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Results WP4: Mouse Models of PAD

TACI-knock-in Mice As A Model For Antibody Deficiencies

Mutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B cell defects observed in patients with common variable immunodeficiency therefore remains unclear. We sought to define the functional consequences of the C104R mutation on B cell function. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis. These data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects. Overall we have been able to show significant abnormalities of B cell homeostasis and humoral function in TACI C76R deficient mice. These data clearly show that the C76R mutation alone in mice has a detrimental effect on B cell function and if extrapolated to the human setting suggests that TACI C104R affects human B cells and contributes to the B cell defects seen in CVID.

SAP Knock-out Mice As A Model For Hypogammaglobulinemia in XLP

Vaccination studies in SAP knockout mice suggest that responses to T cell dependent antigens are severely compromised in SAP deficient mice and further support the argument that T cell help is defective in SAP deficiency rather than there being an intrinsic B cell abnormality. We then investigated whether humoral function could be restored either by standard bone marrow transplant or through gene modification of murine stem cells. SAP knockout mice were subject to lethal irradiated and then reconstituted with either BM from wild type mice or from SAP KO lin-ve cells transduced with lentiviral vectors encoding SAP or a reporter vector as a control. Mice were then sacrificed at 12 weeks and were analysed from total IgG, IgG subclasses and for reconstitution of cytokine production in splenic CD4 T cells. Naive total IgG levels are significantly lower in SAP KO mice than in wild type mice. Following BMT from wild type and gene transfer using SAP encoding vectors we saw an increase in total IgG levels that was statistically significant. No increase was seen following gene transfer encoding vector. Similarly both BMT and SAP gene transfer both resulted in increase in levels of IgG1, IgG2a and IgG2b whereas no increase was seen following reporter gene transfer. To look at whether this increase may have been due to restoration of T cell help, splenic CD4 cells from the different groups of mice were either unstimulated or stimulated with an anti-CD3 antibody and analysed for IL-4 or IL-10 secretion by ELISA. In comparison to SAP KO mice, both BMT and SAP gene transfer resulted in increased levels of IL-4 and IL-10 production whereas no difference in cytokine production was seen in mice receiving reporter gene modified cells. Together, these data suggest that there is a significant defect of Ig production both in the naive state and in response to T dependent antigen stimulation. These and other data suggest the abnormalities are due to an absence of T cell help. Our studies show that this humoral defect can be corrected by BMT and SAP gene therapy and the correction is due to the restoration of effective function in CD4+ T cells. Further experiments vaccinating mice after reconstitution are required to demonstrate that the full humoral function can be restored in these mice by BMT or gene therapy.

Analysis Of Membrane-IgG1- And IgE-signalling In A Murine Knock-in Model

Using stable isotope labelling with amino acids in cell culture (SILAC) in B cells, we have applied this technology to analyse the Spleen tyrosine kinase (Syk) and mapped Syk phosphorilation sites and elucidate the B-lymphoid interactome of human Syk. The major task was the generation and analysis of gene-targeted mice, which express a mutated form of the mIgG1 (IgG1-YF) and mIgE (IgE-YF) B cell receptors. Like with the IgE-YF mice, chimeric IgG1-YF mice have been generated, and germline transmission of the modified allele has been achieved. Both mouse strains have been backcrossed to remove cre or flp recombinase transgenes, transferred to the barried facility by in-vitro fertilisation and bred to homozygosity. Cohorts of both strains have been immunised with model antigens and the immune response has been measured by ELISA. Preliminary results of these studies have been presented on the PADnet meetings. Taken together, the two successfully established mouse models will provide the means to analyse the importance of tyrosine-phosphorylated cytoplasmic domains of mIgG and mIgE in BCR signalling in a physiological setting and to unravel isotype-specific signalling cascades. These results are important for understanding of PAD and, in the case of IgE, allergic diseases.

BCR-intrinsic Costimulation Of Memory B Cells

During the project we have identified and published the adaptor molecule Grb2 as a pivotal factor for the BCR-intrinsic costimulation of memory B cells. We then set out to analyse the Grb2 interactome and identified Bruton's tyrosine kinase (BTK) as an interaction partner of Grb2. For further analysis, we generated a BTK-deficient variant of the human B cell line DG75, which will allow us to test the signalling potential or different BCR-variants in the presence or absence of BTK. We have also performed experiments on Stap1, which is a unique IgE binding partner discovered during the first half of the project. On the one hand, we used the Stap1-deficient human B cell line DG75 generated in the first half to analyse the composition of IgE-interacting proteins in the absence of Stap1. On the other hand, we generated cell lines expressing a modified version of Stap1, which carries an affinity tag and allowed the purification of Stap1-interacting proteins. The subsequent mass spectrometric analysis of the purified complexes revealed several unknown interaction partners, and allowed us to draw first conclusions on the role and function of Stap1 in the BCR-intrinsic costimulation of memory B cells.

