`
`immuno
`iologyo
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Zz
`
`
`
`
`
`
`
`CHARLES A JANEWAY PAUL TRAVERS
`MARK WALPORT
`MARK SHLOMCHIK
`
`Lassen - Exhibit 1039, p. 1
`
`Lassen - Exhibit 1039, p. 1
`
`
`
`————————————
`
`immuno
`iologye
`
`THE IMMUNE SYSTEM IN HEALTH AND DISBEABE
`
`|
`
`|
`
`Charles A. Janeway, Jr.
`Yale University School of Medicine
`<a
`Paul Travers
`
`Anthony Nolan Research Institute, London
`aa
`Mark Walport
`Imperial College School of Medicine, London
`@
`Mark J. Shlomchik
`
`Yale University School of Medicine
`
`
`
`Lassen - Exhibit 1039, p. 2
`
`Lassen - Exhibit 1039, p. 2
`
`
`
`Vice President:
`Text Editors:
`Managing Editor:
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`Denise Schanck
`Penelope Austin, Eleanor Lawrence
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`Michael Morales
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`Liza Furnival
`Blink Studic, London
`Marion Morrow, Rory MacDonald
`
`© 2001 by Garland Publishing.
`All rights reserved. No part of this pubilcation may be reproduced, stored in a retrieval
`system or transmitted in any form or by any means—electronic, mechanical, photocopying,
`recording, or otharwiss—withoullhe prior written permission of the copyright holder.
`
`Distributors:
`Inside North Amatica, Garland Publishing, 29 Wes! 35th Street,
`New York, NY 10001-2299,
`Inside Japar. Nankodo Co. Ltd., 42-6, Hongo 3-Chrome, Bunkyo-ku,
`Tokyo, 113-8410, Japan.
`Outside North America and Japan. Churchill Livingstone, Robert Stevenson House,
`1-3 Baxter's Place, Leith Walk, Edinburgh, EH1 SAF,
`
`ISBN 0 8153 3642 X (paperback) Garland
`ISBN 0 4480 7098 9 (paperback) Churchill Livingstone
`ISBN 0 4430 7099 7 (paperback) Intemational Student Edition
`
`Library of Congress Cataloging-In-Publication Data
`Immunobiology : the immune system in health and disease / Charles A, Janeway,Jr....
`[et al.j.-- Sth ed.
`p. om.
`Includes bibliographical references and index.
`ISBN 0-8159-3642-X (pbk.)
`1. Immunology. 2. immunity. |. Janeway, Charles. 1. Title.
`
`QRi81 .1454 2001
`618.07'9--dc21
`
`2001018039
`
`This book was produced using QuarkXpress 4.11 and Adobe illustrator 9.0
`
`Published by Garland Publishing, a memberof the Taylor & Francis Group,
`29 West 35th Street, Naw York, NY 10001-2299.
`
`Printed In the United States of America.
`168141312 111998765432
`
`Lassen - Exhibit 1039, p. 3
`
`Lassen - Exhibit 1039, p. 3
`
`
`
`|
`
`
`CONTENTS
`
`PARTI|AN INTRODUCTION TO IMMUNOBIOLOGY AND INNATE IMMUNITY
`
`1
`Basic Concepts in Immunology
`Chapter 1
`
`Chapter 2—_Innate Immunity 35
`
`THE RECOGNITION OF ANTIGEN
`
`[
`
`
`
`Chapter 3—Antigen Recognition by B-cell and T-cell Receptors 93
`
`
`Chapter4
`The Generation of Lymphocyte Antigen Receptors
`123
`
`Chapter5
`Antigen Presentation to T Lymphocytes
`155
`
`PART Ill|THE DEVELOPMENT OF MATURE LYMPHOCYTE RECEPTOR
`REPERTOIRES
`
`Chapter 6—Signaling Through Immune System Receptors 187
`
`
`Chapter?
`The Development and Survival of Lymphocytes
`221
` ART
`THE ADAPTIVE IMMUNE RESPONSE
`IV |
`
`
`—_T Cell-Mediated Immunity
`Chapter8
`The Humoral Immune Response
`Chapter9
`Chapter 10 Adaptive Immunity to tnfection
`
`295
`341
`381
`
`
`
`
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Failures of Host Defense Mechanisms
`Chapter 11
`Chapter 12 Allergy and Hypersensitivity
`Chapter 13 Autoimmunity and Transplantation
`Chapter 14 Manipulation of the Immune Response
`
`Afterword Evolution of the Immune System:Past, Present, and Future,
`by Charles A. Janeway, Jr.
