**4. Isotypic exclusion**

A single B-cell never expresses both a κ string and a λ string. The first recombination attempt for the L chains takes place at one of the two κ genes. In case of failure, the κ gene of the other chromosome 2 or the λ genes is used.

### **5. Mature B-cell activation: immunoglobulin class/isotype switching and somatic hypermutation**

#### **5.1 Isotype switching: a process affecting the constant region of the immunoglobulin H chain**

The B-cell that initially produced the IgM isotype will subsequently produce other immunoglobulin isotypes (IgG, IgE, and IgA), thanks to a process termed Ig H chain class switching, isotype switching, or class switch recombination (CSR).

#### **Figure 6.**

*CSR process. Here is an example of switching to the IgG1 isotype. First, AID induces DSB formation after deamination of Sμ and Sγ1 regions. Subsequently, these two regions recombine by an intrachromosomal deletional recombination, and the expressed VDJ segment associates with the Cγ1 gene. AID: activation-induced cytidine deaminase, CSR: class switch recombination (also called immunoglobulin heavy chain class switching/ isotype switching), DSB: double strand-breaks, Eμ: intronic enhancer, S: switch region.*

**33**

(**Figure 6**).

lar B-cells.

**and H chains**

*5.2.1 Brief anatomo-histological overview of B-cells*

*5.2.1.1 Primary follicles and interplay between B-cells and FDCs*

been designed because it contains a high cell density) (**Figure 7**).

*Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation*

Such a phenomenon occurs after activation of a mature B-cell by an appropriate antigen, thereby generating different antibody isotypes that have the same variable domains as the original antibody generated in the immature B-cell during V(D)J

CSR is instigated following conversion of deoxycytidines in S regions-a G rich with high density of WGCW (A/T-G-C-A/T) motifs-to deoxyuracil by activationinduced cytidine deaminase (AID) in the IG loci, which is also required for SHM. The presence of deoxyuracil promotes DNA mutagenesis though a subset of DNA repair proteins. Deoxyuracil residues are subsequently removed from DNA by enzymes of the base excision repair (BER) and mismatch repair (MMR) complex MSH2/6 pathways, leading to mutations, single-strand DNA breaks (SSBs), and the DSBs required for CSR. Recall that, in humans, nine functional CH genes are located downstream of the V, D, and J gene segments of antigen receptor loci. The V(D)J segment, initially rearranged in the bone marrow, can be juxtaposed, during B-cell activation, to one of these functional genes coding for another constant domain, depending on antigen and the cytokine milieu, and occurs between DSBs introduced into the donor μ S (Sμ) region and a downstream/acceptor S region located from ∼65 to 160 kb downstream, which can subsequently recombine with an S region farther downstream (for review, see [31–33]). Finally, Igs resulting from the CSR process have the same specificity for the antigen responsible for the B-cell activation. They have also the same L chains as well as the same variable fragments of the H chains

**5.2 Somatic hypermutation: mutations take place on the variable segments of L** 

Many resting B-cells, agglutinated around follicular dendritic cells (FDCs), harbor primary follicles. Thus, in the adult spleen, about 80% of B-cells are follicu-

A major role is attributed to FDCs, as prominent stromal cell constituents of B-cell follicles. These cells do not express major histocompatibility complex class II (MHC II) molecules nor do they have the capacity to phagocytose and process exogenous antigens for MHC I-restricted presentation [34]. Experiments using cryoimmunogold electron microscopy have demonstrated that the presence of MHC II molecules on their surface is passive and originate from microvesicles/ exosomes they are attached to [35]. Additionally, their ontogeny remains controversial. They are not derived from the bone marrow hematopoietic stem cell, but they could originate from local mesenchymal precursors in lymphoid organs [36]. Moreover, FDCs promote the survival and continuous recirculation of naive B-cells and allow for the attraction of activated B-cells, as well as the selective process for affinity maturation within the GC of lymphoid follicles during humoral adaptive/antibody-mediated immune response, allowing activated B-cells to significantly improve the affinity of their BCR. Thus, they have the ability to retain on their Fc receptor (FcR) antigens in native form combined with antibodies (immune complexes [ICs]), for long periods of time, ranging from months to years, thus making them accessible to the centrocytes (CC) that enter light zone (LZ) and result from the proliferation of blasts in the dark zone (DZ; this zone has

rearrangement, but having distinct constant domains in their H chains.

