SISTEMA INMUNE INMUNOGLOBULINAS RECEPTORES, RESPUESTA INMUNE Y LINFOCITO B.
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! Antígenos y anticuerpos
Antígenos y epitoposEstructura de los anticuerposUnión antígeno-anticuerpoEfectos mediados por anticuerposClases de anticuerpos y efectos biológicosDeterminantes antigénicos de las IgsLa generación de diversidad de los AcsBCRLa superfamilia de las Igs
Immunogenicidad y antigenicidad
2
Caso del hápteno: por si solo no inmunogénico, pero si con un carrier
• Antígeno: es toda sustancia que reacciona con los elementos de defensa de la respuesta inmune, es lo que “detectamos” en el laboratorio con los anticuerpos y las células inmunes.
• Excepciones•No toda sustancia es inmunogénica. •El antígeno no es siempre la substancia inductora de la respuesta
3
antigens by receptors of adaptive immunity, which can leadto autoimmune disorders. Like antibodies and T-cell recep-tors, pattern-recognition receptors are proteins. However,the genes that encode PRRs are present in the germline of theorganism. In contrast, the genes that encode the enormousdiversity of antibodies and TCRs are not present in thegermline. They are generated by an extraordinary process ofgenetic recombination that is discussed in Chapter 5.
Many different pattern-recognition receptors have beenidentified and several examples appear in Table 3-7. Some arepresent in the bloodstream and tissue fluids as soluble circu-lating proteins and others are on the membrane of cells suchas macrophages, neutrophils, and dendritic cells. Mannose-binding lectin (MBL) and C-reactive protein (CRP) are solu-ble pattern receptors that bind to microbial surfaces andpromote their opsonization. Both of these receptors alsohave the ability to activate the complement system when theyare bound to the surface of microbes, thereby making the invader a likely target of complement-mediated lysis. Yet another soluble receptor of the innate immune system,lipopolysaccharide-binding protein, is an important part of the system that recognizes and signals a response tolipopolysaccharide, a component of the outer cell wall ofgram-negative bacteria.
Pattern-recognition receptors found on the cell mem-brane include scavenger receptors and the toll-like receptors.Scavenger receptors (SRs) are present on macrophages andmany types of dendritic cells, and are involved in the bindingand internalization of gram-positive and gram-negative bac-teria, as well as the phagocytosis of apoptotic host cells. Theexact roles and mechanisms of action of the many types ofscavenger receptors known to date are under active investiga-tion. The toll-like receptors (TLRs) are important in recog-nizing many microbial patterns. This family of proteins is
ancient—toll-like receptors mediate the recognition andgeneration of defensive responses to pathogens in organismsas widely separated in evolutionary history as humans andflies. Typically, signals transduced through the TLRs causetranscriptional activation and the synthesis and secretion ofcytokines, which promote inflammatory responses thatbring macrophages and neutrophils to sites of inflammation.
70 P A R T I I Generation of B-Cell and T-Cell Responses
TABLE 3-6 Reactivity of antisera with various haptens
REACTIVITY WITH
Antiserum against Aminobenzene (aniline) o-Aminobenzoic acid m-Aminobenzoic acid p-Aminobenzoic acid
Aminobenzene ! 0 0 0
o-Aminobenzoic acid 0 ! 0 0
m-Aminobenzoic acid 0 0 ! 0
p-Aminobenzoic acid 0 0 0 +
KEY: 0 " no reactivity; ! " strong reactivity
SOURCE: Based on K. Landsteiner, 1962, The Specificity of Serologic Reactions, Dover Press. Modified by J. Klein, 1982, Immunology: The Science of Self-Nonself Discrimination, John Wiley.
NH2 NH2
COOH
NH2
COOH
NH2
COOH
LipoproteinsLipoarabinomannanLPS (Leptospira)LPS (P. gingivalis)PGN (Gram-positive)Zymosan (Yeast)GPI anchor (T. cruzi)
LPS (Gram-negative)Taxol (Plant)F protein (RS virus)hsp60 (Host)Fibronectin (Host)
Flagellin CpG DNA
TLR2 TLR4 TLR5 TLR9TLR6MD-2
FIGURE 3-11 Location and targets of some pattern-recognition re-ceptors. Many pattern-recognition receptors are extracellular and tar-get microbes or microbial components in the bloodstream andtissue fluids, causing their lysis or marking them for removal byphagocytes. Other pattern-recognition receptors are present on thecell membrane and bind to a broad variety of microbes or microbialproducts. Engagement of these receptors triggers signaling path-ways that promote inflammation or, in the case of the scavenger re-ceptors, phagocytosis or endocytosis. dsRNA " double strandedRNA; LPS " lipopolysaccharide. [S. Akira et al., 2001, Nature Im-munology 2:675.]
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Estructura básica de las Ig
4
polypeptides of molecular weight 50,000 or more. Like theantibody molecules they constitute, H and L chains are alsocalled immunoglobulins. Each light chain is bound to aheavy chain by a disulfide bond, and by such noncovalent in-teractions as salt linkages, hydrogen bonds, and hydrophobicbonds, to form a heterodimer (H-L). Similar noncovalent in-teractions and disulfide bridges link the two identical heavyand light (H-L) chain combinations to each other to form thebasic four-chain (H-L)2 antibody structure, a dimer ofdimers. As we shall see, the exact number and precise posi-tions of these interchain disulfide bonds differs among anti-body classes and subclasses.
The first 110 or so amino acids of the amino-terminal re-gion of a light or heavy chain varies greatly among antibodiesof different specificity. These segments of highly variable se-quence are called V regions: VL in light chains and VH in heavy.All of the differences in specificity displayed by different anti-bodies can be traced to differences in the amino acid se-quences of V regions. In fact, most of the differences amongantibodies fall within areas of the V regions called comple-mentarity-determining regions (CDRs), and it is these CDRs,on both light and heavy chains, that constitute the antigen-binding site of the antibody molecule. By contrast, within thesame antibody class, far fewer differences are seen when onecompares sequences throughout the rest of the molecule. Theregions of relatively constant sequence beyond the variable re-gions have been dubbed C regions, CL on the light chain and
CH on the heavy chain. Antibodies are glycoproteins; with fewexceptions, the sites of attachment for carbohydrates are re-stricted to the constant region. We do not completely under-stand the role played by glycosylation of antibodies, but itprobably increases the solubility of the molecules. Inappro-priate glycosylation, or its absence, affects the rate at whichantibodies are cleared from the serum, and decreases the effi-ciency of interaction between antibody and the complementsystem and between antibodies and Fc receptors.
Chemical and Enzymatic Methods RevealedBasic Antibody StructureOur knowledge of basic antibody structure was derived froma variety of experimental observations. When the !-globulinfraction of serum is separated into high- and low-molecular-weight fractions, antibodies of around 150,000-MW, des-ignated as immunoglobulin G (IgG) are found in the low-molecular-weight fraction. In a key experiment, brief diges-tion of IgG with the enzyme papain produced three frag-ments, two of which were identical fragments and a third thatwas quite different (Figure 4-3). The two identical fragments
Antibodies: Structure and Function C H A P T E R 4 77
!"
#
Globulins
Albumin
Abs
orba
nce
Migration distance
+ $
FIGURE 4-1 Experimental demonstration that most antibodies arein the !-globulin fraction of serum proteins. After rabbits were im-munized with ovalbumin (OVA), their antisera were pooled and elec-trophoresed, which separated the serum proteins according to theirelectric charge and mass. The blue line shows the electrophoreticpattern of untreated antiserum. The black line shows the pattern ofantiserum that was incubated with OVA to remove anti-OVA anti-body and then electrophoresed. [Adapted from A. Tiselius and E. A.Kabat, 1939, J. Exp. Med. 69:119, with copyright permission of theRockefeller University Press.]
S SS S
S S
S SS S
S S
CHO CHO
COO–
Light chain% or &
CH 2
CH 3
SSS S
VH
CH 1
S
S
S
SVL
CL
S
S
S
S
C H1
V H
S
S
S
S
C L
V LS
S
S
S
Heavy chainµ,#,",', or "
Hinge
NH 3+ NH
3 +
NH3 +NH 3
+
COO–
COO–COO–
Biologicalactivity
Antigenbinding
CH
2C
H3
446
214
FIGURE 4-2 Schematic diagram of structure of immunoglobulinsderived from amino acid sequencing studies. Each heavy and lightchain in an immunoglobulin molecule contains an amino-terminalvariable (V) region (aqua and tan, respectively) that consists of 100–110 amino acids and differs from one antibody to the next. The re-mainder of each chain in the molecule—the constant (C) regions(purple and red)—exhibits limited variation that defines the twolight-chain subtypes and the five heavy-chain subclasses. Someheavy chains (!, #, and $) also contain a proline-rich hinge region(black). The amino-terminal portions, corresponding to the V re-gions, bind to antigen; effector functions are mediated by the otherdomains. The % and " heavy chains, which lack a hinge region, con-tain an additional domain in the middle of the molecule.
Go to www.whfreeman.com/immunology AnimationImmunoglobulins
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Estructura elemental Igs✦ Bivalentes✦ Fc y Fab✦ Dos cadenas pesadas y
dos ligeras✦ Dominio básico de Ig de
unos 110✦ Puentes disulfuro✦ Bisagra
5
polypeptides of molecular weight 50,000 or more. Like theantibody molecules they constitute, H and L chains are alsocalled immunoglobulins. Each light chain is bound to aheavy chain by a disulfide bond, and by such noncovalent in-teractions as salt linkages, hydrogen bonds, and hydrophobicbonds, to form a heterodimer (H-L). Similar noncovalent in-teractions and disulfide bridges link the two identical heavyand light (H-L) chain combinations to each other to form thebasic four-chain (H-L)2 antibody structure, a dimer ofdimers. As we shall see, the exact number and precise posi-tions of these interchain disulfide bonds differs among anti-body classes and subclasses.
The first 110 or so amino acids of the amino-terminal re-gion of a light or heavy chain varies greatly among antibodiesof different specificity. These segments of highly variable se-quence are called V regions: VL in light chains and VH in heavy.All of the differences in specificity displayed by different anti-bodies can be traced to differences in the amino acid se-quences of V regions. In fact, most of the differences amongantibodies fall within areas of the V regions called comple-mentarity-determining regions (CDRs), and it is these CDRs,on both light and heavy chains, that constitute the antigen-binding site of the antibody molecule. By contrast, within thesame antibody class, far fewer differences are seen when onecompares sequences throughout the rest of the molecule. Theregions of relatively constant sequence beyond the variable re-gions have been dubbed C regions, CL on the light chain and
CH on the heavy chain. Antibodies are glycoproteins; with fewexceptions, the sites of attachment for carbohydrates are re-stricted to the constant region. We do not completely under-stand the role played by glycosylation of antibodies, but itprobably increases the solubility of the molecules. Inappro-priate glycosylation, or its absence, affects the rate at whichantibodies are cleared from the serum, and decreases the effi-ciency of interaction between antibody and the complementsystem and between antibodies and Fc receptors.
Chemical and Enzymatic Methods RevealedBasic Antibody StructureOur knowledge of basic antibody structure was derived froma variety of experimental observations. When the !-globulinfraction of serum is separated into high- and low-molecular-weight fractions, antibodies of around 150,000-MW, des-ignated as immunoglobulin G (IgG) are found in the low-molecular-weight fraction. In a key experiment, brief diges-tion of IgG with the enzyme papain produced three frag-ments, two of which were identical fragments and a third thatwas quite different (Figure 4-3). The two identical fragments
Antibodies: Structure and Function C H A P T E R 4 77
!"
#
Globulins
Albumin
Abs
orba
nce
Migration distance
+ $
FIGURE 4-1 Experimental demonstration that most antibodies arein the !-globulin fraction of serum proteins. After rabbits were im-munized with ovalbumin (OVA), their antisera were pooled and elec-trophoresed, which separated the serum proteins according to theirelectric charge and mass. The blue line shows the electrophoreticpattern of untreated antiserum. The black line shows the pattern ofantiserum that was incubated with OVA to remove anti-OVA anti-body and then electrophoresed. [Adapted from A. Tiselius and E. A.Kabat, 1939, J. Exp. Med. 69:119, with copyright permission of theRockefeller University Press.]
S SS S
S S
S SS S
S S
CHO CHO
COO–
Light chain% or &
CH 2
CH 3
SSS S
VH
CH 1
S
S
S
SVL
CL
S
S
S
S
C H1
V H
S
S
S
S
C L
V LS
S
S
S
Heavy chainµ,#,",', or "
Hinge
NH 3+ NH
3 +
NH3 +NH 3
+
COO–
COO–COO–
Biologicalactivity
Antigenbinding
CH
2C
H3
446
214
FIGURE 4-2 Schematic diagram of structure of immunoglobulinsderived from amino acid sequencing studies. Each heavy and lightchain in an immunoglobulin molecule contains an amino-terminalvariable (V) region (aqua and tan, respectively) that consists of 100–110 amino acids and differs from one antibody to the next. The re-mainder of each chain in the molecule—the constant (C) regions(purple and red)—exhibits limited variation that defines the twolight-chain subtypes and the five heavy-chain subclasses. Someheavy chains (!, #, and $) also contain a proline-rich hinge region(black). The amino-terminal portions, corresponding to the V re-gions, bind to antigen; effector functions are mediated by the otherdomains. The % and " heavy chains, which lack a hinge region, con-tain an additional domain in the middle of the molecule.
Go to www.whfreeman.com/immunology AnimationImmunoglobulins
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Fragmentos típicos de las Igs
6
(each with a MW of 45,000), had antigen-binding activityand were called Fab fragments (“fragment, antigen bind-ing”). The other fragment (MW of 50,000) had no antigen-binding activity at all. Because it was found to crystallizeduring cold storage, it was called the Fc fragment (“frag-ment, crystallizable”). Digestion with pepsin, a different pro-teolytic enzyme, also demonstrated that the antigen-bindingproperties of an antibody can be separated from the rest ofthe molecule. Pepsin digestion generated a single 100,000-MW fragment composed of two Fab-like fragments desig-nated the F(ab!)2 fragment, which binds antigen. The Fcfragment was not recovered from pepsin digestion because ithad been digested into multiple fragments.
A key observation in deducing the multichain structure ofIgG was made when the molecule was subjected to mercap-toethanol reduction and alkylation, a chemical treatmentthat irreversibly cleaves disulfide bonds. If the sample is chro-matographed on a column that separates molecules by sizefollowing cleavage of disulfide bonds, it is clear that the intact150,000-MW IgG molecule is, in fact, composed of subunits.Each IgG molecule contains two 50,000-MW polypeptidechains, designated as heavy (H) chains, and two 25,000-MWchains, designated as light (L) chains (see Figure 4-3).
Antibodies themselves were used to determine how theenzyme digestion products—Fab, F(ab!)2, and Fc—were re-lated to the heavy-chain and light-chain reduction products.
This question was answered by using antisera from goats thathad been immunized with either the Fab fragments or the Fcfragments of rabbit IgG. The antibody to the Fab fragmentcould react with both the H and the L chains, whereas anti-body to the Fc fragment reacted only with the H chain. Theseobservations led to the conclusion that the Fab fragmentconsists of portions of a heavy and a light chain and that Fccontains only heavy-chain components. From these results,and those mentioned above, the structure of IgG shown inFigure 4-3 was deduced. According to this model, the IgGmolecule consists of two identical H chains and two identicalL chains, which are linked by disulfide bridges. The enzymepapain cleaves just above the interchain disulfide bonds link-ing the heavy chains, whereas the enzyme pepsin cleaves justbelow these bonds, so that the two proteolytic enzymes gen-erate different digestion products. Mercaptoethanol reduc-tion and alkylation allow separation of the individual heavyand light chains.
Obstacles to Antibody SequencingInitial attempts to determine the amino acid sequence of theheavy and light chains of antibody were hindered because in-sufficient amounts of homogeneous protein were available.Although the basic structure and chemical properties of differ-
78 P A R T I I Generation of B-Cell and T-Cell Responses
S
Disulfidebonds
L chain
L chains
SHHS
Pepsindigestion
F(ab')2
+
+
Fc fragments
Fc
Fab Fab
Mercaptoethanolreduction
H chain
H chains
SS S
S S S S
S SS S
S S S S
S SS S
SHSH
S S S S
Papaindigestion
SHHS
+
SHSH
+ +
FIGURE 4-3 Prototype structure of IgG, showing chain structureand interchain disulfide bonds. The fragments produced by various
treatments are also indicated. Light (L) chains are in gray and heavy(H) chains in blue.
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http://bcs.whfreeman.com/immunology6e/content/cat_030/immunoglobulin/start.htmlVisión 3
7
across the faces of the ! sheets (Figure 4-8). Interactionsform links between identical domains (e.g., CH2/CH2,CH3/CH3, and CH4/CH4) and between nonidentical do-mains (e.g., VH/VL and CH1/CL). The structure of the im-munoglobulin fold also allows for variable lengths and
sequences of amino acids that form the loops connectingthe ! strands. As the next section explains, some of theloop sequences of the VH and VL domains contain variableamino acids and constitute the antigen-binding site of themolecule.
Antibodies: Structure and Function C H A P T E R 4 81
FIGURE 4-5 Ribbon representation of an intact monoclonal anti-body depicting the heavy chains (yellow and blue) and light chains(red). The domains of the molecule composed of ! pleated sheetsare readily visible as is the extended conformation of the hinge re-
FIGURE 4-6 (a) Heavy and light chains are folded into domains,each containing about 110 amino acid residues and an intrachaindisulfide bond that forms a loop of 60 amino acids. The amino-terminal domains, corresponding to the V regions, bind to antigen;
gion. [The laboratory of A. McPherson provided this image, which isbased on x-ray crystallography data determined by L. J. Harris et al.,1992, Nature 360:369. The image was generated using the computerprogram RIBBONS.]
CHO
SS
SS
SS
SS
SS
SS
SS
SS
SSS
SS SS S
CH2
(a) !, ", # (b) ", #
CH3
CHO
Hinge
261
321
367
425
446
214
200194
144134
22
C H1
V H
C L
V L
SS
SS S
SS
S
Biologicalactivity
No hingeregion
Antigenbinding
88
CH2
CH3
CH4Additionaldomain
effector functions are mediated by the other domains. (b) The " and# heavy chains contain an additional domain that replaces the hinge region.