`
`Appendix!
`
`Immunologists’ Toolbox
`
`Appendix II
`
`CD Antigens
`
`Appendix Ill Cytokines and their Receptors
`
`425
`471
`501
`553
`
`597
`
`613
`
`661
`
`677
`
`680
`681
`682
`
`!
`
`1
`
`
`
`
`
`683 Index
`
`Appendix IV Chemokines and their Receptors
`Appendix V Immunological Constants
`Biographies
`Glossary
`
`
`
`
`
`Lassen - Exhibit 1039, p. 4
`
`Lassen - Exhibit 1039, p. 4
`
`
`
`7 —
`
`THE RECOGNITION
`OF ANTIGEN
`
`
`
`Lassen - Exhibit 1039, p. 5
`
`Lassen - Exhibit 1039, p. 5
`
`
`
`123
`
`The Generation of
`Lymphocyte Antigen
`Receptors
`
`
`
`Lymphocyte antigen receptors, in the form of immunoglobulins on B cells
`and T-cell receptors on T cells, are the means by which lymphocytes sense
`the presence of antigens in their environment. The receptors produced by
`each lymphocyte have a unique antigen specificity, which is determined by
`the structureoftheir antigen-bindingsite, as described in Chapter 3. Because
`each person possesses billions of lymphocytes, these cells collectively pro-
`vide the individual with the ability to respond to a great variety of antigens.
`The wide range of antigen specificities in the antigen receptor repertoire is
`due to variation in the amino acid sequence at the antigen-bindingsite,
`which is made up from the variable (V) regions of the receptor protein chains.
`In each chain the V regionis linked to an invariant constant (C) region, which
`provides effector orsignaling functions,
`
`Given the importance of a diverse repertoire of lymphocyte receptors in the
`defense against infection,
`it is not surprising that a complex and elegant
`genetic mechanism has evolved for generating these highly variable proteins.
`Each receptor chain variant cannot be encodedin full in the genome, as this
`would require more genes for antigen receptors than there are genes in the
`entire genome. Instead,wewill see that the V regions of the receptorchainsare
`encodedin several pieces—so-called gene segments. These are assembled in
`the developing lymphocyte by somatic DNA recombination to form a complete
`V-region sequence, a mechanism known generally as gene rearrangement.
`Each type of gene segment is present in multiple copies in the germline
`genome.Theselection of a gene segment of each type during gene rearrange-
`Ment occurs at random, and the large numberofpossible different combi-
`nations accounts for muchofthe diversity of the receptor repertoire,
`
`In the first two parts of this chapter we will describe the gene rearrangement
`mechanism that generates the V regions of immunoglobulin and T-cell
`receptor genes. The basic mechanism is commonto both B cells and T cells,
`and involves manyif notall of the same enzymes. Wewill describe the details
`of the enzymology of this recombination process, the evolution of which was
`probablycritical to the evolution of the vertebrate adaptive immunesystem.
`In B cells, but not T cells, the rearranged V region undergoes additional
`modification, known as somatic hypermutation. This does not occur until
`afier B cells encounter and becomeactivated by antigen, [n these cells, the
`V regions of the assembled immunoglobulin genes undergo a high rate of
`point mutation that creates additional! diversity within the expanding clone
`of B cells responding to antigen.
`In the third part of the chapter we consider the limited, but functionally
`important, diversity of immunogiobulin C regions. The C regions of T-cell
`Teceptors do not show such diversity as they function only as part of a
`Membrane-bound antigen receptor. Theirrole is to anchor and support the
`
`Lassen - Exhibit 1039, p. 6
`
`Lassen - Exhibit 1039, p. 6
`
`
`
`
`
`| Chapler4:TheGenerationofLymphocyte Antigen Receptors
`
`i-
`
`
`
`V regions at the cell surface as well as linking the binding of antigen by the
`V regions to the receptor-associated intracellular signaling complex. The
`C regions of immunoglobulins also serve these functions but in addition the
`C regions of the heavy chain are responsible for the effector functions of the
`secreted immunoglobulins, or antibodies, made by activatedB cells. These Cy
`regions comein several different versions, or isotypes, each of which has a
`different effector function, In B cells that have becomeactivated by antigen. the
`heavy-chain V region can become associated with a different C), region bya fur.
