B Cell Maturation and Development

• Before birth, B cell maturation happens in the yolk sac, fetal liver, and fetal bone marrow.
• After birth, B cell maturation occurs only in the bone marrow.
• B cell maturation depends on the rearrangement of immunoglobulin (Ig) DNA in lymphoid stem cells.
• B cell development can be measured in bone marrow fractions, and developmental stages are reflected in Ig heavy and light chain rearrangement and surface expression.
• Pluripotent hematopoietic stem cells give rise to lymphoid or myeloid progenitor cells.
• Only lymphocytes (T and B cells) and plasma cells are antigen-specific and possess diversity, specificity, memory, and non-self-recognition.
• B cell development begins as lymphoid precursor cells differentiate and remain in the bone marrow, requiring transcription factors and the surface protein C-kit.
• Pro-B cells rearrange the Ig heavy chain genes and express a surrogate light chain.
• Several transcription factors activate adhesion molecules for attachment to stromal cells in the bone marrow.
• Stromal cells secrete cytokines, most notably IL-7, which supports B cell development.
• Pro-B cell interaction with stromal cells activates C-kit, leading to differentiation into pre-B cells.
• IL-7 from stromal cells binds to surface receptors and drives maturation toward a pre-B cell, eventually downregulating adhesion molecules.
• Light chain genes rearrange and replace the surrogate light chain.
• Cells retain many markers as they progress from the Pro to pre-B cell stage but cease C-kit expression and begin expressing CD25, the Alpha chain for the IL-2 receptor.
• Each pre-B cell undergoes six to eight divisions, creating as many as 256 descendants and increasing antibody repertoire diversity.
• Immature B cells express mu heavy chains plus kappa or lambda light chains on their surface.
• Immature B cells express membrane IgM, Ig alpha, and Ig beta, forming a B cell receptor (BCR) capable of signaling antigen engagement.
• Immature B cells leaving the bone marrow differentiate into naive B cells.
• Mature B cells co-express IgM and IgD on their cell surface; while IgD function is unclear, the IgM-bearing immature B cell is not fully functional until IgD and IgM are co-expressed, which involves a change in RNA expression, important for immunoglobulin production.

Pro-B Cells

• Pro-B cells are the earliest stage of B cell development in the bone marrow.
• The primary role of Pro-B cells is to rearrange the genes encoding the B cell receptor (BCR), consisting of heavy and light chains.
• V(D)J recombination results in the generation of diverse BCRs that can recognize a wide range of antigens.
• Pro-B cells express surface markers, including CD19 and CD34, and do not yet have a functional BCR on their surface.

Pre-B Cells

• Pre-B cells represent the next stage of B cell development.
• They have successfully rearranged the genes encoding the heavy chain of the BCR and now express a functional IgM heavy chain on their surface.
• Pre-B cells are tested for their ability to assemble a functional light chain (kappa or lambda) to complete the BCR.
• Pre-B cells express a functional IgM heavy chain, but their BCR still lacks a light chain
• This stage is characterized by the expression of markers such as CD19, CD25, and a pre-BCR complex.

Immature B Cells

• Immature B cells represent the final stages of B cell development within the bone marrow.
• At this point, they have successfully assembled both heavy and light chains to form a complete and functional BCR.
• Immature B cells undergo negative selection to ensure that they do not recognize self-antigens too strongly.
• If they pass this selection, they leave the bone marrow and enter the peripheral lymphoid organs (e.g., lymph nodes and spleen) as mature B cells.
• Immature B cells express both IgM and IgD on their surface.
• Their BCRs are tested for self-reactivity during negative selection.

RAG-1, RAG-2, and TdT

• RAG-1, RAG-2, and TdT (terminal deoxynucleotidyl transferase) are enzymes crucial in B cell maturational development, particularly during V(D)J recombination.
• These enzymes are involved in genetically rearranging the genes encoding the B cell receptor (BCR), consisting of heavy and light chains.
• RAG-1 and RAG-2 initiate and catalyze V(D)J recombination, rearranging variable (V), diversity (D), and joining (J) gene segments to generate diverse BCRs, allowing B cells to recognize a wide range of antigens.
• RAG-1 and RAG-2 are active in early B cell development, specifically in pro-B cells and pre-B cells and are crucial for the initial recombination events that lead to the assembly of the heavy chain of the BCR.
• Once these events are successful, pre-B cells express a functional heavy chain.
• TdT adds non-templated (N) nucleotides at the junctions between rearranged gene segments, contributing to the BCR repertoire's diversity by introducing random nucleotide sequences.
• TdT is primarily active in the pro-B cell and early pre-B cell stages.
• The addition of N nucleotides is particularly important in generating diversity in the complementarity-determining regions (CDR3) of the BCR, which are critical for antigen recognition.
• Once the heavy chain rearrangement is complete, the light chain rearrangement proceeds, involving similar processes and enzymes, including TdT.

