− mice. Gut microbial composition differed significantly and might therefore have added to the observed phenotype. Conclusions FcRγ-chain deficiency reduces the development of diet-induced obesity, as well as associated AT inflammation and IR at 11 weeks of HFD.

Background & Aims Although tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis. Methods We generated Rag1 −/− mice that lack all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR −/− Rag1 −/− mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RB hi to FcγR −/− Rag1 −/− or Rag1 −/− littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206 + (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay. Results Rag1 −/− mice with colitis given anti-TNF had near complete mucosal healing and Rag1 −/− mice given an isotype control antibody developed severe colitis. In contrast, FcγR −/− Rag1 −/− mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR −/− Rag1 −/− mice given a control antibody. Colons from Rag1 −/− mice that received anti-TNF had an increased number of CD206 + macrophages compared with Rag1 −/− mice given control antibody; in FcγR −/− Rag1 −/− mice given anti-TNF these numbers were as low as FcγR −/− Rag1 −/− given the control antibody. In human PBMCs, anti-TNF increased the number of CD206 + macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206 + macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206 + macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206 + macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology. Conclusions In a study of the in vitro and in vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206 + macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206 + macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease.

The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-β-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.

Background FcγRIIB-deficient C57BL/6 (B6) mice spontaneously develop severe lupus nephritis in combination with Yaa locus (TLR7-duplication). Objectives The aim of this study is to clarify the cell type-specific roles of FcγRIIB for the pathogenesis of Yaa-related lupus. Methods We established B cell-specific (CD19Cre.Yaa), myeloid-derived cell-specific (C/EBPαCre.Yaa), and dendritic cell (DC)-specific (CD11cCre.Yaa) FcγRIIB-deficient mice on B6.Yaa background, and compared the disease features of these mice with full FcγRIIB-deficient B6.FcγRIIB-/-.Yaa mice. Results CD19Cre.Yaa mice developed milder lupus nephritis compared to B6.FcγRIIb-/-.Yaa mice, indicating that FcγRIIB deficiency on only B cells is not sufficient for the development of severe disease. Surprisingly, C/EBPαCre.Yaa mice developed similar mild disease as CD19Cre.Yaa mice whereas CD11cCre.Yaa stayed disease free. These observations indicate that, in B6. FcγRIIB-/-.Yaa mice, FcγRIIB deficiency on both B cells and myeloid cells, but not on DCs, contribute to the development of severe lupus with high autoantibody titers. Flow cytometric analysis showed that the frequency of peripheral Gr-1-, but not Gr-1+, monocytes was increased and correlated positively with the frequency of splenic PNA+ activated B cells in B6.FcγRIIB-/-.Yaa and C/EBPaCre.Yaa, but not CD19Cre.Yaa, mice. This suggests a link between FcγRIIB deficiency on monocytes, the high frequency of Gr-1- monocytes and B cell activation. Transcriptome analysis of Gr-1+ and Gr-1- monocytes revealed that B cell-stimulating factor-3 (BSF-3), IL-10, and IL-1β were all up-regulated in Gr-1- monocytes. Conclusions FcγRIIB on B cells and monocytes controls B cell activation and autoimmune responses via different but synergistic pathways in Yaa-related lupus nephritis. References Boross P, et al. J. Immunol. 187:1304–1313, 2011. Disclosure of Interest None declared