`ther somatic recombination event, in the process knownas isotype switching,
`This enables the different heavy-chain C regions, each with a different func.
`tion, to be represented among antibodiesof the same antigenspecificity.
`
`The generation of diversity in immunoglobulins.
`
`Virtually any substance can elicit an antibody response. Furthermore, the
`response evento a simple antigen bearing a single antigenic determinant is
`diverse, comprising manydifferent antibody molecules each with a unique
`affinity, or binding strength, for the antigen anda subily different specificity,
`The total number ofantibodyspecificities available to an individualis known
`as the antibodyrepertoire, or immunoglobulin repertoire, and in humansis
`at Jeast 10'', perhaps many more, The numberof antibody specificities pre-
`sent at any one timeis, however, limited by the total number of J} cells in an
`individual, as well as by eachindividual's encounters with antigens.
`Before it was possible to examine the immunoglobulin genes directly, there
`were two main hypotheses for the origin of this diversity. The germline theory
`held that there is a separate gene for each different immunoglubulin chain
`and that the antibody repertoire is targely inherited. By contrast, somatic
`diversification theories proposed that the observed repertoire is generated
`from a limited number of inherited V-region sequences that undergo alter-
`ation within B cells during the individual’s lifetime. Cloning of the
`immunoglobulin genes revealed that the antibody repertoireis, in tact, gen-
`erated by DNA rearrangements during B-cell development. As we will sec in
`this part of the chapter, a DNA sequence encoding a V regionis assembled
`at each locus by selection from a relatively small group of inherited gene
`segments. Diversity is further enhanced by the process of somatic hyper-
`mutation in mature activated B cells, hus the somatic diversification theory
`was essentially correct, although the concept of multiple germline genes
`embodied in the germline theory also proved true.
`
`4-1
`
`Immunoglobulin genes are rearranged in antibody-producing cells.
`
`sequences. In germline DNA, from nonlymphoid cells, the V- and C-region
`
`in nonlymphoid cells, the gene segments encoding the greater part of the Vv
`region of an immunoglobulin chain are some considerable distance away from
`the sequence encoding the C region. In mature B lymphocytes, however, the
`assembled V-region sequence lies much nearer the C region, as a consequence
`of gene rearrangement. Rearrangement within the immunoglobulin genes was
`originally discovered 25 years ago, whenit first became possible to study the
`organization of the immunoglobulin genes in both B cells and nonlymphoid
`cells using restriction enzyme analysis and Southern blotting, In this proce
`dure, chromosomal DNAisfirst cut with a restriction enzyme, and the DNA
`fragments containing particular V- and C-region sequences are identified by
`hybridization with radiolabeled DNA probes specific for the relevant DNA
`
`Lassen - Exhibit 1039, p. 7
`
`Lassen - Exhibit 1039, p. 7
`
`
`
`The generation of diversity inimmunoglobullns|125
`
`
`
`Flg. 4.1 Immunoglobulin genes are
`rearranged In B cells. The two photo-
`graphs ontheleft (germline DNA) show
`a Southern blot of a restriction enzyme
`digest of DNA from nonlymphoid celts
`from a normal person. The locations of
`Immunoglobulin DNA sequences are
`identified by hybridization with V- and
`C-region probes. The V and C regions
`are found in cistinct DNA fragments in
`the nonlymphoid ONA. The two photo-
`graphs onthe right (B-cell DNA) are of
`the samerestriction digest of DNA from
`peripheral blood lymphocytes from a
`patient with chronic lymphocytic
`leukemia (see Chapter 7), in which a
`single clone of B cells is greatly
`
`expanded, The malignant B cells
`express the V region from which the
`V-region probe was obtained and,
`owing to their predominancein the cell
`population, this unique rearrangement
`can be detected. In this DNA, the V and
`C regions are found in the same
`fragment, which is a different size from
`either the C- or the V-region germline
`fragments. Although nol shown in this
`figure, a population of normal B lympho-
`cytes has manydifferent rearranged
`genes, so they yield a smear of ONA
`fragmentsizes, which are noi visible as
`a crisp band. Photograph courtesyof
`S. Wagnerand L. Luzzatto,
`
`sequencesidentified by the probes are on separate DNA fragments. However,
`in DNA from an antibody-producing B cell these V- and C-region sequences
`are on the same DNA fragment, showing that a rearrangement of the DNA has
`occured.A typical experiment using human DNAis shownin Fig.4.1.
