**3. The secondary antibody repertoire**

#### **3.1. Somatic hypermutation**

After the assembly of the V region of the heavy and light chain and cell surface expression of a functional BCR, naive B cells migrate to the secondary lymphatic organs, for example, to the lymph nodes. In the germinal center of the lymph node, the primary antibody repertoire is further diversified by introducing mutations in the V domains of the heavy and light chain mediated by the activation-induced cytidine deaminase (AID) [27–29]. This enzyme is only expressed and active in germinal center-activated mature B cells and is the key enzyme for the somatic hypermutation (SHM). The anatomical structure of the germinal center in the lymph node is divided macroscopically in two parts, the light zone and the dark zone. Somatic hypermutation mediated by AID activity takes place in the dark zone. Cells which produce a nonfunctional B-cell receptor (BCR) upon mutation are dying by apoptosis, whereby the B cells with a functional BCR will migrate into the light zone. In the light zone, positive selected B cells with a low-affinity B-cell receptor were stimulated for survival, proliferation, and reentry to the dark zone for a next round of affinity maturation. At this point, after several rounds of affinity maturation, B cells can leave the germinal center and differentiate into antibody producing plasma cells or B memory cells. B cells with very low affinity are suffering for survival signals, and before they can reenter the dark zone, they die by apoptosis [30].

On the other hand, B-cell clones with a BCR of high affinity toward the antigen receive growth signals, for example, from the follicular T helper cells, and are expended. This principle is called positive selection. Selected B cells which have undergone affinity maturation are showing more mutations in the critical regions for the antigen binding, namely, the CDRs. A mutation in the CDR, which produces an amino acid change, very likely alters the antigen affinity. B cells with sufficient affinity to the antigen which is presented by follicular dendritic cells (FDCs) in the light zone can capture it, process it, and present the antigen peptide via the major histocompatibility complex II (MHC II) to the T cells. Then, B-cell clones get survival and mitogenic signals through the T-cell receptor (TCR) recognition, CD40-CD40L interaction, and cytokine stimulation of T cells (**Figure 4**). As a consequence, B-cell receptors and CD40 cluster together and promote thereby positive selection signaling. Follicular dendritic cells present foreign antigens on their dendritic surface in form of iccosomes (immune complex-coated bodies) [33–35]. Iccosomes are antigen/antibody/complement complexes bound to Fc and complement receptors on FDCs. When B cells recognize antigens presented by iccosomes, they can take them up and process them for MHC II-mediated T-cell presentation. The efficiency and amount of iccosome uptake can also influence the fate of the B cell. B-cell clones with higher affinity for the antigen can capture more from the iccosome-presented antigen, which resulted in more representation of the processed antigen peptide on the B-cell surface, complexed in the MHC II molecule. Therefore, these clones get more surviving and prolifera-

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tion signals in the light zone from the recognizing follicular T helper cell (TFH).

noglobulin class-switching process by acting on the residues in switch regions.

usually only present in RNA molecules.

The process of somatic hypermutation (SHM) has not only a cellular dimension; it also has a molecular dimension, which can be characterized by the details of the mechanism of SHM and affinity maturation. The central enzyme in SHM is the activation-induced cytidine deaminase (AID). AID catalyzes the deamination of the DNA nucleotide cytosine to uracil, which is

The expression of AID is tightly restricted to germinal center B cells; this protects other cells from somatic hypermutation. Furthermore, it cannot act on predominantly doublestranded genomic DNA. To protect the majority of the genomic DNA from mutation, AID has developed a clever mechanism [36–38]. AID can act specifically only on single-stranded DNA molecules. The genomic DNA is released during transcription as a single strand by the RNA polymerase, which granted access of the AID for deamination. The immunoglobulin V region genes are actively transcribed in germinal center B cells, and somatic hypermutation can occur. Beside of the immunoglobulin V region, also some other transcribed genes can be affected by AID, fortunately by a lower frequency. AID has not only the function of somatic hypermutation by acting on the immunoglobulin V region loci; it can also activate the immu-

The deamination of cytidine to uracil by AID is the initiation step of SHM or class-switch recombination (CSR). Further mutation of the DNA around the initial deamination is executed by two different DNA repair pathways [39–41]. For example, the DNA mismatch repair process recognizes the wrong base pairing of uracil (U) to guanosine (G). Mismatch repair proteins MSH2 and MSH6 (mutS homolog 2/6) detect the wrong U/G base pairing, which then recruits DNA nucleases to remove the uracil and the adjacent nucleotides. The following

