One of the most intriguing features of the specific immune system is the generation of mature B and T lymphocytes that carry immunoglobulin (Ig) molecules and T-cell receptors (TCR), which are highly specific for antigens, even when these cells have not encountered the antigens before. The generation and selection of a diverse and flexible specificity repertoire by lymphocytes is dependent on a series of developmental cell fate decisions, including the induction and regulation of Ig and TCR gene rearrangements. These decisions are implemented by the activity of specific transcription factors and enzyme systems and occur at specific checkpoints, controlled by antigen receptors and their immature forms.

Gene expression profiles determine the differentiation lineage, developmental stage, and activation stage of the involved cells. Just like in any other cell type, regulation of gene expression in lymphocytes is largely controlled at the level of transcriptional initiation by transcription factors and transcriptional repressors.

In particular, the research aims of this program comprise:

• To study the role of lymphoid-specific transcription factors that control the in vivo developmental program of lymphoid cells.
• To delineate the transcription factors and signaling routes that are controlling the most immature steps of lymphoid differentiation.
• To unravel the essential steps in the induction and execution of Ig / TCR gene rearrangements, e.g. using immunodeficiencies as a model.
• To characterise the signal transduction pathways that are downstream of the antigen receptors and are essential for survival, selection and developmental progression of lymphoid cells.
• To investigate how defects in the regulation of differentiation and proliferation steps during lymphoid development result in immunodeficiencies or lymphoid malignancies.
• To translate the obtained knowledge on normal lymphoid differentiation and gene defects into novel diagnostics and opportunities for gene therapy in primary immunodeficiencies (PID).
• To study immunogenetic defects in primary immunodeficiencies.

Lymphoid differentation

The research projects included the generation of transgenic and knock-out mice as well as the precise characterization of human PID, particularly agammaglobulinemia and severe combined immunodeficiencies (SCID).

• We demonstrated that Wnt signaling via Tcf-1 is required at the earliest stages of T cell development in the thymus. Tcf-1 deficient mice specifically have a thymic defect, not in other lineages and surprisingly develop thymic lymphomas. This indicates that Tcf-1 first functions as transcription factor later as tumor suppressor gene. Therefore, Tcf-1 functions as molecular switch during T cell development.
• These thymic lymphomas arose from deregulated Wnt signaling via Lef-1, followed in most instances by activating Notch1 mutations.
• We showed that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis (Luis et al, Blood 2009).
• Wnt3a deficiency leads to a complete abolishment of canonical Wnt signaling in fetal liver stem cells, providing genetic proof that Wnt signalling is required for self-renewal of HSC (Luis et al, Blood 2009; Staal et al, Nat Rev Immunol 2008).