`This simple experiment showed that segments of genomic DNA within the
`immunoglobulin genes are rearrangedin cells of the B-lymphocyte lineage,
`but not in othercells. This process of rearrangement is known as somatic
`recombination, to distinguish it from the meiotic recombination that takes
`place during the productionof gametes.
`
`4-2
`
`The DNA sequence encoding a complete V region is generated by the
`somatic recombination of separate gene segments.
`
`The V region, or V domain, of an immunoglobulin heavy or light chain is
`encoded by more than one gene segment.For thelight chain, the V domain
`is encoded by two separate DNA segments. The first segment encodes the
`first 95-101 amino acids of the light chain and is termed a V gene segment
`because it encodes moatof the V domain. ‘The second segment encodes the
`remainder of the V domain (up to 13 amino acids) and is termed a joining or
`J gene segment.
`
`The rearrangements that Jead to the production of a complete immunoglobulin
`light-chain gene are showninFig. 4.2 (center panel). The joining of a Vand a
`J gene segmentcreates a continuous exon that encodes the wholeof the iight-
`chain V region, In the unrearranged DNA, the V gene segments are located
`relatively far away (rom the C region. The | gene segments are located close
`to the C region, however, and joining of a V segmentto a) gene segment also
`brings the V gene close to a C-region sequence. The J genc segmentof the
`rearranged V region is separated from a C-region sequence only by an intron.
`'n the experiment shownin Fig, 4.1, the germline DNA fragmentidentified by
`the ‘V-region probe’ contains the V gene segment, and that identified by the
`‘C-regionprobe’ actually contains both the J gene segment and the C-region
`sequence. To make a complete immunoglobulin light-chain messenger RNA,
`the V-region exonis joined to the C-region sequence by RNA splicing after
`transcription (see Fig. 4.2).
`A heavy-chain V region is encodedin three gene segments. [n additionto the
`Vand J gene segments (denoted Vj, and Jy; to distinguish them fromthelight-
`chain Vand Jj), there Is a third zene segmentcalled the diversity or Dy gene
`Segment, which lies between the Vj, and Jy gene segments. The process of
`fecombination that generates a complete heavy-chain V region is shown in
`
`Lassen - Exhibit 1039, p. 8
`
`Lassen - Exhibit 1039, p. 8
`
`
`
`126
`
`Chapter4: The Generation of Lymphocyte Antigen Receptors
`
`Germline DNA
`
`Somatic
`recombination
`
`Splicing
`
`arranged DNA
`Somatic
`fecombination
`
`rearranged D)
`
`Transcription
`Prima
`transcriptRNA
`
`the L-to-V and the J-to-C introns. Heavy-chaln V regione are
`Fig. 4.2 V-region genes are constructed from gene
`segments. Light-chain V-ragion genes are constructed trom two—_—constructed from three gene segments(right panel). Firat, the
`segments (canter panel). A variable (V) and a joining (J) gene
`diversity (D) and J gene segmentsjoin, then the V gene
`segmentin the genomic ONAare joined to torm & complete
`segment joins lo the combined DJ sequence, forming a complete
`light-chain V-region exon. Immunoglobulin chains are exira-
`Vx exon. A heavy-chain C-ragion gene is encoded by several
`cellular proteins and the V gene segmentis preceded by an
`exons. The C-region exons, together with the leader sequence,
`exon encoding a leader peplide (L), which directs the protein
`are spliced to the V-domain sequence during processing of the
`Into the cell's secretory pathways and Is then cleaved. The light-—heavy-chain RNAtranscript. The leader sequence is removed
`chain C region is encoded in a separate exon andis joined to
`after translation and the disullide bonds thatlink the polypeptide
`the V-region exon by splicing of the light-chain RNA to remove
`chains are formed. The hinge region is shownin purple.
