The development of B cells defined by immunoglobulin gene rearrangements

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In this article, I briefly explain the development of B cells defined by the rearrangements of immunoglobulin genes.

The development of B cell begins with the pre-pro-B cell

The stages of B-cell development begin with the first cell committed to the B cell lineage, the pre-pro B cell. The developing common lymphoid progenitor gets an entry into the pre-pro-B-cell stage with the accession of B220 (CD45R), the B-cell lineage-specific marker and the expression of increasing levels of the transcription factor EBF1. The transcription factor EBF1, plays an important role in the lymphoid development. The transcription of the Ebf1 gene is controlled by multiple transcription factors like STAT5, E2A, Foxo1, and Runx1.

The immunoglobulin gene

Protein sequencing of mouse and human antibody heavy and light chains, showed that approximately the first 110 amino terminal amino acids of antibody heavy and light chains are the most variable in nature among different antibody molecules. Thus, this region was designated as variable (V) region of the heavy and light chains. The remaining region of the light and heavy chains was classified as constant (C) region.

The Theories about the arrangements of genes of light and heavy chain

Many immunologists proposed many theories about the arrangement of genes of light and heavy chain.

In 1965, William Dreyer and J. Claude Bennett proposed that the heavy chains and the light chains of antibody are each encoded in two separate segments in the germ-line genome.

In the early 1970s, the innovative somatic hypermutation theory arrived. This theory suggested that only B cells undergo a mutational process. Thus, they alter the antibodies in an individual animal and increase the total number of different antibodies available to it. Such mutations would not be passed on to offspring as these mutations are not affecting the germ line genes.

According to Tonegawa and colleagues, the variable and constant regions of the antibody light chain gene were encoded in segments, located in two different places in the germ line. In mature antibody producing cells, these segments were brought together to form an intact light chain gene by the process of DNA recombination.

Tonegawa and colleagues, then further sequenced mouse and human light-chain variable and constant region genes. They reached a confirmation that the variable and constant regions of antibody light chains are encoded by different DNA segments.

They also showed that the variable regions of the light chain are themselves encoded by two separate gene segments, i.e., the V and the J segments. After the gene rearrangements take place during the B cell development, these segments are made contiguous. The greatest diversity is found at the V-J gene junction.

Sequencing of the immunoglobulin heavy-chain genes

Lee Hood and group cloned and sequenced the immunoglobulin heavy-chain genes. The results showed that the variable region of the heavy chain was encoded in three separate gene segments in the germ line, rather than in two segments. During the development of B cell, these segments were recombined and create the contiguous coding information for the heavy chain variable region.

It was found that the heavy chain variable region was encoded by a germ-line heavy-chain variable (VH) region gene fragment. This (VH) region encodes 1-100 amino acid residues of the antibody heavy chain. A second fragment, which included a heavy-chain joining (JH) region gene segment determines the the amino acid sequence, 107-123 of the heavy chain.

The amino acid residues, 102-106 of the heavy chain were encoded by the DNA segment located at 5′ of the J region in the mouse embryonic DNA. The gene segment is variable in its length. So, due to its diversity it is known as the D gene segment. There is no D region in the light chain. Thus, it is always denoted to the heavy chain.

Recombination between three gene segments, i.e., the VH, D, and JH takes place and the variable region of the heavy chain of the immunoglobulin molecule is encoded. The D-region and the V-D and D-J junctions, encode the greatest sequence diversity regions in the antibody heavy chain that corresponds to the CDR3 regions of the antibody heavy chains.

The immunoglobulin proteins consists of two identical heavy chains and two identical light chains. The light chains can be classified as kappa (k) light chains, or lambda (λ) light chains. The human and mouse k-chain loci are arranged in groups of V and J segments, located upstream from a Ck gene segment.

Rearrangement of the immunoglobulin genes in pre-pro-B cells

In pre-pro-B-cell, the transcription factors EBF1 and E2A get attached to the immunoglobulin heavy-chain locus. In this way, preparing the cells for the primary step of immunoglobulin gene recombination by promoting the accessibility of the D-JH gene segments. Immunoglobulin gene rearrangements are also controlled by epigenetic chromatin modifications.

The B-lineage genes were previously made silent epigenetically through functional interactions with the SWI/SNF chromatin remodeling complex. Their reactivation is facilitated by the transcription factor EBF1. There are two transcription factors, i.e., Notch 1 and GATA-3, which support T-cell development. Notch 1 and GATA-3, are inhibited by EBF1, which helps movement of the cells towards the B-cell lineage.

Rearrangement of the immunoglobulin genes in progenitor B cells (PRO-B cells)

In the pro-B cells or progenitor B cell, the cell starts preparing for VH-to-DJH joining as D-to-JH recombination is completed. The vital B cell transcription factor PAX5 is expressed until the mature B cell is activated by antigen, and get differentiated into antibody secreting plasma cells.

The transcription factors Ikaros, PU.1, and E2A support the expression of PAX5, which strengthens EBF1 expression. Expression of PAX5 makes the cells to move from pro-B-cell stage to late pro-B-cell stage of development. The B cell transcription factor, PAX5 controls the transcription of genes.

At the late pro-B-cell stage, the expression of non-B-lineage genes is permanently ceased. The transcription factor PAX5, blocks the expression of Notch1 gene. In this way, prohibiting any remaining potential of the pro-B-cell to move along T-cell lineage.

At the pro-B-cell stage, many important B-cell genes are activated under the transcriptional control of PAX5 and other transcription factors. In early pro-B-cells, two such genes encoding Igα and Igβ, are turned on. These are the signaling chains of the membrane immunoglobulin B-cell receptors and CD19 ( a component of B cell co-receptor). The expression of CD19 is controlled epigenetically.