Contains fulltext : 177187.pdf (Publisher’s version ) (Open Access) Deficiency of the cysteine protease inhibitor cystatin M/E (Cst6) in mice leads to disturbed epidermal cornification, impaired barrier function, and neonatal lethality. We report the rescue of the lethal skin phenotype of ichq (Cst6-deficient; Cst6-/-) mice by transgenic, epidermis-specific, reexpression of Cst6 under control of the human involucrin (INV) promoter. Rescued Tg(INV-Cst6)Cst6ichq/ichq mice survive the neonatal phase, but display severe eye pathology and alopecia after 4 mo. We observed keratitis and squamous metaplasia of the corneal epithelium, comparable to Cst6-/-Ctsl+/- mice, as we have reported in other studies. We found the INV promoter to be active in the hair follicle infundibulum; however, we did not observe Cst6 protein expression in the lower regions of the hair follicle in Tg(INV-Cst6)Cst6ichq/ichq mice. This result suggests that unrestricted activity of proteases is involved in disturbance of hair follicle biology, eventually leading to baldness. Using quenched activity-based probes, we identified mouse cathepsin B (CtsB), which is expressed in the lower regions of the hair follicle, as an additional target of mouse Cst6. These data suggest that Cst6 is necessary to control CtsB activity in hair follicle morphogenesis and highlight Cst6-controlled proteolytic pathways as targets for preventing hair loss.-Oortveld, M. A. W., van Vlijmen-Willems, I. M. J. J., Kersten, F. F. J., Cheng, T., Verdoes, M., van Erp, P. E. J., Verbeek, S., Reinheckel, T., Hendriks, W. J. A. J., Schalkwijk, J., Zeeuwen, P. L. J. M. Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type–specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell–specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell–specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility.

Objective Arthritis and valvular carditis coexist in several human rheumatic diseases, including systemic lupus erythematosus, rheumatic fever, and rheumatoid arthritis. T cell receptor–transgenic K/BxN mice develop spontaneous autoantibody-associated arthritis and valvular carditis. The common Fc receptor γ (FcRγ) signaling chain is required for carditis to develop in K/BxN mice. FcRγ pairs with numerous receptors in a variety of cells. The aim of this study was to identify the FcRγ-associated receptors and Fcγ receptor (FcγR)–expressing cells that mediate valvular carditis in this model. Methods We bred K/BxN mice lacking the genes that encode activating Fcγ receptors (FcγRI, FcγRIII, and FcγRIV), and we assessed these mice for valvular carditis. We similarly evaluated complement component C3–deficient K/BxN mice. Immunohistochemistry, bone marrow transplantation, and macrophage depletion were used to define the key FcRγ-expressing cell type. Results Genetic deficiency of only one of the activating Fcγ receptors did not prevent carditis, whereas deficiency of all 3 activating Fcγ receptors did. Further analysis demonstrated that FcγRIII and FcγRIV were the key drivers of valve inflammation; FcγRI was dispensable. C3 was not required. FcRγ expression by radioresistant cells was critical for valvular carditis to develop, and further analysis indicated that macrophages were the key candidate FcγR-expressing effectors of carditis. Conclusion FcγRIII and FcγRIV act redundantly to promote valvular carditis in K/BxN mice with systemic autoantibody-associated arthritis. Macrophage depletion reduced the severity of valve inflammation. These findings suggest that pathogenic autoantibodies engage Fcγ receptors on macrophages to drive valvular carditis. Our study provides new insight into the pathogenesis of cardiovascular inflammation in the setting of autoantibody-associated chronic inflammatory diseases.

Motivation: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive or uncharacterized regions. Results: We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by transcription activator-like effector nucleases (TALENs) and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics. Availability and implementation: We have implemented TSSV as a Python package that can be installed through the command-line using pip install TSSV command. Its source code and documentation are available at https://pypi.python.org/pypi/tssv and http://www.lgtc.nl/tssv. Contact: S.Y.Anvar@lumc.nl Supplementary information: Supplementary data are available at Bioinformatics online.