During the migration, B cells change their expression pattern depending on their location in the light or dark zone of the germinal center. The C-X-C chemokine receptor type 4 (CXCR4) is one of the classical markers, which changes the expression level in response of the migration to the other germinal zone. In the dark zone, the B-cell CXCR4 expression is strong and is reduced in the light zone. CXCL12 is a ligand of CXCR4 and expressed on the cell surface of reticular cells in the dark zone. The CXCL12/CXCR4 signaling of B cells in the dark zone is regarded as a homing signal to keep B cells in the dark zone, if CXCR4 expression is high [31, 32]. CXCR4 deficiency in germinal B cells restricted the B cells to the light zone, but the deletion is not sufficient alone for functional transition of dark zone B cells (centroblasts) to light zone B cells (centrocytes) [31].

The process of affinity maturation is also known as the cyclic reentry model (**Figure 4**). It starts with the introduction of mutations in the V region initiated by AID. The induced mutation rate is about one nucleotide per 10,000 nucleotides after each cell cycle division [4]. This is much higher than the normal mutation rate of about 1010 mutations per cell cycle. Only a slight change in one or a few amino acids in the CDRs or frameworks of the V region can change dramatically the antigen affinity and specificity. Mutations can have detrimental effects and produce lower affinity B-cell receptors, especially when the complementarity-determining regions (CDRs) are affected with mutations leading to antibodies which cannot anymore recognize the antigen-binding site. At this stage, negative selection of B cells occurs. When B cells are affected by negative changes and were not able to produce a functional receptor presented on the B cell surface or lost antigen affinity, cell death by apoptosis is initiated. Subsequently, phagocytic clearance of apoptotic B cells is executed by tingible body macrophages (TBM).

On the other hand, B-cell clones with a BCR of high affinity toward the antigen receive growth signals, for example, from the follicular T helper cells, and are expended. This principle is called positive selection. Selected B cells which have undergone affinity maturation are showing more mutations in the critical regions for the antigen binding, namely, the CDRs. A mutation in the CDR, which produces an amino acid change, very likely alters the antigen affinity.

**3. The secondary antibody repertoire**

After the assembly of the V region of the heavy and light chain and cell surface expression of a functional BCR, naive B cells migrate to the secondary lymphatic organs, for example, to the lymph nodes. In the germinal center of the lymph node, the primary antibody repertoire is further diversified by introducing mutations in the V domains of the heavy and light chain mediated by the activation-induced cytidine deaminase (AID) [27–29]. This enzyme is only expressed and active in germinal center-activated mature B cells and is the key enzyme for the somatic hypermutation (SHM). The anatomical structure of the germinal center in the lymph node is divided macroscopically in two parts, the light zone and the dark zone. Somatic hypermutation mediated by AID activity takes place in the dark zone. Cells which produce a nonfunctional B-cell receptor (BCR) upon mutation are dying by apoptosis, whereby the B cells with a functional BCR will migrate into the light zone. In the light zone, positive selected B cells with a low-affinity B-cell receptor were stimulated for survival, proliferation, and reentry to the dark zone for a next round of affinity maturation. At this point, after several rounds of affinity maturation, B cells can leave the germinal center and differentiate into antibody producing plasma cells or B memory cells. B cells with very low affinity are suffering for survival signals, and before they can reenter the dark zone, they die

During the migration, B cells change their expression pattern depending on their location in the light or dark zone of the germinal center. The C-X-C chemokine receptor type 4 (CXCR4) is one of the classical markers, which changes the expression level in response of the migration to the other germinal zone. In the dark zone, the B-cell CXCR4 expression is strong and is reduced in the light zone. CXCL12 is a ligand of CXCR4 and expressed on the cell surface of reticular cells in the dark zone. The CXCL12/CXCR4 signaling of B cells in the dark zone is regarded as a homing signal to keep B cells in the dark zone, if CXCR4 expression is high [31, 32]. CXCR4 deficiency in germinal B cells restricted the B cells to the light zone, but the deletion is not sufficient alone for functional transition of dark zone B cells (centroblasts) to light zone

The process of affinity maturation is also known as the cyclic reentry model (**Figure 4**). It starts with the introduction of mutations in the V region initiated by AID. The induced mutation rate is about one nucleotide per 10,000 nucleotides after each cell cycle division [4]. This is much higher than the normal mutation rate of about 1010 mutations per cell cycle. Only a slight change in one or a few amino acids in the CDRs or frameworks of the V region can change dramatically the antigen affinity and specificity. Mutations can have detrimental effects and produce lower affinity B-cell receptors, especially when the complementarity-determining regions (CDRs) are affected with mutations leading to antibodies which cannot anymore recognize the antigen-binding site. At this stage, negative selection of B cells occurs. When B cells are affected by negative changes and were not able to produce a functional receptor presented on the B cell surface or lost antigen affinity, cell death by apoptosis is initiated. Subsequently, phagocytic clearance of apoptotic B cells is executed by tingible body macrophages (TBM).