*DOI: http://dx.doi.org/10.5772/intechopen.90637*

#### *Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation DOI: http://dx.doi.org/10.5772/intechopen.90637*

*Normal and Malignant B-cell*

**4. Isotypic exclusion**

**and somatic hypermutation**

**immunoglobulin H chain**

mediated by this pre-BCR block the accessibility of the RAG recombinases on the second allele of the nonrecombinant μ H chain and redirect them toward the Lκ chain locus to initiate the first recombinations. The formation of a complete BCR

Importantly, it has been shown, using genetically engineered mice that carry two functional IGH alleles that are completely recombined and different, that the expression of IG loci does not appear to be monoallelic and that B-cells could have

A single B-cell never expresses both a κ string and a λ string. The first recombination attempt for the L chains takes place at one of the two κ genes. In case of

**5. Mature B-cell activation: immunoglobulin class/isotype switching** 

The B-cell that initially produced the IgM isotype will subsequently produce other immunoglobulin isotypes (IgG, IgE, and IgA), thanks to a process termed Ig H chain class switching, isotype switching, or class switch recombination (CSR).

*CSR process. Here is an example of switching to the IgG1 isotype. First, AID induces DSB formation after deamination of Sμ and Sγ1 regions. Subsequently, these two regions recombine by an intrachromosomal deletional recombination, and the expressed VDJ segment associates with the Cγ1 gene. AID: activation-induced cytidine deaminase, CSR: class switch recombination (also called immunoglobulin heavy chain class switching/*

*isotype switching), DSB: double strand-breaks, Eμ: intronic enhancer, S: switch region.*

combining H and L chain blocks recombinations on other L chain alleles.

the ability to express H chains by both alleles [29] (for review, see [30]).

failure, the κ gene of the other chromosome 2 or the λ genes is used.

**5.1 Isotype switching: a process affecting the constant region of the** 

**32**

**Figure 6.**

Such a phenomenon occurs after activation of a mature B-cell by an appropriate antigen, thereby generating different antibody isotypes that have the same variable domains as the original antibody generated in the immature B-cell during V(D)J rearrangement, but having distinct constant domains in their H chains.

CSR is instigated following conversion of deoxycytidines in S regions-a G rich with high density of WGCW (A/T-G-C-A/T) motifs-to deoxyuracil by activationinduced cytidine deaminase (AID) in the IG loci, which is also required for SHM. The presence of deoxyuracil promotes DNA mutagenesis though a subset of DNA repair proteins. Deoxyuracil residues are subsequently removed from DNA by enzymes of the base excision repair (BER) and mismatch repair (MMR) complex MSH2/6 pathways, leading to mutations, single-strand DNA breaks (SSBs), and the DSBs required for CSR. Recall that, in humans, nine functional CH genes are located downstream of the V, D, and J gene segments of antigen receptor loci. The V(D)J segment, initially rearranged in the bone marrow, can be juxtaposed, during B-cell activation, to one of these functional genes coding for another constant domain, depending on antigen and the cytokine milieu, and occurs between DSBs introduced into the donor μ S (Sμ) region and a downstream/acceptor S region located from ∼65 to 160 kb downstream, which can subsequently recombine with an S region farther downstream (for review, see [31–33]). Finally, Igs resulting from the CSR process have the same specificity for the antigen responsible for the B-cell activation. They have also the same L chains as well as the same variable fragments of the H chains (**Figure 6**).

### **5.2 Somatic hypermutation: mutations take place on the variable segments of L and H chains**

#### *5.2.1 Brief anatomo-histological overview of B-cells*

#### *5.2.1.1 Primary follicles and interplay between B-cells and FDCs*

Many resting B-cells, agglutinated around follicular dendritic cells (FDCs), harbor primary follicles. Thus, in the adult spleen, about 80% of B-cells are follicular B-cells.

A major role is attributed to FDCs, as prominent stromal cell constituents of B-cell follicles. These cells do not express major histocompatibility complex class II (MHC II) molecules nor do they have the capacity to phagocytose and process exogenous antigens for MHC I-restricted presentation [34]. Experiments using cryoimmunogold electron microscopy have demonstrated that the presence of MHC II molecules on their surface is passive and originate from microvesicles/ exosomes they are attached to [35]. Additionally, their ontogeny remains controversial. They are not derived from the bone marrow hematopoietic stem cell, but they could originate from local mesenchymal precursors in lymphoid organs [36]. Moreover, FDCs promote the survival and continuous recirculation of naive B-cells and allow for the attraction of activated B-cells, as well as the selective process for affinity maturation within the GC of lymphoid follicles during humoral adaptive/antibody-mediated immune response, allowing activated B-cells to significantly improve the affinity of their BCR. Thus, they have the ability to retain on their Fc receptor (FcR) antigens in native form combined with antibodies (immune complexes [ICs]), for long periods of time, ranging from months to years, thus making them accessible to the centrocytes (CC) that enter light zone (LZ) and result from the proliferation of blasts in the dark zone (DZ; this zone has been designed because it contains a high cell density) (**Figure 7**).