The CD19 locus enhancer undergoes chromatin remodeling. This finally activates Cd19 gene transcription by binding of E2A with EBF1 and PAX5. The development of B-cell progresses by the expression of Igα, Igβ, and CD19. The expression of these genes is also required for signaling through membrane Ig receptors.

The transcription factor PAX5, contracts the IgH locus and enhances the chances of recombination of VH -to-DJH gene segments. PAX5 brings the distant VH gene segments closer to the already rearranged DJH gene segments. The VH -to-DJH Ig gene segment recombination is completed with the onset of precursor B cell (pre-B-cell) stage.

Rearrangement of the immunoglobulin genes in pre-B-cells

The pre-B-cell receptor is formed by the two surrogate light chain pair (SLC pair) with two μ heavy chains. The surrogate light chain is formed by VpreB and λ5, encoded by the transcription factors EBF1 and PAX5.

Both VpreB and λ5 have extra amino acid sequences. VpreB has additional 25 amino acid residues at its C terminus, whereas, λ5 has extra 50 amino acid residues at its N terminus. These extra polypeptide sequences, contribute to the β sheets in the immunoglobulin like domains of the surrogate light chain. These extend over the heavy-chain complementary determining region 3 (CDR3), thus blocking it from catching any antigen.

According to studies, multiple pre-B cell receptors aggregate with each other on the pre-B-cell surface in the endoplasmic reticulum. The oppositely charged tails of VpreB and λ5 interact with each other to form the aggregation.

Initiation of signaling

Signaling is initiated through the Igα/Igβ signaling chains by the ligand-independent self-aggregation. A sequence of events essential for B-cell development is initiated by signal from pre-B-cell receptor. To Progress to the pre-B-cell stage, it is necessary for animals to express the pre-B-cell receptor or the signaling components Igα/Igβ.

The pre-B-cell receptor gives stop signals to the VH gene rearrangement. It gives a survival signal activating several rounds of cell division, followed by light-chain gene rearrangements.

The pre-B cells that express heavy-chain, proliferate to generate a pool of daughter cells. The daughter cells have the same heavy chain configuration, but each with a distinct light-chain rearrangement. So, each successful heavy-chain rearrangement has many different receptor specificities. The pre-BCR expression and initiation of these events account for the pre-B-cell (first) checkpoint in the development of B cell.

The formation of a functional BCR prohibits further heavy-chain recombination

The signal from the pre-B-cell receptor results in inhibiting transcription of RAG genes. The productive rearrangement of one heavy chain gene forms a functional BCR. This will prohibit any further heavy-chain recombination.

The phenomenon of allelic exclusion, thus arise where in a single B cell the genes of only one of the two heavy-chain alleles can be expressed. Thus, this pre B-cell receptor signaling, force the chromatin at the unrearranged heavy-chain locus to undergo many physical changes. These changes make it unfit for participation in rearrangement events ahead.

The incipient recombination of VH, D, and JH was the result of coming closure of VH, D, and JH loci after getting a signal from IL7. The reduction of expression of IL7 happens due to changes in transcription factors at the pre-B-cell stage. This causes physical separation of the three loci in the unrearranged heavy-chain locus. This event, is followed by chromatin deacetylation events that make the unused heavy-chain locus deactivate and get back it to an inactive and closed (heterochromatic) configuration.

The cells at the small pre-B-cell stage

Many changes at the end of the pre- BCR activated cell proliferation, lead to the lack of pre-BCR expression. Through the pre-B-cell receptor, a negative feedback round of signaling ceases surrogate light-chain gene transcription. This in result causes the displacement of EBF1 from the λ5 and VpreB promoters. Gradual loss of IL-7R signaling also takes place. When the cells stop proliferating, they get an entry into the late or small pre-B-cell stage.

The cells at the small pre-B-cell stage start light-chain gene rearrangement. In mouse, V and J gene segments had gone through pre-BCR induced epigenetic changes. The newly expressed transcription factors IRF4, IRF8, and FOXO1 carry out the stimulation for initiation of light chain rearrangement. This becomes possible by the re-expression of the RAG1/2 proteins.

The gene segment rearrangement is started with the k-chain. If the rearrangement is failed with two k-chains, then rearrangement is done through each of the λ chain chromosomes. In contrast, human beings undergo gene rearrangement in any of the two either k or λ loci.

After the completion of light-chain rearrangement, the cell is signaled to stop any further light-chain gene rearrangements by the IgM B-cell receptor expressed on the cell surface. The B cell expressing membrane IgM, is defined as an immature B cell.

The light-chain immunoglobulin gene rearrangement, at both the chains of k and λ loci causes the death of budding cell. It dies through apoptosis and this accounts for the second or immature B cell checkpoint. In the case of unproductive light chain rearrangement with a chance for editing the same, B cells successfully rearrange their heavy chains. The heavy chains will express membrane IgM and continue forming immature B cells.

Conclusion

The development of B cell can be defined by rearrangements of immunoglobulin genes. The first cell committed to the B cell lineage, the pre-pro B cell from which the B-cell development begins. Multiple theories are proposed about the arrangement of genes of light and heavy chain.

The transcription factor EBF1, plays an important role in lymphoid development. The process is controlled by multiple transcription factors like STAT5, E2A, Foxo1, and Runx1. The vital B cell transcription factor PAX5, strengthens the expression of the transcription factor EBF1.

Cells start light chain gene rearrangement at the small pre-B-cell stage. The IgM B cell receptor expressed on the cell surface gives stop signal to any further light chain gene rearrangements after the completion of light-chain rearrangement.

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