1. Kindt • Goldsby • Osborne
Kuby IMMUNOLOGY
Sixth Edition
Chapter 5

2. FIGURE 5-1 B-Cell Development
The events that occur during maturation in the bone marrow do not require antigen, whereas activation and differentiation of mature B cells in peripheral lymphoid organs require antigen.
The labels mIgM and mIgD refer to membrane-associated Igs. IgG, IgA, and IgE are secreted immunoglobulins.

3. Diversing a Genetic Model Compatible with Ig Structure
The vast diversity of antibody specificities
The presence in Ig heavy and light chains of a variable region at the amino terminal end and a constant region at the carboxyl-terminal end.
The existence of isotypes with the same antigenic specificity, which result from the association of a given variable region with different heavy-chain constant regions.

4. Genetic Model Compatible with Ig Structure
Germ line theory : Genome contributed by germ line cells, egg and sperm, contains a large repertoire of Ig genes =>
Somatic variation theory: Genome contains a relatively small number of Ig genes, from which a large number of Abs are generated in the somatic cells by mutation or recombination. =>
Identical variable region sequences were associated with both r and m heavy chain constant regions
이들 두 제안은 항체 구조적인 특성 설명에는 부적절
Two gene model (Dreyer and Bennett): Two separate genes encode a single Ig H or L chain, one gene for the V region and the other for the C region. Two genes must come together at the DNA level to form a continuous message that can be transcribed and translated into single Ig H or L chain. Thounds of V region genes are located in the germ line, whereas only single copies of C-region class subclass genes need exist.
이론적인 가설이 당시 기술이 따르지 못해 증명하지를 못하였다.
이 가설을 Tonegawa가 증명함으로 항체의 다양성을 이해

5. 1976년, Tonegawa의 Southern blotting 실험 :
The first direct evidence that separate genes encode the V and C regions of Igs and that the genes are rearranged in the course of B-cell differentiation.
 배아세포 (embryonic cell) 의 항체 유전자와 adult myeloma B cell의 항체유전자의 구조가 다름을 증명
배아세포와 B cell에서 얻은 DNA를 제한 효소로 절단한 후, agarose gel을 이용한 전기영동 하고, probe를 이용하여 항체 유전자가 어떤 크기의 조각과 결합하는 지를 조사
두 세포에서 같은 probe에 hybridize하는 DNA의 조각의 크기가 다름을 확인 (restriction fragment length polymorphism, RFLP)
전구세포에서 B cell로 성숙되는 동안 항체유전자의 구조에 변화
B cell은 다양한 종류의 항체 유전자를 보유
    : 전구세포단계의 항체유전자 즉 재배열되기 전의 항체 유전자 (germ line DNA)
    : B 림프구에 존재하는 완성된 항체유전자 (재배열된 유전자)
    : 항체 유전자의 전사된 mRNA 유전자 (cDNA 유전자)
항체 유전자는 V 지역과 C 지역을 구성하는 유전자가 따로 여러 개 존재

6. FIGURE 5-2 Experimental basis for diagnosis of rearrangement at an immunoglobulin locus.
The number and size of restriction fragments generated by the treatment of DNA with a restriction enzyme are determined by the sequence of the DNA. The digestion of rearranged DNA with a restriction enzyme(RE) yields a pattern of restriction fragments that differ from those obtained by digestion of an unrearranged locus with the same RE. Typically, the fragments are analyzed by the technique of Southern blotting. In this example, a probe that includes a J gene segment is used to identify RE digestion fragments that include all or portions of this segment. As shown, rearrangement results in the deletion of a segment of germ-line DNA and the loss of the restriction sites that it includes. It also results in the joining of gene segments, in this case a V and a J segment, that are separated in the germ line. Consequently, fragments dependent on the presence of this segment for their generation are absent from the restriction-enzyme digest of DNA from the rearranged locus. Furthermore, rearranged DNA gives rise to novel fragments that are absent from digests of DNA in the germ-line configuration. This can be useful because both B cells and non-B cells have two immunoglobulin loci. One of these is rearranged, and the other is not. Consequently, unless a genetic accident has resulted in the loss of the germ-line locus, digestion of DNA from a myeloma or normal B-cell clone will produce a pattern of restriction that includes all of those in a germ-line digest plus any novel fragments that are generated from the change in DNA sequence that accompanies rearrangement. Note that only one of the several J gene segments present is shown.