#### **Figure 7.**

*Schematic representation of the organization of the germinal center. The dark zone is occupied mainly by proliferating centroblasts. These are cells in which SHM would take place. After proliferation, B-cells, now called centrocytes, are in the adjacent LZ, which also contains Tfh cells and FDCs. Centrocytes that poorly link the antigen will die by apoptosis. Those that can bind the antigen and receive survival signals from the BCR and Tfh cells can either return to the dark zone for another cycle of proliferation, mutation, and selection or become memory B-cells or plasma cells that migrate to bone marrow to ensure prolonged antibody secretion [3]. BCR: B-cell antigen receptor complex, DZ: dark zone, FDC: follicular dendritic cell, LZ: light zone, M: mantle zone, SHM: somatic hypermutation, TBM: tingible body macrophages, Tfh: CD4+ T follicular helper cell.*

#### *5.2.1.2 Immune response, B-cell fates, and GC formation*

Most protein antigens induce T-dependent (TD) antigen humoral immune responses (responses to T-independent [TI] antigen are not evoked in this chapter). Such responses require cooperation between the antigen-specific B-cell and the T-cell carrying a specific TCR of the same antigen. Nevertheless, the two cell types have different localizations within ganglion, and the probability for a B-cell to meet a T-cell with the same antigen specificity is extremely low and is about 1 ⁄10 8 to 1 ⁄10 12, knowing that the proportion of naive B-cell and T-cell that are specific for a given antigen is about 1 ⁄10 4 to 1 ⁄10 6 [37].

The search for the antigen by the appropriate B-cells is done through an immutable path. The blood enables the naive B-cells, *i.e.*, the mature peripheral B-cells (known as follicular B-cells), to be transported to the lymph nodes where they enter through the high endothelial venules (HEV), migrate across the T-cell area, and spend about 24 hours in the follicles before exiting through the efferent lymph and returning to the circulation [38] (for review, see [39]).

When B-cells expressing antigen-specific BCR encounter the appropriate antigen (acquired from FDCs) in secondary lymphoid organs (SLOs), within lymph nodes for the antigen that is carried into them from the tissues, or within spleen for the antigen that reach it from the bloodstream, they increase the expression of the C-C chemokine receptor type 7 (CCR7) on their surface and migrate to the T/B border [38] (comment in [40]) in the spleen and in the interfollicular region in lymph nodes, after both T-cells and B-cells have been primed with antigen. The activated B-cells follow one of the following two fates to trigger a TD humoral immune response to protein antigen (for review, see [41–44]):

a.either they migrate to **extrafollicular foci (EF)** of SLOs, in the medullary cords of lymph nodes or in foci in the red pulp of the spleen [45], and

**35**

*Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation*

differentiate rapidly, as plasmablasts, in **short-lived antibody-secreting plasma cells (CLPCs)** [ 41], synthesizing IgM and IgG, which makes it possible to have rapidly circulating antigen-specific antibodies. Of note, the development of EFs in TD immune responses requires the help of CD4+

b.or they migrate to a primary **lymphoid follicle** (B-cell follicle) of the lymph nodes or spleen, where they undergo clonal expansion-a strong oligoclonal proliferation-to form, after a few days, the **GCs**, **then undergo somatic mutation to generate high-affinity memory B-cells and long-lived** 

**plasma cells (LLPCs)**. GC formation requires about 7 days during a primary immune response and about 36 h during a secondary response, which occurs following immunization and activation of memory B-cell with the same

proliferate, and undergo CSR. GCs persist until a few weeks. Of note, similar to naïve B-cells, memory B-cells can also be recruited into EF and give rise to immune responses that are associated with CSR but, at the most, only

TD antigen. The activated B-cells receive signals from the CD4<sup>+</sup>

*5.2.1.3 Immune responses to protein antigens and B-cell cooperation with CD4+*

Only CCs that express a high affinity receptor for epitopes of the antigen presented in its native form by the FDCs and that can capture it are selected efficiently. These selected CCs process the antigen and present antigen-derived peptides bound

cells in T-cell zones [46] (comment in [47]). The Tfh cells then give survival and differentiation signals to B-cells, which can then undergo CSR and mature either in

• *GCs*. The GCs consist essentially of blast cells that divide every 6 h, reaching a number of 60,000 blasts after about 60 h, thus considerably increasing the number of antigen-specific B-cells. They are polarized into two cellular areas:

i.*Centroblasts* (*DZ*), which no longer produce surface Igs because their genes

ii.*CC* (*LZ*), which correspond to smaller B-cells, expressing their new surface Ig, no longer proliferating, and are entangled in a large network of FDCs.