`
`Fig. 4.2 (right panel), and occurs in two separate stages. In the first, a Dy gene
`segment is joined to a J, gene segment; then a Vj, gene segment rearranges
`to Dj) to make a complete Vjj-region exon, As with the light-chain genes,
`RNA splicing joins the assembled V-region sequence to the neighboring
`C-region gene.
`
`4-3
`
`There are muitiple different V-region gene segments.
`
`For simplicity, we have so far discussed the formation of a complete
`immunoglobulin V-region sequence as though there were only a single copy
`of each gene segment.In fact, there are multiple copiesofall of the gene seg-
`ments in germline DNA,It is the random selection of just one gene segment
`of each type to assemble a V region that makes possible the great diversity of
`Vregions among immunoglobulins, The numbers offunctional gene segments
`
`Lassen - Exhibit 1039, p. 9
`
`Lassen - Exhibit 1039, p. 9
`
`
`
`
`
`Thegenerationofdiversityin immunoglobullns | 127
`
`of each type in the human genome, as determined by gene cloning and
`sequencing, are shownin Fig. 4.3, Notall the gene segments discovered are
`functional, as a proportion have accumulated mutations (hat prevent them
`from encoding a functional protein. These are termed ‘pseudogenes.’
`RBecuuse there are many V, D, and J] gene segments in germline DNA, no
`single one is essential. This reduces the evolutionary pressure on each gene
`segment to remain intact, and has resulted in a relatively large number of
`pseudogenes.Since someof these pseudogenes can undergo rearrangement
`just
`like a normal functional gene segment, a significant proportion of
`rearrangements will incorporate a pseudogene and thus be nonfunctional.
`
`
`
`The immunoglobulin gene segments are organized into three clusters or
`genetic locithex, 1, and heavy-chain loci. These are on different chromo-
`somes and each is organized slightly differently, as shown in Fig. 4.4 for
`humans.At theAlight-chain locus, located on chromosome22, a cluster of V4
`gene segments is followed by foursets ofJ, gene segments eachlinked to a
`Fig. 4.3 The numbers of functional
`single C;, gene.In the x light-chain locus, on chromosome2,the cluster ofV,,
`gene segmentsfor the V regions of
`human heavy and light chains. These
`gene segments is followed by a cluster of J, gene segments, and then by a
`numbers are derived from exhaustive
`single Cy gene. The organization of the heavy-chain locus, on chromosome
`cloning and sequencing of ONA from
`Id, resembles that of the k Jocus, with separate clusters of Vj), Diy. and Jn gene
`one individual and exclude al] pseudo-
`segments and of Cy genes. The heavy-chain locus differs in one important
`genes (mutated and nonfunctional
`way: instead of a single C-region, it contains a series of C regions arrayed one
`versions of a gene sequence). Owing
`after the other, each of which corresponds to a different isotype. Generally, a
`to genetic polymorphism, the numbers
`will not be the sameforall people.
`cell expresses only one al a time, beginning with IgM. The expressionof other
`isotypes, such as IgG, can occur through isotype switching, as will be
`described in Section 4-16.
`
`The human V gene segments can be grouped into families in which each
`membershates at least 80% DNA sequence identity with all others in the
`family. Both the heavy-chain and x-chain V gene segments can be subdivided
`into seven such families, whereas there are eight families of Vi, gene seg-
`ments. ‘The families can be grouped into clans, made upoffamilies that are
`
`A.
`
`light-chaln locus
`Li V)1
`
`L2 V2
`
`« light-chain locus
`LI Vy
`L2 Vy2
`
`LB YS Ven
`
`Jnl-5
`
`tx
`
`
`| LV,-30
`dod
`C11
`Jy2
`C2
`J24
`C4
`
`
`
`
`Heavy-chain locus
`
`Li Vit
`
`
`
`L2 VL Vd ga |
`
`D,,1-27
`
`|
`
`Jyt-6
`
`Cy
`
`Fig. 4.4 The germilne organization of tha Immunoglobulin
`heavy- and light-chain loci In the human genome. The
`genetic locus for the A light chain (chromosome 22) has about
`30 functional V, gene segments and four pairs of functional
`J;, gene segments and C,, genes. The « locus (chromosome 2)
`'s organized In a similar way, with about 40 functional V,. gene
`Segments accompanied by a cluster of five J, gene segments
`but with a single C,, gene. In approximately 50%of individuals,
`the entire cluster of k V gene segments has undergone an
`increase by duplication (not shownfor simplicity), The heavy-
`Chain jocus (chromosome 14) has about 65 functional Vy gene
`
`segments and a cluster of around 27 D segments lying between
`these Vy gene segments and six J gene segments. The
`heavy-chain locus also contains a large cluster of Gy genes that
`are described in Fig. 4.18. For simplicity we have shown only a
`single Cy gene in this diagram withoutillustrating its separate
`exons, have omitted pseudogenes, and have shownall V gene
`sagments in the sameorientation. L, leader sequence. This
`diagram is not to scale: the total langth ot the heavy-chain locus
`|s over 2 megabases (2 million bases}, whereas someof the
`OD segments are only six bases long.