8. Multigene Organization of Ig Genes
Cloning and sequencing of the light- and heavy-chain DNA was accomplished.
Each multigene family has distinct features
The k and λ Light chain families contain V, J, and C gene segments
Heavy chain family contains V, D, J, and C gene segments.
L---V------D-----J C

9. FIGURE 5-3 Organization of Immunoglobulin Germ-Line Gene Segments in the Mouse : (a) λ Light Chain, (b) κ Light Chain, and (c) Heavy Chain
The λ and κ light chains are encoded by V, J and C gene segments. The heavy chain is encoded by V, D, J and C gene segments. The distances in (kb) separating the various gene segments in mouse germ-line DNA are shown below each chain diagram.

10. In this example, rearrangement joins 𝑉 κ 23 and 𝐽 κ 4.
FIGURE 5-4 Kappa light-chain gene rearrangement and RNA processing events required to generate a κ light-chain protein
In this example, rearrangement joins 𝑉 κ 23 and 𝐽 κ 4.

11. Heavy-chain gene rearrangement and RNA processing events required to generate finished μ or δ heavy-chain protein.
Two DNA joinings are necessary to generate a functional heavy-chain gene : a 𝐷 H to 𝐽 H joining and a 𝑉 H to 𝐷 H 𝐽 H joining. In this example, 𝑉 H 21, 𝐷 H 7, and are 𝐽 H joined. Expression of functional heavy-chain genes, although generally similar to expression of light-chain genes, involves differential RNA processing, which generates several different products, including μ or δ heavy chains. Each C gene is drawn as a single coding sequence ; in reality, each is organized as a series of exons and introns.

12. FIGURE Two conserved sequences in light-chain and heavy-chain DNA function as recombination signal sequences(RSSs).
(a)
(b)
(a) Both signal sequences consist of a conserved palindromic heptamer and conserved AT-rich nonamer ; these are separated by nonconserved spacers of 12 or 23 base pairs.
(b) The two types of RSS-designated one-turn RSS and two-turn RSS-have characteristic locations within λ-chain, κ-chain, and heavy-chain germ-line DNA. During DNA rearrangement, gene segments adjacent to the one-turn RSS can join only with segments adjacent to the two-turn RSS.

13. Model depicting the general process of recombination of immunoglobulin gene segments is illustrated with 𝑉 κ and 𝐽 κ .
Deletional joining occurs when the gene segments to be joined have the same transcriptional orientation(indicated by horizontal blue arrows). This process yields two products : a rearranged VJ unit that includes the coding joint and a circular excision product consisting of the recombination signal sequences(RSSs), signal joint, and intervening DNA.
Inversional joining occurs when the gene segments have opposite transcriptional orientations. In this case, the RSSs, signal joint, and intervening DNA are retained, and the orientation of one of the joined segments is inverted.
In both types of recombination, a few nucleotides may be deleted from or added to the cut ends of the coding sequences before they are rejoined.

14. Circular DNA isolated from thymocytes in which the DNA encoding the chains of the T-cell receptor(TCR) undergoes rearrangement in a process like that involving the immunoglobulin genes.
Isolation of this circular excision product is direct evidence for the mechanism of deletional joining shown in Figure 5-7

15. FIGURE 5-9 Junctional flexibility in the joining of immunoglobulin gene segments is illustrated with 𝑉 κ and 𝐽 κ .
In-phase joining(arrows 1, 2 and 3) generates a productive rearrangement, which can be translated into protein. Out-of-phase joining(arrows 4 and 5) leads to a nonproductive rearrangement that contains stop codons and is not translated into protein.