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Isotipos Inmunoglobulinas
http://bcs.whfreeman.com/immunology6e/content/cat_030/immunoglobulin/start.html
8
Clase Cadena pesada Subclase Cadena LigeraIgG gamma gamma-1, 2, 3, 4 kappa o lambda
IgM mu No Kappa o lambda
IgA alfa alfa-1; alfa 2 Kappa o lambda
IgD delta No Kappa o lambda
IgE épsilon No Kappa o lambda
Isotipos Inmunoglobulinas
Formación del lugar de unión al Ag: CDRs
9
Diversity in the Variable-Region Domain Is Concentrated in CDRsDetailed comparisons of the amino acid sequences of a largenumber of VL and VH domains revealed that the sequencevariation is concentrated in a few discrete regions of thesedomains. The pattern of this variation is best summarized bya quantitative plot of the variability at each position of thepolypeptide chain. The variability is defined as:
# of different amino acids at a given positionVariability !
Frequency of the most common amino acidat given position
Thus if a comparison of the sequences of 100 heavy chainsrevealed that a serine was found in position 7 in 51 of the se-quences (frequency 0.51), it would be the most commonamino acid. If examination of the other 49 sequences showedthat position 7 was occupied by either glutamine, histidine,proline, or tryptophan, the variability at that position wouldbe 9.8 (5/0.51). Variability plots of VL and VH domains of hu-man antibodies show that maximum variation is seen inthose portions of the sequence that correspond to the loopsthat join the " strands (Figure 4-9). These regions were orig-inally called hypervariable regions in recognition of theirhigh variability. Hypervariable regions form the antigen-binding site of the antibody molecule. Because the antigenbinding site is complementary to the structure of the epitope,
82 P A R T I I Generation of B-Cell and T-Cell Responses
FIGURE 4-7 (a) Diagram of an immunoglobulin light chain depict-ing the immunoglobulin-fold structure of its variable and constantdomains. The two " pleated sheets in each domain are held togetherby hydrophobic interactions and the conserved disulfide bond. The "strands that compose each sheet are shown in different colors. Theamino acid sequences in three loops of each variable domain showconsiderable variation; these hypervariable regions (blue) make upthe antigen-binding site. Hypervariable regions are usually called
(a)
(b)
CL domain
Disulfide bond
! strands
!-strand arrangement
Loops
VL domain
NH2
NH2
COOH
COOH
COOH
CDRs
CDRs
NH2
CDRs (complementarity-determining regions). Heavy-chain do-mains have the same characteristic structure. (b) The " pleatedsheets are opened out to reveal the relationship of the individual "strands and joining loops. Note that the variable domain containstwo more " strands than the constant domain. [Part (a) adaptedfrom M. Schiffer et al., 1973, Biochemistry 12:4620; reprinted withpermission; part (b) adapted from Williams and Barclay, 1988, Annu.Rev. Immunol. 6:381.]
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Complementariedad
10
Diferentes tipos de unión
Tamaño area de unión, de una cadena lateral de 15 aminoácidos
Antigens C H A P T E R 3 65
V I S U A L I Z I N G C O N C E P T S
FIGURE 3-6 Protein antigens usually contain both sequentialand nonsequential B-cell epitopes. (a) Diagram of sperm whalemyoglobin showing locations of five sequential B-cell epitopes(blue). (b) Ribbon diagram of hen egg-white lysozyme showingresidues that compose one nonsequential (conformational) epi-tope. Residues that contact antibody light chains, heavy chains, or
both are shown in red, blue, and white, respectively. Theseresidues are widely spaced in the amino acid sequence but arebrought into proximity by folding of the protein. [Part (a) adaptedfrom M. Z. Atassi and A. L. Kazim. 1978, Adv. Exp. Med. Biol. 98:9;part (b) from W. G. Laver et al., 1990, Cell 61:554.]
Heme
(145) 146 !151COOH
(a) (b)
NH2
15 ! 21 (22)
56 ! 62
113 ! 119
FIGURE 3-5 Computer simulation of an interaction between anti-body and influenza virus antigen, a globular protein. (a) The antigen(yellow) is shown interacting with the antibody molecule; the variableregion of the heavy chain is red, and the variable region of the light
Antigen Antibody
(a) (b)
chain is blue. (b) The complementarity of the two molecules is re-vealed by separating the antigen from the antibody by 8 Å. [Based onx-ray crystallography data collected by P. M. Colman and W. R. Tulip.From G. J. V. H. Nossal, 1993, Sci. Am. 269(3):22.]
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Variabilidad en los CDRs y unión
11
large globular protein antigens or with a number of smallerantigens including carbohydrates, nucleic acids, peptides,and small haptens. In addition, complete structures havebeen obtained for several intact monoclonal antibodies. X-ray diffraction analysis of antibody-antigen complexes has
shown that several CDRs may make contact with the antigen,and a number of complexes have been observed in which allsix CDRs contact the antigen. In general, more residues in theheavy-chain CDRs appear to contact antigen than in thelight-chain CDRs. Thus the VH domain often contributes
84 P A R T I I Generation of B-Cell and T-Cell Responses
Residue position number
VL domain
150
12020 10060400 80
Var
iabi
lity
100
50
0
CDR2 CDR3CDR1
Var
iabi
lity
150
1007550250
120
60
30
0
Residue position number
VH domain
CDR1 CDR2 CDR3
FIGURE 4-9 Variability of amino acid residues in the VL and VH do-mains of human antibodies with different specificities. Three hyper-variable (HV) regions, also called complementarity-determiningregions (CDRs), are present in both heavy- and light-chain V do-mains. As shown in Figure 4-7 (right), the three HV regions in the
light-chain V domain are brought into proximity in the folded struc-ture. The same is true of the heavy-chain V domain. [Based on E. A.Kabat et al., 1977, Sequence of Immunoglobulin Chains, U.S. Dept.of Health, Education, and Welfare.]
(a) (b)
FIGURE 4-10 (a) Side view of the three-dimensional structure ofthe combining site of an angiotensin II–Fab complex. The peptide isin red. The three heavy-chain CDRs (H1, H2, H3) and three light-chain CDRs (L1, L2, L3) are each shown in a different color. All sixCDRs contain side chains, shown in yellow, that are within van der
Waals contact of the angiotensin peptide. (b) Side view of the vander Waals surface of contact between angiotensin II and Fab frag-ment. [From K. C. Garcia et al., 1992, Science 257:502; courtesy of M. Amzel, Johns Hopkins University.]
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Visión de la unión de un péptido (angiotensina) a un fragmento Fab. Dos regiones determinantes de complementariedad de la cadena ligera (L1 y L2 y tres de la cadena pesada (H1, H2, H3) forman la cavidad. A la derecha representación volumétrica
Regiones Hipervariables y CDRs
12
large globular protein antigens or with a number of smallerantigens including carbohydrates, nucleic acids, peptides,and small haptens. In addition, complete structures havebeen obtained for several intact monoclonal antibodies. X-ray diffraction analysis of antibody-antigen complexes has
shown that several CDRs may make contact with the antigen,and a number of complexes have been observed in which allsix CDRs contact the antigen. In general, more residues in theheavy-chain CDRs appear to contact antigen than in thelight-chain CDRs. Thus the VH domain often contributes
84 P A R T I I Generation of B-Cell and T-Cell Responses
Residue position number
VL domain
150
12020 10060400 80
Var
iabi
lity
100
50
0
CDR2 CDR3CDR1
Var
iabi
lity
150
1007550250
120
60
30
0
Residue position number
VH domain
CDR1 CDR2 CDR3
FIGURE 4-9 Variability of amino acid residues in the VL and VH do-mains of human antibodies with different specificities. Three hyper-variable (HV) regions, also called complementarity-determiningregions (CDRs), are present in both heavy- and light-chain V do-mains. As shown in Figure 4-7 (right), the three HV regions in the
light-chain V domain are brought into proximity in the folded struc-ture. The same is true of the heavy-chain V domain. [Based on E. A.Kabat et al., 1977, Sequence of Immunoglobulin Chains, U.S. Dept.of Health, Education, and Welfare.]
(a) (b)
FIGURE 4-10 (a) Side view of the three-dimensional structure ofthe combining site of an angiotensin II–Fab complex. The peptide isin red. The three heavy-chain CDRs (H1, H2, H3) and three light-chain CDRs (L1, L2, L3) are each shown in a different color. All sixCDRs contain side chains, shown in yellow, that are within van der
Waals contact of the angiotensin peptide. (b) Side view of the vander Waals surface of contact between angiotensin II and Fab frag-ment. [From K. C. Garcia et al., 1992, Science 257:502; courtesy of M. Amzel, Johns Hopkins University.]
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Epitopos: estructura proteíca
13
Epitopos continuos y no continuos
14
Antigens C H A P T E R 3 65
V I S U A L I Z I N G C O N C E P T S
FIGURE 3-6 Protein antigens usually contain both sequentialand nonsequential B-cell epitopes. (a) Diagram of sperm whalemyoglobin showing locations of five sequential B-cell epitopes(blue). (b) Ribbon diagram of hen egg-white lysozyme showingresidues that compose one nonsequential (conformational) epi-tope. Residues that contact antibody light chains, heavy chains, or
both are shown in red, blue, and white, respectively. Theseresidues are widely spaced in the amino acid sequence but arebrought into proximity by folding of the protein. [Part (a) adaptedfrom M. Z. Atassi and A. L. Kazim. 1978, Adv. Exp. Med. Biol. 98:9;part (b) from W. G. Laver et al., 1990, Cell 61:554.]
Heme
(145) 146 !151COOH
(a) (b)
NH2
15 ! 21 (22)
56 ! 62
113 ! 119
FIGURE 3-5 Computer simulation of an interaction between anti-body and influenza virus antigen, a globular protein. (a) The antigen(yellow) is shown interacting with the antibody molecule; the variableregion of the heavy chain is red, and the variable region of the light
Antigen Antibody
(a) (b)
chain is blue. (b) The complementarity of the two molecules is re-vealed by separating the antigen from the antibody by 8 Å. [Based onx-ray crystallography data collected by P. M. Colman and W. R. Tulip.From G. J. V. H. Nossal, 1993, Sci. Am. 269(3):22.]
8536d_ch03_065 9/10/02 10:12 AM Page 65 mac46 mac46:385_REB:
Mioglobina de ballena, los anticuerpos reaccionan con varios epitopos a lo largo de la cadena
Lisozima de la albúmina de huevo de gallina, el epitopo esta formado por aa de diferentes parte de la cadena
A la formación de los epitopos B contribuyen la estructura secundaria, terciaria y cuaternaria de las proteínas
Un antisuero contiene múltiples anticuerpos que reconocen varios epitopos
!
Interacciones Ag-Ac
Tipos de unión (enlace)Medida de la afinidad
Naturaleza de la reacción Ag-Ac
✦ Puentes de hidrógeno✦ Fuerzas electrostáticas (iones)✦ Interacciones hidrofóbicas✦ Fuerzas de van der Waals✦ Todas estas fuerzas actúan a muy corta distancias por
lo que tiene que haber complementaridad estérica (3D)
✦ No hay enlaces covalentes
16
Reacción Ag-Ac
17
Ag + Ac Ag-Abk1
k-1
k1
k-1
Constante afinidad Ka
where k1 is the forward (association) rate constant and k!1 isthe reverse (dissociation) rate constant. The ratio k1/k!1 is the association constant Ka (i.e., k1/k!1 " Ka), a measure ofaffinity. Because Ka is the equilibrium constant for the abovereaction, it can be calculated from the ratio of the molar con-centration of bound Ag-Ab complex to the molar concentra-tions of unbound antigen and antibody at equilibrium asfollows:
Ka "
The value of Ka varies for different Ag-Ab complexes anddepends upon both k1, which is expressed in units ofliters/mole/second (L/mol/s), and k!1, which is expressed inunits of 1/second. For small haptens, the forward rate con-stant can be extremely high; in some cases, k1 can be as highas 4 # 108 L/mol/s, approaching the theoretical upper limitof diffusion-limited reactions (109 L/mol/s). For larger pro-tein antigens, however, k1 is smaller, with values in the rangeof 105 L/mol/s.
The rate at which bound antigen leaves an antibody’sbinding site (i.e., the dissociation rate constant, k!1) plays amajor role in determining the antibody’s affinity for anantigen. Table 6-1 illustrates the role of k!1 in determining
[Ag-Ab]$[Ab][Ag]
the association constant Ka for several Ag-Ab interactions.For example, the k1 for the DNP-L-lysine system is aboutone fifth that for the fluorescein system, but its k!1 is 200times greater; consequently, the affinity of the antifluores-cein antibody Ka for the fluorescein system is about 1000-fold higher than that of anti-DNP antibody. Low-affinityAg-Ab complexes have Ka values between 104 and 105
L/mol; high-affinity complexes can have Ka values as highas 1011 L/mol.
For some purposes, the dissociation of the antigen-anti-body complex is of interest:
Ag-Ab 334 Ab % Ag
The equilibrium constant for that reaction is Kd, the recipro-cal of Ka
Kd " [Ab][Ag]&[Ab-Ag] " 1&Ka
and is a quantitative indicator of the stability of an Ag-Abcomplex; very stable complexes have very low values of Kd,and less stable ones have higher values.
The affinity constant, Ka, can be determined by equilib-rium dialysis or by various newer methods. Because equilib-rium dialysis remains for many the standard against which
138 P A R T I I Generation of B-Cell and T-Cell Responses
V I S U A L I Z I N G C O N C E P T S
FIGURE 6-1 The interaction between an antibody and an anti-gen depends on four types of noncovalent forces: (1) hydrogenbonds, in which a hydrogen atom is shared between two elec-tronegative atoms; (2) ionic bonds between oppositely chargedresidues; (3) hydrophobic interactions, in which water forces hy-
drophobic groups together; and (4) van der Waals interactionsbetween the outer electron clouds of two or more atoms. In anaqueous environment, noncovalent interactions are extremelyweak and depend upon close complementarity of the shapes ofantibody and antigen.
ANTIGEN
CH2
ANTIBODY
OH ••• O C CH2 CH2
NH2
Hydrogen bond
CH2 CH2 NH3+ –O
CH2 CH2C Ionic bond
O
CH2
CH3
CH
CH3 CH3
+H3N
CH2CHCH3van der Waals interactionsCH CHCH3
CH CH3
O
O–CH2 C CH2 Ionic bond
CH3 Hydrophobic interactions
8536d_ch06_137-160 8/1/02 12:41 PM Page 138 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
En
Ley de acción de masas aplicada a la reacción Ag con
Naturaleza de la reaccion Ag-Ac
18
where k1 is the forward (association) rate constant and k!1 isthe reverse (dissociation) rate constant. The ratio k1/k!1 is the association constant Ka (i.e., k1/k!1 " Ka), a measure ofaffinity. Because Ka is the equilibrium constant for the abovereaction, it can be calculated from the ratio of the molar con-centration of bound Ag-Ab complex to the molar concentra-tions of unbound antigen and antibody at equilibrium asfollows:
Ka "
The value of Ka varies for different Ag-Ab complexes anddepends upon both k1, which is expressed in units ofliters/mole/second (L/mol/s), and k!1, which is expressed inunits of 1/second. For small haptens, the forward rate con-stant can be extremely high; in some cases, k1 can be as highas 4 # 108 L/mol/s, approaching the theoretical upper limitof diffusion-limited reactions (109 L/mol/s). For larger pro-tein antigens, however, k1 is smaller, with values in the rangeof 105 L/mol/s.
The rate at which bound antigen leaves an antibody’sbinding site (i.e., the dissociation rate constant, k!1) plays amajor role in determining the antibody’s affinity for anantigen. Table 6-1 illustrates the role of k!1 in determining
[Ag-Ab]$[Ab][Ag]
the association constant Ka for several Ag-Ab interactions.For example, the k1 for the DNP-L-lysine system is aboutone fifth that for the fluorescein system, but its k!1 is 200times greater; consequently, the affinity of the antifluores-cein antibody Ka for the fluorescein system is about 1000-fold higher than that of anti-DNP antibody. Low-affinityAg-Ab complexes have Ka values between 104 and 105
L/mol; high-affinity complexes can have Ka values as highas 1011 L/mol.
For some purposes, the dissociation of the antigen-anti-body complex is of interest:
Ag-Ab 334 Ab % Ag
The equilibrium constant for that reaction is Kd, the recipro-cal of Ka
Kd " [Ab][Ag]&[Ab-Ag] " 1&Ka
and is a quantitative indicator of the stability of an Ag-Abcomplex; very stable complexes have very low values of Kd,and less stable ones have higher values.
The affinity constant, Ka, can be determined by equilib-rium dialysis or by various newer methods. Because equilib-rium dialysis remains for many the standard against which
138 P A R T I I Generation of B-Cell and T-Cell Responses
V I S U A L I Z I N G C O N C E P T S
FIGURE 6-1 The interaction between an antibody and an anti-gen depends on four types of noncovalent forces: (1) hydrogenbonds, in which a hydrogen atom is shared between two elec-tronegative atoms; (2) ionic bonds between oppositely chargedresidues; (3) hydrophobic interactions, in which water forces hy-
drophobic groups together; and (4) van der Waals interactionsbetween the outer electron clouds of two or more atoms. In anaqueous environment, noncovalent interactions are extremelyweak and depend upon close complementarity of the shapes ofantibody and antigen.
ANTIGEN
CH2
ANTIBODY
OH ••• O C CH2 CH2
NH2
Hydrogen bond
CH2 CH2 NH3+ –O
CH2 CH2C Ionic bond
O
CH2
CH3
CH
CH3 CH3
+H3N
CH2CHCH3van der Waals interactionsCH CHCH3
CH CH3
O
O–CH2 C CH2 Ionic bond
CH3 Hydrophobic interactions
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Bond = enlace
Ejemplos de afinidades
19
Anticuerpos Ligando K1asociación
K-1separación
Ka afinidad Kd disociación
Anti-DNP e-DNP-Lisina 8x107 1 1x107 1x10--7
Anti-fluoresceina
Fluorescein 4x108 5x10-3 1x1011 1x10-11
Anti-BSA BSA 3x105 2x10-3 1,7x108 5,9x10-9
Medida experimental de la afinidad
20
other methods are evaluated, it is described here. This proce-dure uses a dialysis chamber containing two equal compart-ments separated by a semipermeable membrane. Antibody isplaced in one compartment, and a radioactively labeled lig-and that is small enough to pass through the semipermeablemembrane is placed in the other compartment (Figure 6-2).Suitable ligands include haptens, oligosaccharides, and oligo-peptides. In the absence of antibody, ligand added to com-partment B will equilibrate on both sides of the membrane(Figure 6-2a). In the presence of antibody, however, part
of the labeled ligand will be bound to the antibody at equi-librium, trapping the ligand on the antibody side of the ves-sel, whereas unbound ligand will be equally distributed inboth compartments. Thus the total concentration of ligandwill be greater in the compartment containing antibody (Fig-ure 6-2b). The difference in the ligand concentration in thetwo compartments represents the concentration of ligandbound to the antibody (i.e., the concentration of Ag-Ab com-plex). The higher the affinity of the antibody, the more ligandis bound.