**3.1. Somatic hypermutation**

10 Antibody Engineering

by apoptosis [30].

B cells (centrocytes) [31].

B cells with sufficient affinity to the antigen which is presented by follicular dendritic cells (FDCs) in the light zone can capture it, process it, and present the antigen peptide via the major histocompatibility complex II (MHC II) to the T cells. Then, B-cell clones get survival and mitogenic signals through the T-cell receptor (TCR) recognition, CD40-CD40L interaction, and cytokine stimulation of T cells (**Figure 4**). As a consequence, B-cell receptors and CD40 cluster together and promote thereby positive selection signaling. Follicular dendritic cells present foreign antigens on their dendritic surface in form of iccosomes (immune complex-coated bodies) [33–35]. Iccosomes are antigen/antibody/complement complexes bound to Fc and complement receptors on FDCs. When B cells recognize antigens presented by iccosomes, they can take them up and process them for MHC II-mediated T-cell presentation. The efficiency and amount of iccosome uptake can also influence the fate of the B cell. B-cell clones with higher affinity for the antigen can capture more from the iccosome-presented antigen, which resulted in more representation of the processed antigen peptide on the B-cell surface, complexed in the MHC II molecule. Therefore, these clones get more surviving and proliferation signals in the light zone from the recognizing follicular T helper cell (TFH).

The process of somatic hypermutation (SHM) has not only a cellular dimension; it also has a molecular dimension, which can be characterized by the details of the mechanism of SHM and affinity maturation. The central enzyme in SHM is the activation-induced cytidine deaminase (AID). AID catalyzes the deamination of the DNA nucleotide cytosine to uracil, which is usually only present in RNA molecules.

The expression of AID is tightly restricted to germinal center B cells; this protects other cells from somatic hypermutation. Furthermore, it cannot act on predominantly doublestranded genomic DNA. To protect the majority of the genomic DNA from mutation, AID has developed a clever mechanism [36–38]. AID can act specifically only on single-stranded DNA molecules. The genomic DNA is released during transcription as a single strand by the RNA polymerase, which granted access of the AID for deamination. The immunoglobulin V region genes are actively transcribed in germinal center B cells, and somatic hypermutation can occur. Beside of the immunoglobulin V region, also some other transcribed genes can be affected by AID, fortunately by a lower frequency. AID has not only the function of somatic hypermutation by acting on the immunoglobulin V region loci; it can also activate the immunoglobulin class-switching process by acting on the residues in switch regions.

The deamination of cytidine to uracil by AID is the initiation step of SHM or class-switch recombination (CSR). Further mutation of the DNA around the initial deamination is executed by two different DNA repair pathways [39–41]. For example, the DNA mismatch repair process recognizes the wrong base pairing of uracil (U) to guanosine (G). Mismatch repair proteins MSH2 and MSH6 (mutS homolog 2/6) detect the wrong U/G base pairing, which then recruits DNA nucleases to remove the uracil and the adjacent nucleotides. The following DNA polymerase Polη has no exonuclease activity and is error prone in B cells. The polymerase preferentially misincorporates thymidine (T), regardless of the template sequence, which leads to a preference of adenosine (A)-thymidine (T) mutations at the original targeted cytosine and the adjacent nucleotides by the mismatch repair pathway.

Unlike in the parallel expression of IgM and IgD, the class switch is a chromosomal DNA rearrangement, leading to only one ultimate antibody isotype in the affected B cell. The process is guided by conserved switch region (S) upstream of the heavy chain constant genes, coding for the respective constant domains. The switch regions are repetitive stretches of DNA placed in introns upstream to the C region genes [28, 42]. The initial activation of CSR is done by the enzyme activation-induced cytidine deaminase (AID), which has also an essential role in the somatic hypermutation process. This produces a single-stranded DNA break (nick) at two switch regions, and the DNA between both switch sites were irreversible excised. The removal includes always the μ and δ chain constant region. Both DNA strands were brought together by the non-homologous end joining (NHEJ) mechanism; this rearranges the variable region with the constant region of the chosen immunoglobulin isotype. The decision which isotype will be produced is influenced by different cytokines, secreted

CSR is induced by the enzyme activation-induced cytidine deaminase (AID) acting on the switch regions (S) of the respective constant region gene. The non-homologous end joining

constant region gene, which is next to the V region, is then expressed together with the V(D)

[1] Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition.

