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Introduction to WP4: Mouse Models of PAD

Animal studies make it possible to analyze several aspects of PAD, which cannot be addressed in humans. Although in vitro systems have undisputable advantages (e.g. costs, no use of animal experiments, high through-put) and go some way addressing the mechanisms of disease pathogenesis, in vitro systems lack the complexities of an in vivo system. We will generate and study a variety of murine models that will allow a greater understanding of human antibody deficiency syndromes.
The following examples will directly address key aspects of PAD in animal models:
1. When the molecular basis is not understood, such as in patients with heterozygous TACI mutations (CVID) or XLP, the generation of knock-in and knockout models will provide new insights.
2. When the defect is known, but when the precise mechanism is not understood, the generation of cell-specific in vivo knockdowns, such as Btk-knockdown in B cells will elucidate cell specific functions of a given gene product.
3. When experimental in vitro data suggest novel genetic disease mechanisms, the generation of the corresponding knock-in mouse models (membrane IgG mutations and docking molecules) will test these hypotheses.
4. Developing "humanised mice" will provide us with a model system allowing us to test the capabilities of the transferred hematopoietic cells in any given patient (e.g. for patients with CVID).
The projects are based upon the study of the following diseases: CVID, XLA, AR agamma, XLP, PAD of unknown cause The animal studies will be based upon the following technology platforms: Knockout, knock-in, shRNA (lentiviral), dominant negative (lentivir), humanized mouse

Project 1: Knock-in model of TACI deficiency in CVID

The aim of this work package was to generate a murine model of the C104R mutation and to determine whether this resulted in a murine humoral deficiency. After the successful generation of the respective mouse mutant C76R, we were able to undertake some phenotypic analysis of the mutant. In summary, our data suggest that the C76R homozygous mutant mice do show humoral abnormalities that are similar to human TACI null mutants with a hyper-expansive B cell phenotype and defects in immunoglobulin production. To date no major differences have been observed in heterozygous mutants. Further analysis including immunisation experiments will be undertaken in the latter half of the project.

Project 2: Knockout model of SAP deficiency in X-linked lymphoproliferative disease (XLP)

XLP is a complex immunodeficiency in which mutations in the gene encoding the adaptor protein SAP (SLAM associated protein) lead to severe immunodysregulatory phenomena [1]. Boys with XLP present typically but not always after EBV infection with fulminant infectious mononucleosis, B cell lymphoma or hypogammaglobulinaemia which is highly reminiscent of a CVID phenotype. The pathogenesis of hypogammaglobulinaemia in XLP is unclear and has been until very recently been thought to arise due to a lack of T cell help.

[1] Gaspar HB, Sharifi R, Gilmour KC, Thrasher AJ. X-linked lymphoproliferative disease: clinical, diagnostic and molecular perspective. Br.J.Haematol. 2002;119:585-95.

Project 3: Knock-in model for antigen receptor signalling

Establishment of IgG-switched memory B cells and their costimulation-independent activation during secondary immune responses is thought to be dependent on isotype-specific antigen receptor signals. Since the start of the project we have made significant progress in the identification of intracellular signalling proteins that are recruited to tyrosine-phosphorilated cytoplasmic domains of mIgG and mIgE. The first interaction partner that was detected by co-immunoprecipitation and confirmed by Western blot, the adaptor molecule Grb2, could already be published in Nature Immunology in September 2009 (Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B-cells. Engels N, König LM, Heemann C, Lutz J, Tsubata T, Griep S, Schrader V, Wienands J. Nat Immunol. 2009 Sep;10(9):1018-25). Based on these findings functional studies of Grb2 and its potential binding partners are in progress, which include the generation and analysis of Grb2 mutants that lack either the N- or C-terminal SH3 domain or carry amino acid substitutions in the binding sties for known interaction partners. At the same time we are trying to generate Grb2-deficient cell lines.