Antigen-Antibody Interactions: Principles and Applications C H A P T E R 6 139
TABLE 6-1Forward and reverse rate constants (k1 and k!1) and association and dissociation constants (Ka and Kd) for three ligand-antibody interactions
Antibody Ligand k1 k!1 Ka Kd
Anti-DNP "-DNP-L-lysine 8 # 107 1 1 # 108 1 # 10!8
Anti-fluorescein Fluorescein 4 # 108 5 # 10!3 1 # 1011 1 # 10!11
Anti-bovine serum albumin (BSA) Dansyl-BSA 3 # 105 2 # 10!3 1.7 # 108 5.9 # 10!9
SOURCE: Adapted from H. N. Eisen, 1990, Immunology, 3rd ed., Harper & Row Publishers.
FIGURE 6-2 Determination of antibody affinity by equilibrium dial-ysis. (a) The dialysis chamber contains two compartments (A and B)separated by a semipermeable membrane. Antibody is added to onecompartment and a radiolabeled ligand to another. At equilibrium,the concentration of radioactivity in both compartments is mea-
sured. (b) Plot of concentration of ligand in each compartment withtime. At equilibrium, the difference in the concentration of radioac-tive ligand in the two compartments represents the amount of ligandbound to antibody.
(a)
Radiolabeledligand
A B A B
(b)
Con
cent
rati
on o
f lig
and,
M
100
50
100
50
ControlControl: No antibody present(ligand equilibrates on both sides equally)
ExperimentalExperimental: Antibody in A(at equilibrium more ligand in A due to Ab binding)
Ligand boundby antibody
2 4 6 8Time, h
Initial state Equilibrium
A B A B
Initial state Equilibrium
DAntibody
A
B
A
B
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other methods are evaluated, it is described here. This proce-dure uses a dialysis chamber containing two equal compart-ments separated by a semipermeable membrane. Antibody isplaced in one compartment, and a radioactively labeled lig-and that is small enough to pass through the semipermeablemembrane is placed in the other compartment (Figure 6-2).Suitable ligands include haptens, oligosaccharides, and oligo-peptides. In the absence of antibody, ligand added to com-partment B will equilibrate on both sides of the membrane(Figure 6-2a). In the presence of antibody, however, part
of the labeled ligand will be bound to the antibody at equi-librium, trapping the ligand on the antibody side of the ves-sel, whereas unbound ligand will be equally distributed inboth compartments. Thus the total concentration of ligandwill be greater in the compartment containing antibody (Fig-ure 6-2b). The difference in the ligand concentration in thetwo compartments represents the concentration of ligandbound to the antibody (i.e., the concentration of Ag-Ab com-plex). The higher the affinity of the antibody, the more ligandis bound.
Antigen-Antibody Interactions: Principles and Applications C H A P T E R 6 139
TABLE 6-1Forward and reverse rate constants (k1 and k!1) and association and dissociation constants (Ka and Kd) for three ligand-antibody interactions
Antibody Ligand k1 k!1 Ka Kd
Anti-DNP "-DNP-L-lysine 8 # 107 1 1 # 108 1 # 10!8
Anti-fluorescein Fluorescein 4 # 108 5 # 10!3 1 # 1011 1 # 10!11
Anti-bovine serum albumin (BSA) Dansyl-BSA 3 # 105 2 # 10!3 1.7 # 108 5.9 # 10!9
SOURCE: Adapted from H. N. Eisen, 1990, Immunology, 3rd ed., Harper & Row Publishers.
FIGURE 6-2 Determination of antibody affinity by equilibrium dial-ysis. (a) The dialysis chamber contains two compartments (A and B)separated by a semipermeable membrane. Antibody is added to onecompartment and a radiolabeled ligand to another. At equilibrium,the concentration of radioactivity in both compartments is mea-
sured. (b) Plot of concentration of ligand in each compartment withtime. At equilibrium, the difference in the concentration of radioac-tive ligand in the two compartments represents the amount of ligandbound to antibody.
(a)
Radiolabeledligand
A B A B
(b)
Con
cent
rati
on o
f lig
and,
M
100
50
100
50
ControlControl: No antibody present(ligand equilibrates on both sides equally)
ExperimentalExperimental: Antibody in A(at equilibrium more ligand in A due to Ab binding)
Ligand boundby antibody
2 4 6 8Time, h
Initial state Equilibrium
A B A B
Initial state Equilibrium
DAntibody
A
B
A
B
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binding = unión,
Avidez y afinidad
✦ Avidez, es la fuerza con la que al Ag se une al Ac➡ Además de la afinidad de cada lugar de unión,
depende de la valencia➡ Es el producto de las afinidades individuales
✦ Es mejor medida de la capacidad total de un Ac al antígeno
✦ IgM : Ka x10, medir experimentalmente!
21
Efectos mediados por los Acs
22
these areas are now more widely called complementarity de-termining regions (CDRs). The three heavy-chain and threelight-chain CDR regions are located on the loops that con-nect the ! strands of the VH and VL domains. The remainderof the VL and VH domains exhibit far less variation; thesestretches are called the framework regions (FRs). The widerange of specificities exhibited by antibodies is due to varia-tions in the length and amino acid sequence of the six CDRsin each Fab fragment. The framework region acts as a scaf-fold that supports these six loops. The three-dimensionalstructure of the framework regions of virtually all antibodies
analyzed to date can be superimposed on one another; incontrast, the hypervariable loops (i.e., the CDRs) have differ-ent orientations in different antibodies.
CDRs Bind AntigenThe finding that CDRs are the antigen-binding regions ofantibodies has been confirmed directly by high-resolution x-ray crystallography of antigen-antibody complexes. Crys-tallographic analysis has been completed for many Fab fragments of monoclonal antibodies complexed either with
Antibodies: Structure and Function C H A P T E R 4 83
FIGURE 4-8 Interactions between domains in the separate chainsof an immunoglobulin molecule are critical to its quaternary struc-ture. (a) Model of IgG molecule, based on x-ray crystallographicanalysis, showing associations between domains. Each solid ball rep-resents an amino acid residue; the larger tan balls are carbohydrate.The two light chains are shown in shades of red; the two heavychains, in shades of blue. (b) A schematic diagram showing the in-
VL domain
Antigen–binding site
CL domain
Heavy chains
Carbohydrate chain
Carbohydrate
Antigen–binding site
VH domain
(a)
(b)
S S
VHCL
VL
C!2
C!3
C!1VH
C!2
VL
VL domain
VH domainCH1
CH2
CH3
teracting heavy- and light-chain domains. Note that the CH2/CH2domains protrude because of the presence of carbohydrate (tan) inthe interior. The protrusion makes this domain more accessible, en-abling it to interact with molecules such as certain complementcomponents. [Part (a) from E. W. Silverton et al., 1977, Proc. Nat.Acad. Sci. U.S.A. 74:5140.]
Go to www.whfreeman.com/immunology Molecular VisualizationAntibody Recognition of Antigen
8536d_ch04_076-104 9/6/02 9:02 PM Page 83 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e:
Fijación de complemento
Unión a receptores Fc
✴ Neutralización✴ Opsonización✴ Fijación de complemento✴ Transcitosis (intestino, placenta)✴ Citotoxicidad celular dependiente de Ac
A A
Estructura Igs diméricas
23
polypeptide (see Figure 4-13d). The IgA of external secre-tions, called secretory IgA, consists of a dimer or tetramer, aJ-chain polypeptide, and a polypeptide chain called secre-tory component (Figure 4-15a, page 93). As is explained be-low, secretory component is derived from the receptor that isresponsible for transporting polymeric IgA across cell mem-branes. The J-chain polypeptide in IgA is identical to thatfound in pentameric IgM and serves a similar function in fa-
cilitating the polymerization of both serum IgA and secre-tory IgA. The secretory component is a 70,000-MW polypep-tide produced by epithelial cells of mucous membranes. Itconsists of five immunoglobulin-like domains that bind tothe Fc region domains of the IgA dimer. This interaction isstabilized by a disulfide bond between the fifth domain of thesecretory component and one of the chains of the dimericIgA.
Antibodies: Structure and Function C H A P T E R 4 91
(b) IgD
VL
C!1CL
C!2
VH
C!3
(d) IgA (dimer)
Hinge region
VL
C"1CL
C"2
VH
C"3
(c) IgE
C#1
C#3
VH
C#4
VL
CL
C#2
Cµ1
Cµ3
VH
Cµ4
VL
CL
Cµ2
J chain
Disulfidebond
(e) IgM (pentamer)
J chain
VL
C$1
C$2
VH
C$3
Hinge region
(a) IgG
CL
FIGURE 4-13 General structures of the five major classes of se-creted antibody. Light chains are shown in shades of pink, disulfidebonds are indicated by thick black lines. Note that the IgG, IgA, andIgD heavy chains (blue, orange, and green, respectively) contain fourdomains and a hinge region, whereas the IgM and IgE heavy chains(purple and yellow, respectively) contain five domains but no hingeregion. The polymeric forms of IgM and IgA contain a polypeptide,
called the J chain, that is linked by two disulfide bonds to the Fc re-gion in two different monomers. Serum IgM is always a pentamer;most serum IgA exists as a monomer, although dimers, trimers, andeven tetramers are sometimes present. Not shown in these figuresare intrachain disulfide bonds and disulfide bonds linking light andheavy chains (see Figure 4-2).
8536d_ch04_076-104 9/5/02 6:19 AM Page 91 mac76 mac76:385 Goldsby et al./Immunology5e:
Intravascular & Membrana linfocito B Mastocito
The daily production of secretory IgA is greater than thatof any other immunoglobulin class. IgA-secreting plasmacells are concentrated along mucous membrane surfaces.Along the jejunum of the small intestine, for example, thereare more than 2.5 ! 1010 IgA-secreting plasma cells—anumber that surpasses the total plasma cell population of thebone marrow, lymph, and spleen combined! Every day, a hu-man secretes from 5 g to 15 g of secretory IgA into mucoussecretions.
The plasma cells that produce IgA preferentially migrate(home) to subepithelial tissue, where the secreted IgA bindstightly to a receptor for polymeric immunoglobulin mole-cules (Figure 4-15b). This poly-Ig receptor is expressed onthe basolateral surface of most mucosal epithelia (e.g., thelining of the digestive, respiratory, and genital tracts) and onglandular epithelia in the mammary, salivary, and lacrimalglands. After polymeric IgA binds to the poly-Ig receptor, thereceptor-IgA complex is transported across the epithelialbarrier to the lumen. Transport of the receptor-IgA complexinvolves receptor-mediated endocytosis into coated pits anddirected transport of the vesicle across the epithelial cell tothe luminal membrane, where the vesicle fuses with theplasma membrane. The poly-Ig receptor is then cleaved en-zymatically from the membrane and becomes the secretorycomponent, which is bound to and released together withpolymeric IgA into the mucous secretions. The secretorycomponent masks sites susceptible to protease cleavage in thehinge region of secretory IgA, allowing the polymeric mole-cule to exist longer in the protease-rich mucosal environ-ment than would be possible otherwise. Pentameric IgM isalso transported into mucous secretions by this mechanism,although it accounts for a much lower percentage of anti-body in the mucous secretions than does IgA. The poly-Ig re-ceptor interacts with the J chain of both polymeric IgA andIgM antibodies.
Secretory IgA serves an important effector function atmucous membrane surfaces, which are the main entry sites
for most pathogenic organisms. Because it is polymeric, se-cretory IgA can cross-link large antigens with multiple epi-topes. Binding of secretory IgA to bacterial and viral surfaceantigens prevents attachment of the pathogens to the mu-cosal cells, thus inhibiting viral infection and bacterial colo-nization. Complexes of secretory IgA and antigen are easilyentrapped in mucus and then eliminated by the ciliated ep-ithelial cells of the respiratory tract or by peristalsis of thegut. Secretory IgA has been shown to provide an importantline of defense against bacteria such as Salmonella, Vibriocholerae, and Neisseria gonorrhoeae and viruses such as polio,influenza, and reovirus.
Breast milk contains secretory IgA and many other mole-cules that help protect the newborn against infection duringthe first month of life (Table 4-3). Because the immune sys-tem of infants is not fully functional, breast-feeding plays animportant role in maintaining the health of newborns.
Immunoglobulin E (IgE)The potent biological activity of IgE allowed it to be identi-fied in serum despite its extremely low average serum con-centration (0.3 "g/ml). IgE antibodies mediate the immediatehypersensitivity reactions that are responsible for the symp-toms of hay fever, asthma, hives, and anaphylactic shock. Thepresence of a serum component responsible for allergic reac-tions was first demonstrated in 1921 by K. Prausnitz and H. Kustner, who injected serum from an allergic person intra-dermally into a nonallergic individual. When the appropriate antigen was later injected at the same site, awheal and flare reaction (analogous to hives) developedthere. This reaction, called the P-K reaction (named for itsoriginators, Prausnitz and Kustner), was the basis for the firstbiological assay for IgE activity.
Actual identification of IgE was accomplished by K. and T. Ishizaka in 1966. They obtained serum from an allergic in-
92 P A R T I I Generation of B-Cell and T-Cell Responses
IgG3 IgG4IgG2IgG1
Disulfidebond
FIGURE 4-14 General structure of the four subclasses of humanIgG, which differ in the number and arrangement of the interchain
disulfide bonds (thick black lines) linking the heavy chains. A notablefeature of human IgG3 is its 11 interchain disulfide bonds.
8536d_ch04_076-104 9/5/02 6:19 AM Page 92 mac76 mac76:385 Goldsby et al./Immunology5e:
Estructura Igs poliméricas
24polypeptide (see Figure 4-13d). The IgA of external secre-tions, called secretory IgA, consists of a dimer or tetramer, aJ-chain polypeptide, and a polypeptide chain called secre-tory component (Figure 4-15a, page 93). As is explained be-low, secretory component is derived from the receptor that isresponsible for transporting polymeric IgA across cell mem-branes. The J-chain polypeptide in IgA is identical to thatfound in pentameric IgM and serves a similar function in fa-
cilitating the polymerization of both serum IgA and secre-tory IgA. The secretory component is a 70,000-MW polypep-tide produced by epithelial cells of mucous membranes. Itconsists of five immunoglobulin-like domains that bind tothe Fc region domains of the IgA dimer. This interaction isstabilized by a disulfide bond between the fifth domain of thesecretory component and one of the chains of the dimericIgA.
Antibodies: Structure and Function C H A P T E R 4 91
(b) IgD
VL
C!1CL
C!2
VH
C!3
(d) IgA (dimer)
Hinge region
VL
C"1CL
C"2
VH
C"3
(c) IgE
C#1
C#3
VH
C#4
VL
CL
C#2
Cµ1
Cµ3
VH
Cµ4
VL
CL
Cµ2
J chain
Disulfidebond
(e) IgM (pentamer)
J chain
VL
C$1
C$2
VH
C$3
Hinge region
(a) IgG
CL
FIGURE 4-13 General structures of the five major classes of se-creted antibody. Light chains are shown in shades of pink, disulfidebonds are indicated by thick black lines. Note that the IgG, IgA, andIgD heavy chains (blue, orange, and green, respectively) contain fourdomains and a hinge region, whereas the IgM and IgE heavy chains(purple and yellow, respectively) contain five domains but no hingeregion. The polymeric forms of IgM and IgA contain a polypeptide,
called the J chain, that is linked by two disulfide bonds to the Fc re-gion in two different monomers. Serum IgM is always a pentamer;most serum IgA exists as a monomer, although dimers, trimers, andeven tetramers are sometimes present. Not shown in these figuresare intrachain disulfide bonds and disulfide bonds linking light andheavy chains (see Figure 4-2).
8536d_ch04_076-104 9/5/02 6:19 AM Page 91 mac76 mac76:385 Goldsby et al./Immunology5e:
Mucosas y secrecionesResistente a digestión
Intravascular
Menor afinidad mas valencias- avidez suficiente
length and amino acid composition of the CDR loops, theability of these loops to significantly change conformationupon antigen binding enables antibodies to assume a shapemore effectively complementary to that of their epitopes.
As already indicated, conformational changes followingantigen binding need not be limited to the antibody. Al-though it is not shown in Figure 4-11, the conformation ofthe protease peptide bound to the Fab shows no structuralsimilarity to the corresponding epitope in the native HIVprotease. It has been suggested that the inhibition of proteaseactivity by this anti-HIV protease antibody is a result of itsdistortion of the enzyme’s native conformation.
Constant-Region DomainsThe immunoglobulin constant-region domains take part invarious biological functions that are determined by theamino acid sequence of each domain.
CH1 AND CL DOMAINS
The CH1 and CL domains serve to extend the Fab arms of theantibody molecule, thereby facilitating interaction with anti-gen and increasing the maximum rotation of the Fab arms.In addition, these constant-region domains help to hold theVH and VL domains together by virtue of the interchaindisulfide bond between them (see Figure 4-6). Also, the CH1and CL domains may contribute to antibody diversity by al-lowing more random associations between VH and VL do-mains than would occur if this association were driven by the
VH/VL interaction alone. These considerations have impor-tant implications for building a diverse repertoire of anti-bodies. As Chapter 5 will show, random rearrangements ofthe immunoglobulin genes generate unique VH and VL se-quences for the heavy and light chains expressed by each Blymphocyte; association of the VH and VL sequences thengenerates a unique antigen-binding site. The presence of CH1and CL domains appears to increase the number of stable VH
and VL interactions that are possible, thus contributing to theoverall diversity of antibody molecules that can be expressedby an animal.