• In in vivo experiments, we showed that EFS and CD19 coBTK almost fully restores the mild block in B cell development in Btk-/- mice, restores IgM^-IgD⁺ B cells, as well as IgM and IgG3 serum titres and fully restores CD5⁺ B1 cells in the peritoneal cavity. The in vivo vaccination response to T cell independent antigens (normally absent in these mice) is fully restored with normal or close to normal antigen specific IgM and IgG3 production.
• Safety studies were done on a total of 41 mice using EFS-coBTK and CD19 coBTK viral batches. Both in primary and secondary transplantations (4 months plus 4 months follow-up) no single leukemia was detected in CD19-BTK nor EFS-BTK treated mice, indicating that the developed SIN-LV have a much improved safety profile compared to the previously developed vectors for future clinical applications. As B cell development and Ig titers are restored in secondary recipients, true stem cell gene correction has occurred.
• For both RAG1 and RAG2, lentiviral transduction of autologous stem cells in RAG1 or RAG2 deficient mice leads to functional reconstitution of T and B lymphocytes that are capable of in vitro proliferation to mitogenic stimuli and production of IgM and IgG.
• We obtained proof of principle for lentiviral gene therapy of both RAG1 and RAG2 deficiency with production of functional T and B cells (Pike et al, Mol Therapy 2007).
• We have found that SLC pre-B cell receptor components had the capacity to induce constitutive B cell receptor internalization, secondary immunoglobulin light-chain rearrangement, and a severe developmental arrest of immature B cells, dependent on the downstream adaptor protein Slp65. During B cell development silencing of surrogate light chain genes is not essential for the limitation of pre-B cell proliferation, but is required for the prevention of constitutive activation of B cells (Van Loo et al, Immunity 2007).
• We have defined roles for the transcription factor CTCF in T cell development in the thymus, as well as in differentiation of various T helper subsets (Heath et al, 2008; Ribeiro de Almeida et al, 2009). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Using conditional targeting in vivo we showed that CTCF is essential for Gata3-dependent regulation of Th2 cytokine gene expression (Ribeiro de Almeida et al, J Immunol 2009). 
• Gene expression profiling in mice with enforced Gata3 expression revealed putative targets of Gata3 in double positive thymocytes (Van Hamburg et al, Mol Immunol 2009). We found that enforced Gata3 expression affected in vitro T helper cell differentiation, as well as Th17 activity in vivo (Van Hamburg et al, Eur J Immunol 2008; Van Hamburg et al, Arthr Rheumat 2009). We also were able to show that Gata3 controls the expression of CD5 and the T cell receptor during CD4 T cell lineage development (Ling et al, Eur J Immunol 2007) and that Gata3-driven Th2 responses inhibit TGF-beta1-induced FoxP3 expression and the formation of regulatory T cells (Mantel et al, PloS Biol 2007).
• We found in mouse models that Bruton’s tyrosine kinase and SLP-65 regulate pre-B cell differentiation and the induction of immunoglobulin light chain gene rearrangement (Kersseboom et al, J Immunol 2006). In addition, Bruton’s tyrosine kinase and Phospholipase C-gamma mediate chemokine-controlled B cell migration and homing. (De Gorter et al, Immunity 2007).