Project 4: Lentiviral short hairpin (sh) RNA-induced down-regulation as a tool for validation of Btk-dependent transcripts

Although the genetic defect for several human PADs has been identified, the molecular basis is still not understood. As an example Btk, BLNK (SLP65), CD79a and -b deficiency all result in impaired intracellular signalling causing the defect. However, it is currently not known, which genes are the crucial targets of this signalling. We have started to analyze down-stream transcriptional targets by the use of expression profiling using Btk and Btk/Tec double-deficient mice. Both mouse strains show defective growth and differentiation of B lymphocytes. This analysis has resulted in the identification of many potentially, biologically relevant targets. However, it is unclear, which of these genes are the key effectors, responsible for the growth and differentiation defect.
Owing to that inactivation of such target genes in non-hematopoietic cells may cause lethality, conventional knock-out technology may not be applicable (and is also highly laborious). However, if the corresponding gene is inactivated in the hematopoietic population only [note that Btk and Tec double knockout phenotype is confined to B lymphocytes], this problem is circumvented. Such inactivation can be carried out using conditional knock-outs (also highly laborious) or, alternatively, by using the shRNA technology.
We will aim to identify the biologically relevant genes, which are affected by defective Btk (XLA) signalling.

Project 5: Development of knockdowns and humanized mice

Development of human B cells in reconstituted double knockout Rag2/IL2RG deficient mice.
Early steps in human B cells in reconstituted double knockout Rag2/IL2RG deficient mice developing from engrafted CD34+ hematopoietic stem cells were found in bone marrow and blood but rarely in the spleen or in other secondary lymphoid organs. To improve the development of human lymphocytes in a murine cytokine environment Rag2/IL2RG mice transgenic for human IL7 were generated. To facilitate the construction of mice expressing human cytokines and growth factors supporting the development of human hematopoietic cells we also started to generate ES cell lines on the Rag2/IL2RG background. In addition, CD34+ HSC were engrafted into Rag2/IL2RG transgenic for human BAFF. The analysis of these mice is under way.

Project 6: Humanized mice to study memory and plasma cells in CVID Responsible partner: R Carsetti

IgM memory B cells play a fundamental role in the defence against encapsulated bacteria producing anti-bacterial IgM antibodies [1-3]. These bind to the capsular polysaccharides of rendering them targets of monocyte-macrophage cells.
Therefore, IgM antibodies are indispensable for the removal of bacteria circulating in the blood, collected by the filtering system of the spleen and invading tissues.
Antibodies, however, also play an important role before tissue invasion, forming a protective layer on mucosal surfaces. Bronchial and intestinal epithelia are both covered by a film of antibodies of IgA isotype. Secretory IgA (sIgA) is secreted by mucosal plasma cells and then transported from the basal to the apical side of epithelial cells by the poly-Ig receptor. In the mouse most of the IgA at epithelial surfaces is secreted in a T-independent manner and originate from a defined type of B cells, the B-1a. This population is ontogenetically and functionally distinct from conventional B-2 B cells [4].
In humans the important question of the origin of mucosal plasma cells has never been addressed.

[1] Kruetzmann S., Rosado M.M., Weber H., Germing U., Tournilhac O., Peter H.H., Berner R., Peters A., Boehm T., Plebani A., Quinti I., and Carsetti R. 2003. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J. Exp. Med. 197:939-945.
[2] Carsetti R., Rosado M.M., Donnanno S., Guazzi V., Soresina A., Meini A., Plebani A., Aiuti F., and Quinti I. 2005. The loss of IgM memory B cells correlates with clinical disease in common variable immunodeficiency. J. Allergy Clin. Immunol. 115:412-7.
[3] Weller S., Braun M.C., Tan B.K., Rosenwald A., Cordier C., Conley M.E., Plebani A., Kumararatne D.S., Bonnet D., Tournilhac O., Tchernia G., Steiniger B., Staudt L.M., Casanova J.L., Reynaud C.A., and Weill J.C. 2004. Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood. 104:3647-3654.
[4] Rosado MM, Aranburu A, Giorda E, Cascioli S, Cenci F, Miller E, Leanderson T, Bottazzo GF, Natali PG and Carsetti R. Bone marrow-independent development of B cells producing secretory IgA in the intestine (submitted).