HINGE REGION
The !, ", and # heavy chains contain an extended peptide se-quence between the CH1 and CH2 domains that has no ho-mology with the other domains (see Figure 4-8). This region,called the hinge region, is rich in proline residues and is flex-ible, giving IgG, IgD, and IgA segmental flexibility. As a result,the two Fab arms can assume various angles to each otherwhen antigen is bound. This flexibility of the hinge regioncan be visualized in electron micrographs of antigen-anti-body complexes. For example, when a molecule containingtwo dinitrophenol (DNP) groups reacts with anti-DNP anti-body and the complex is captured on a grid, negativelystained, and observed by electron microscopy, large com-plexes (e.g., dimers, trimers, tetramers) are seen. The anglebetween the arms of the Y-shaped antibody molecules differsin the different complexes, reflecting the flexibility of thehinge region (Figure 4-12).
86 P A R T I I Generation of B-Cell and T-Cell Responses
(a)
DNP ligand O2N
NO2
N N NO2
NO2
25Å
S SS S S S
S S
S S S S
S S
SS S
S
Anti-DNP
Ag-Ab Trimer
Hingeregion
DNPligand
(b)
FIGURE 4-12 Experimental demonstration of the flexibility of thehinge region in antibody molecules. (a) A hapten in which two dini-trophenyl (DNP) groups are tethered by a short connecting spacergroup reacts with anti-DNP antibodies to form trimers, tetramers,and other larger antigen-antibody complexes. A trimer is shownschematically. (b) In an electron micrograph of a negatively stainedpreparation of these complexes, two triangular trimeric structures
are clearly visible. The antibody protein stands out as a light struc-ture against the electron-dense background. Because of the flexibilityof the hinge region, the angle between the arms of the antibody mol-ecules varies. [Photograph from R. C. Valentine and N. M. Green,1967, J. Mol. Biol. 27:615; reprinted by permission of Academic PressInc. (London) Ltd.]
8536d_ch04_076-104 9/5/02 6:19 AM Page 86 mac76 mac76:385 Goldsby et al./Immunology5e:
IgA
25
dividual and immunized rabbits with it to prepare anti-isotype antiserum. The rabbit antiserum was then allowed toreact with each class of human antibody known at that time(i.e., IgG, IgA, IgM, and IgD). In this way, each of the knownanti-isotype antibodies was precipitated and removed fromthe rabbit anti-serum. What remained was an anti-isotypeantibody specific for an unidentified class of antibody. Thisantibody turned out to completely block the P-K reaction.The new antibody was called IgE (in reference to the E anti-gen of ragweed pollen, which is a potent inducer of this classof antibody).
IgE binds to Fc receptors on the membranes of blood ba-sophils and tissue mast cells. Cross-linkage of receptor-bound IgE molecules by antigen (allergen) induces basophilsand mast cells to translocate their granules to the plasmamembrane and release their contents to the extracellular en-vironment, a process known as degranulation. As a result, avariety of pharmacologically active mediators are releasedand give rise to allergic manifestations (Figure 4-16). Local-ized mast-cell degranulation induced by IgE also may releasemediators that facilitate a buildup of various cells necessaryfor antiparasitic defense (see Chapter 15).
Antibodies: Structure and Function C H A P T E R 4 93
FIGURE 4-15 Structure and formation of secretory IgA. (a) Secre-tory IgA consists of at least two IgA molecules, which are covalentlylinked to each other through a J chain and are also covalently linkedwith the secretory component. The secretory component containsfive Ig-like domains and is linked to dimeric IgA by a disulfide bondbetween its fifth domain and one of the IgA heavy chains. (b) Secre-
Plasma cell
(a) Structure of secretory IgA
J chain
Secretorycomponent
(b) Formation of secretory IgA
Dimeric IgA
Poly-Igreceptor
Vesicle
Enzymaticcleavage
SecretoryIgA
Epithelial cells
Lumen
Submucosa
tory IgA is formed during transport through mucous membrane epithelial cells. Dimeric IgA binds to a poly-Ig receptor on the baso-lateral membrane of an epithelial cell and is internalized by receptor-mediated endocytosis. After transport of the receptor-IgA complex to the luminal surface, the poly-Ig receptor is enzymatically cleaved,releasing the secretory component bound to the dimeric IgA.
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✦Transcitosis✦Producida por cels plasmatica de la mucosa y médula ósea. Cada día se producen de 5 a 15 g más que todas las otras Igs✦IgA de la leche materna protege al recién nacido✦Varios grados de polimerización
IgE mediadora de las reacciones alérgicas
26
Immunoglobulin D (IgD)IgD was first discovered when a patient developed a multiplemyeloma whose myeloma protein failed to react with anti-isotype antisera against the then-known isotypes: IgA, IgM,and IgG. When rabbits were immunized with this myelomaprotein, the resulting antisera were used to identify the sameclass of antibody at low levels in normal human serum. Thenew class, called IgD, has a serum concentration of 30 !g/mland constitutes about 0.2% of the total immunoglobulin inserum. IgD, together with IgM, is the major membrane-bound immunoglobulin expressed by mature B cells, and itsrole in the physiology of B cells is under investigation. No bi-ological effector function has been identified for IgD.
Antigenic Determinants on ImmunoglobulinsSince antibodies are glycoproteins, they can themselves func-tion as potent immunogens to induce an antibody response.Such anti-Ig antibodies are powerful tools for the study ofB-cell development and humoral immune responses. Theantigenic determinants, or epitopes, on immunoglobulinmolecules fall into three major categories: isotypic, allotypic,and idiotypic determinants, which are located in characteris-tic portions of the molecule (Figure 4-17).
IsotypeIsotypic determinants are constant-region determinants thatcollectively define each heavy-chain class and subclass and
94 P A R T I I Generation of B-Cell and T-Cell Responses
TABLE 4-3 Immune benefits of breast milk
Antibodies of Bind to microbes in baby’s digestive tract and thereby prevent their attachment to the walls of the gut and their secretory IgA class subsequent passage into the body’s tissues.
B12 binding protein Reduces amount of vitamin B12, which bacteria need in order to grow.
Bifidus factor Promotes growth of Lactobacillus bifidus, a harmless bacterium, in baby’s gut. Growth of such nonpathogenic bacteria helps to crowd out dangerous varieties.
Fatty acids Disrupt membranes surrounding certain viruses and destroy them.
Fibronectin Increases antimicrobial activity of macrophages; helps to repair tissues that have been damaged by immune reactions in baby’s gut.
Hormones and Stimulate baby’s digestive tract to mature more quickly. Once the initially “leaky” membranes lining the gut growth factors mature, infants become less vulnerable to microorganisms.
Interferon (IFN-") Enhances antimicrobial activity of immune cells.
Lactoferrin Binds to iron, a mineral many bacteria need to survive. By reducing the available amount of iron, lactoferrinthwarts growth of pathogenic bacteria.
Lysozyme Kills bacteria by disrupting their cell walls.
Mucins Adhere to bacteria and viruses, thus keeping such microorganisms from attaching to mucosal surfaces.
Oligosaccharides Bind to microorganisms and bar them from attaching to mucosal surfaces.
SOURCE: Adapted from J. Newman, 1995, How breast milk protects newborns, Sci. Am. 273(6):76.
FIGURE 4-16 Allergen cross-linkage of receptor-bound IgE onmast cells induces degranulation, causing release of substances(blue dots) that mediate allergic manifestations.
Mast cell
Allergen
Granule
Histamine andother substancesthat mediateallergic reactions
IgE
Fc receptorspecific for IgE
Degranulationand release ofgranule contents
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★El mastocito amplifica la consecuencias de la reacción Ag-Ac
★Contribuye a la eliminación de parásitos
★Media las reacciones alérgicas
27
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocito
sLinfocitos
Isotipos de las inmunoglobulinas y funciones
28
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocito
sLinfocitos
Isotipos de las inmunoglobulinas y funciones
29
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocito
sLinfocitos
Isotipos de las inmunoglobulinas y funciones
30
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocito
sLinfocitos
Isotipos de las inmunoglobulinas y funciones
31
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocito
sLinfocitos
Isotipos de las inmunoglobulinas y funciones
32
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocito
sLinfocitos
Isotipos de las inmunoglobulinas y funciones
33
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocito
sLinfocitos
Isotipos de las inmunoglobulinas y funciones
34
IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Peso molecular 150.000 150.000 150.000 150.000 150.000600.000
150.000600.00
900.000 190.000 150.000
Cadena pesada γ1 γ2 γ3 γ4 α1 α2 µ ε δ
Conc en suero (mg/dl)
500-900 200-500 20-70 20-80 70-40070-400 40-230 0,03<100 KU
<10
Vida media 23 23 8 23 6 6 5 2,5 3
Activación complemento
+ +/- ++ - - - ++ - -
Cruza la placenta + +/- + + - - - - -
Parte del BCR cel B madura
- - - - - - + - +
Transcitosis mucosa
- - - - ++ ++ + - -
Degranulación mastocitos
- - - - - - - + -
Compartimento vascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelularvascular y liquido extracelular Mucosas y secrecionesMucosas y
secrecionesIntravasc
ularMastocitos Linfocitos
Isotipos de las inmunoglobulinas y funciones
Isotipo, alotipo e idiotipo
35
each light-chain type and subtype within a species (see Fig-ure 4-17a). Each isotype is encoded by a separate constant-region gene, and all members of a species carry the sameconstant-region genes (which may include multiple alleles).Within a species, each normal individual will express all iso-types in the serum. Different species inherit different con-stant-region genes and therefore express different isotypes.Therefore, when an antibody from one species is injectedinto another species, the isotypic determinants will be recog-nized as foreign, inducing an antibody response to the iso-typic determinants on the foreign antibody. Anti-isotype
antibody is routinely used for research purposes to deter-mine the class or subclass of serum antibody produced dur-ing an immune response or to characterize the class ofmembrane-bound antibody present on B cells.
AllotypeAlthough all members of a species inherit the same set of iso-type genes, multiple alleles exist for some of the genes (seeFigure 4-17b). These alleles encode subtle amino acid differ-ences, called allotypic determinants, that occur in some, butnot all, members of a species. The sum of the individual allo-typic determinants displayed by an antibody determines itsallotype. In humans, allotypes have been characterized forall four IgG subclasses, for one IgA subclass, and for the !light chain. The "-chain allotypes are referred to as Gmmarkers. At least 25 different Gm allotypes have been identi-fied; they are designated by the class and subclass followed bythe allele number, for example, G1m(1), G2m(23), G3m(11),G4m(4a). Of the two IgA subclasses, only the IgA2 sub-class has allotypes, as A2m(1) and A2m(2). The ! light chain has three allotypes, designated !m(1), !m(2), and!m(3). Each of these allotypic determinants represents dif-ferences in one to four amino acids that are encoded by different alleles.
Antibody to allotypic determinants can be produced byinjecting antibodies from one member of a species into an-other member of the same species who carries different allo-typic determinants. Antibody to allotypic determinantssometimes is produced by a mother during pregnancy in re-sponse to paternal allotypic determinants on the fetal im-munoglobulins. Antibodies to allotypic determinants canalso arise from a blood transfusion.
IdiotypeThe unique amino acid sequence of the VH and VL domainsof a given antibody can function not only as an antigen-bind-ing site but also as a set of antigenic determinants. The idio-typic determinants arise from the sequence of the heavy- andlight-chain variable regions. Each individual antigenic deter-minant of the variable region is referred to as an idiotope(see Figure 4-17c). In some cases an idiotope may be the ac-tual antigen-binding site, and in some cases an idiotope maycomprise variable-region sequences outside of the antigen-binding site. Each antibody will present multiple idiotopes;the sum of the individual idiotopes is called the idiotype ofthe antibody.
Because the antibodies produced by individual B cells de-rived from the same clone have identical variable-region se-quences, they all have the same idiotype. Anti-idiotypeantibody is produced by injecting antibodies that have mini-mal variation in their isotypes and allotypes, so that the idio-typic difference can be recognized. Often a homogeneousantibody such as myeloma protein or monoclonal antibodyis used. Injection of such an antibody into a recipient who is
Antibodies: Structure and Function C H A P T E R 4 95
FIGURE 4-17 Antigenic determinants of immunoglobulins. Foreach type of determinant, the general location of determinants withinthe antibody molecule is shown (left) and two examples are illus-trated (center and right). (a) Isotypic determinants are constant-region determinants that distinguish each Ig class and subclasswithin a species. (b) Allotypic determinants are subtle amino acid differences encoded by different alleles of isotype genes. Allotypic differences can be detected by comparing the same antibody classamong different inbred strains. (c) Idiotypic determinants are gen-erated by the conformation of the amino acid sequences of theheavy- and light-chain variable regions specific for each antigen. Eachindividual determinant is called an idiotope, and the sum of the indi-vidual idiotopes is the idiotype.
(a) Isotypic determinants
Mouse IgG1 Mouse IgM
!1 µ "
(b) Allotypic determinants
Mouse IgG1(strain A)
Mouse IgG1(strain B)
!1 "
(c) Idiotypic determinants
Mouse IgG1against antigen a
"
"
!1 "
Mouse IgG1against antigen b
!1
Idiotopes
"
!1
Idiotopes
8536d_ch04_076-104 9/5/02 6:19 AM Page 95 mac76 mac76:385 Goldsby et al./Immunology5e:
Variantes presentes en cada indivíduo
Variantes entre individuosReacciones transfusionales menores
Variantes entre clonasProblema terapia con anticuerpos monoclonales
! La pregunta
¿Como se genera una variedad tan elevada de anticuerpos a partir de un número limitado de genes?
Las respuestasHipotesis instructivaHipotesis genética
Base del experimento de Tonegawa
37
Tonegawa’s Bombshell—ImmunoglobulinGenes RearrangeIn 1976, S. Tonegawa and N. Hozumi found the first directevidence that separate genes encode the V and C regions ofimmunoglobulins and that the genes are rearranged in thecourse of B-cell differentiation. This work changed the fieldof immunology. In 1987, Tonegawa was awarded the NobelPrize for this work.
Selecting DNA from embryonic cells and adult myelomacells—cells at widely different stages of development—Tonegawa and Hozumi used various restriction endonucle-ases to generate DNA fragments. The fragments were thenseparated by size and analyzed for their ability to hybridizewith a radiolabeled mRNA probe. Two separate restrictionfragments from the embryonic DNA hybridized with themRNA, whereas only a single restriction fragment of theadult myeloma DNA hybridized with the same probe. Tone-gawa and Hozumi suggested that, during differentiation oflymphocytes from the embryonic state to the fully differenti-ated plasma-cell stage (represented in their system by the
myeloma cells), the V and C genes undergo rearrangement.In the embryo, the V and C genes are separated by a largeDNA segment that contains a restriction-endonuclease site;during differentiation, the V and C genes are brought closertogether and the intervening DNA sequence is eliminated.
The pioneering experiments of Tonegawa and Hozumiemployed a tedious and time-consuming procedure that hassince been replaced by the much more powerful approach ofSouthern-blot analysis. This method, now universally used toinvestigate the rearrangement of immunoglobulin genes,eliminates the need to elute the separated DNA restrictionfragments from gel slices prior to analysis by hybridizationwith an immunoglobulin gene segment probe. Figure 5-2shows the detection of rearrangement at the ! light-chain lo-cus by comparing the fragments produced by digestion ofDNA from a clone of B-lineage cells with the pattern ob-tained by digestion of non-B cells (e.g., sperm or liver cells).The rearrangement of a V gene deletes an extensive section ofgerm-line DNA, thereby creating differences between re-arranged and unrearranged Ig loci in the distribution andnumber of restriction sites. This results in the generation of
108 P A R T I I Generation of B-Cell and T-Cell Responses
FIGURE 5-2 Experimental basis for diagnosis of rearrangement atan immunoglobulin locus. The number and size of restriction frag-ments generated by the treatment of DNA with a restriction enzymeis determined by the sequence of the DNA.The digestion of re-arranged DNA with a restriction enzyme (RE) yields a pattern of re-striction fragments that differ from those obtained by digestion of anunrearranged locus with the same RE. Typically, the fragments are an-alyzed by the technique of Southern blotting. In this example, a probethat includes a J gene segment is used to identify RE digestion frag-ments that include all or portions of this segment. As shown, re-arrangement results in the deletion of a segment of germ-line DNAand the loss of the restriction sites that it includes. It also results inthe joining of gene segments, in this case a V and a J segment, that
are separated in the germ line. Consequently, fragments dependenton the presence of this segment for their generation are absent fromthe restriction-enzyme digest of DNA from the rearranged locus. Fur-thermore, rearranged DNA gives rise to novel fragments that are ab-sent from digests of DNA in the germ-line configuration. This can beuseful because both B cells and non-B cells have two immunoglobu-lin 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 lo-cus, digestion of DNA from a myeloma or normal B-cell clone willproduce a pattern of restriction that includes all of those in a germ-line digest plus any novel fragments that are generated from thechange in DNA sequence that accompanies rearrangement. Notethat only one of the several J gene segements present is shown.
3!5!Vn
RE
V2
RE
J C
V1
RE RE RE
3!5!Vn
RE
V2
RE
V1
RE RE
REGerm line Rearranged
Germ line Rearranged
Deleted
Rearrangement
J C
Probe Probe
RE digestion RE digestion
Southernblot
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Base del experimento de Tonegawa
38
Tonegawa’s Bombshell—ImmunoglobulinGenes RearrangeIn 1976, S. Tonegawa and N. Hozumi found the first directevidence that separate genes encode the V and C regions ofimmunoglobulins and that the genes are rearranged in thecourse of B-cell differentiation. This work changed the fieldof immunology. In 1987, Tonegawa was awarded the NobelPrize for this work.
Selecting DNA from embryonic cells and adult myelomacells—cells at widely different stages of development—Tonegawa and Hozumi used various restriction endonucle-ases to generate DNA fragments. The fragments were thenseparated by size and analyzed for their ability to hybridizewith a radiolabeled mRNA probe. Two separate restrictionfragments from the embryonic DNA hybridized with themRNA, whereas only a single restriction fragment of theadult myeloma DNA hybridized with the same probe. Tone-gawa and Hozumi suggested that, during differentiation oflymphocytes from the embryonic state to the fully differenti-ated plasma-cell stage (represented in their system by the
myeloma cells), the V and C genes undergo rearrangement.In the embryo, the V and C genes are separated by a largeDNA segment that contains a restriction-endonuclease site;during differentiation, the V and C genes are brought closertogether and the intervening DNA sequence is eliminated.