16. Allelic Exclusion ensures a single antigenic specificity
항체 유전자의 재배열은 항체의 대립유전자 (alleles) 모두에서 동시에 나타나지 않는다 (allelic exclusion)
만일 B cell 의 한 allele가 nonproductive 한 rearrangement 를 했다면 다른 allele에서 rearrangement 가 일어난다
 항체 유전자의 한 allele의  heavy chain gene에서 재배열이 제대로 일어나 정상적인 heavy chain 단백질이 만들어지면, 다른 allele의  heavy chain gene에서 재배열은 일어나지 않는다
light chain gene의 재배열에서도 같은 현상이 나타난다
그 결과 하나의 B cell에는 한 가지의 heavy chain 단백질과 한가지의 light chain 단백질만이 만들어진다
재배열이 잘못되어 발현이 안되는 B cell은 apoptosis 기작에 의해 사멸
 하나의 B cell에서는 한 가지의 항체만 생산

17. FIGURE 5-10 Because of allelic exclusion, the immunoglobulin heavy-and light-chain genes of only one parental chromosome are expressed per cell.
This process ensures that B cells possess a single antigenic specificity. The allele selected for rearrangement is chosen randomly. Thus, the expressed immunoglobulin may contain one maternal and one paternal chain or both chains may derive from only one parent. Only B cells and T cells exhibit allelic exclusion. Asterisks(*) indicate the expressed alleles.

18. FIGURE 5-11 Model to account for allelic exclusion.
Heavy-chain genes rearrange first, and once a productive heavy-chain gene rearrangement occurs, the μ protein product prevents rearrangement of the other heavy-chain allele and initiates light-chain gene rearrangement. In the mouse, rearrangement of κ light-chain genes precedes rearrangement of the λ genes, as shown here. In humans, either κ or λ rearrangement can proceed once a productive heavy-chain rearrangement has occurred. Formation of a complete immunoglobulin inhibits further light-chain gene rearrangement. If a nonproductive rearrangement occurs for one allele, then the cell attempts rearrangement of the other allele.

19. Generation of Antibody Diversity
항체의 다양한 특이성은 몇 개 안 되는 항체 유전자의 효율적인 활용으로 가능
Germ-line 상태에서 유전자조각을 여러 개 가지고 있다 (multiple germ-line gene segments)
 VDJ 유전자 조각이 다양하게 재배열되어 조합 (combinatorial diversity)  
재조합 시 합쳐지는 부위가 다양하다 (junctional flexibility)   
합쳐질 때 새로운 염기들이 첨가된다 (P/N nucleotide addition)
체세포 돌연변이에 의하여 V 지역의 염기배열이 바뀐다 (somatic hypermutation)
 heavy chain과 light chain이 서로 임의로 결합한다 (heavy and light chain combination)

21. FIGURE 5-12 Experimental evidence for junctional flexibility in immunoglobulin-gene rearrangement.
The nucleotide sequences flanking the coding joints between 𝑉 K 21 and 𝐽 K 1 and the corresponding signal joint sequences were determined in four pre-B cell lines. The sequence constancy in the signal joints contrasts with the sequence variability in the coding joints. Pink and yellow shading indicate nucleotides derived from 𝑉 K 21 and 𝐽 K 1, respectively, and purple and orange shading indicate nucleotides from the two RSSs.

22. FIGURE 5-13 P-nucleotide and N-nucleotide addition during joining.
If cleavage of the hairpin intermediate yields a double-stranded end on the coding sequence, then P-nucleotide addition does not occur. In many cases, however, cleavage yields a single-stranded end. During subsequent repair, complementary nucleotides are added, called P-nucleotide, to produce palindromic sequences(indicated by brackets). In this example, four extra base pairs(blue) are present in the coding joint as the result of P-nucleotide addition.
Besides P-nucleotide addition, addition of random N-nucleotides(light red) by a terminal deoxynucleotidyl transferase(TdT) can occur during joining of heavy-chain coding sequences.