Murine Knock-in Model For Antigen Receptor Signalling

Optimised splice correcting oligonucleotides (SCO) for gene correction of XLA in a mouse model

The ability to correct the aberrant splicing varied for different SCOs. However, splice correcting LNA based oligos are not able to efficiently be taken up by lymphoid primary cells in our system (mouse B cells and human monocytes). Only by the use of electroporation techniques we were able to successfully correct the aberrant BTK splicing in those cell types. As such a new oligonucleotide chemistry - morpholino - was used in combination with a covalently attached cell penetrating peptide. Cell penetrating peptides covalently attached to antisense morpholino splice correcting oligomers (CPP-SCO) were used without the need for electroporation and were extremely efficient in correction of BTK aberrant splicing in patient monocytes. These splice correcting oligos equiped with the CPP are able, by the use of the membrane translocation properties of the peptide, to accumulate inside the cell when added exogenously to the medium. These cell uptake properties of the peptide-oligo conjugates makes them ideal candidates for further testing in in vivo situation. The main objective of our work within the consortium is to improve our understanding on molecular basis of the BTK deficiency. We have developed a new model: BAC-transgenic mice carrying the human genomic BTK. We have two mouse strains; one carrying the WT human BTK gene and another carrying mutated human BTK gene. At the moment the transgenic mice are bred onto the Btk-/- C57BL6 mice and we assume that they will provide a new excellent model for studying different aspects of Btk deficiency. Furthermore, we aimed to correct the mutated BTK in the BAC-transgenic mouse as a proof of concept for the treatment possibilities. We achieved gene correction of the mutated BTK gene in a transgenic mouse model. The transgenic mice are now bred on the Btk-/- mice, providing an excellent humanised model to study different aspects of Btk and XLA disease. We have been able to successfully correct the mutated human BTK gene in our animal model and we could prove that both at the RNA and protein level. Gene correction by using the same means of monocytes from XLA patients carrying similar mutations to prove the efficacy of the method in human primary cells was achieved. Gene expression profiling in Btk-/- and WT B cells stimulated with CpG as an important component in our studies on molecular basis of Btk deficiency. Our preliminary data indicate that the CpG stimulation alters the phenotype of Btk-/- cells making them more alike WT B cells (an important factor in XLA is that B-lymphocytes do not survive). This work is submitted under: Moreno P et al. "Correction of splicing mutation in X-linked agammaglobulinemia (XLA) using an exon skipping strategy". Importantly, XLA has a very decisive feature, namely that the end-stage cells, the plasma cells, do not express any BTK protein. This means that if regular B-lymphocytes can develop as a result of the treatment, subsequent immunisation is anticipated to generate long-lived plasma cells. This also means that a short-term treatment can mediate a long-term clinical outcome. If the animal model shows an in vivo treatment effect, the personalised medicines developed in this program could therefore hopefully, in the not too distant future, also be used as experimental drugs in the affected patients.

Development Of Human B Cells In Humanised Mice

In the humanised mouse model the co-transplantation of mesenchymal stem cells (MSC) may improve engraftment and development of human hematopoietic cells. We found that MSCs isolated from adult bone marrow are a heterogeneous cell population and therefore we are currently trying to identify those cells which provide the strongest support for HSC. We studied the development of human B cells in humanised mice of different ages. In total, 273 mice were transplanted at UKL-FR with human CD34+HSC. Bone marrow engraftment was found in 1/4 of the mice. For comparison, bone marrow B cell development was analysed in BM samples from CVID patients. In total, bone marrow samples of 48 patients and 15 controls were analysed by immunohistology. From 25 patients, fresh aspirates were available and studied by immunophenotyping. In 94% of the patients' BM plasma cells were either absent or significantly reduced and correlated with serum IgG levels. Biopsies from CVID patients had significantly more diffuse and nodular T cell infiltrates than biopsies from controls. These infiltrates correlated with autoimmune cytopenia but not with other clinical symptoms nor with disease duration and peripheral B cell counts. Nodular T cell infiltrates correlated significantly with circulating memory T cells, elevated soluble IL2-receptor and neopterin serum levels indicating an activated T cell compartment in most patients. Nine out of 25 patients had a partial block in B cell development at the pre-B-I to pre-B-II stage. Since the development block correlates with lower transitional and mature B cell counts in the periphery, we propose that these patients might form a new subgroup of CVID patients.