The pioneering experiments of Tonegawa and Hozumiemployed a tedious and time-consuming procedure that hassince been replaced by the much more powerful approach ofSouthern-blot analysis. This method, now universally used toinvestigate the rearrangement of immunoglobulin genes,eliminates the need to elute the separated DNA restrictionfragments from gel slices prior to analysis by hybridizationwith an immunoglobulin gene segment probe. Figure 5-2shows the detection of rearrangement at the ! light-chain lo-cus by comparing the fragments produced by digestion ofDNA from a clone of B-lineage cells with the pattern ob-tained by digestion of non-B cells (e.g., sperm or liver cells).The rearrangement of a V gene deletes an extensive section ofgerm-line DNA, thereby creating differences between re-arranged and unrearranged Ig loci in the distribution andnumber of restriction sites. This results in the generation of
108 P A R T I I Generation of B-Cell and T-Cell Responses
FIGURE 5-2 Experimental basis for diagnosis of rearrangement atan immunoglobulin locus. The number and size of restriction frag-ments generated by the treatment of DNA with a restriction enzymeis determined by the sequence of the DNA.The digestion of re-arranged DNA with a restriction enzyme (RE) yields a pattern of re-striction fragments that differ from those obtained by digestion of anunrearranged locus with the same RE. Typically, the fragments are an-alyzed by the technique of Southern blotting. In this example, a probethat includes a J gene segment is used to identify RE digestion frag-ments that include all or portions of this segment. As shown, re-arrangement results in the deletion of a segment of germ-line DNAand the loss of the restriction sites that it includes. It also results inthe joining of gene segments, in this case a V and a J segment, that
are separated in the germ line. Consequently, fragments dependenton the presence of this segment for their generation are absent fromthe restriction-enzyme digest of DNA from the rearranged locus. Fur-thermore, rearranged DNA gives rise to novel fragments that are ab-sent from digests of DNA in the germ-line configuration. This can beuseful because both B cells and non-B cells have two immunoglobu-lin 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 lo-cus, digestion of DNA from a myeloma or normal B-cell clone willproduce a pattern of restriction that includes all of those in a germ-line digest plus any novel fragments that are generated from thechange in DNA sequence that accompanies rearrangement. Notethat only one of the several J gene segements present is shown.
3!5!Vn
RE
V2
RE
J C
V1
RE RE RE
3!5!Vn
RE
V2
RE
V1
RE RE
REGerm line Rearranged
Germ line Rearranged
Deleted
Rearrangement
J C
Probe Probe
RE digestion RE digestion
Southernblot
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two signal sequences and the adjacent coding sequences(gene segments) are brought into proximity
! Cleavage of one strand of DNA by RAG-1 and RAG-2 atthe junctures of the signal sequences and coding sequences
! A reaction catalyzed by RAG-1 and RAG-2 in which thefree 3!-OH group on the cut DNA strand attacks thephosphodiester bond linking the opposite strand to thesignal sequence, simultaneously producing a hairpinstructure at the cut end of the coding sequence and aflush, 5!-phosphorylated, double-strand break at thesignal sequence
! Cutting of the hairpin to generate sites for the additionof P-region nucleotides, followed by the trimming of afew nucleotides from the coding sequence by a single-strand endonuclease
! Addition of up to 15 nucleotides, called N-regionnucleotides, at the cut ends of the V, D, and J codingsequences of the heavy chain by the enzyme terminaldeoxynucleotidyl transferase
! Repair and ligation to join the coding sequences and tojoin the signal sequences, catalyzed by normal double-strand break repair (DSBR) enzymes
Recombination results in the formation of a coding joint,falling between the coding sequences, and a signal joint, be-tween the RSSs. The transcriptional orientation of the genesegments to be joined determines the fate of the signal jointand intervening DNA. When the two gene segments are inthe same transcriptional orientation, joining results in dele-tion of the signal joint and intervening DNA as a circular ex-cision product (Figure 5-8). Less frequently, the two genesegments have opposite orientations. In this case joining oc-curs by inversion of the DNA, resulting in the retention of
114 P A R T I I Generation of B-Cell and T-Cell Responses
(a) Deletional joining
3!5!V" J"
RSS
3!5!V" J"
(b) Inversional joining
3!
5!
3!
Recognition of RSSsby RAG-1/2 and synapsis
V"L J"
Coding joint
5! 3!
Signal joint
Signaljoint Coding
joint
Single-strandDNA cleavageby RAG-1/2
Hairpin formationand double-strandDNA break byRAG-1/2
Random cleavage of hairpin by endonuclease generatessites for the additionof P-nucleotides
Optional additionto H-chain segments of N-nucleotides by TdT
Repair and ligationof coding and signal sequencesto form joints by DSBR enzymes
1
2
3
4
5
= Two-turn RSS
= One-turn RSS
L L
+
FIGURE 5-7 Model depicting the general process of recombina-tion of immunoglobulin gene segments is illustrated with V" and J".(a) Deletional joining occurs when the gene segments to be joinedhave the same transcriptional orientation (indicated by horizontalblue arrows). This process yields two products: a rearranged VJ unitthat includes the coding joint, and a circular excision product con-sisting of the recombination signal sequences (RSSs), signal joint,and intervening DNA. (b) Inversional joining occurs when the genesegments have opposite transcriptional orientations. In this case, theRSSs, signal joint, and intervening DNA are retained, and the orien-tation of one of the joined segments is inverted. In both types of re-combination, a few nucleotides may be deleted from or added to thecut ends of the coding sequences before they are rejoined.
FIGURE 5-8 Circular DNA isolated from thymocytes in which theDNA encoding the chains of the T-cell receptor (TCR) undergoes re-arrangement in a process like that involving the immunoglobulingenes. Isolation of this circular excision product is direct evidence forthe mechanism of deletional joining shown in Figure 5-7. [From K.Okazaki et al., 1987, Cell 49:477.]
8536d_ch05_105-136 8/22/02 2:47 PM Page 114 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
Pregunta ¿Cómo se genera la enorme variedad de anticuerpos?
✦ Respuesta, combinando elementos, como en un alfabeto
✦ Se comprende cuando se analiza la estructura de los genes de las Igs
✦ Hay un elemento de azar
39
Mecánica de la reordenación
40
two signal sequences and the adjacent coding sequences(gene segments) are brought into proximity
! Cleavage of one strand of DNA by RAG-1 and RAG-2 atthe junctures of the signal sequences and coding sequences
! A reaction catalyzed by RAG-1 and RAG-2 in which thefree 3!-OH group on the cut DNA strand attacks thephosphodiester bond linking the opposite strand to thesignal sequence, simultaneously producing a hairpinstructure at the cut end of the coding sequence and aflush, 5!-phosphorylated, double-strand break at thesignal sequence
! Cutting of the hairpin to generate sites for the additionof P-region nucleotides, followed by the trimming of afew nucleotides from the coding sequence by a single-strand endonuclease
! Addition of up to 15 nucleotides, called N-regionnucleotides, at the cut ends of the V, D, and J codingsequences of the heavy chain by the enzyme terminaldeoxynucleotidyl transferase
! Repair and ligation to join the coding sequences and tojoin the signal sequences, catalyzed by normal double-strand break repair (DSBR) enzymes
Recombination results in the formation of a coding joint,falling between the coding sequences, and a signal joint, be-tween the RSSs. The transcriptional orientation of the genesegments to be joined determines the fate of the signal jointand intervening DNA. When the two gene segments are inthe same transcriptional orientation, joining results in dele-tion of the signal joint and intervening DNA as a circular ex-cision product (Figure 5-8). Less frequently, the two genesegments have opposite orientations. In this case joining oc-curs by inversion of the DNA, resulting in the retention of
114 P A R T I I Generation of B-Cell and T-Cell Responses
(a) Deletional joining
3!5!V" J"
RSS
3!5!V" J"
(b) Inversional joining
3!
5!
3!
Recognition of RSSsby RAG-1/2 and synapsis
V"L J"
Coding joint
5! 3!
Signal joint
Signaljoint Coding
joint
Single-strandDNA cleavageby RAG-1/2
Hairpin formationand double-strandDNA break byRAG-1/2
Random cleavage of hairpin by endonuclease generatessites for the additionof P-nucleotides
Optional additionto H-chain segments of N-nucleotides by TdT
Repair and ligationof coding and signal sequencesto form joints by DSBR enzymes
1
2
3
4
5
= Two-turn RSS
= One-turn RSS
L L
+
FIGURE 5-7 Model depicting the general process of recombina-tion of immunoglobulin gene segments is illustrated with V" and J".(a) Deletional joining occurs when the gene segments to be joinedhave the same transcriptional orientation (indicated by horizontalblue arrows). This process yields two products: a rearranged VJ unitthat includes the coding joint, and a circular excision product con-sisting of the recombination signal sequences (RSSs), signal joint,and intervening DNA. (b) Inversional joining occurs when the genesegments have opposite transcriptional orientations. In this case, theRSSs, signal joint, and intervening DNA are retained, and the orien-tation of one of the joined segments is inverted. In both types of re-combination, a few nucleotides may be deleted from or added to thecut ends of the coding sequences before they are rejoined.
FIGURE 5-8 Circular DNA isolated from thymocytes in which theDNA encoding the chains of the T-cell receptor (TCR) undergoes re-arrangement in a process like that involving the immunoglobulingenes. Isolation of this circular excision product is direct evidence forthe mechanism of deletional joining shown in Figure 5-7. [From K.Okazaki et al., 1987, Cell 49:477.]
8536d_ch05_105-136 8/22/02 2:47 PM Page 114 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
Dependiendo de la orientación RSS se genera o no un circulo de DNA (TREC)
two signal sequences and the adjacent coding sequences(gene segments) are brought into proximity
! Cleavage of one strand of DNA by RAG-1 and RAG-2 atthe junctures of the signal sequences and coding sequences
! A reaction catalyzed by RAG-1 and RAG-2 in which thefree 3!-OH group on the cut DNA strand attacks thephosphodiester bond linking the opposite strand to thesignal sequence, simultaneously producing a hairpinstructure at the cut end of the coding sequence and aflush, 5!-phosphorylated, double-strand break at thesignal sequence
! Cutting of the hairpin to generate sites for the additionof P-region nucleotides, followed by the trimming of afew nucleotides from the coding sequence by a single-strand endonuclease
! Addition of up to 15 nucleotides, called N-regionnucleotides, at the cut ends of the V, D, and J codingsequences of the heavy chain by the enzyme terminaldeoxynucleotidyl transferase
! Repair and ligation to join the coding sequences and tojoin the signal sequences, catalyzed by normal double-strand break repair (DSBR) enzymes
Recombination results in the formation of a coding joint,falling between the coding sequences, and a signal joint, be-tween the RSSs. The transcriptional orientation of the genesegments to be joined determines the fate of the signal jointand intervening DNA. When the two gene segments are inthe same transcriptional orientation, joining results in dele-tion of the signal joint and intervening DNA as a circular ex-cision product (Figure 5-8). Less frequently, the two genesegments have opposite orientations. In this case joining oc-curs by inversion of the DNA, resulting in the retention of
114 P A R T I I Generation of B-Cell and T-Cell Responses
(a) Deletional joining
3!5!V" J"
RSS
3!5!V" J"
(b) Inversional joining
3!
5!
3!
Recognition of RSSsby RAG-1/2 and synapsis
V"L J"
Coding joint
5! 3!
Signal joint
Signaljoint Coding
joint
Single-strandDNA cleavageby RAG-1/2
Hairpin formationand double-strandDNA break byRAG-1/2
Random cleavage of hairpin by endonuclease generatessites for the additionof P-nucleotides
Optional additionto H-chain segments of N-nucleotides by TdT
Repair and ligationof coding and signal sequencesto form joints by DSBR enzymes
1
2
3
4
5
= Two-turn RSS
= One-turn RSS
L L
+
FIGURE 5-7 Model depicting the general process of recombina-tion of immunoglobulin gene segments is illustrated with V" and J".(a) Deletional joining occurs when the gene segments to be joinedhave the same transcriptional orientation (indicated by horizontalblue arrows). This process yields two products: a rearranged VJ unitthat includes the coding joint, and a circular excision product con-sisting of the recombination signal sequences (RSSs), signal joint,and intervening DNA. (b) Inversional joining occurs when the genesegments have opposite transcriptional orientations. In this case, theRSSs, signal joint, and intervening DNA are retained, and the orien-tation of one of the joined segments is inverted. In both types of re-combination, a few nucleotides may be deleted from or added to thecut ends of the coding sequences before they are rejoined.
FIGURE 5-8 Circular DNA isolated from thymocytes in which theDNA encoding the chains of the T-cell receptor (TCR) undergoes re-arrangement in a process like that involving the immunoglobulingenes. Isolation of this circular excision product is direct evidence forthe mechanism of deletional joining shown in Figure 5-7. [From K.Okazaki et al., 1987, Cell 49:477.]
8536d_ch05_105-136 8/22/02 2:47 PM Page 114 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
Opciones de la unión de segmentos
41
both the coding joint and the signal joint (and interveningDNA) on the chromosome. In the human ! locus, about halfof the V! gene segments are inverted with respect to J! andtheir joining is thus by inversion.
Ig-Gene Rearrangements May Be Productive or NonproductiveOne of the striking features of gene-segment recombinationis the diversity of the coding joints that are formed betweenany two gene segments. Although the double-strand DNAbreaks that initiate V-(D)-J rearrangements are introducedprecisely at the junctions of signal sequences and coding se-quences, the subsequent joining of the coding sequences isimprecise. Junctional diversity at the V-J and V-D-J codingjoints is generated by a number of mechanisms: variation incutting of the hairpin to generate P-nucleotides, variation intrimming of the coding sequences, variation in N-nucleotideaddition, and flexibility in joining the coding sequences. Theintroduction of randomness in the joining process helps gen-erate antibody diversity by contributing to the hypervariabil-ity of the antigen-binding site. (This phenomenon is coveredin more detail below in the section on generation of antibodydiversity.)
Another consequence of imprecise joining is that genesegments may be joined out of phase, so that the triplet read-ing frame for translation is not preserved. In such a nonpro-ductive rearrangement, the resulting VJ or VDJ unit is likelyto contain numerous stop codons, which interrupt transla-tion (Figure 5-9). When gene segments are joined in phase,the reading frame is maintained. In such a productive re-arrangement, the resulting VJ or VDJ unit can be translatedin its entirety, yielding a complete antibody.
If one allele rearranges nonproductively, a B cell may stillbe able to rearrange the other allele productively. If an in-phase rearranged heavy-chain and light-chain gene are notproduced, the B cell dies by apoptosis. It is estimated thatonly one in three attempts at VL-JL joining, and one in threesubsequent attempts at VH-DHJH joining, are productive. Asa result, less than 1/9 (11%) of the early-stage pre-B cells inthe bone marrow progress to maturity and leave the bonemarrow as mature immunocompetent B cells.
Allelic Exclusion Ensures a Single Antigenic SpecificityB cells, like all somatic cells, are diploid and contain both ma-ternal and paternal chromosomes. Even though a B cell is
Organization and Expression of Immunoglobulin Genes C H A P T E R 5 115
J!
V!
C A C T G T G G T G G A C T A G G
G A G G A T G C T C C C A C A G T G
RSS
RSS
2
34
5
G A G G A T G C G A C T A G G
Glu Asp Ala Thr Arg
1
G A G G A T G G G A C T A G G
Glu Asp Gly Thr Arg
G A G G A T T G G A C T A G G
Glu Asp Trp Thr Arg
Productive rearrangements
2
3
G A G G A T G C G G A C T A G G
Glu Asp Ala Asp Stop
G A G G T G G A C T A G G
Glu Val Asp Stop
Nonproductive rearrangements
4
5
1
Joiningflexibility
FIGURE 5-9 Junctional flexibility in the joining of immunoglobulingene segments is illustrated with V! and J!. In-phase joining (arrows1, 2, and 3) generates a productive rearrangement, which can betranslated into protein. Out-of-phase joining (arrows 4 and 5) leadsto a nonproductive rearrangement that contains stop codons and isnot translated into protein.
!! "" HH
! " H
** * *
! " H
Paternalchromosomes
Gene rearrangement
Maternalchromosomes
Maternal H chain
Maternal! chain
MaternalH chain
Paternal" chain
FIGURE 5-10 Because of allelic exclusion, the immunoglobulinheavy- and light-chain genes of only one parental chromosome areexpressed per cell. This process ensures that B cells possess a singleantigenic specificity. The allele selected for rearrangement is chosenrandomly. Thus the expressed immunoglobulin may contain one ma-ternal and one paternal chain or both chains may derive from onlyone parent. Only B cells and T cells exhibit allelic exclusion. Asterisks(#) indicate the expressed alleles.
8536d_ch05_105-136 8/22/02 2:47 PM Page 115 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
Reordenamientos productivos
Reordenamientos fallidos
Imprecisión de unión: nucleotidos P y N
Reordenamiento en la otra copia del cromosoma
Exclusión alélica
42
both the coding joint and the signal joint (and interveningDNA) on the chromosome. In the human ! locus, about halfof the V! gene segments are inverted with respect to J! andtheir joining is thus by inversion.
Ig-Gene Rearrangements May Be Productive or NonproductiveOne of the striking features of gene-segment recombinationis the diversity of the coding joints that are formed betweenany two gene segments. Although the double-strand DNAbreaks that initiate V-(D)-J rearrangements are introducedprecisely at the junctions of signal sequences and coding se-quences, the subsequent joining of the coding sequences isimprecise. Junctional diversity at the V-J and V-D-J codingjoints is generated by a number of mechanisms: variation incutting of the hairpin to generate P-nucleotides, variation intrimming of the coding sequences, variation in N-nucleotideaddition, and flexibility in joining the coding sequences. Theintroduction of randomness in the joining process helps gen-erate antibody diversity by contributing to the hypervariabil-ity of the antigen-binding site. (This phenomenon is coveredin more detail below in the section on generation of antibodydiversity.)
Another consequence of imprecise joining is that genesegments may be joined out of phase, so that the triplet read-ing frame for translation is not preserved. In such a nonpro-ductive rearrangement, the resulting VJ or VDJ unit is likelyto contain numerous stop codons, which interrupt transla-tion (Figure 5-9). When gene segments are joined in phase,the reading frame is maintained. In such a productive re-arrangement, the resulting VJ or VDJ unit can be translatedin its entirety, yielding a complete antibody.