23. Somatic Hypermutation adds Diversity in Already-rearranged Gene Segments
성숙된 B cell은 항원과 반응하게되면 항체를 생산하게 되며, 항원자극이 되풀이되면 될수록, 만들어지는 항체의 항원 친화력은 이전 보다 점점 좋아지게 된다  
친화력의 성숙 (affinity maturation)
항체의 친화력의 성숙은 항원에 의하여 활성화된 B cell이 증식 분화하는 동안 항체의 H chain과 L chain 유전자의 V 지역에 돌연변이가 유도되어 나타난다
     : 체세포 돌연변이 (somatic mutation)
     : 항체의 V지역 중 특히 hypervariable region (CDR)에 집중
     : 친화력이 높은 항체들은 항원에 의하여 자연선택
항원 침입이 잦으면 잦을수록 점점 더 좋은 항체들이 만들어져, 자주 침입하는 항원에 대해 보다 효과적인 방어가 가능
10-3 per base pair per generation

24. FIGURE 5-14 Experimental evidence for somatic mutation in variable regions of immunoglobulin genes.
The diagram compares the mRNA sequences of heavy chains and of light chains from hybridomas specific for the phOx hapten. The horizontal solid lines represent the germ-line 𝑉 H and 𝑉 K Ox-1 sequences ; dashed lines represent sequences derived from other germ-line genes. Blue shading shows the areas where mutations clustered ; the blue circles with vertical lines indicate locations of mutations that encode a different amino acid than the germ-line sequence. These data show that the frequency of mutation (1) increases in the course of the primary response(day 7 vs. day 14) and (2) is higher after secondary and tertiary immunizations than after primary immunization. Moreover, the dissociation constant ( 𝐾 d ) of the anti-phOx antibodies decreases during the transition from the primary to tertiary response, indicating an increase in the overall affinity of the antibody. Note also that most of the mutations are clustered within CDR1 and CDR2 of both the heavy and the light chains.

25. A final source of diversity is combinatorial assciation of heavy and light chains
In human, there is the potential to generate 6624 heavy chain genes and 375 light genes as a result of variable region gene rearrangements.
H and L combinations is 2,484,000.

26. FIGURE 5-15 Immunoglobulin diversification occurs by gene conversion in chickens.
In the chicken germ line, the single functional 𝑉 H and 𝑉 λ immunoglobulin genes are preceded by many pseudogenes.
Rearrangement creates a single functional rearranged V-(D)-J. Gene conversion introduces diversity into the V segments of rearranged V-(D)-J genes using upstream V pseudogenes as a template.

27. Class Switching among Constant-Region Genes
항원이 최초로 숙주에게 들어왔을 때에는 IgM 급의 항체가 주로 만들어지나, 같은 항원이 다시 들어왔을 때에는 다른 급, 주로 IgG급의 항체가 만들어진다 (항체의 급의 전환)
heavy chain의 C 지역이 바뀌어 나타나는 현상
heavy chain의 C지역의 전환은 항체 유전자의 재조합 (DNA recombination)을 통하여 이루어지나 아직 mechanism은 잘 규명되어있지 않다.
Switch region: δ chain의 C 지역유전자를 제외한 모든 C 지역 유전자의 5' (2-3 kb upstram )끝에 있는 보존되어 있는 염기배열 (conserved sequence, GAGCT and TGGG)에 의한 재조합
Recombinase and switch factor (IL-4)
항체 유전자의 급의 전환에는 IL-4와 같은 helper T cell이 만들어낸 cytokine이 관여하는 것으로 알려져 있다. 
 Overall, class switching depends on the interplay of
Four elements: switch region,
switch recombinase,
cytokine signals (IL-4)
activation-induced cytidine deaminase (AID)

28. FIGURE 5-16 Proposed mechanism for class switching induced by interleukin-4 in rearranged immunoglobulin heavy-chain genes.
A switch site is located upstream from each 𝐶 H segment except 𝐶 δ . Identification of the indicated circular excision products containing portions of the switch sites suggested that IL-4 induces sequential class switching from 𝐶 μ to 𝐶 γ 1 to 𝐶 ε .

29. FIGURE 5-17 Experimental demonstration of the role of the enzyme AID in class switching and somatic hypermutation.
(a)
(b)
AID-expressing (+/-) and AID knockout (-/-) mice were immunized twice with a hapten-carrier conjugate and the antihapten antibody responses measured and plotted in arbitrary units. IgM responses were detected in both types of mice. Production of IgG, which requires class switching, occurred only in AID-expressing (+/-) mice.
Messenger RNA encoding the variable regions of antigen-reactive antibodies in immunized AID-expressing and AID knockout mice was sequenced and the position and frequency of mutations plotted. Many mutations are seen in the AID-expressing mice ; only background levels of mutation are seen in the AID knockout mice.