FcγRIIb is the sole inhibitory FcR for IgG in humans and mice, where it is involved in the negative regulation of Ab production and cellular activation. FcγRIIb-deficient mice show exacerbated disease following the induction of nephrotoxic nephritis (NTN). In this study, we determined the cellular origin of the FcγRIIb-knockout phenotype by inducing NTN in mice with a deficiency of FcγRIIb on either B cells alone (FcγRIIBfl/fl/CD19Cre+) or myeloid cells (FcγRIIBfl/fl/CEBPαCre+). Deletion of FcγRIIb from B cells did not increase susceptibility to NTN, compared with wild-type (WT) mice, despite higher Ab titers in the FcγRIIBfl/fl/CD19Cre+ mice compared with the WT littermate controls. In contrast, mice lacking FcγRIIb on myeloid cells had exacerbated disease as measured by increased glomerular thrombosis, glomerular crescents, albuminuria, serum urea, and glomerular neutrophil infiltration when compared with WT littermate controls. The role for FcγRIIb expression on radioresistant intrinsic renal cells in the protection from NTN was then investigated using bone marrow chimeric mice. FcγRIIb−/− mice transplanted with FcγRIIb−/− bone marrow were more susceptible to NTN than WT mice transplanted with FcγRIIb−/− bone marrow, indicating that the presence of WT intrinsic renal cells protects from NTN. These results demonstrate that FcγRIIb on myeloid cells plays a major role in protection from NTN, and therefore, augmentation of FcγRIIb on these cells could be a therapeutic target in human Ab-mediated glomerulonephritis. Where there was a lack of FcγRIIb on circulating myeloid cells, expression of FcγRIIb on intrinsic renal cells provided an additional level of protection from Ab-mediated glomerulonephritis.

Human intravenous immune globulin (IVIg), a purified IgG fraction composed of ~60% IgG1 and obtained from the pooled plasma of thousands of donors, is clinically used for a wide range of diseases. The biological actions of IVIg are incompletely understood and have been attributed both to the polyclonal antibodies therein and also to their IgG (IgG) Fc regions. Recently, we demonstrated that multiple therapeutic human IgG1 antibodies suppress angiogenesis in a target-independent manner via FcγRI, a high-affinity receptor for IgG1. Here we show that IVIg possesses similar anti-angiogenic activity and inhibited blood vessel growth in five different mouse models of prevalent human diseases, namely, neovascular age-related macular degeneration, corneal neovascularization, colorectal cancer, fibrosarcoma and peripheral arterial ischemic disease. Angioinhibition was mediated by the Fc region of IVIg, required FcγRI and had similar potency in transgenic mice expressing human FcγRs. Finally, IVIg therapy administered to humans for the treatment of inflammatory or autoimmune diseases reduced kidney and muscle blood vessel densities. These data place IVIg, an agent approved by the US Food and Drug Administration, as a novel angioinhibitory drug in doses that are currently administered in the clinical setting. In addition, they raise the possibility of an unintended effect of IVIg on blood vessels.

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

Fc receptors (FcRs) play a key role in regulating and coordinating responses from both innate and adaptive arms of the immune system. The inhibitory Fc gamma receptor II (FcγRIIB; CD32) is central to this regulation with FcγRIIB−/− mice demonstrating augmented responses to mAb immunotherapy, elevated incidence and severity of auto-immunity, and increased response to mAb-mediated cancer therapy. To date, these observations have remained unexploited therapeutically, partly through a lack of specific mAb reagents capable of exclusively binding mouse FcγRIIB. Thus almost all of the FcγRIIB-binding mAb currently available, such as 2.4G2, also bind FcγRIII (CD16), and polyclonal reagents have limited availability and are of unproven specificity and avidity, making in vivo manipulation of FcγRIIB impossible. Following an extensive immunisation protocol using FcγRIIB−/− mice, we recently produced three unique mAb that are suitable for this purpose. Here we characterise these novel reagents and demonstrate that they fall into two distinct categories; those which cause phosphorylation and subsequent activation of FcγRIIB (agonistic) and those that block receptor phosphorylation (antagonistic). These two types of mAb exhibit different characteristics in a range of biochemical, cellular, and functional assays relevant to FcγRIIB activity and mAb therapy.