If one allele rearranges nonproductively, a B cell may stillbe able to rearrange the other allele productively. If an in-phase rearranged heavy-chain and light-chain gene are notproduced, the B cell dies by apoptosis. It is estimated thatonly one in three attempts at VL-JL joining, and one in threesubsequent attempts at VH-DHJH joining, are productive. Asa result, less than 1/9 (11%) of the early-stage pre-B cells inthe bone marrow progress to maturity and leave the bonemarrow as mature immunocompetent B cells.
Allelic Exclusion Ensures a Single Antigenic SpecificityB cells, like all somatic cells, are diploid and contain both ma-ternal and paternal chromosomes. Even though a B cell is
Organization and Expression of Immunoglobulin Genes C H A P T E R 5 115
J!
V!
C A C T G T G G T G G A C T A G G
G A G G A T G C T C C C A C A G T G
RSS
RSS
2
34
5
G A G G A T G C G A C T A G G
Glu Asp Ala Thr Arg
1
G A G G A T G G G A C T A G G
Glu Asp Gly Thr Arg
G A G G A T T G G A C T A G G
Glu Asp Trp Thr Arg
Productive rearrangements
2
3
G A G G A T G C G G A C T A G G
Glu Asp Ala Asp Stop
G A G G T G G A C T A G G
Glu Val Asp Stop
Nonproductive rearrangements
4
5
1
Joiningflexibility
FIGURE 5-9 Junctional flexibility in the joining of immunoglobulingene segments is illustrated with V! and J!. In-phase joining (arrows1, 2, and 3) generates a productive rearrangement, which can betranslated into protein. Out-of-phase joining (arrows 4 and 5) leadsto a nonproductive rearrangement that contains stop codons and isnot translated into protein.
!! "" HH
! " H
** * *
! " H
Paternalchromosomes
Gene rearrangement
Maternalchromosomes
Maternal H chain
Maternal! chain
MaternalH chain
Paternal" chain
FIGURE 5-10 Because of allelic exclusion, the immunoglobulinheavy- and light-chain genes of only one parental chromosome areexpressed per cell. This process ensures that B cells possess a singleantigenic specificity. The allele selected for rearrangement is chosenrandomly. Thus the expressed immunoglobulin may contain one ma-ternal and one paternal chain or both chains may derive from onlyone parent. Only B cells and T cells exhibit allelic exclusion. Asterisks(#) indicate the expressed alleles.
8536d_ch05_105-136 8/22/02 2:47 PM Page 115 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
Asegura que cada clona de células B expresa una sola especie de molécula de Ig
43
Diversidad de los anticuerpos por combinación al azar de segmentos génicosDiversidad de los anticuerpos por combinación al azar de segmentos génicosDiversidad de los anticuerpos por combinación al azar de segmentos génicosDiversidad de los anticuerpos por combinación al azar de segmentos génicos
Cadena pesada Cadena ligeraCadena ligera
Segmento kappa lambda
V 48 41 34
D 23 0 0
J 6 5 5
Combinaciones 48x23x6=6624 41x5=205 34x5=170
Total 6624x(205+170)=2,48x1066624x(205+170)=2,48x1066624x(205+170)=2,48x106
Mecanismo de generación de diversidad
✦ Múltiples de segmentos génicos (variabilidad individual)
✦ Combinación aleatoria de segmentos V-(D)-J✦ Combinación de cadenas pesadas y ligeras✦ Flexibilidad punto de unión✦ Adición de nucleotidos P y N (TdT transferasa
terminal de dinucleótidos)✦ Hipermutación somática
44
Hipermutación somática
45
Organization and Expression of Immunoglobulin Genes C H A P T E R 5 121
flexibility, P-nucleotide addition, and N-nucleotide additionare within the third CDR, they are positioned to influencethe structure of the antibody binding site. In addition tothese sources of antibody diversity, the phenomenon of so-matic hypermutation contributes enormously to the reper-toire after antigen stimulation.
Class Switching among Constant-Region GenesAfter antigenic stimulation of a B cell, the heavy-chain DNAcan undergo a further rearrangement in which the VHDHJH
unit can combine with any CH gene segment. The exactmechanism of this process, called class switching or iso-type switching, is unclear, but it involves DNA flanking sequences (called switch regions) located 2–3 kb upstreamfrom each CH segment (except C!). These switch regions,though rather large (2 to 10 kb), are composed of multiplecopies of short repeats (GAGCT and TGGGG). One hy-pothesis is that a protein or system of proteins that consti-tute the switch recombinase recognize these repeats andupon binding carry out the DNA recombination that resultsin class switching. Intercellular regulatory proteins knownas cytokines act as “switch factors” and play major roles indetermining the particular immunoglobulin class that is ex-pressed as a consequence of switching. Interleukin 4 (IL-4),
FIGURE 5-14 Experimental evidence for somatic mutation in vari-able regions of immunoglobulin genes. The diagram compares themRNA sequences of heavy chains and of light chains from hybrido-mas specific for the phOx hapten. The horizontal solid lines repre-sent the germ-line VH and V" Ox-1 sequences; dashed lines representsequences derived from other germ-line genes. Blue shading showsthe areas where mutations clustered; the blue circles with verticallines indicate locations of mutations that encode a different aminoacid than the germ-line sequence. These data show that the fre-
quency of mutation (1) increases in the course of the primary re-sponse (day 7 vs. day 14) and (2) is higher after secondary and ter-tiary immunizations than after primary immunization. Moreover, thedissociation constant (Kd) of the anti-phOx antibodies decreases dur-ing the transition from the primary to tertiary response, indicating anincrease in the overall affinity of the antibody. Note also that most ofthe mutations are clustered within CDR1 and CDR2 of both the heavyand the light chains. [Adapted from C. Berek and C. Milstein, 1987, Im-munol. Rev. 96:23.]
Heavy–chain V regions
Day
7
Prim
ary
!1!1!1!1!1!1!1!1!1!1!1!1
!1!3!1!1µµµ!1
!1!1!1!1!1!1!1!1
!1!1!1!1!1!1
CDR1 CDR3CDR2CDR1 CDR2 CDR3J3
3.7
(D)
Hybridomaclonesubclass
Light–chain V regions Kd " 10–7M
J5
Day
14
Seco
ndar
yT
erti
ary
2.82.82.8
3.64.03.30.56.04.00.93.4
0.70.40.10.2
1.40.6
0.90.021.1
0.10.4
# 0.021.0
# 0.03# 0.03# 0.03
0.150.2
J4J4J4
J2
J2J2J4J4
J2J4
J4
J4J4
J4
8536d_ch05_105-136 8/22/02 2:48 PM Page 121 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
Hipermutación somática
✦ Frecuencia de mutaciones en el genoma 1/108 nucleótidos por división
✦ Región VDJ 1/103 -> 1 en las dos regiones V por cada dos divisiones celulares
✦ En los anticuerpos hay una concentración de mutaciones somáticas en las zonas que codifican la regiones determinantes de complementariedad (CDRs) de los segmentos V de las cadenas ligera y pesada
46
47
La respuesta B en el centro germinal
✦ Las FDC contienen Ics con Ag en su superficie y producen CXCL13 que atrae a las células B
✦ Los linfocitos B (centrocitos) que maduran junto a las FDC forman la zona clara mientras que los que proliferan rápidamente constituyen la zona oscura (centroblastos)
✦ A nivel molecular tiene lugar la hipermutación somática y la maduración por afinidad bajo la influencia de las células Th y el cambio de isotipo
✦
MantoCentrocitos
TCD4+
linfosT
Centroblastos
Zona ClaraCD21, FDC
L
DCentroblastos
FDCL
D
Centrocitos
Fases respuesta inmune
✦ Primeros 5-7 días, inmunidad innata✦ 7 en adelante adaptativa primaria (específica)✦ Nueva exposición, respuesta secundaria
IgM Ig
IgM e IgD: procesamiento alternativos
50
plasma cells and subsequent splicing will yield the secretedform of the ! or " heavy chains, respectively (see Figure 5-16b).
Synthesis, Assembly, and Secretion ofImmunoglobulinsImmunoglobulin heavy- and light-chain mRNAs aretranslated on separate polyribosomes of the rough endo-plasmic reticulum (RER). Newly synthesized chains con-tain an amino-terminal leader sequence, which serves toguide the chains into the lumen of the RER, where the sig-nal sequence is then cleaved. The assembly of light (L) andheavy (H) chains into the disulfide-linked and glycosylatedimmunoglobulin molecule occurs as the chains passthrough the cisternae of the RER. The complete moleculesare transported to the Golgi apparatus and then into
secretory vesicles, which fuse with the plasma membrane(Figure 5-18).
The order of chain assembly varies among the im-munoglobulin classes. In the case of IgM, the H and L chainsassemble within the RER to form half-molecules, and thentwo half-molecules assemble to form the complete molecule.In the case of IgG, two H chains assemble, then an H2L inter-mediate is assembled, and finally the complete H2L2 mole-cule is formed. Interchain disulfide bonds are formed, andthe polypeptides are glycosylated as they move through theGolgi apparatus.
If the molecule contains the transmembrane sequence ofthe membrane form, it becomes anchored in the membraneof a secretory vesicle and is inserted into the plasma mem-brane as the vesicle fuses with the plasma membrane (seeFigure 5-18, insert). If the molecule contains the hydrophilicsequence of secreted immunoglobulins, it is transported as afree molecule in a secretory vesicle and is released from thecell when the vesicle fuses with the plasma membrane.
Organization and Expression of Immunoglobulin Genes C H A P T E R 5 125
FIGURE 5-17 Expression of membrane forms of ! and " heavychains by alternative RNA processing. (a) Structure of rearrangedheavy-chain gene showing C! and C" exons and poly-A sites. (b)Structure of !m transcript and !m mRNA resulting from poly-
adenylation at site 2 and splicing. (c) Structure of "m transcript and"m mRNA resulting from polyadenylation at site 4 and splicing.Both processing pathways can proceed in any given B cell.
3!
(a) H-chain primary transcript
VDJJ M1
5!M2µ1 µ2 µ3 µ4
Poly-Asite 1
Poly-Asite 2
Cµ
"1
C"
M1M2
Poly-Asite 4
Poly-Asite 3
"2 "3
#6.5kb
S SL
(A)n
J S
(A)n
µ1 µ2 µ3 µ4
Splicing
M1 M2
M1 M2
µm transcript 5!
VDJ
µm mRNA 5!
VDJ
(b) Polyadenylation of primary transcript at site 2 µm$Cµ
L
L
(A)n
Splicing
M2VDJ
"1 "2 "3
(c) Polyadenylation of primary transcript at site 4 $"m
VDJ
5! (A)n
M1
"m transcript
"m mRNA
Cµ C"
J M1 M2µ1 µ2 µ3 µ4 "1 "2 "3S S M1M2
L
L
8536d_ch05_105-136 8/22/02 3:07 PM Page 125 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e:
Generación formas de membrana o secretadas
51
124 P A R T I I Generation of B-Cell and T-Cell Responses
FIGURE 5-16 Expression of secreted and membrane forms ofthe heavy chain by alternative RNA processing. (a) Amino acid sequence of the carboxyl-terminal end of secreted and membrane! heavy chains. Residues are indicated by the single-letter aminoacid code. Hydrophilic and hydrophobic residues and regions areindicated by purple and orange, respectively, and charged aminoacids are indicated with a " or #. The white regions of the
(b)
PrimaryH-chaintranscript
VDJJ
(A)n
Poly-Asite 1
Poly-Asite 2
Cµ C!
Poly-Asite 3
Poly-Asite 4
Polyadenylation
RNA transcript for secreted µD J µ1 µ2 µ3 µ4
µ1 µ2 µ3 µ4 S
JV S
(A)n
RNA transcript for membrane µD J µ1 µ2 µ3 µ4JV S
(A)n
D µ1 µ2 µ3 µ4JV S
(A)n
D µ1 µ2 µ3 µ4JV
RNA splicing
mRNA encoding secreted µ chain mRNA encoding membrane µ chain
M1 M2
M1 M2
M1 M2
Site 1 Site 2
L
L
L
L
L
Outside
(a) Key:
Hydrophilic
Hydrophobic
T
G
VNAEEEGF
NL
F IV
S AT
LF
S T TYF L S
VT
LFKVK
LL
WT
T
Membrane
Cytoplasm
+
–
556
563
575576
SS bridge
COOH
Cµ4
556
Secreted µ
CHO
Membrane µ
COOH COOH
COOH
594597
576576
568
556
556
568
E–
E–
––
–
E–
+
+
597
594
TGKPTLYNVSLIMSDTGGTCY
Cµ4
Encoded by S exon of Cµ
Encoded by M1 and M2 exons of Cµ
sequences are identical in both forms. (b) Structure of the pri-mary transcript of a rearranged heavy-chain gene showing the C!
exons and poly-A sites. Polyadenylation of the primary transcriptat either site 1 or site 2 and subsequent splicing (indicated by V-shaped lines) generates mRNAs encoding either secreted ormembrane ! chains.
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Cambio de isotipo
52
for example, induces class switching from C! to C"1 or C#.In some cases, IL-4 has been observed to induce class switch-ing in a successive manner: first from C! to C"1 and thenfrom C"1 to C# (Figure 5-15). Examination of the DNA ex-cision products produced during class switching from C! toC"1 showed that a circular excision product containing C!
together with the 5$ end of the "1 switch region (S"1) andthe 3$ end of the ! switch region (S!) was generated. Fur-thermore, the switch from C"1 to C# produced circular exci-sion products containing C"1 together with portions of the!, ", and # switch regions. Thus class switching dependsupon the interplay of three elements: switch regions, aswitch recombinase, and the cytokine signals that dictate theisotype to which the B cell switches. A more complete de-
scription of the role of cytokines in class switching appearsin Chapter 11.
Expression of Ig GenesAs in the expression of other genes, post-transcriptionalprocessing of immunoglobulin primary transcripts is required to produce functional mRNAs (see Figures 5-4and 5-5). The primary transcripts produced from re-arranged heavy-chain and light-chain genes contain inter-vening DNA sequences that include noncoding introns andJ gene segments not lost during V-(D)-J rearrangement.In addition, as noted earlier, the heavy-chain C-gene
122 P A R T I I Generation of B-Cell and T-Cell Responses
FIGURE 5-15 Proposed mechanism for class switching inducedby interleukin 4 in rearranged immunoglobulin heavy-chain genes. Aswitch site is located upstream from each CH segment except C%.
Identification of the indicated circular excision products containingportions of the switch sites suggested that IL-4 induces sequentialclass switching from C! to C"1 to C#.
5! 3!Cµ C"3V D J C# C"1 C"2b
S"2b
C"2a
S"2a
C$
S$
C%
S%
3!
S"1S"3Sµ
S"1
5!S"1
3!S"15!Sµ
3!Sµ
Recombination atSµ and S"1
DNA looping
S"3
C#C"3
Cµ
S"3
C#C"3
Cµ
5!S$3!S"1
S"2a
C"2a
C"2b
S"2b
5! 3! +
+
V D J C"1 C"2b
S"2b
C"2a
S"2a
C$
S$
C%
S%
DNA looping and recombinationat S"1 and S$
5! 3!V D J C$ C%
S%
5!V D J
Sµ
C"1
C"1
C"2b
S"2b
C"2a
S"2a
C$
S$
C%
S%
L
L
L
L
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• Durante la maduración de la respuesta
• Dirigida por citocinas• Orientada a la función biológica
del Ac
looping = formación de
each light-chain type and subtype within a species (see Fig-ure 4-17a). Each isotype is encoded by a separate constant-region gene, and all members of a species carry the sameconstant-region genes (which may include multiple alleles).Within a species, each normal individual will express all iso-types in the serum. Different species inherit different con-stant-region genes and therefore express different isotypes.Therefore, when an antibody from one species is injectedinto another species, the isotypic determinants will be recog-nized as foreign, inducing an antibody response to the iso-typic determinants on the foreign antibody. Anti-isotype
antibody is routinely used for research purposes to deter-mine the class or subclass of serum antibody produced dur-ing an immune response or to characterize the class ofmembrane-bound antibody present on B cells.
AllotypeAlthough all members of a species inherit the same set of iso-type genes, multiple alleles exist for some of the genes (seeFigure 4-17b). These alleles encode subtle amino acid differ-ences, called allotypic determinants, that occur in some, butnot all, members of a species. The sum of the individual allo-typic determinants displayed by an antibody determines itsallotype. In humans, allotypes have been characterized forall four IgG subclasses, for one IgA subclass, and for the !light chain. The "-chain allotypes are referred to as Gmmarkers. At least 25 different Gm allotypes have been identi-fied; they are designated by the class and subclass followed bythe allele number, for example, G1m(1), G2m(23), G3m(11),G4m(4a). Of the two IgA subclasses, only the IgA2 sub-class has allotypes, as A2m(1) and A2m(2). The ! light chain has three allotypes, designated !m(1), !m(2), and!m(3). Each of these allotypic determinants represents dif-ferences in one to four amino acids that are encoded by different alleles.
Antibody to allotypic determinants can be produced byinjecting antibodies from one member of a species into an-other member of the same species who carries different allo-typic determinants. Antibody to allotypic determinantssometimes is produced by a mother during pregnancy in re-sponse to paternal allotypic determinants on the fetal im-munoglobulins. Antibodies to allotypic determinants canalso arise from a blood transfusion.
IdiotypeThe unique amino acid sequence of the VH and VL domainsof a given antibody can function not only as an antigen-bind-ing site but also as a set of antigenic determinants. The idio-typic determinants arise from the sequence of the heavy- andlight-chain variable regions. Each individual antigenic deter-minant of the variable region is referred to as an idiotope(see Figure 4-17c). In some cases an idiotope may be the ac-tual antigen-binding site, and in some cases an idiotope maycomprise variable-region sequences outside of the antigen-binding site. Each antibody will present multiple idiotopes;the sum of the individual idiotopes is called the idiotype ofthe antibody.