[2] Stein LD. Human genome: End of the beginning. Nature. 2004;**431**(7011):915-916. DOI:

[3] Pertea M, Salzberg SL. Between a chicken and a grape: Estimating the number of human

[4] Murphy K et al. Janeway's Immunobiology. 8th ed. Vol. xix. New York: Garland Science;

[5] IMGT.The international ImMunoGeneTics information system: IMGT Repertoire (IG and TR) – Table Functional IG genes. Available from: http://www.imgt.org/IMGTrepertoire/

LocusGenes/genetable/human/geneNumber.html [Accessed: February 19, 2017]

International Immunology. 2009;**21**(4):317-337. DOI: 10.1093/intimm/dxp017

genes. Genome Biology. 2010;**11**(5):206. DOI: 10.1186/gb-2010-11-5-206

2b). The

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(NHEJ) machinery joins the chosen constant gene segment to the V region (here C<sup>y</sup>

Address all correspondence to: olli.backhaus@googlemail.com

Institute of Pathology, University Clinic of RWTH Aachen, Aachen, Germany

by T cells [43, 44].

J gene sequence.

**Author details**

Oliver Backhaus

**References**

10.1038/431915a

2012. 868 p

Alternatively, in the base excision repair pathway, the uracil DNA glycosylase (UNG) cleaves the uracil nucleobase from the uridine and leaves an abasic site in the DNA strand. During the following DNA replication, a random DNA base will be inserted in the opposite DNA strand of the abasic nucleotide. This is mediated by an error-prone DNA polymerase used in translesion DNA synthesis for damaged DNA caused by UV radiation.

As mentioned before, AID can also initiate class-switch recombination, by acting of apurinic/ apyrimidinic endonuclease 1 (APE1) upon UNG-mediated introduction of an abasic nucleotide in the switch region. APE1 cleaves the DNA strand at the abasic site and produces a single-strand nick. In the switch regions, upstream of the constant region genes, the DNA nick is further cleaved which produces a double-strand break (DSB). This leads to a joint of another constant region gene to the V region, produced by the double-strand break repair machinery.

#### **3.2. Class-switch recombination**

In naive B cells, which had already rearranged their V region by somatic DNA recombination, two antibody isotypes are co-expressed at the same time. The V region and the μ chain (IgM) together with the δ chain (IgD) were transcribed on the same RNA transcript. By alternative splicing, either the μ chain or the δ chain is chosen, which produces two different messenger RNAs (**Figure 1**). Upon antigen contact and B-cell activation, B cells switch their antibody isotypes from IgM/IgD to IgG, IgA, or IgE. This is achieved by a process called class-switch recombination (CSR) or isotype switching. The antibody isotype is changed by an exchange of the constant region of the heavy chain locus. Only the constant region is replaced by CSR, which means the V region stays the same, but class switch confers the antibody the ability to interact with different effector molecules by their fragment crystallizable (Fc) region (**Figure 5**).

**Figure 5.** Mechanism of switch recombination from IgM to IgG2b.

Unlike in the parallel expression of IgM and IgD, the class switch is a chromosomal DNA rearrangement, leading to only one ultimate antibody isotype in the affected B cell. The process is guided by conserved switch region (S) upstream of the heavy chain constant genes, coding for the respective constant domains. The switch regions are repetitive stretches of DNA placed in introns upstream to the C region genes [28, 42]. The initial activation of CSR is done by the enzyme activation-induced cytidine deaminase (AID), which has also an essential role in the somatic hypermutation process. This produces a single-stranded DNA break (nick) at two switch regions, and the DNA between both switch sites were irreversible excised. The removal includes always the μ and δ chain constant region. Both DNA strands were brought together by the non-homologous end joining (NHEJ) mechanism; this rearranges the variable region with the constant region of the chosen immunoglobulin isotype. The decision which isotype will be produced is influenced by different cytokines, secreted by T cells [43, 44].

CSR is induced by the enzyme activation-induced cytidine deaminase (AID) acting on the switch regions (S) of the respective constant region gene. The non-homologous end joining (NHEJ) machinery joins the chosen constant gene segment to the V region (here C<sup>y</sup> 2b). The constant region gene, which is next to the V region, is then expressed together with the V(D) J gene sequence.
