I have spent most of my adult life and all of my career, studying the development of B cells and antibodies. If you have read any of my previous “The Biology of Antibodies” blog posts, you are likely already well aware of my fascination and amazement of all things antibody. Of particular interest, is the development of B lymphocytes, the cells that produce antibodies. In this post I will provide an overview of B cell development with the goal of providing an understanding of how antibodies are generated in vivo. My review will be focused primarily on mouse B cell development (although there are great similarities with humans), as that has been the primary focus of my work as well as the host for monoclonal antibody development here at MBS.
B cells differentiate from the hematopoietic stem cell (HSC) in the bone marrow. Approximately 10-20 million B cells are estimated to be produced each day in the mouse bone marrow, however only about 10% of these B lymphocytes or B cells, leave the bone marrow and enter into circulation. While HSCs can be found in circulation they are predominantly found in the bone marrow. All red blood cells and leukocytes (white blood cells) are differentiated from HSCs, and with the notable exception of T lymphocytes all of the differentiation occurs in the bone marrow. The different hematopoietic lineages (red cells, lymphocytes, myeloid cells) all require different microenvironments or niches that provide distinct cytokines and cellular support required for distinct differentiation programs to be regulated.
II. B Cell Commitment and Differentiation
HSCs give rise to two predominant populations, the common myeloid progenitor (CMP) and the common lymphocyte progenitor (CLP). CMP differentiation results in the production of: neutrophils, basophils, eosinophils, monocyte/macrophages, erythrocytes (red blood cells) and myeloid dendritic cells. The CLP is responsible for the production of: B lymphocytes, T lymphocytes, natural killer cells, and lymphoid dendritic cells. In addition to the generation of all of these lineages the HSC, CMP, and CLP all undergo self-renewal to provide a stable stem cell population for continual renewal of all of the leukocyte (and erythrocyte) cell populations throughout the life of the host.
It is from the CLP from which B cells arise (see Figure 1), commitment to the B cell lineage is a result of contact with the stromal cells as well as growth factor and cytokine production that results in initiating gene regulation that drives B cell commitment. This becomes a progenitor B (or pro-B) cell. In these early differentiation stages, IL-7, stem cell factor, Flt3 ligand, and specific adhesion molecules are important to support growth and survival of the pro-B cells. In the pro-B cells heavy chain (HC) immunoglobulin (Ig) rearrangement begins which is the beginning of antibody production. Once HC Ig gene rearrangement has successfully initiated, the cells become precursor B (or pre-B) cells. During the pre-B cell stage (which can be divided into different phases) HC Ig gene rearrangement is completed and tested (I’ll get back to this in a moment) and light chain (LC) gene rearrangement takes place to generate a fully functional B cell receptor (BCR, which is a membrane bound antibody). At this stage the cell is called an immature B cell.
During the pro-B and pre-B stages not only is HC and LC Ig rearrangement occur, but each stage is tested to make sure that it is a functional rearrangement (to insure the generation of a fully functional antibody). This initially tests the HC Ig gene rearrangement through the production of the surrogate light chain (SLC) which will interact with a complete HC and if this results in the generation of an appropriate signal, similar to a fully functional BCR, then differentiation can continue. Once the LC gene is rearranged and produced it also must pair with the HC and be transported to the plasma membrane and interact with all the appropriate components signaling a fully formed BCR. At this point the immature B cells are “tested” for auto-reactivity: if the immature B cell encounters an antigen and it generates a strong signal, indicating moderate to high affinity towards the antigen, then this would be a result of interacting with self antigens as these cells are only seeing self cells and serum proteins. These cells will most likely be removed as they would be harmful to the host and not released into the periphery.
III. Maturation of B cells in the Spleen
The immature B cells that successfully make it through this process (only about 10%) enter into the bloodstream and migrate to the spleen. The immature B cells have both IgM and IgG (BCRs) expressed on their surface at this point and once they enter into the spleen are called transitional type 1 (T1) B cells. In the spleen B cells, T cells, and follicular dendritic cells, form what is known as a primary follicle or sometimes the white pulp (See Figure 2). The T1 B cells are located outside of the follicle (extrafollicular) in an area known as the red pulp (as this is where all of the red cells are flowing through the spleen). At this point, the T1 B cells are exposed to more self cells and circulating proteins and if they respond strongly it would indicate autoreactivity and the cells are typically induced to become T3 B cells, which are anergic (which means they become non-responsive to antigen), and will likely die off.
If the T1 B cells survive through this they can then migrate into the follicle and become T2 B cells (See Figure 1). At this point they will be responsive to antigen, and become naive follicular B cells, which are fully mature B cells that can participate in immune responses. The mature B cells will circulate through all lymphoid tissues (spleen, lymph nodes, and lymphoid associated tissue) being exposed to antigens. If they encounter antigens that stimulate a response then they will migrate to the border of the B cell/T cell boundary and interact with stimulated T cells. B cells present antigen to the T cells and those that are specific for that antigen will activate the B cell to become an antibody producing B cell, as well as generate a germinal center, where B cells undergo affinity maturation and class switching. This will generate B cells that can produce antibodies with increased specificity to the antigen and also memory B cells, which will be able to respond much more quickly upon subsequent exposures to the same antigen.
The terminal differentiation of a B cell is becoming a plasma cell which is essentially a cell devoted to secreting antibodies. From the germinal center or the memory B cell population, antigen specific B cells will proliferate (in the presence of the antigen) and become plasmablasts and then plasma cells. Plasma cells that are located in the spleen (or lymph node) are short-term plasma cells which only produce antibody for a limited period of time. Plasma cells that migrate to the bone marrow are long-lived plasma cells and can persist for extended periods of time (perhaps decades) and secrete antibodies into the bloodstream.
From the initial HSC through to the terminally differentiated plasma cell, there are many morphological and gene expression changes which are supported by stromal cells, dendritic cells, and/ or T cells and regulated by antigen. All of this, including the removal of strongly responsive autoreactive B cells, and generation of highly specific isotype switched antibodies is part of the overall immune response and will be regulated according to the specific pathogen.
IV. Implications for Monoclonal Antibody Development
When developing hybridomas for the production of monoclonal antibodies, it is important to consider the antigen’s stimulation of the immune response to properly activate B cells. As we collect the spleen for isolating antibody producing cells for fusion, the B cells of interest (mostly the activated mature B cells, germinal center B cells, and plasmablasts) must be localized to the spleen in order to achieve a successful hybridoma. Antigen dose as well as route of immunization should be taken into consideration to try to increase the specific B cell population of interest and maximize their collection in the spleen.
In the next edition of “The Biology of Antibodies” I will discuss the genetics of Ig gene rearrangement and how this leads to generating antibody diversity.