`
`Lassen - Exhibit 1039, p. 10
`
`Lassen - Exhibit 1039, p. 10
`
`
`
`128
`
`Chapter 4: The Generation of Lymphocyte Antigen Receptors
`
`more similar to each other than to families in other clans. Human V}; gene
`segments fall into three suchclans. All of the Vj; gene segments identifiect
`from amphibians, reptiles, and mammals alsofall into the same three clans,
`suggesting that these clans existed in a common ancestor of these modern
`animal groups.
`
`4-4
`
`Rearrangement of V, D, and J gene segmentsis guided by flanking
`DNA sequences.
`
`A system is required to ensure that DNA rearrangements take place at the
`correct locations relative to the V, D, or J gene segment coding regions. In
`addition, joins must be regulated such that a V gene segment joins to a D or J
`and notto another V. DNA rearrangementsare in fact guided by conserved
`noncoding DNA sequences that are found adjacent to the points at which
`recombination takes place. These sequences consist of a conserved block of
`seven nucleotides—the heptamer 5‘CACAGTG3’—whichis always contiguous
`with the coding sequence, followed by a nonconserved region knownas the
`spacer, whichis either (2 or 23 nucleotides long. This is followed by a second
`conserved block of nine nucleotides—the nonamer 5’ACAAAAACC3’ (Fig.
`4.5). The spacer varies in sequence but its conserved length corresponds to
`oneor twoturns of the DNA doublehelix. This brings the heptamer and non-
`amer sequencesto the sameside of the DNA helix, where they can be bound
`by the complex of proteins
`that
`catalyzes
`recombination, The
`heptamer-spacer—nonameris called a recombination signal sequence (RSS).
`
`Recombination only occurs between gene segments located on the same
`chromosome.It generally follows the rule that only a gene segmentflanked
`by a RSS with a 12-base pair (bp) spacer can be joined to one Nanked by a 23
`bp spacer RSS. This is known as the 12/23 rule. Thus, for the heavy chuin, a
`Dy gene segment canbe joined to a Ji gene segment and a Vj) gene segment
`to a Dy, gene segment, but Vj) gene segments cannot be joined to )); gene seg-
`mentsdirectly, as both V,; and Jj; gene segments are Nanked by 23 bp spacers
`and the Dy gene segments have 12 bp spacers on both sides (see Fig, 4.5).
`
`It is now apparent, however, that, even thoughit violates the 12/23 rule, direct
`joining of one D gene segment to another can occtir in most species. In humans,
`D-D fusionis found in approximately 5% of antibodies and is the major mech-
`anism accounting for the unusually long CDR3 loops found in some heavy
`chains, By creating extra-long CDR3s and unusual amino acid combinations,
`these D-D fusions add furtherto the diversity of the antibody repertoire.
`
`The mechanism of DNA rearrangementis similar for the heavy- and light-
`chain loci, although only one joining eventis needed to generate a light-chain
`gene whereas two are needed to generate a complete heavy-chain gene.
`The commonest mode of rearrangement (Fig. 4.6, left panels) involves the
`
`racombination signal sequence.
`
`Fig. 4.5 Conserved heptamer and
`nonamer sequences flank the gene
`segmente encoding the V regions of
`heavy (H) and light (A and x) chains.