Because the antibodies produced by individual B cells de-rived from the same clone have identical variable-region se-quences, they all have the same idiotype. Anti-idiotypeantibody is produced by injecting antibodies that have mini-mal variation in their isotypes and allotypes, so that the idio-typic difference can be recognized. Often a homogeneousantibody such as myeloma protein or monoclonal antibodyis used. Injection of such an antibody into a recipient who is
Antibodies: Structure and Function C H A P T E R 4 95
FIGURE 4-17 Antigenic determinants of immunoglobulins. Foreach type of determinant, the general location of determinants withinthe antibody molecule is shown (left) and two examples are illus-trated (center and right). (a) Isotypic determinants are constant-region determinants that distinguish each Ig class and subclasswithin a species. (b) Allotypic determinants are subtle amino acid differences encoded by different alleles of isotype genes. Allotypic differences can be detected by comparing the same antibody classamong different inbred strains. (c) Idiotypic determinants are gen-erated by the conformation of the amino acid sequences of theheavy- and light-chain variable regions specific for each antigen. Eachindividual determinant is called an idiotope, and the sum of the indi-vidual idiotopes is the idiotype.
(a) Isotypic determinants
Mouse IgG1 Mouse IgM
!1 µ "
(b) Allotypic determinants
Mouse IgG1(strain A)
Mouse IgG1(strain B)
!1 "
(c) Idiotypic determinants
Mouse IgG1against antigen a
"
"
!1 "
Mouse IgG1against antigen b
!1
Idiotopes
"
!1
Idiotopes
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Secreción de las Igs
53
Regulation of Ig-Gene TranscriptionThe immunoglobulin genes are expressed only in B-lineagecells, and even within this lineage, the genes are expressed atdifferent rates during different developmental stages. As withother eukaryotic genes, three major classes of cis regulatorysequences in DNA regulate transcription of immunoglobu-lin genes:
! Promoters: relatively short nucleotide sequences,extending about 200 bp upstream from the transcriptioninitiation site, that promote initiation of RNAtranscription in a specific direction
! Enhancers: nucleotide sequences situated some distanceupstream or downstream from a gene that activatetranscription from the promoter sequence in anorientation-independent manner
! Silencers: nucleotide sequences that down-regulatetranscription, operating in both directions over adistance.
The locations of the three types of regulatory elements ingerm-line immunoglobulin DNA are shown in Figure 5-19.All of these regulatory elements have clusters of sequencemotifs that can bind specifically to one or more nuclear pro-teins.
Each VH and VL gene segment has a promoter located justupstream from the leader sequence. In addition, the J!cluster and each of the DH genes of the heavy-chain locus are preceded by promoters. Like other promoters, the immunoglobulin promoters contain a highly conserved AT-rich sequence called the TATA box, which serves as a site forthe binding of a number of proteins that are necessary for theinitiation of RNA transcription. The actual process of tran-scription is performed by RNA polymerase II, which startstranscribing DNA from the initiation site, located about 25bp downstream of the TATA box. Ig promoters also containan essential and conserved octamer that confers B-cell speci-ficity on the promoter. The octamer binds two transcriptionfactors, oct-1, found in many cell types, and oct-2, foundonly in B cells.
While much remains to be learned about the function ofenhancers, they have binding sites for a number of proteins,many of which are transcription factors. A particularly im-portant role is played by two proteins encoded by the E2Agene which can undergo alternate splicing to generate twocollaborating proteins, both of which bind to the " and ! in-tronic enhancers. These proteins are essential for B-cell de-velopment and E2A knockout mice make normal numbers ofT cells but show a total absence of B cells. Interestingly, trans-fection of these enhancer-binding proteins into a T cell lineresulted in a dramatic increase in the transcription of " chainmRNA and even induced the T cell to undergo DH # JH !DHJH rearrangement. Silencers may inhibit the activity of Ig
126 P A R T I I Generation of B-Cell and T-Cell Responses
Secreted Ig
Secretoryvesicles
Heavy-chaintranslation
Light-chaintranslation
Leader
NascentIg (leadercleaved)
RER
Cis Golgi
Trans Golgi
Trans Golgireticulum
Oligosaccharides
Membrane Ig
Fusion withmembrane
Secretory vesicle
Transmembranesegment
FIGURE 5-18 Synthesis, assembly, and secretion of the im-munoglobulin molecule. The heavy and light chains are synthesizedon separate polyribosomes (polysomes). The assembly of thechains to form the disulfide-linked immunoglobulin molecule oc-curs as the chains pass through the cisternae of the rough endo-plasmic reticulum (RER) into the Golgi apparatus and then intosecretory vesicles. The main figure depicts assembly of a secretedantibody. The inset depicts a membrane-bound antibody, whichcontains the carboxyl-terminal transmembrane segment. This formbecomes anchored in the membrane of secretory vesicles and thenis inserted into the cell membrane when the vesicles fuse with themembrane.
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Las cadenas se sintetizan y las molécula de anticuerpo se monta en RERLos anticuerpos se glicosilan en el aparato de GolgiEn las células plasmáticas neoplásicas puede fallar la coordinación y generarse cadenas ligeras libres: proteínas de Bences-Jones
Receptor de la célula B
54
genetically identical to the donor will result in the formationof anti-idiotype antibody to the idiotypic determinants.
The B-Cell ReceptorImmunologists have long been puzzled about how mIg me-diates an activating signal after contact with an antigen. Thedilemma is that all isotypes of mIg have very short cytoplas-mic tails: the mIgM and mIgD cytoplasmic tails contain only3 amino acids; the mIgA tail, 14 amino acids; and the mIgGand mIgE tails, 28 amino acids. In each case, the cytoplasmictail is too short to be able to associate with intracellular sig-naling molecules (e.g., tyrosine kinases and G proteins).
The answer to this puzzle is that mIg does not constitutethe entire antigen-binding receptor on B cells. Rather, the B-cell receptor (BCR) is a transmembrane protein complexcomposed of mIg and disulfide-linked heterodimers calledIg-!/Ig-". Molecules of this heterodimer associate with anmIg molecule to form a BCR (Figure 4-18). The Ig-! chain
has a long cytoplasmic tail containing 61 amino acids; the tailof the Ig-" chain contains 48 amino acids. The tails in bothIg-! and Ig-" are long enough to interact with intracellularsignaling molecules. Discovery of the Ig-!/Ig-" heterodimerby Michael Reth and his colleagues in the early 1990s hassubstantially furthered understanding of B-cell activation,which is discussed in detail in Chapter 11.
Fc Receptors Bond to Fc Regions of AntibodiesMany cells feature membrane glycoproteins called Fc recep-tors (FcR) that have an affinity for the Fc portion of the anti-body molecule. These receptors are essential for many of thebiological functions of antibodies. Fc receptors are responsi-ble for the movement of antibodies across cell membranesand the transfer of IgG from mother to fetus across the pla-centa. These receptors also allow passive acquisition of anti-body by many cell types, including B and T lymphocytes,neutrophils, mast cells, eosinophils, macrophages, and nat-ural killer cells. Consequently, Fc receptors provide a meansby which antibodies—the products of the adaptive immunesystem—can recruit such key cellular elements of innate im-munity as macrophages and natural killer cells. Engagementof antibody-bound antigens by the Fc receptors of macro-phages or neutrophils provides an effective signal for the efficient phagocytosis (opsonization) of antigen-antibodycomplexes. In addition to triggering such effector functionsas opsonization or ADCC, crosslinking of Fc receptors byantigen-mediated crosslinking of FcR-bound antibodies cangenerate immunoregulatory signals that affect cell activation,induce differentiation and, in some cases, downregulate cel-lular responses.
There are many different Fc receptors (Figure 4-19). Thepoly Ig receptor is essential for the transport of polymericimmunoglobulins (polymeric IgA and to some extent, pen-tameric IgM) across epithelial surfaces. In humans, theneonatal Fc receptor (FcRN) transfers IgGs from mother tofetus during gestation and also plays a role in the regulationof IgG serum levels. Fc receptors have been discovered for allof the Ig classes. Thus there is an Fc!R receptor that bindsIgA, an Fc#R that binds IgE (see Figure 4-16 also), an Fc$Rthat binds IgD, IgM is bound by an Fc%R, and several vari-eties of Fc&R receptors capable of binding IgG and its sub-classes are found in humans. In many cases, the crosslinkingof these receptors by binding of antigen-antibody complexesresults in the initiation of signal-transduction cascades thatresult in such behaviors as phagocytosis or ADCC. The Fc re-ceptor is often part of a signal-transducing complex that in-volves the participation of other accessory polypeptidechains. As shown in Figure 4-19, this may involve a pair of &chains or, in the case of the IgE receptor, a more complex as-semblage of two & chains and a " chain. The association of anextracellular receptor with an intracellular signal-transduc-ing unit was seen in the B cell receptor (Figure 4-18) and is acentral feature of the T-cell-receptor complex (Chapter 9).
96 P A R T I I Generation of B-Cell and T-Cell Responses
FIGURE 4-18 General structure of the B-cell receptor (BCR). Thisantigen-binding receptor is composed of membrane-bound im-munoglobulin (mIg) and disulfide-linked heterodimers called Ig-!/Ig-". Each heterodimer contains the immunoglobulin-foldstructure and cytoplasmic tails much longer than those of mIg. As depicted, an mIg molecule is associated with one Ig-!/Ig-"heterodimer. [Adapted from A. D. Keegan and W. E. Paul, 1992, Im-munol. Today 13:63, and M. Reth, 1992, Annu. Rev. Immunol. 10:97.]
S S
S
S
S
S
S
S
S
S
S
S
S
S
SS
SS
SS
SS
SS
SS
SS
SS
S S
SS S
S
mIg
S
S
S
S
Ig-!Ig-"
48-aa tail 6l-aa tail
Cytoplasmic tails
Plasmamembrane
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Igα e Igβ hacen posible la señalización por la ocupación de la Ig de membrana
both the coding joint and the signal joint (and interveningDNA) on the chromosome. In the human ! locus, about halfof the V! gene segments are inverted with respect to J! andtheir joining is thus by inversion.
Ig-Gene Rearrangements May Be Productive or NonproductiveOne of the striking features of gene-segment recombinationis the diversity of the coding joints that are formed betweenany two gene segments. Although the double-strand DNAbreaks that initiate V-(D)-J rearrangements are introducedprecisely at the junctions of signal sequences and coding se-quences, the subsequent joining of the coding sequences isimprecise. Junctional diversity at the V-J and V-D-J codingjoints is generated by a number of mechanisms: variation incutting of the hairpin to generate P-nucleotides, variation intrimming of the coding sequences, variation in N-nucleotideaddition, and flexibility in joining the coding sequences. Theintroduction of randomness in the joining process helps gen-erate antibody diversity by contributing to the hypervariabil-ity of the antigen-binding site. (This phenomenon is coveredin more detail below in the section on generation of antibodydiversity.)
Another consequence of imprecise joining is that genesegments may be joined out of phase, so that the triplet read-ing frame for translation is not preserved. In such a nonpro-ductive rearrangement, the resulting VJ or VDJ unit is likelyto contain numerous stop codons, which interrupt transla-tion (Figure 5-9). When gene segments are joined in phase,the reading frame is maintained. In such a productive re-arrangement, the resulting VJ or VDJ unit can be translatedin its entirety, yielding a complete antibody.
If one allele rearranges nonproductively, a B cell may stillbe able to rearrange the other allele productively. If an in-phase rearranged heavy-chain and light-chain gene are notproduced, the B cell dies by apoptosis. It is estimated thatonly one in three attempts at VL-JL joining, and one in threesubsequent attempts at VH-DHJH joining, are productive. Asa result, less than 1/9 (11%) of the early-stage pre-B cells inthe bone marrow progress to maturity and leave the bonemarrow as mature immunocompetent B cells.
Allelic Exclusion Ensures a Single Antigenic SpecificityB cells, like all somatic cells, are diploid and contain both ma-ternal and paternal chromosomes. Even though a B cell is
Organization and Expression of Immunoglobulin Genes C H A P T E R 5 115
J!
V!
C A C T G T G G T G G A C T A G G
G A G G A T G C T C C C A C A G T G
RSS
RSS
2
34
5
G A G G A T G C G A C T A G G
Glu Asp Ala Thr Arg
1
G A G G A T G G G A C T A G G
Glu Asp Gly Thr Arg
G A G G A T T G G A C T A G G
Glu Asp Trp Thr Arg
Productive rearrangements
2
3
G A G G A T G C G G A C T A G G
Glu Asp Ala Asp Stop
G A G G T G G A C T A G G
Glu Val Asp Stop
Nonproductive rearrangements
4
5
1
Joiningflexibility
FIGURE 5-9 Junctional flexibility in the joining of immunoglobulingene segments is illustrated with V! and J!. In-phase joining (arrows1, 2, and 3) generates a productive rearrangement, which can betranslated into protein. Out-of-phase joining (arrows 4 and 5) leadsto a nonproductive rearrangement that contains stop codons and isnot translated into protein.
!! "" HH
! " H
** * *
! " H
Paternalchromosomes
Gene rearrangement
Maternalchromosomes
Maternal H chain
Maternal! chain
MaternalH chain
Paternal" chain
FIGURE 5-10 Because of allelic exclusion, the immunoglobulinheavy- and light-chain genes of only one parental chromosome areexpressed per cell. This process ensures that B cells possess a singleantigenic specificity. The allele selected for rearrangement is chosenrandomly. Thus the expressed immunoglobulin may contain one ma-ternal and one paternal chain or both chains may derive from onlyone parent. Only B cells and T cells exhibit allelic exclusion. Asterisks(#) indicate the expressed alleles.
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Receptores Fc
55
The Immunoglobulin SuperfamilyThe structures of the various immunoglobulin heavy andlight chains described earlier share several features, suggest-ing that they have a common evolutionary ancestry. In particular, all heavy- and light-chain classes have the immunoglobulin-fold domain structure (see Figure 4-7).The presence of this characteristic structure in all im-munoglobulin heavy and light chains suggests that the genesencoding them arose from a common primordial gene en-coding a polypeptide of about 110 amino acids. Gene dupli-cation and later divergence could then have generated thevarious heavy- and light-chain genes.
Large numbers of membrane proteins have been shown topossess one or more regions homologous to an im-munoglobulin domain. Each of these membrane proteins isclassified as a member of the immunoglobulin superfamily.The term superfamily is used to denote proteins whose corre-sponding genes derived from a common primordial gene en-coding the basic domain structure. These genes have evolvedindependently and do not share genetic linkage or function.The following proteins, in addition to the immunoglobulinsthemselves, are representative members of the immunoglob-ulin superfamily (Figure 4-20):
! Ig-!/Ig-" heterodimer, part of the B-cell receptor
! Poly-Ig receptor, which contributes the secretorycomponent to secretory IgA and IgM
! T-cell receptor
! T-cell accessory proteins, including CD2, CD4, CD8,CD28, and the #, $, and % chains of CD3
! Class I and class II MHC molecules
! "2-microglobulin, an invariant protein associated withclass I MHC molecules
! Various cell-adhesion molecules, including VCAM-1,ICAM-1, ICAM-2, and LFA-3
! Platelet-derived growth factor
Numerous other proteins, some of them discussed in otherchapters, also belong to the immunoglobulin superfamily.
X-ray crystallographic analysis has not been accom-plished for all members of the immunoglobulin superfamily.Nevertheless, the primary amino acid sequence of these proteins suggests that they all contain the typical immuno-globulin-fold domain. Specifically, all members of the immunoglobulin superfamily contain at least one or morestretches of about 110 amino acids, capable of arrangement
Antibodies: Structure and Function C H A P T E R 4 97
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S!2m
Fc"RI
FcRN
Poly IgR
S
S
S
S
S
S Fc"RII
""
CD64
CD32
S
S
S
S
""
CD16
Fc"RIIIA
!
S
S
S
S
""
Fc&RI
S
S
S
S
!""
CD89
Fc#R
FIGURE 4-19 The structure of a number of human Fc-receptors.The Fc-binding polypeptides are shown in blue and, where present,accessory signal-transducing polypeptides are shown in green. Theloops in these structures represent portions of the molecule with the characteristic immunoglobulin-fold structure. These molecules
appear on the plasma membrane as cell-surface antigens and, as in-dicated in the figure, many have been assigned CD designations (forclusters of differentiation; see Appendix). [Adapted from M. Daeron,1999, in The Antibodies, vol. 5, p. 53. Edited by M. Zanetti and J. D.Capra.]
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Activadores, inhibidores, moduladores,
Receptores Fc
56
Nature Reviews | Immunology
Common -chain
Cell membrane
Immunoglobulindomain
Name
Function Activating Inhibitory
Structure
Fc RI
Not expressed Not expressed
Not expressed
Not expressed Not expressed
Not expressed
Not expressedMonocyte, DCand macrophage
Monocyte, DC, plateletand macrophage
Monocyte, DCand macrophage
Monocyte, DCand macrophage
NK cell NK cell B cell and plasma cell
Neutrophil andeosinophil
Neutrophil, mast cell and eosinophil
Neutrophil, basophiland mast cell Neutrophil
Fc RIIA Fc RIIIA Fc RIIIB
GPI anchor
Fc RIIBFc RIIC
IgG1 IgG2, IgG3 and IgG4
IgG1 and IgG3IgG2 IgG4
IgG1 IgG3IgG4 IgG2
IgG1 IgG2 and IgG3 IgG4
IgG1 and IgG3IgG2 and IgG4
IgG1 IgG3IgG4 IgG2
Lymphoid
Myeloid
Granulocyte
IgG binding affinity
ITIM
Expression
ITAM
Acute-phase proteinsA group of proteins, the plasma concentration of which changes in response to trauma, inflammation and infection. C-reative protein is the prototypical acute-phase protein and binds phosphocholine on pathogens and apoptotic cells, opsonizing them for disposal by phagocytes by Fc!R binding.
FcR common !-chain A membrane-associated signal adaptor protein that contains an ITAM. It is shared by Fc!RI and Fc!RIIIA, as well as other receptors, including collagen receptor glycoprotein IV, NKp46, ILT1 (also known as LIR7) and osteoclast-associ-ated immunoglobulin-like receptor (OSCAR).
Follicular DCs (FDCs). Cells with a dendritic morphology that are present within B cell follicles in secondary lymphoid tissues such as lymph nodes and spleen. They display intact antigens that are held in immune complexes on their surface, accessible to B cells. FDCs are of non-haematopoietic origin and are not related to ‘conventional’ DCs.