Primary immunodeficiencies

During the past 5 years, four new primary immunodeficiencies were described by our research group. In addition, our studies on primary immunodeficiencies gave new insights in the underlying immunological defects of SCID and antibody deficiencies.

• We identified a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). So far, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. Our studies that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects (Van der Burg et al, J Clin Invest 2006).
• Artemis is involved in opening of hairpin-sealed coding ends. We found that residual DH-JH junctions from patients' bone marrow precursor B cells show high numbers of palindromic (P)-nucleotides. In 25% of junctions, microhomology was observed in the P-nucleotide regions. We utilized this difference between Artemis-deficient cells and normal controls to develop a V(D)J recombination assay to determine hairpin-opening activity. Mutational analysis of the Artemis gene confirmed and extended the mapping of an N-terminal nuclease active site (Van der Burg et al, Eur J Immunol 2007).
• In a T-B- SCID patient with an unknown defect, a detailed study resulted in the identification of a mutation in the DNA-PKcs gene. Interestingly, the DNA-PKcs mutation did not affect the kinase activity or DNA end binding activity and consequently the DNA-PKcs gene was initially not regarded as candidate gene. However, in vivo analysis of V(D)J recombination resulted in the identification of long P-nucleotide stretches, which is characteristic for inadequete activation of the Artemis nuclease (Van der Burg et al, Eur J Immunol 2007). As Artemis activity is dependent on DNA-PKcs function, we hypothezised that DNA-PKcs should be the candidate gene and genetic analysis resulted in the identification of the genetic defect. This is the first example of a DNA-PKcs defect that results in SCID in human (Van der Burg et al, J Clin Invest 2009).
• In a 10-yr old girl with a mild form of radiosensitive (RS) T-/B- SCID resulting in severe hypogammaglobulinemia and reduced absolute numbers of B- and T-lymphocytes we identified a unique splice-site mutation in the Artemis gene. Clinical symptoms include bronchiectasis and progressive granulomatous skin lesions. This study gave new insights in the concept of the diagnostic strategy for RS-SCID showing the importance of combination of DNA, RNA, protein, function (in vitro and in vivo) based data and the clinical phenotype. A manuscript on this topic is in preparation.
• We identified four patients, from two unrelated families, who had hypogammaglobulinemia and defective CD19. These patients had normal levels of B cells in blood but had undetectable or very low levels of surface CD19, owing to a homozygous mutation in the CD19 gene. Immunological characterization of the B-cell compartment showed that CD19 gene disruption causes a type of hypogammaglobulinemia in which the response of mature B cells to antigenic stimulation is defective (Van Zelm et al, N Eng J Med 2006).
• We evaluated a 7-year-old girl from consanguineous parents, who presented with recurrent infections and acute nephrotic syndrome. Flow cytometric immunophenotyping of blood showed lack of CD19 expression on B-lymphocytes, but the CD19 gene did not contain mutations. Subsequent analysis of the other components of the CD19 complex (CD21, CD81, CD225) showed a deficiency of CD81 on all leukocytes but normal CD21 and CD225 expression. The patient was homozygous for a CD81 splice site mutation. Retroviral transduction of CD81 in a B-cell line of the patient resulted in expression of both CD81 and CD19. The CD81 immunodeficiency resembles that of the CD19 deficiency, and is characterized by poor terminal B-cell differentiation into memory B-cells and Ig-producing plasma cells (Van Zelm et al, submitted).
• We provided evidence that a high TE content, irrespective of the type of element, results in the increased incidence of gross deletions as gene disruption underlying human disease including primary immunodeficiencies (van Zelm et al, Am J Human Genet 2008).
• Based on the combined Ig gene rearrangement status and gene expression profiles of consecutive precursor B cell subsets sorted from bone marrow samples of healthy individuals, we identified 16 candidate genes that are supposed to be involved in initiation and/or regulation of Ig gene rearrangements. These analyses provide new insights into early human precursor B cell differentiation steps and represent an excellent template for studies on oncogenic transformation in precursor B acute lymphoblastic leukemia and B cell differentiation blocks in primary antibody deficiencies (Van Zelm et al, J Immunol 2005).
• We developed an assay to quantify the replication history of mouse and human B lymphocyte subsets by calculating the ratio between genomic coding joints and signal joints on kappa-deleting recombination excision circles (KREC) of the IGK-deleting rearrangement. This allowed us to determine the contribution of proliferation to B lymphocyte homeostasis and antigen responses. These new insights will support the understanding of peripheral B cell regeneration after hematopoietic stem cell transplantation or B cell–directed antibody therapy, and the identification of defects in homeostatic or antigen-induced B cell proliferation in patients with common variable immunodeficiency or another antibody deficiency (van Zelm et al, J Exp Med 2007).
• Most patients with common variable immunodeficiency (CVID) have a defect in B-cell differentiation/maturation. We hypothesized that within the heterogeneous group of CVID patients, homogeneous subgroups can be identified with specific B-cell differentiation/maturation defects in one or more of the critical B-cell differentiation/maturation steps. With our intergrated approach, which is a combination of immunophenotypic, functional and molecular assays, we were able to define several CVID subgroups. Data has been collected from our Erasmus MC CVID cohort of 60 patients. (Driessen et al, manuscript is in preparation).
• Three-dimensional FISH techniques were used for comparison of distance distributions of the VH,  DH and JH gene segments in precursor-B-cell subsets to understand the spatial organization of the IGH gene during early B-cell differentiation, when rearrangements in the locus take place. The results indicated that the IGH locus is organized into compartments consisting of multiple loops which are separated by a flexible linker. This multi-loop-containing compartments and the flexibility of the intervening sequences permit long-range genomic interaction between the multiple VH, DH and JH gene segments (Jhunjhunwala, Van Zelm et al, Cell 2008 and Cell 2009.