Increasing evidence suggests that antibodies can have stimulatory effects on T-cell immunity. However, the contribution of circulating antigen-specific antibodies on MHC class I cross-priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross-presentation of antigen to CD8(+) T cells. Mice with hapten-specific circulating antibodies, but naϊve for the T-cell antigen, were infused with haptenated antigen and CD8(+) T-cell induction was measured. Mice with circulating hapten-specific antibodies showed significantly enhanced cross-presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross-presentation in mice with circulating antigen-specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c(+) APCs were responsible for the enhanced and sustained cross-presentation, although CD11c(-) APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen-antibody immune complexes improves MHC class I cross-presentation, and CD8(+) T-cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor-associated antigens intensively used in the clinic nowadays.

Fc gamma R ligation by Ag-Ab immune complexes (IC) not only mediates effective Ag uptake, but also strongly initiates dendritic cell (DC) maturation, a requirement for effective T cell activation. Besides the activating Fc gamma RI, Fc gamma RIII, and Fc gamma RIV, the inhibitory Fc gamma RIIb is expressed on DCs. It is unclear how the ratio between signals from the activating Fc gamma R and the inhibitory Fc gamma RIIb determines the outcome of Fc gamma R ligation on DCs. By microarray analysis, we compared the transcriptomes of steady state and IC-activated bone marrow-derived wild-type (WT) DCs expressing all Fc gamma R or DCs expressing only activating Fc gamma R (Fc gamma RIIb knockout [KO]) or only the inhibitory Fc gamma RIIb (FcR gamma-chain KO). In WT DCs, we observed a gene expression profile associated with effective T cell activation, which was absent in FcR gamma-chainKO, but strikingly more pronounced in Fc gamma RIIb KO bone marrow-derived DCs. These microarray results, confirmed at the protein level for many cytokines and other immunological relevant genes, demonstrate that the transcriptome of IC-activated DCs is dependent on the presence of the activating Fc gamma R and that the modulation of the expression of the majority of the genes was strongly regulated by Fc gamma RIIb. Our data suggest that Fc gamma RIIb-deficient DCs have an improved capacity to activate naive T lymphocytes. This was confirmed by their enhanced Fc gamma R-dependent Ag presentation and in vivo induction of CD8(+) T cell expansion compared with WT DCs. Our findings underscore the potency of Fc gamma R ligation on DCs for the effective induction of T cell immunity by ICs and the strong regulatory role of Fc gamma RIIb. The Journal of Immunology, 2012, 189: 92-101.

Introduction CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to toxic NOTCH3 protein accumulation in the small- to medium sized arterioles. The accumulation is systemic but most pronounced in the brain vasculature where it leads to clinical symptoms of recurrent stroke and dementia. There is no therapy for CADASIL, and therapeutic development is hampered by a lack of feasible clinical outcome measures and biomarkers, both in mouse models and in CADASIL patients. To facilitate pre-clinical therapeutic interventions for CADASIL, we aimed to develop a novel, translational CADASIL mouse model. Results We generated transgenic mice in which we overexpressed the full length human NOTCH3 gene from a genomic construct with the archetypal c.544C > T, p.Arg182Cys mutation. The four mutant strains we generated have respective human NOTCH3 RNA expression levels of 100, 150, 200 and 350 % relative to endogenous mouse Notch3 RNA expression. Immunohistochemistry on brain sections shows characteristic vascular human NOTCH3 accumulation in all four mutant strains, with human NOTCH3 RNA expression levels correlating with age at onset and progression of NOTCH3 accumulation. This finding was the basis for developing the ‘NOTCH3 score’, a quantitative measure for the NOTCH3 accumulation load. This score proved to be a robust and sensitive method to assess the progression of NOTCH3 accumulation, and a feasible biomarker for pre-clinical therapeutic testing. Conclusions This novel, translational CADASIL mouse model is a suitable model for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation, using the NOTCH3 score as a biomarker. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0268-1) contains supplementary material, which is available to authorized users.