6. Antibody Generation by B cells


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The two phases of immunoglobulin generation in B cells: the antigen-independent phase and the antigen dependent phase

  • The development of B cells in ontogeny occurs as a series of genetically determined adaptive cellular transformations in response to an ever-changing environment.
  • B cell development occurs in two phases: 1) in uterine development prior to exposure to foreign antigens and, subsequently, 2) after birth following exposure to the myriad of foreign substances which comprise the external environment.
  • Figure 1 portrays a schematic representation of the total cellular events occurring during B cell development prior to and after birth showing the division of the lifespan of a B cell into these two phases: 1) the antigen-independent phase and 2) the antigen-dependent phase.
Figure 1

Figure 1. Schematic representation of the developmental stages of B-cells showing the antigen-independent and antigen-dependent phases. Deletion of auto-reactive B lymphocytes and tolerance induction occurs during B cell development. During the antigen-dependent phase, thymic-independent antigens (i.e, polysaccharides) react with B1 lymphocytes without Th2 help to form predominantly IgM antibody; the B2 lymphocytes that react with thymic-dependent antigens (i.e., proteins) require Th2 help for isotype switching and somatic mutation of VH/VL genes to produce switching from IgM→ IgG→IgA→IgE antibody. [Reproduced with permission from Bellanti, JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012].

 

  • In the human, B lymphocytes are generated from hematopoietic stem cells (HSC) in the bone marrow (Chapter 2, Bellanti, JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012).
  • B cells in different stages of development can be identified based upon their phenotype and function.
  • The progression from one stage of B cell differentiation to the next is tightly regulated and early B cell development is dependent on the orchestrated control of sequential immunoglobulin gene rearrangements, and expression of receptors, cell surface markers and other gene products leading to survival, proliferation and further differentiation.
  • In the antigen-independent phase, a hematopoietic stem cell undergoes a series of divisions giving rise to the generation of a large and diverse repertoire of daughter clones of B cells of increasing maturity
  • These consist of pro B-cell, pre B-cell, immature B cell and are characterized by the acquisition of certain surface markers, e.g., CD19 and ultimately with the expression of a cell surface IgM receptor (Figure 1).
  • This sets the stage for, the antigen dependent phase, during which binding of antigen to the surface immunoglobulin in peripheral lymphoid tissues leads to a series of proliferative and differentiative steps leading ultimately to the terminal differentiation of the B cell into plasma cell, the source of secreted immunoglobulin (Figure 1).

 

 

B cells are generated in the bone marrow in the absence of specific antigen.

  • In the antigen-independent phase, a hematopoietic stem cell undergoes a series of divisions giving rise to the generation of a large and diverse repertoire of daughter clones of B cells of increasing maturity
  • These consist of pro B-cell, pre B-cell, immature B cell and are characterized by the acquisition of certain surface markers, e.g., CD19 and ultimately with the expression of a cell surface IgM receptor (Figure 1).
  • This sets the stage for, the antigen dependent phase, during which binding of antigen to the surface immunoglobulin in peripheral lymphoid tissues leads to a series of proliferative and differentiative steps leading ultimately to the terminal differentiation of the B cell into plasma cell, the source of secreted immunoglobulin (Figure 1).

 

The genes responsible for immunoglobulin synthesis must be first assembled in the developing B cell

  • The genes responsible for immunoglobulin synthesis are found in all cells of the body arranged in gene segments
  • These are sequentially situated along the chromosome, each of which is responsible for the synthesis of a specific piece of the immunoglobulin molecule: i.e., the variable (V) and constant (C) portions of both the light and heavy chains.
  • These segments occur at considerable distance from one another and therefore cannot be readily joined and be functionally expressed.
  • The immunoglobulin genes found in these segments are inherited through a germline configuration.
  • It is during the antigen-independent phase seen in the B cell that the process of gene rearrangement of the scattered gene segments into a functional gene (i.e., gene rearrangement, somatic recombination) occurs which is required for expression of the gene product, i.e., surface IgM and IgD.
  • This process of gene rearrangement continues into the antigen dependent phase when antigen is encountered during which immunoglobulin genes undergo additional genetic modifications (somatic mutation and isotype switching) (Figure 1).
  • During the antigen-independent phase, a library of antigen binding sites on B cells is actually generated by the process of immunoglobulin gene rearrangement prior to any antigen encounter (Figure 1).
  • Each B lymphocyte produces antibodies of single epitope specificity dictated by the sequence of rearranged genes.
  • The size of the library of antigen binding sites is limited both by the number of B cells as well as by the variety of rearranged immunoglobulin genes found in the body at any one time.
  • As new B cells replace old B cells, the antigen binding sites found in the library change.
  • Clones of B cells bearing BCRs specific for a particular antigen are then stimulated to proliferate upon exposure to that antigen during the antigen-specific phase which ultimately culminates in the sequential production of IgM, IgG, IgA and IgE during isotype switching.
  • How is the immune system able to generate literally millions of different antigen binding sites during the antigen-independent phase? The answer lies in the genes that specify the light and the heavy chains genes.

