Inducible B Cell Development

A Novel Mouse Model Reveals Insights Into B1 Ontogeny

  • Copyright: Helmholtz Centre for Infection ResearchCopyright: Helmholtz Centre for Infection Research
  • Copyright: Helmholtz Centre for Infection Research
  • Fig. 1: Principle of inducible B cell development. (A) In the recombinant mouse, B cell development is blocked due to the inversion of the coding sequence (CDS) of the Rag1 gene - no functional Rag1 protein can be produced. The Cre recombinase fused to the estrogen receptor is already expressed but stays in the cytoplasm due to binding of heat shock proteins (hsp90). (B) Tamoxifen binds to the estrogen receptor. This leads to a change in the conformation and dissociation from the heat shock proteins. The recombinase can now enter the nucleus and mediate the recombination process. (C) The Rag1-CDS has been turned into its original orientation, functional Rag1 protein can be expressed and B cell development can take place.
  • Fig. 2: Serum levels of IgM and IgG. Induced and uninduced mice were compared with wild-type and Rag1-/- mice as controls.
  • Fig. 3: B1 cells in the peritoneal cavity of induced mice. Cells of the peritoneal cavity were stained with antibodies specific for B1 cells and analyzed by flow cytometry. Mice with and without induction were compared with wild-type control mice.
  • Dr. Sandra Düber and Dr. Siegfried Weiß, Helmholtz Centre for Infection Research, Braunschweig, Germany

Lymphocytes develop in the fetal liver and in the bone marrow after birth. It was strongly debated whether B1 cells, important for the first line of antibody defense, can be generated in adult bone marrow. We now established a new recombinant mouse, which is lymphopenic but allows induction of B cell development at any time point. With this mouse we could prove the generation of B1 cells from adult sources. Extension of this system e.g. to T cells will add important knowledge on developmental constraints of lymphocytes.

Introduction


The immune system protects our body against a huge range of pathogens. To be able to do so, it consists of many different types of cells with distinct effector functions. All of these immune cells are generated from hematopoietic stem cells in the fetal liver or the bone marrow after birth. One particular cell type are B lymphocytes or B cells. Their task is to produce antibodies that recognize and bind invading microorganisms. This way they can either neutralize them or mark them for phagocytic cells. The B cells express these antibodies as surface receptors and upon encounter of their specific antigen, like a structure on a pathogen, can differentiate to secrete them.

B cells can be divided into B1 and B2 cells [1]. B1 cells are already generated in the fetal liver, while B2 cells are generated from bone marrow after birth. They show distinct functional characteristics. B1 cells belong to the first line of immune defense and produce so‑called ‘natural antibodies'. These antibodies are present in serum even without antigen stimulation like in germfree or antigen-free mice [2].

Antibodies derived from B1 cells often recognize common structures on bacterial cell walls and play an important role in the initial phase of infections. On the other hand, B2 cells are able to produce highly specific antibodies against various antigenic structures. They are involved in the final clearance of infections and can mediate livelong memory against a pathogen encountered before thus creating the basis for vaccination.

Up to now, it has been suggested that generation of B1 cells ceases shortly after birth and B1 cells are maintained in the body by their longevity and occasional cell divisions.

With our new mouse model, we were able to disprove this and could show now that these B1 cells can also develop in the adult animal [3].

Generation of Mice with Inducible B Cell Development

Beforehand, it was always difficult to differentiate between newly generated cells and long-lived cells. Therefore, we wanted to generate a mouse in which we could influence the B cell development. First it should be turned off and at any desired time-point, like in the adult mouse, it should be activatable. To realize this, we used murine embryonic stem (ES) cells in which it is possible to insert targeted changes into genes of interest. When injected into blastocysts, such ES cells can contribute cells to the developing mouse including germ cells. Thus, the targeted genetic alterations will be passed on to the offspring.

In our case, we inactivated the recombination activating gene 1 (Rag1) that is essential for the development of B cells by inverting its coding sequence (fig. 1). This results in a complete block of B cell development. The inverted fragment was flanked by inverted recognition sequences (loxP sites) for the recombinase Cre. A transgenic Cre recombinase driven by the B cell-specific mb-1 promoter was introduced as a fusion protein between Cre and a mutated estrogen receptor. This recombinase is expressed in an inactive form but can be activated by the oral administration of the estrogen analogue tamoxifen. Thus, after administration of tamoxifen the recombinase can mediate the recombination. Due to the inverted loxP sites, the inverted Rag1 gene will flip back into its original orientation. Now the Rag1 gene will work properly and B cell development takes place. Tamoxifen can be administered at any desired time-point. Usually we waited until the age of 8 weeks, when a mouse is classified as adult.

Development of B1 Cells from Adult Bone Marrow

The development of newly formed B cells in tamoxifen treated mice could be easily monitored. They were generated in bone marrow, migrated via blood to the spleen where they completed maturation - like in normal mice. One month after onset of B cell development, serum levels of antibodies in induced mice had reached normal levels indicating that these B cells were fully functional (fig. 2).

Interestingly, not only B2 cells were found in the periphery. In the peritoneal cavity, the main location of B1 cells, substantial numbers of B cells with the typical B1 phenotype could be detected (fig. 3).

Since phenotypic markers could be influenced by the environment or inflammatory events, we proved the B1 nature of these B cells also by functional tests. For instance, the newly generated B1 cells exhibited receptors with typical B1 cell specificities. They responded very fast by antibody production to lipopolysaccharide, a component of bacterial cell walls that acts as mitogen. This is an important characteristic for their function as first line of immune defense.
With this mouse model we could demonstrate for the first time that the adult bone marrow is able to generate substantial numbers of B1 cells. Since the primary repertoire of B1 cells from fetal and adult mice is different this demonstrates an unexpected plasticity of B1 cells.

Further Perspectives for This New Mouse Model

The new mouse model has much more potential than studying B cell development. With a change in the transgenic recombinase fusion protein, it is also possible to induce the development of T cells in addition to B cells. Thus, we can investigate T cell subpopulations, which are claimed to develop only in the fetal thymus. We also can test the potential of a naive adult thymus to support the development of such T cells and establish the conditions that such T cells require to develop. Similarly, since we are now able to influence the immune system from the outside, it should be possible to influence the development of autoimmune diseases like diabetes or SLE that spontaneously develop in susceptible mice. In the long run, this will lead to specific therapies.

References
[1] Allman D. et al.: Curr Opin Immunol 20, 149-157 (2008)
[2] Baumgarth N. et al.: Springer Semin Immunopathol 26, 347-362 (2005)
[3] Düber S. et al.: Blood 114, 4960-4967 (2009)

Authors

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Helmholtz Centre for Infection Research
Inhoffenstr. 7
38124 Braunschweig
Germany
Phone: +49 531 6181 0
Telefax: +49 531 6181 2655

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