Germinal centreA lymphoid structure that arises in B cell follicles in secondary lymphoid organs, such as spleen and lymph node, after immunization with, or exposure to, a T cell- dependent antigen. It is specialized for facilitating the development of high-affinity, long-lived plasma cells and memory B cells.
isoform and is expressed by B cells and, at lower levels, by monocytes18. It has inhibitory functions and is not thought to mediate immune complex internalization. Fc!RIIB2 is the main isoform expressed by myeloid-derived cells and can mediate endocytosis19, a proc-ess dependent on a dileucine motif in its cytoplasmic domain20,21. Although Fc!RIIB1 and Fc!RIIB2 are encoded by the same gene, the Fc!RIIB1 isoform is generated by alternative mRNA splicing that results in a 47-amino acid cytoplasmic insertion upstream of the ITIM, which inhibits endocytosis by preventing recep-tor accumulation in clathrin-coated pits22. Fc!RIIB3 is a soluble isoform that lacks the transmembrane and first cytoplasmic domains23, and it can inhibit the pres-entation of IgG-complexed antigen24. Another soluble form of Fc!RIIB can be generated by cleavage of the two extracellular domains25 and can suppress plasma-blast production in vitro26. Thus, there are two soluble forms of Fc!RIIB that can inhibit immune complex-mediated immunity in vitro. However, remarkably lit-tle is known about their physiological role in vivo, and the remainder of this review will therefore focus on the membrane-bound isoforms. When studies do not dif-ferentiate between the isoforms (as is usually the case) they are referred to collectively as Fc!RIIB.
In this Review, the regulation of Fc!RIIB expression and function, as well as its role in controlling immune responses, is discussed. The signalling pathways downstream of Fc!RIIB have been recently reviewed elswhere1, and therefore we focus on the influence of Fc!RIIB on cellular immunity, autoimmunity and responses to infection. Although Fc!RIIB has a con-served and important role in regulating immunity in mice and humans, common genetic variants have been reported that result in diverse receptor expression and function within populations of both species. The effect of these variants on the predisposition to both auto-immunity and infection, and the potential evolution-ary effects of the interplay between them, is discussed. Finally, we consider the effect that our increasing knowl-edge of the biology and function of Fc!RIIB might have on the optimal use of current biological therapies, such as intravenous immunoglobulin (IVIG), and on the development of new therapeutic approaches.
The functions of Fc!RIIBThe main function of Fc!RIIB is to inhibit activating sig-nals, which is achieved through co-ligation of Fc!RIIB with either activating Fc!Rs or with the BCR by immune complexes (FIG. 2). This leads to phosphorylation of the
Figure 1 | Structure, cellular distribution and IgG isotype-binding affinity of human activating and inhibitory Fc!Rs. Human Fc receptors for IgG (Fc!Rs) differ in function, affinity for the Fc fragment of antibody and in cellular distribution. There are five activating Fc!Rs: the high-affinity receptor Fc!RI, which can bind monomeric IgG, and four low-affinity receptors (Fc!RIIA, Fc!RIIC, Fc!RIIIA and Fc!RIIIB), which bind only immune-complexed IgG. Cross-linking of activating Fc!Rs by immune complexes results in the phosphorylation of immunoreceptor tyrosine-based activating motifs (ITAMs) that are present either in the cytoplasmic domain of the receptor (Fc!RIIA and Fc!RIIC), or in the associated FcR common !-chain (Fc!RI and Fc!RIIIA), resulting in an activating signalling cascade. Fc!RIIIB is a glycosylphosphatidylinositol (GPI)-linked receptor that has no cytoplasmic domain. Fc!RIIB is the only inhibitory Fc!R. It is a low affinity receptor that binds immune-complexed IgG and contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. Fc!RIIB cross-linking by immune complexes results in ITIM phosphorylation and inhibition of the activating signalling cascade. Fc!Rs differ in their cellular expression; myeloid cells express Fc!RI, Fc!RIIA and Fc!RIIIA, whereas granulocytes express Fc!RI, Fc!RIIA and Fc!RIIIB. In such cells, immune complex-mediated activation of these receptors is negatively regulated by Fc!RIIB. Fc!RIIB is the only Fc!R expressed by B cells and negatively regulates B cell receptor activation by immune-complexed antigen. Fc!Rs bind different IgG subtypes with differing affinity. For example, in the case of Fc!RIIB, binding affinity is highest for IgG1, followed by IgG3, which in turn has higher affinity than IgG4 followed by IgG2. The ratio of binding of an IgG subtype to activating Fc!Rs and inhibitory Fc!RIIB is known as the A/I ratio, and it determines the activation threshold of the cell. DC, dendritic cell; NK, natural killer.
R E V I E W S
NATURE REVIEWS | IMMUNOLOGY VOLUME 10 | MAY 2010 | 329
F O C U S O N T H E R A P E U T I C A N T I B O D I E S
© 20 Macmillan Publishers Limited. All rights reserved10
Nature Reviews | Immunology
CD4+ T cell
CD4+ T cell
CD8+ T cell
B cellFc RIIB
Fc R
Fc R
T cell-dependent
a Humoral immunity b Antigen presentation by DCs c Innate immunity
d Hypersensitivity
Inhibition of activating signals
Suppression ofantigen presentation
Immune complextrapping
Fc R-mediated phagocytosis, superoxide production, adhesion, rolling and migration?
Fc R-mediated phagocytosis, superoxide production and cytokinerelease
IgE-mediated degranulation, IL-4 production and histamine release
SCF-inducedproliferation
TLR-mediated activation
CD40L
Inhibition of DC maturation
ImmatureDC
Neutrophil
Macrophage
FDC
CD40
CD40
CD28
CD86
Suppression of antigen internalizationand presentation
MHC class II
MHC class II
TCR
IgG BCR
Apoptosis
Antigen uptake
Presentation of intactantigen to B cells?
Germinal centre
Plasma cell
Mast cell
IgE
Figure 2 | The functions of Fc!RIIB. a | Fc receptor IIB for IgG (Fc!RIIB) has an important role in controlling humoral immunity by regulating B cell activation, localization of B cells in the germinal centres, as well as plasma cell survival. Fc!RIIB regulates B cell activation by increasing the B cell receptor (BCR) activation threshold and suppressing B cell-mediated antigen presentation to T cells. Follicular dendritic cells (FDCs) express Fc!RIIB, which is thought to be important for trapping immune-complexed antigen for presentation to germinal centre B cells. The absence of Fc!RIIB on germinal centre FDCs results in impaired antibody and memory responses. Terminally differentiated plasma cells express little or no BCR but express high levels of Fc!RIIB, and cross-linking Fc!RIIB with immune complexes in vitro can induce apoptosis. b | Fc!RIIB influences antigen presentation by inhibiting Fc!R-dependent internalization of immune-complexed antigen by DCs, as well antigen presentation to both CD4+ and CD8+ T cells (cross-presentation). Fc!RIIB is also thought to provide a basal level of inhibition to DC maturation, as blockade of immune complex binding to Fc!RIIB results in DC maturation and type I interferon production. There is also a possibility that Fc!RIIB may deliver intact antigen to a non-degradative compartment, allowing its recycling to the cell surface where it could interact with the BCR and activate B cells. c | Fc!RIIB can also influence innate immunity: in macrophages, Fc!RIIB cross-linking inhibits Fc!R-mediated phagocytosis and cytokine release (including tumour necrosis factor, interleukin-6 (IL-6) and IL-1"), as well as Toll-like receptor 4 (TLR4)-mediated activation. In neutrophils, cross-linking of activating Fc!Rs results in phagocytosis, superoxide production and enhanced neutrophil adhesion, rolling and migration, all of which are probably inhibited by ligating Fc!RIIB. d | Fc!RIIB also inhibits IgE-induced mast cell and basophil degranulation, thus contributing to hypersensitivity responses. Fc#R, Fc recpetor for IgE; SCF, stem cell factor.
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330 | MAY 2010 | VOLUME 10 www.nature.com/reviews/immunol
REVIEWS
© 20 Macmillan Publishers Limited. All rights reserved10
Las múltiples facetas de la inhibición por FcgammaRIIB
Nature Reviews | Immunology
CD4+ T cell
CD4+ T cell
CD8+ T cell
B cellFc RIIB
Fc R
Fc R
T cell-dependent
a Humoral immunity b Antigen presentation by DCs c Innate immunity
d Hypersensitivity
Inhibition of activating signals
Suppression ofantigen presentation
Immune complextrapping
Fc R-mediated phagocytosis, superoxide production, adhesion, rolling and migration?
Fc R-mediated phagocytosis, superoxide production and cytokinerelease
IgE-mediated degranulation, IL-4 production and histamine release
SCF-inducedproliferation
TLR-mediated activation
CD40L
Inhibition of DC maturation
ImmatureDC
Neutrophil
Macrophage
FDC
CD40
CD40
CD28
CD86
Suppression of antigen internalizationand presentation
MHC class II
MHC class II
TCR
IgG BCR
Apoptosis
Antigen uptake
Presentation of intactantigen to B cells?
Germinal centre
Plasma cell
Mast cell
IgE
Figure 2 | The functions of Fc!RIIB. a | Fc receptor IIB for IgG (Fc!RIIB) has an important role in controlling humoral immunity by regulating B cell activation, localization of B cells in the germinal centres, as well as plasma cell survival. Fc!RIIB regulates B cell activation by increasing the B cell receptor (BCR) activation threshold and suppressing B cell-mediated antigen presentation to T cells. Follicular dendritic cells (FDCs) express Fc!RIIB, which is thought to be important for trapping immune-complexed antigen for presentation to germinal centre B cells. The absence of Fc!RIIB on germinal centre FDCs results in impaired antibody and memory responses. Terminally differentiated plasma cells express little or no BCR but express high levels of Fc!RIIB, and cross-linking Fc!RIIB with immune complexes in vitro can induce apoptosis. b | Fc!RIIB influences antigen presentation by inhibiting Fc!R-dependent internalization of immune-complexed antigen by DCs, as well antigen presentation to both CD4+ and CD8+ T cells (cross-presentation). Fc!RIIB is also thought to provide a basal level of inhibition to DC maturation, as blockade of immune complex binding to Fc!RIIB results in DC maturation and type I interferon production. There is also a possibility that Fc!RIIB may deliver intact antigen to a non-degradative compartment, allowing its recycling to the cell surface where it could interact with the BCR and activate B cells. c | Fc!RIIB can also influence innate immunity: in macrophages, Fc!RIIB cross-linking inhibits Fc!R-mediated phagocytosis and cytokine release (including tumour necrosis factor, interleukin-6 (IL-6) and IL-1"), as well as Toll-like receptor 4 (TLR4)-mediated activation. In neutrophils, cross-linking of activating Fc!Rs results in phagocytosis, superoxide production and enhanced neutrophil adhesion, rolling and migration, all of which are probably inhibited by ligating Fc!RIIB. d | Fc!RIIB also inhibits IgE-induced mast cell and basophil degranulation, thus contributing to hypersensitivity responses. Fc#R, Fc recpetor for IgE; SCF, stem cell factor.
REVIEWS
330 | MAY 2010 | VOLUME 10 www.nature.com/reviews/immunol
REVIEWS
© 20 Macmillan Publishers Limited. All rights reserved10
Las múltiples facetas de la inhibición por FcgammaRIIB
Nature Reviews | Immunology
CD4+ T cell
CD4+ T cell
CD8+ T cell
B cellFc RIIB
Fc R
Fc R
T cell-dependent
a Humoral immunity b Antigen presentation by DCs c Innate immunity
d Hypersensitivity
Inhibition of activating signals
Suppression ofantigen presentation
Immune complextrapping
Fc R-mediated phagocytosis, superoxide production, adhesion, rolling and migration?
Fc R-mediated phagocytosis, superoxide production and cytokinerelease
IgE-mediated degranulation, IL-4 production and histamine release
SCF-inducedproliferation
TLR-mediated activation
CD40L
Inhibition of DC maturation
ImmatureDC
Neutrophil
Macrophage
FDC
CD40
CD40
CD28
CD86
Suppression of antigen internalizationand presentation
MHC class II
MHC class II
TCR
IgG BCR
Apoptosis
Antigen uptake
Presentation of intactantigen to B cells?
Germinal centre
Plasma cell
Mast cell
IgE
Figure 2 | The functions of Fc!RIIB. a | Fc receptor IIB for IgG (Fc!RIIB) has an important role in controlling humoral immunity by regulating B cell activation, localization of B cells in the germinal centres, as well as plasma cell survival. Fc!RIIB regulates B cell activation by increasing the B cell receptor (BCR) activation threshold and suppressing B cell-mediated antigen presentation to T cells. Follicular dendritic cells (FDCs) express Fc!RIIB, which is thought to be important for trapping immune-complexed antigen for presentation to germinal centre B cells. The absence of Fc!RIIB on germinal centre FDCs results in impaired antibody and memory responses. Terminally differentiated plasma cells express little or no BCR but express high levels of Fc!RIIB, and cross-linking Fc!RIIB with immune complexes in vitro can induce apoptosis. b | Fc!RIIB influences antigen presentation by inhibiting Fc!R-dependent internalization of immune-complexed antigen by DCs, as well antigen presentation to both CD4+ and CD8+ T cells (cross-presentation). Fc!RIIB is also thought to provide a basal level of inhibition to DC maturation, as blockade of immune complex binding to Fc!RIIB results in DC maturation and type I interferon production. There is also a possibility that Fc!RIIB may deliver intact antigen to a non-degradative compartment, allowing its recycling to the cell surface where it could interact with the BCR and activate B cells. c | Fc!RIIB can also influence innate immunity: in macrophages, Fc!RIIB cross-linking inhibits Fc!R-mediated phagocytosis and cytokine release (including tumour necrosis factor, interleukin-6 (IL-6) and IL-1"), as well as Toll-like receptor 4 (TLR4)-mediated activation. In neutrophils, cross-linking of activating Fc!Rs results in phagocytosis, superoxide production and enhanced neutrophil adhesion, rolling and migration, all of which are probably inhibited by ligating Fc!RIIB. d | Fc!RIIB also inhibits IgE-induced mast cell and basophil degranulation, thus contributing to hypersensitivity responses. Fc#R, Fc recpetor for IgE; SCF, stem cell factor.
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330 | MAY 2010 | VOLUME 10 www.nature.com/reviews/immunol
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© 20 Macmillan Publishers Limited. All rights reserved10
Las múltiples facetas de la inhibición por FcgammaRIIB
El efecto de IVIG en las enfermedades autoinmunes
✦ Dependientes de Fab: ✦ ant-idiotipo✦ modulación de citocinas✦ neutralización de C3a y C5a
✦ Aumento de expresión de FCgammaRIIB en Macrófagos
✦ Efectos a través de CSF✦ Efectos dependiento del nivel de glicosilación✦ Polimorfismos FcgammaRIIB → SLE
Fc!RIIB as a therapeutic target. A monoclonal anti-body against Fc!RIIB (hu2B6-3.5; MacroGenics) has been used to direct monocyte- or macrophage-induced cytotoxicity against B cell lymphoma cells150 and plasma cells from patients with systemic light-chain amyloidosis151. Fc!RIIB cross-linking on B cells, plasma cells and myeloma cell lines40 can induce apop-tosis in vitro, and thus Fc!RIIB-specific antibodies might show efficacy in B and plasma cell malignan-cies. These antibodies might also provide a means of targeting plasma cells in antibody-driven autoimmune diseases.
Cross-linking Fc!RIIB activating receptors could therapeutically harness its inhibitory effect. This is being investigated in allergy, where Fc!RIIB cross-linking with IgE–antigen complexes or Fc"RI has been used to inhibit IgE-mediated histamine release from human mast cells and basophils in vitro and in mouse models152,153. These observations have recently extended to non-human primates154,155. Moreover, the possibility that bispecific
antibodies could co-ligate specific antigen and Fc!RIIB raises the prospect of autoantigen-specific suppression of humoral immunity.
The soluble Fc!RIIB3 isoform has been shown to inhibit the effects of immune complexes in vitro24,26. A more recent study of soluble Fc!RIIB in (NZB # NZW) F1 mice showed therapeutic efficacy, slowing the progression of nephritis and prolonging survival156 and prompting its use in human trials in SLE.
Conclusion and future directionFc!RIIB, identified as the mediator of Fc fragment-induced B cell suppression over 30 years ago, is now known to be widely expressed and to control key aspects of immunity. Variation in its complex and often subtle regulation is crucially involved in determining susceptibil-ity to autoimmunity and defence against infection. Better understanding of this variation is beginning to provide insight into the evolution of disease susceptibility and to open new opportunities for therapy.
Table 1 | Current and future therapeutic exploitation of Fc!RIIB
Therapeutic strategy Clinical effect Refs
Modulation of Fc!RIIB expression on effector cells
IVIG indirectly increases Fc!RIIB expression on ‘effector’ cells 138,139
Infliximab (Remicade; Centocor/Merck) blocks TNF, leading to increased Fc!RIIB expression on monocytes
142
Modulation of IgG-binding to Fc!RIIB
Decrease in affinity of depleting monoclonal antibodies for Fc!RIIB increases efficacy
45,47
Use of Fc!RIIB as a direct therapeutic target
Monoclonal antibody directed against Fc!RIIB allowing co-crosslinking of Fc!RIIB may induce apoptosis of B cells and plasma cells in lymphoma, myeloma or autoimmunity
40,150,151
A bispecific antibody that binds antigen-specific BCR and Fc!RIIB potentially allows targeted inhibition of autoreactive B cells
–
Therapeutic antibody targeting IgE receptor can inhibit mast cell degranulation and may be useful in the treatment of allergy
152–155
Soluble Fc!RIIB may inhibit immune complex-mediated immune activation and has shown efficacy in a mouse model of SLE
156
BCR, B cell receptor; Fc!R, Fc receptor for IgG; IVIG, intravenous immunoglobulin; SLE, systemic lupus erythematosus; TNF, tumour necrosis factor.
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2. Perez, L. G., Costa, M. R., Todd, C. A., Haynes, B. F. & Montefiori, D. C. Utilization of immunoglobulin G Fc receptors by human immunodeficiency virus type 1: a specific role for antibodies against the membrane-proximal external region of gp41. J. Virol. 83, 7397–7410 (2009).
3. Verma, A. et al. Analysis of the Fc! receptor-dependent component of neutralization measured by anthrax toxin neutralization assays. Clin. Vaccine Immunol. 16, 1405–1412 (2009).
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15. Brooks, D. G., Qiu, W. Q., Luster, A. D. & Ravetch, J. V. Structure and expression of human IgG FcRII (CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes. J. Exp. Med. 170, 1369–1385 (1989).
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20. Matter, K., Yamamoto, E. M. & Mellman, I. Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J. Cell Biol. 126, 991–1004 (1994).
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