B7/CD28 family members are critical positive and negative immune regulators. Recently, a novel ligand /receptor pair of this family was described: BTLA/B7-H4. BTLA is expressed on a variety of immune cells, most prominently on B-cells and after activation on T-cells. The molecular signature of BTLA predicts it to transmit negative signals, and it has been speculated that the molecule might have a critical function in the negative regulation of adaptive immune responses. We have identified BTLA independently as a gene differentially expressed during T-cell development and are studying its function in vivo using mice over-expressing BTLA in T-lymphocytes as well as BTLA-deficient mice. Both mutant strains do not display any gross abnormalities in their lymphoid compartments. Mice constitutively expressing BTLA on T-cells can still mount effective antibody responses indicating that signaling through BTLA is not shutting off T-cells. Aged BTLA-deficient mice show no apparent susceptibility to autoimmune inflammatory conditions, which is in stark contrast to mice deficient in CTLA-4 and PD-1, two other negative regulators of the CD28 family. Based on these observations we are now investigating the role of BTLA in the modulation rather than termination of immune responses. Our current focus is on two aspects: detailed intracellular wiring of BTLA and the role of BTLA in modulation of adaptive immune responses.

Bone Morphogenetic Proteins: Novel Regulators in the Immune System?

Bone Morphogenetic Proteins (BMPs) constitute a family of secreted molecules belonging to the TGFβ superfamily. First isolated from bone, they took their name for their ability to ectopically induce bone formation. Studies on lower vertebrates and Drosophila have established that BMPs are morphogens providing positional information to individual cells. Not surprisingly BMPs regulate many diverse aspects of embryonic development and tissue specification. Only more recently have they been implicated in homeostatic regulation of various adult tissues.

BMP signaling is complex and highly regulated both in the extracellular and intracellular space. Our focus is on the extracellular space where secreted BMP antagonists and modifiers such as Noggin, Gremlin, Chordin, and Twisted Gastrulation regulate receptor binding, thereby establishing an extracellular BMP-signaling network.

In previous work we have identified a role for BMP-2/4 in the T-cell development. BMP-2/4 restrict the transition of immature CD4-CD8- double negative (DN) thymocytes to the CD4+CD8+ double positive (DP) stage. Individual thymocytes that have received a pre T-cell receptor (pTCR) dependent maturation signal can overcome this BMP brake by up-regulating Twisted Gastrulation (Tsg), which in synergy with Chordin inactivates BMP-2/4.

Expression and function of BMPs in the adult immune system

Following this first description of a role for BMPs in T-lymphocytes, we are now probing for the wider function of these molecules in the immune system. Our area of interest includes not only lymphocyte development, but also the mature immune system and immune disorders such as leukemias and chronic inflammation.

For our studies we are employing a two-pronged approach. First, we are determining in detail time and location of BMP / BMP antagonist expression. We believe that it is crucial to study the BMP signaling network in its entity to be able to understand BMP function. Second, we manipulate the BMP signaling network on the molecular level using conditional gene targeting tools.

For expression studies, we mainly make use of reporter mice that express lacZ or a fluorescent marker in lieu of the gene of interest. Simple visualization of the reporter in combination with a suitable histochemical marker gives us a precise picture where and when a particular gene is expressed. This is exemplified in figure 3, depicting a lymphoid follicle in the spleen with BMP-4 expressing cells (blue, indicated by black arrow) in relation to B220+ cells B-cells (brown).

For functional studies, we mainly use conditional alleles for BMPs or BMP antagonists of interest, as the straight null mice are often embryonic or neonatal lethal. Cre-mediated cell specific ablation or over-expression of individual members of the signaling network allows us to address their role on development and homeostasis of the immune system. We are now in the process of analyzing the function of BMP or BMP antagonists through gene ablation in individual cell types of the immune system using appropriate Cre-drivers. In combination with the expression studies this will allow us to dissect and understand the role of BMP in the immune system, and to assess whether distorting this signaling network is a contributory factor for the development of a variety of immune disorders such as leukemias or chronic inflammation.

Novel gene targeting technologies for precise allele construction

Traditionally, the assembly of gene targeting cassettes was time-consuming and cumbersome. Recently, an alternative approach became possible using Bacterial artificial chromosomes (BACs). BACs have the capacity to e carry large inserts (100-300kb), are clonally stable, have a low rate of chimerism, and can be handled with ease. As part of the genome-sequencing project, annotated BACs for virtually all mouse genes are available. Modifications or mutagenesis of BACs is now straightforward using the technology of bacterial homologous recombination (BHR). In this technology, generic targeting cassettes are flanked with short pieces of DNA homologous to the endogenous sequence. The DNA is transformed into E. coli that carry the BAC of interest and are proficient for BHR. Recombination between the BAC and the introduced donor DNA allows the introduction of the generic cassette into the BAC at the desired sites with nucleotide precision. To facilitate the rapid generation of conditional alleles, we have developed a set of generic targeting cassettes for the introduction of a pair of loxP sites. The use of generic targeting cassettes allows processing of several targeting constructs in parallel. We have been applying this technology to a variety of genes of the BMP signaling pathway.