30. Expression of membrane or secreted Ig
분비된 항체(sIg)와 세포막과 결합된 항체(mIg)도 항원결합부위는 서로 완전히 같기 때문에 항원 특이성에 차이가 없다
     : 세포막에 결합되어 있던 항체단백질이 단백질분해효소에 의하여 절단되어 sIg가 만들어지는 것도 아니라 mIg 와 sIg형의 항체는 각각의 mRNA로부터 번역되어 만들어진다.
primary transcript를 조작하는 방법으로 분비형과 새포막형의 항체를 만든다 (alternative splicing)
     : heavy chain 유전자를 δ chain의 C 지역 유전자 뒤까지 전사하여 Cμ와 Cδ chain 유전자들이 모두 포함된 일차 전사체 RNA를 얻는다
     : 이 primary transcript 에 있는 Cμ 전사체의 2개의 poly A site와 alternative splicing을 이용하여 IgM의 memb.과 secreted 형 생산
     : 이들 mRNA를 번역하여 각각의 분비형과 세포막형의 heavy chain 단백질 생산
‧ mRNA 모두 기존의 재배열된 VDJ 유전자의 구조가 변하지 않기 때문에 생성된 항체의 항원 특이성은 변하지 않는다.

31. (a)
(b)
FIGURE 5-18 Expression of secreted and membrane forms of the heavy chain by alternative RNA processing.
Amino acid sequence of the carboxyl-terminal end of secreted and membrane μ heavy chains. Residues are indicated by the single-letter amino acidcode. Hydrophilic and hydrophobic residues and regions are indicated by purple and orange, respectively, and charged amino acids are indicated with a + or - . The white regions of the sequences are identical in both forms.
Structure of the primary transcript of a rearranged heavy-chain gene showing the 𝐶 μ exons and poly-A sites. Polyadenylation of the primary transcript at either site 1 or site 2 and subsequent splicing(indicated by V-shaped lines) generates mRNAs encoding either secreted or membrane μ chains.

32. (a)
(b)
(c)
FIGURE 5-19 Expression of membrane forms of μ and δ heavy chains by alternative RNA processing.
Structure of rearranged heavy-chain gene showing 𝐶 μ and 𝐶 δ exons and poly-A sites.
Structure of μ 𝑚 transcript and μ 𝑚 mRNA resulting from polyadenylation at site 2 and splicing.
Structure of δ 𝑚 transcript and δ 𝑚 mRNA resulting from polyadenylation at site 4 and splicing. Both processing pathways can proceed in any given B cell.

33. Synthesis, Assembly, and Secretion of Igs
전사된 항체의 heavy chain과 light chain의 mRNA는 모두 소포체 (endoplasmic reticulum, ER)과 결합되어 있는 polyribosome에서 translation (synthesis)
     : 5‘쪽에 leader sequence, signal peptide를 coding 하는 L 염기배열이 있어서, translation 과 동시에 만들어지는 polypeptide chains들은 ER의 막에 결합, ER 안쪽으로 이동
     : ER안쪽으로 이동하면서 leader peptide는 잘려져 나가고, ER이나 Golgi body에서 heavy chain과 light chain의 assembly가 일어나 완전한 Ig 이 만들어진다
     : secretory vesicle을 통하여 세포막 쪽으로 이동하고, 세포막과 vesicle의 융합을 통하여 세포 밖으로 secretion

34. FIGURE 5-20 Synthesis, assembly, and secretion of the immunoglobulin molecule.
The heavy and light chains are synthesized on separate polyribosomes(polysomes). The assembly of the chains, the formation of intrachain and interchain disulfide linkages, and the addition of carbohydrate all take place in the rough endoplasmic reticulum(RER). Vesicular transport brings the Ig to the Golgi, which it transits, departing in vesicles that fuse with the cell membrane. The main figure depicts the assembly of a secreted antibody. The inset depicts a membrane-bound antibody, which contains the carboxyl-terminal transmembrane segment. This form becomes anchored in the membrane of secretory vesicles and is retained in the cell membrane when the vesicles fuse with the cell membrane.