 

Genes for heavy, kappa, and lambda chains are located on three separate human chromosomes.

  • In humans, the immunoglobulin genes are found at three chromosomal locations:
    • 1) the κ light chain (LC) locus on chromosome 2
    • 2) the λ light-chain (LC) on chromosome 22
    • 3) the heavy-chain (HC) locus found on chromosome 14.
  • Figure 2 is a schematic representation of chromosomal locations of these various gene loci together with the chromosomal arrangement of gene loci in the germ line, followed by gene rearrangement and protein synthesis of the immunoglobulin molecule in the subsequent antigen-dependent phase.
Figure 2

Figure 2. A schematic representation of the chromosomal locations of genetic loci which control immunoglobulin synthesis together with the progressive steps of gene rearrangement, transcription and translation involved in the synthesis and assembly of the various parts of an immunoglobulin molecule. The loci for light chains are found on chromosome 2 (kappa) and chromosome 22 (lambda) and for the heavy chain on chromosome 14. [Reproduced with permission from Bellanti, JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012].

 

  • Table 1 shows the major genetic loci which control the synthesis of various segments of the immunoglobulin molecule
  • These include heavy chain, light chain kappa and lambda chain loci.
  • Also shown in this table is the PAX5 gene which is a member of the paired box (PAX) family of transcription factors, a set of a novel, highly conserved DNA-binding motifs which encode proteins which are important regulators in early development, and which when altered are thought to contribute to neoplastic transformation, e.g., lymphomas.
  • The PAX5 gene encodes the B-cell lineage specific activator protein (BSAP) that is expressed at early, but not late stages of B-cell differentiation.
  • Its expression not only is important in B-cell differentiation, but also in neural development and spermatogenesis.
  • The human IGH locus is located on the chromosome 14 at band 14q32.33, at the telomeric extremity of the long arm; the orientation of the locus has been determined by the analysis of translocations, involving the IGH locus, in leukemia and lymphoma

 

Table 1. Major gene loci, chromosomal locations and functions of gene products involved in immunoglobulin synthesis

LocusNameChromosomal LocationGene product/Function
IGH@ Immunoglobulin heavy chain locus 14encodes heavy chains of human Ig
IGL@ Immunoglobulin lambda locus22encodes lambda light chains of Ig
IGLC2Immunoglobulin lambda constant 2 locus22encodes constant domain of lambda
light chains of Ig
IGK@ Immunoglobulin kappa locus2encodes kappa light chains of Ig
IGKCImmunoglobulin kappa constant 2 locus2encodes constant domain of kappa
light chains of Ig
PAX5 Paired box gene 59p13B-cell lineage specific activator
(B-cell lineage specific activator)protein (BSAP) that is expressed at early, but not late stages of B-cell differentiation. Its expression has also been detected in developing CNS and testis, therefore, PAX5 gene product may not only play an important role in B-cell differentiation, but also in neural development and
spermatogenesis.

 

The genes that encode the heavy chains

  • The gene segments encoding the heavy chains (HC) are found on chromosome 14 (Figure 2) and are similar to those encoding the light chain.
  • As with the light chain gene loci, there are multiple gene segments that encode the variable region of heavy chains (VH).
  • The heavy chain locus, however, includes an additional set of diversity (D) gene segments that are found between the V and J gene segments.
  • The HC locus contains approximately 51 V gene segments each also preceded by a leader exon.
  • There are about 27 D segments, and 6 J segments which are followed by the constant region exons of each of the classes and subclasses of heavy chain beginning with mu (µ), then delta (δ), and then followed by the others.
  • This is the germline gene organization (Figure 2).

 

The diversity of antibody variable regions is generated by somatic recombination

  • During the development of B cells in the antigen-independent phase the arrays of V, D and J segments are cut and joined by DNA recombination (Figure 2).
  • This process is called somatic recombination because it occurs in cells of the soma (Gr, soma, body) a term which encompasses all of the body cells except the germ cells.
  • Outlined in Figure 2 are the events occurring during the synthesis of an immunoglobulin molecule showing both the germline genes and the phases of rearrangement at both the LC and HC loci.

 