`The spacer (white) between the
`heptamer (orange) and nonamer
`(purple) sequencesis always either
`approximately 12 bp or approximately
`23 bp, and joining almost always
`involves a 12 bp and a 23 bp
`
`
`
`_QGTTTITGT
`CCAAAAACA
`
`_CACTGTS |
`GTGACAC
`
`Lassen - Exhibit 1039, p. 11
`
`Lassen - Exhibit 1039, p. 11
`
`
`
`The generation of diversity in immunoglobulins|129
`
`
`
`
`
`
`
`
`looping-out and deletion of the DNA between two gene segments. This occurs
`when the coding sequences of the two gene segments are in the sameorienta-
`tion in the DNA. A second mode of recombination can occur between two
`gene segments that have opposite transcriptional orientations. This mode of
`recombination is less common, although such rearrangements accountfor
`abouthalf of all Vx to J joins; the transcriptional orientation of half of the
`human V, gene segments is opposite to that of the J, gene segments. The
`mechanism of recombination is essentially the same, but the DNA thatlies
`between the two gene segments meetsa differentfate (Fig. 4.6, right panels).
`When the RSSs in such cases are brought together and recombination takes
`Place, the intervening DNAis not lost from the chromosome but is retained
`in an Inverted orientation.
`
`4-5
`
`The reaction that recombinesV, D, and J gene segments involves
`both lymphocyte-specific and ubiquitous DNA-modifying enzymes.
`
`The molecular mechanism ofV-region DNA rearrangement, or V(D)J recombi-
`nation,is illustrated in Fig. 4.7. The 12 bp spaced and 23 bp spaced RSSs are
`brought together by interactions between proteins that specifically recognize
`the length of spacer and thus enforce the 12/23 rule for recombination, The
`DNA molecule is then broken in two places and rejoined in a different
`Contiguration. The ends of the heptamer sequencesare joined precisely in a
`
`Flg. 4.6 V-region gene segments are
`joined by recombination. In every
`V-region recombination event, the
`signals flanking the gene segments are
`broughttogetherto allow recombination
`to take place. Forsimplicity, the
`recombination of a !ight-chain gene is
`MMustrated; for the heavy-chain gene,
`two separate recombination events are
`required to generate a functional
`V region. In some cases, as shownin
`the left panels, the V and J gene
`segments have the sametranscriptional
`orientation. Juxtaposition of the
`recombination signal sequencesresults
`in the looping out of the intervening
`DNA. Heptamers are shownIn orange,
`nonamers in purple, and ihe arrows
`represent the diractlons of the heptamer
`and nonamerrecombination signals
`(see Fig. 4.5). Recombination occurs at
`the ends of the heptamer sequences,
`creating a signal joint and releasing the
`intervening DNAin the form of a closed
`circle. Subgequently, the joining of the
`V and J gene segments creates the
`coding joint. In other cases,illustrated
`in the right panels, the V and J gene
`segments are initially oriented in
`opposite transcriptional directions.
`Bringing together the signal sequences
`in this case requires a more complex
`looping of the DNA.Joining the ends of
`the two heptamer sequences now
`results in the inversion and integration
`of the intervening DNA. Again, the
`joining of the V and J segments creates
`a functional V-region exon.
`
`Lassen - Exhibit 1039, p. 12
`
`Lassen - Exhibit 1039, p. 12
`
`
`
`10| Chapter4:TheGenerationofLymphocyteAntigen Receptors
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`
`
`Fig. 4.7 Enzymatic steps in the
`rearrangement of immunoglobulin
`gene segments. Rearrangement begins
`with the binding of RAG-1, RAG-2, and
`high mobility group (HMG)proteina
`(not shown). These RAG-1:RAG-2
`complexes (domes, colored green or
`purple for clarity although they are
`identical at each recombination site)
`recognize the recombination signai
`sequences (arrows) flanking the coding
`sequencesto be joined (red and yellow
`reciangles). These are then brought
`together (second panel), following which
`the RAG complexis activated to cut one
`strand of the double-stranded DNA
`precisely at the end of the heptamer
`sequences(third panel). The 5’ cut end
`of this DNA strand then reacts with the
`complementary uncut strand, breaking it
`
`to leave a double-stranded break at the
`end of the heptamer sequence, and
`forming a hairpin by Joining to the cut
`end of its complementary strand on the
`other side of the break. Subsequently,
`through the action of additional essential
`proteins such as Ku70:Ku80 (indicated
`in blue) that joln the complex (fourth
`pane!) along with the RAG protelns, the
`DNAhairpin is cleaved at a random site
`to yield a single-stranded DNA end. This
`end is then modifled by the action of
`TdT and exonuclease(indicatedin pink,
`fifth panel), which randomly creates
`diverse, Imprecise ends. Finally (sixth
`pane!) the two heptamer sequences,
`which are not modified, are ligated to
`form the precise signaljoint, while the
`coding joint is also ligated, both by the
`action of DNAligase IV.