35. FIGURE 5-21 Quality control during antibody synthesis.
Ig molecules that fail to fold or assemble properly remain bound to the chaperone protein BiP. Interaction with BiP and other factors causes the malformed antibody to be disassembled, exported form the ER, marked for degradation by conjugation with ubiquitin, and degraded by the proteosome.

36. FIGURE Location of promoters(dark red) and enhancers(green) in mouse heavy-chain, κ light-chain, and λ light-chain germ-line DNA.
Variable-region DNA rearrangement moves an enhancer close enough to a promoter that the enhancer can activate transcription from the promoter. The promoters that precede the DH cluster, a number of the C genes, and the 𝐽 λ cluster are omitted from this diagram for clarity.

37. Antibody Genes and Antibody Engineering
항체 단백질을 조작하는 항체 공학 : 단클론 항체의 생산과 변형
사람 항체 제조의 필요성 (human antibody)
     : 단클론항체는 생쥐의 항체이어서, 인간에게 임상적으로 투여하게 되면, 이들 항체들이 사람에서 항원으로 인식되어 투여한 항체에 대하여 면역반응이 나타나게되며, 그 결과 혈액내 단클론 항체를 빠르게 제거시키거나 allegic response를 일으켜 주사 받은 사람을 괴롭히거나 위험하게 만들 수도 있다 . 이를 해결하는 방법은 사람 항체 사용
항체공학 (antibody engineering)을 이용한 humanized antibody 의 제조
     : 생쥐 단클론항체의 V지역 유전자를 사람의 IgG 항체의 C 지역에 결합   - 잡종항체 (chimeric antibody)
     : chimeric antibody의 V 지역의 아미노산 배열을 CDR 지역만을 제외하고 모두 사람의 V지역 아미노산 배열로 바꾸어 주어 보다 사람의 항체에 가까운 항체 제조

39. FIGURE 5-23 Antibodies engineered by recombinant DNA technology.
(a) Chimeric mouse-human monoclonal antibody containing the 𝑉 H and 𝑉 L domains of a mouse monoclonal antibody(blue) and 𝐶 L and 𝐶 H domains of a human monoclonal antibody(gray).
(b) A humanized monoclonal antibody containing only the CDRs of a mouse monoclonal antibody(blue bands) grafted into the framework regions of a human monoclonal antibody.
(c) A chimeric monoclonal antibody in which the terminal Fc domain is replaced by toxin chains(white).
(d) A heteroconjugate in which one half of the mouse antibody molecule is specific for a tumor antigen and the other half is specific for the CD3/T-cell receptor complex.

40. FIGURE 5-24 Human antibody from mice bearing a human artificial chromosome(HAC) that includes entire human heavy-and light-chain loci.
A human artificial chromosome bearing the entire unrearranged human heavy-chain and λ light-chain loci was introduced into mouse embryonic stem(ES) cells in which the mouse heavy-chain and κ and λ light-chain loci had been knocked out. The modified ES cells were introduced into blastocysts, which were transferred to surrogate mothers and allowed to generate chimeric mice. Interbreeding of the chimeric mice produced mice that made human antibodies but not mouse Ig. Immunization of these mice allowed the generation of antigen-specific antiserum that contained only human antibodies or the generation of hybridomas that secreted antigen-specific human monoclonal antibodies.

41. (a)
(b)
FIGURE Generation of phage libraries containing antibody binding sites.
Derivation of single chain fragment variable(scFv) library.
The scFv library is cloned into phages that are used to infect e.coli. Growth of the phage-infected bacteria generates a phage library that is screened on antigen-coated plates for the presence of phage that bind to the desired antigen. Repeated cycles of binding-elution-regrowth results in the enrichment of the phage isolates for antigen specificity. Clones of antigen-specific phage can be isolated and the techniques of recombinant DNA technology used to graft the genes for 𝑉 H and 𝑉 L domains from selected phage onto the constant-region scaffolding of an antibody to engineer a monoclonal antibody of the desired antigen specificity.