Omenn Syndrome

• Omenn syndrome is a rare genetic disorder resulting from mutations in the RAG-1 or RAG-2 genes, which are critical for developing a functional immune system.
• The RAG-1 and RAG-2 genes encode proteins essential for V(D)J recombination, which generates the diverse repertoire of antigen receptors on B and T lymphocytes.
• Without functional RAG-1 or RAG-2 proteins, B and T cells cannot properly develop, and the immune system fails to respond effectively to pathogens.
• Mutations in RAG-1 or RAG-2 that only partially impair function result in Omenn syndrome. This means some V(D)J recombination occurs, but it is inefficient and leads to:
  • A limited and oligoclonal T cell repertoire.
  • Absence or severe reduction of B cells.
  • Aberrant immune activation.
• Omenn syndrome is characterized by:
  • Autoimmune-like symptoms: Widespread skin rash (erythroderma), failure to thrive, hepatosplenomegaly, lymphadenopathy.
  • Immunodeficiency: Recurrent infections due to the lack of effective B and T cells.
  • Elevated IgE and eosinophilia: Paradoxical immune activation caused by the dysfunctional T cells.

Omenn Syndrome Autoimmune Issues

• Characterized by a unique interplay of immune system dysfunction caused by incomplete V(D)J recombination.
• Omenn syndrome is caused by mutations in the RAG-1 or RAG-2 genes that lead to partial or "leaky" function of the recombination-activating genes (RAGs).
• These genes are essential for the proper rearrangement of B and T cell receptors during immune cell development.
• The partial loss of function means that V(D)J recombination still occurs, but it’s inefficient and leads to an oligoclonal T cell repertoire.
• The restricted T cell repertoire results in autoimmune-like reactions, despite the relatively low number of functional T cells.
• These limited T cells are often self-reactive because they were not adequately selected during development due to the imperfect recombination.
• This limited and abnormal T cell population can become activated against the body's own tissues, contributing to autoimmune reactions.
• While B cells are also impaired in Omenn syndrome due to defective V(D)J recombination, B cells can still develop to some extent but fail to function properly.
• The lack of mature B cells and normal antibody production means that the body is unable to mount effective immune responses against infections.
• Despite the lack of functional B cells, autoimmune activation still occurs because of dysfunctional T cells.
• These T cells are less likely to be suppressed by regulatory mechanisms that would usually prevent autoimmunity.
• In Omenn syndrome, dysregulated immune responses lead to the production of autoantibodies and tissue damage, especially in organs like the skin, liver, and spleen.
• The autoimmune symptoms in Omenn syndrome (e.g., skin rashes, hepatosplenomegaly, lymphadenopathy, and elevated IgE) result from these abnormal immune responses, where the T cells are not properly regulated and cause tissue damage through inflammation and autoimmunity.

B Cell Surface Markers

• Changing immunoglobulin development informs us of the B cell maturation stage.
• Each B cell is specific in producing immunoglobulin of only one antibody specificity that recognizes only one epitope similar to T cells.
• The extreme diversity among individual B cells creates the overall diversity of the immunoglobulin or IG response.
• Early in the pro-B cell stage, cells do not display the heavy or light chains.
• Molecules such as IG alpha and iG beta, important for signal transduction through the BCR, are found at the surface.
• The BCR is composed of a membrane IG molecule associated with elite IG alpha and iG beta that is primarily within the cell.
• In pre-B cells, the surrogate light chain is formed by two proteins, V pre-b and lambda five; the membrane-bound complex of mu, heavy chain, and surrogate light chain is important for Ig development.
• The associated IG alpha, and IG beta complex remains and provides the signaling capability for the IG molecule burdens tyrosine kinase or vtk, is a critical downstream protein required for transmitting signals from the pre-B cell receptor and differentiation to immature B cell.
• Only B cells that express these IG and BCR molecules can proceed toward maturity.
• The immature B cell no longer expresses the surrogate light chain; rather, this phenotype expresses kappa or lambda light chain together with the mu heavy chain.

B Cell Development Video

• During B cell development, the developing cells interact closely with the stromal cells of the bone marrow, which are largely composed of mesenchymal stem cells.
• Mesenchymal stem cells are multi-potent and can differentiate into various cells, including macrophages and endothelial cells.
• Mesenchymal cells provide B cells and petition molecules they can use to attach, as well as important growth factors like interleukin-7, which they can use to grow and proliferate as they develop.
• B cells go through six stages; they start as common lymphoid progenitor cells, then become early pro-B cells, then late pro-B cells, then large pre-B cells and small pre-B cells, and finally, immature B cells.
• The cell activates permitted changes in this DNA, so that, by the time it's an immature B cell, it has DNA that uniquely codes for a B cell receptor that can bind to foreign antigens but isn't self-reactive.
• The B cell receptor has two chains, a heavy chain and a light chain.
• The heavy chain contains regions that determine the type of antibody it'll become, as well as one of the B cell receptor will be surface bound or secreted like an antibody. .
• The heavy and light chains come together and form a unique protein structure capable of binding proteins, carbohydrates, or lipids the B cell might eventually encounter; this is called the antigen-binding site.

B cell maturation occurs in the yolk sac, fetal liver, and bone marrow before birth, and exclusively in the bone marrow after birth. This process relies on Ig DNA rearrangement in lymphoid stem cells. B cell development can be tracked in bone marrow fractions through Ig heavy and light chain rearrangement and surface expression.