`
`head-to-head fashion to form a signal joint in a circular piece of extra-
`chromosomal DNA,whichis iost from the genome when thecell divides. The
`Vand J gene segments, which remain on the chromosome, join to form what
`is called the coding joint. This junction is imprecise, and consequently
`generates much additional variability in the V-region sequence.
`
`The complex of enzymesthat act in concert to effect somatic V(D)J recombi-
`nation is termed the V(D)J recombinase. The products of the two genes
`RAG-1 and RAG-2 (recombination-activating genes) comprise the lymphoid-
`specific componentsof the recombinase.This pair of genes is only expressed
`in developing lymphocytes while they are engaged in assembling their anti-
`gen receptors, as is described in more detail in Chapter 7, They are essential
`for V(D)J recombination. Indeed, these genes, when expressed together, are
`sufficient to confer on nonlymphoid ceils such as fibroblasts the capacity to
`rearrange exogenous segments of DNA that contain appropriate RSSs; this is
`how RAG-1 and RAG-2 wereinitially discovered.
`Although the RAG proteins are required for V(D)J recombination,they are not
`the only enzymesin the recombinase. The remaining enzymes are ubiqui-
`tously expressed DNA-modifying proteins that are involved in double-stranded
`DNA repair, DNA bending, or the modification of the ends of the broken DNA
`strands. They include the enzyme DNAligaseIV, the enzyme DNA-dependent
`protein kinase (DNA-PK), and Ku, a well-known autoantigen, which is a
`heterodimer (Ku 70:Ku 80) that associates tightly with DNA-PK.
`V(D)J recombination is a multistep enzymatic process in which the first
`reaction is an endonucleolytic cleavage requiring the coordinated activity of
`both RAG proteins. Initially, two RAG protein complexes, each containing
`RAG-1, RAG-2, and high-mobility group proteins, recognize and align the two
`RSSs that are guiding the join (see Fig. 4.7). RAG-1 is thought to specifically
`recognize the nonamerofthe RSS. At this stage, the 12/23 rule is established
`through mechanisms that are still poorly understood. The endonuclease
`activity of the RAG protein complexes then makes two single-strand DNA
`breaks atsites just 5’ of each bound RSS,leaving a free 3’-OH group at the end
`of each coding segment. This 3’-OH group then hydrolyzes the phosphodi-
`ester bond on the otherstrand, sealing the end of the double-stranded DNA
`to create a DNA‘hairpin’out of the gene segmentcoding region. This process
`simultaneously creates a flush double-stranded break at the ends of the two
`heptamersignal sequences. The DNA ends donotfloat apart, however, but
`are held tightly in a complex by the RAG proteins and other associated DNA
`
`Lassen - Exhibit 1039, p. 13
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`Lassen - Exhibit 1039, p. 13
`
`
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`The generation of diversity In immunoglobulins|1374
`
`yepair enzymesuntil the join is completed. The two RSSsarepreciselyjoined
`to form the signal joint. Coding joint formation is more complex.First, the
`DNAhairpin is nicked open by a single-stranded break, again by the RAG
`proteins. The nicking can happenat various points along the hairpin, which
`leads to sequencevariability in the eventualjoint. The DNA repair enzymes
`in the complex then modify the openedhairpins by removing nucleotides (by
`exonuclease activity) and by randomly adding nucleotides (by terminal
`deoxynucleotidyl transferase, TdT). It is not known if addition and deletion of
`nucleotides at the ends of coding regions occurs simultaneously or in a
`defined order. Finally, ligases such as DNAligase IV join the processed ends
`together to generate a continuous double-stranded DNA,thus reconstituting
`a chromosomethat includes the rearranged gene. This enzymatic process
`seems to create diversity in the joint between gene segments, while ensuring
`that the RSS ends are ligated wi