• In the Btk project we aim to identify the downstream molecular targets of pre-BCR signalling that control the developmental progression of pre-B cells in vivo. These studies should put our knowledge on Btk, BLNK/SLP-65 and pre-BCR signalling – largely derived from experiments in cultured B cell lines or transfected cells – into a new in vivo perspective of pre-B cell development. The key objectives are to define the downstream nuclear events that direct (1) the cellular maturation of pre-B cells, (2) the loss of IL-7 responsiveness of pre-B cells, and (3) the initiation of Ig L chain rearrangement.
• We want to identify GATA-3 targets genes, using DNA micro array technology, in a comparison of gene expression in wild-type and GATA-3 overexpressing cells at various stages of T cell development. In addition, the in vivo function of GATA-3 will be studied in a conditional knock-out mouse model, as well as in GATA-3 overexpressing transgenic mice that have been crossed with TCR transgenic mouse models to study positive and negative selection and CD4/CD8 commitment.  
• Development of new diagnostic tools for PID patients is an ongoing process. Based on new insights, more genes will become known as candidate genes that can be affected in PID patients. We will focus on identification of new genetic defects in patients with T-B- SCID and agammaglobulinemia/antibody deficiencies and common variable immunodeficiency (CVID).
• We will study V(D)J recombination in the context of B-cell differentiation focussing on the core factors involved in this process and factors facilitating this process using primary immunodeficiecies as model systems.
• The gene therapy field is focusing on eliminating unwanted side effects of retroviral integration (insertional mutagenesis) and employing new lentiviral instead of oncoretroviral vectors. We actively participate in these areas.  Special attention will be paid to RAG and BTK gene therapy.

1.            van der Burg M, van Veelen LR, Verkaik NS, Wiegant WW, Hartwig NG, Barendregt BH, Brugmans L, Raams A, Jaspers NG, Zdzienicka MZ, van Dongen JJM,  van Gent DC. A new type of radiosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation. J Clin Invest 2006;116:137-145. IF 16.6 - 5%

2.            van Zelm MC, Reisli I, Van der Burg M, Castano D, van Noesel CJ, van Tol MJ, Woellner C, Grimbacher B, Patino PJ, van Dongen JJM,  Franco JL. An antibody-deficiency syndrome due to mutations in the CD19 gene. N Engl J Med 2006;354:1901-1912. IF 50.0 - 5%

3.            van Zelm MC, Szczepanski T, van der Burg M, van Dongen JJM. Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion. J Exp Med 2007;204:645-655. IF 15.2 - 5%

4.            van Zelm MC, Geertsema C, Nieuwenhuis N, de Ridder D, Conley ME, Schiff C, Tezcan I, Bernatowska E, Hartwig NG, Sanders EA, Litzman J, Kondratenko I, van Dongen JJM,  van der Burg M. Gross deletions involving IGHM, BTK, or Artemis: a model for genomic lesions mediated by transposable elements. Am J Hum Genet 2008;82:320-332. IF 10.2 - 5%

5.            Jhunjhunwala S, van Zelm MC, Peak MM, Cutchin S, Riblet R, van Dongen JJM, Grosveld FG, Knoch TA, Murre C. The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Cell 2008;133:265-279. IF 31.3 - 5%

6.            van der Burg M, IJspeert H, Verkaik NS, Turul T, Wiegant WW, Morotomi-Yano K, Mari PO, Tezcan I, Chen DJ, Zdzienicka MZ, van Dongen JJM,  van Gent DC. A DNA-PKcs mutation in a radiosensitive T-B- SCID patient inhibits Artemis activation and nonhomologous end-joining. J Clin Invest 2009;119:91-98. IF 16.6 - 5%

7.            Jhunjhunwala S, van Zelm MC, Peak MM, Murre C. Chromatin architecture and the generation of antigen receptor diversity. Cell 2009;138:435-448. IF 31.3 - 5%

8.            De Gorter DJJ, Beuling EA, Kersseboom R, Middendorp S, Van Gils JM, Hendriks RW, Pals ST and Spaargaren M. Bruton’s tyrosine kinase and Phospholipase Cgamma mediate chemokine-controlled B cell migration and homing. Immunity 2007;26:93-104. IF 20.6 - 5%

9.            Van Loo PF, Dingjan GM, Maas A and Hendriks RW. Surrogate-light-chain silencing is not critical for the limitation of pre-B cell expansion but is for the termination of constitutive signaling. Immunity 2007;27:468-680. IF 20.6 - 5%

10.        Luis TC, Weerkamp F, Naber BA, Baert MR, de Haas EF, Nikolic T, Heuvelmans S, De Krijger RR, van Dongen JJM, Staal FJT. Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation. Blood 2009;113:546-554. IF 10.4 - 5%