• *CCs*. The few CCs selected after contact with the antigen retained by the FDCs, allowing their affinity maturation, migrate to the apical part of the LZ, then undergo the CSR, and differentiate, as mentioned above, into memory B-cells or in LLPCs, through their cooperation with Tfh cells, which are present specifically within the LZ. DCs can also be selected for apoptosis elimination, if centroblast stage mutations that occur during the SHM process do not modify or even decrease the affinity of their BCR (see below Mechanisms of SHM process and Impacts of mutations on affinity of BCR

T follicular helper (Tfh) cells;

T-cells

Tfh cells,

Tfh cells, which have been shown to

T-cells during antigen priming by dendritic

*DOI: http://dx.doi.org/10.5772/intechopen.90637*

that share characteristics of CD4+

low-level SHM [45].

to MHC II molecules to antigen-specific CD4<sup>+</sup>

develop immediately from naive CD4+

LLPCs or in memory B-cells.

*5.2.1.4 Blast cell differentiation*

undergo SHM.

and secreted Ig).

*Tfh cells*

*Normal and Malignant B-cell*

**Figure 7.**

*5.2.1.2 Immune response, B-cell fates, and GC formation*

*zone, SHM: somatic hypermutation, TBM: tingible body macrophages, Tfh: CD4+*

returning to the circulation [38] (for review, see [39]).

response to protein antigen (for review, see [41–44]):

Most protein antigens induce T-dependent (TD) antigen humoral immune responses (responses to T-independent [TI] antigen are not evoked in this chapter). Such responses require cooperation between the antigen-specific B-cell and the T-cell carrying a specific TCR of the same antigen. Nevertheless, the two cell types have different localizations within ganglion, and the probability for a B-cell to meet a T-cell with the same antigen specificity is extremely low and is about 1 ⁄10 8 to 1 ⁄10 12, knowing that the proportion of naive B-cell and T-cell that are specific for a given antigen is about 1 ⁄10 4 to 1 ⁄10 6 [37]. The search for the antigen by the appropriate B-cells is done through an immutable path. The blood enables the naive B-cells, *i.e.*, the mature peripheral B-cells (known as follicular B-cells), to be transported to the lymph nodes where they enter through the high endothelial venules (HEV), migrate across the T-cell area, and spend about 24 hours in the follicles before exiting through the efferent lymph and

 *T follicular helper cell.*

*Schematic representation of the organization of the germinal center. The dark zone is occupied mainly by proliferating centroblasts. These are cells in which SHM would take place. After proliferation, B-cells, now called centrocytes, are in the adjacent LZ, which also contains Tfh cells and FDCs. Centrocytes that poorly link the antigen will die by apoptosis. Those that can bind the antigen and receive survival signals from the BCR and Tfh cells can either return to the dark zone for another cycle of proliferation, mutation, and selection or become memory B-cells or plasma cells that migrate to bone marrow to ensure prolonged antibody secretion [3]. BCR: B-cell antigen receptor complex, DZ: dark zone, FDC: follicular dendritic cell, LZ: light zone, M: mantle* 

When B-cells expressing antigen-specific BCR encounter the appropriate antigen (acquired from FDCs) in secondary lymphoid organs (SLOs), within lymph nodes for the antigen that is carried into them from the tissues, or within spleen for the antigen that reach it from the bloodstream, they increase the expression of the C-C chemokine receptor type 7 (CCR7) on their surface and migrate to the T/B border [38] (comment in [40]) in the spleen and in the interfollicular region in lymph nodes, after both T-cells and B-cells have been primed with antigen. The activated B-cells follow one of the following two fates to trigger a TD humoral immune

a.either they migrate to **extrafollicular foci (EF)** of SLOs, in the medullary cords of lymph nodes or in foci in the red pulp of the spleen [45], and

**34**

differentiate rapidly, as plasmablasts, in **short-lived antibody-secreting plasma cells (CLPCs)** [ 41], synthesizing IgM and IgG, which makes it possible to have rapidly circulating antigen-specific antibodies. Of note, the development of EFs in TD immune responses requires the help of CD4+ T-cells that share characteristics of CD4+ T follicular helper (Tfh) cells;

b.or they migrate to a primary **lymphoid follicle** (B-cell follicle) of the lymph nodes or spleen, where they undergo clonal expansion-a strong oligoclonal proliferation-to form, after a few days, the **GCs**, **then undergo somatic mutation to generate high-affinity memory B-cells and long-lived plasma cells (LLPCs)**. GC formation requires about 7 days during a primary immune response and about 36 h during a secondary response, which occurs following immunization and activation of memory B-cell with the same TD antigen. The activated B-cells receive signals from the CD4<sup>+</sup> Tfh cells, proliferate, and undergo CSR. GCs persist until a few weeks. Of note, similar to naïve B-cells, memory B-cells can also be recruited into EF and give rise to immune responses that are associated with CSR but, at the most, only low-level SHM [45].