Temporal order of gene rearrangement for light and heavy chains

  • The rearrangement of gene segments required for the synthesis of light and heavy chains of an immunoglobulin follows an ordered process and occurs in a sequential fashion (Figure 2).
  • Rearrangement at the HC locus (chromosome 14) takes place first prior to rearrangement of the LC locus.
  • Rearrangement starts at the LC only after a complete HC is produced.
  • The process begins at the pro-B cell stage with the joining of one D and one JH gene segment at the heavy chain locus on chromosome 14 to form a DJH
  • Any residual DNA between these two genes is deleted.
  • Rearragement occurs randomly and can engage any of several combinatorial possibilities involving the joining of any of the 27 D segments with one of the 6 J joining segments.
  • This is next followed by the joining of one of the 51 upstream VH segments with the DJH complex forming a rearranged VDJH
  • All other genes between the V and D segments of the newly formed VDJH gene are removed.
  • A primary transcript is next generated containing the VDJ region of the heavy chain and both the constant mu and delta chains (Cμ and Cδ). (i.e. the primary transcript contains the segments: V-D-J-Cμ-Cδ).
  • The primary RNA is processed to not only add a polyadenylation (poly-A) tail at the end of the Cμ chain but also to remove sequence between the VDJ segment and this constant gene segment.
  • Translation of this mRNA leads to the production of the Ig μ heavy chain protein.
  • The rearrangement at the LC loci takes place next, first at the kappa (chromosome 2) and later at the lambda locus (chromosome 22).
  • The kappa (κ) and lambda (λ) chains of the immunoglobulin light chain loci rearrange in a very similar way, except the light chains lack a D segment.
  • The first step of recombination for the light chains involves the joining of the V and JL chains to give a VJL complex before the addition of the constant chain gene during primary transcription.
  • Translation of the spliced mRNA for either the kappa or lambda chains results in formation of the Ig κ or Ig λ light chain protein, respectively.
  • Assembly of the Ig μ heavy chain and one of the light chains results in the formation of the fully assembled membrane bound form of the immunglobulin IgM that is expressed on the surface of the immature B cell (Figure 1).

 

The germinal center (GC) reaction

  • Germinal centers (GCs) are regions within secondary lymphoid organs which develop following T cell–dependent antigen exposure. These areas are sources of high-affinity antibodies.
  • One of the fundamental pathways needed for the GC reaction is the interaction between activated follicular helper CD4T (Tfh) cells and B cells.
  • Tfh cells are essential for the GC reactiom as they play a role in increasing the efficiency of antibody responses
  • GC Tfh cells expresse IL-21 at high levels. This cytokine is important for the differentiation and functioning of Tfh cells. These cells also produce IL-4, IFN-γ and IL-2 at moderate/variable levels. Bcl-6 is a transcription factor and an important marker of Tfh cells.  This transcription factor is important for the development of Tfh and germincal center B cells.
  • Tfh cells have been shown to play crucial roles in the production of high-affinity, long-lasting antibody responses to vaccines, autoimmune diseases, allergies and even cancer. These cells are important in HIV because they help in the development of broadly neutralising antibodies which can prevent entry of viruses across the different HIV subtypes.
  • GC-committed B cells move to the center follicle due to loss of EBI2 and initiate the GC reaction.
  • The GC reaction represents a checkpoint where the antigen-specific BCR repertoire is randomly altered in heavily proliferating GC B cells in the dark zone (DZ) through somatic hypermutation (SHM) induced by activation induced cytidine deaminase (AID).
  • Mutated B cells having acquired affinity-enhancing mutations are selected by T cells in the light zone (LZ) of the GC or may migrate back into the DZ and undergo another round of SHM when their affinity is not competitive.
  • Thus, B cells are positively selected through iterative cycles and elucidation of the underlying mechanisms will provide important information for vaccine development.
  • The GC reaction is also required for class switch recombination (CSR). High-affinity plasma cells that migrate back into the BM are the endpoint of the GC reaction.

 

Figure 3: Heterogeneity and differentiation of germinal center T cells. The commitment of T cells in promoting germinal center B-cell responses against immunizing antigens is largely dependent at least initially on TCR engagement and CD28 costimulation by antigen-presenting dendritic cells. Upon transient BCL6 induction after activation, CD4+ naive T cells (Tnaive) give rise to pre-germinal center follicular helper T (pre-Tfh) cells that subsequently require contact with cognate B cells to receive re-enforcement signals via ICOS, PD1, and SAP signaling for BCL6 stabilization and commitment to the germinal center (GC) Tfh cell differentiation pathway. B-cell conjugation and SAP signaling is also important for NK Tfh cell formation from invariant NKT (iNKT) cells and their response toward glycolipid-containing antigens. T-cell-mediated suppression of autoreactive and/or or non-antigen-specific germinal center B cells is mediated by Qa-1-restricted CD8+ regulatory T (CD8+ Treg) cells (that directly inhibit Tfh cells) and follicular regulatory (Tfr) cells. Tfr cells develop from BCL6-dependent natural CD4+ regulatory T cells (nTregs) that receive TCR and CD28 signals and also and SAP-mediated signals during cognate B-cell interactions. Fo B, follicular B cells; GC B, germinal center B cell; memory B, memory B cell. [R, Ramiscal & C, Vinuesa. Immunological Reviews 2013 Vol.252: 146–155]

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For more detailed mechanisms of recombination and development of the B cell lineage, see Chapter 6, Bellanti, JA (Ed). Immunology IV: Clinical Applications in Health and Disease. I Care Press, Bethesda, MD, 2012

 
 
 
 
 
 
International Union of Immunological SocietiesUniversity of South AfricaInstitute of Infectious Disease and Molecular MedicineScience Education PrizesElizabeth Glazer Pediatric Aids FoundationAlere