`
`immuno
`iO ogyo
`
`THE IMMUNE BYITBM IN HEALTH AND DIBIABE
`
`IV"
`
`
`
`
`
`
`
`CHARLES A JANEWAY PAUL THAVEFIB
`
`MARK WALPDHT
`
`MARK BHLDMCHIK
`
`Lassen — Exhibit 1039, p. 1
`
`Lassen - Exhibit 1039, p. 1
`
`

`

`r—————.
`
`,
`
`I
`
`i
`
`l
`
`immuno
`iologye
`
`THE IMMUNE SYSTEM IN HIALTH AND DISEAII
`
`Charles A. Janaway, Jr.
`
`Yale University School of Medicine
`
`Paul Travers
`
`I
`
`Anthony Nolan Research Institute. London
`
`I
`
`Mark Walpor-t
`
`Imperial College School of Medicine. London
`
`I
`
`Mark J. Shlomchik
`
`Yale University School of Medicine
`
`
`
`Lassen — Exhibit 1039, p. 2
`
`Lassen - Exhibit 1039, p. 2
`
`

`

`Vice President:
`Text Editors:
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`Blink Studio. London
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`
`0 2001 by Garland Publishing.
`All rights reserved. No part oi this publication may be reproduced. stored in a retrieval
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`
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`
`iSBN 0 8153 3642 X (paperback) Garland
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`
`Library oi Congress Cataloging-in-Publication Date
`lmmunobiology : the immune system in health and disease I Charles A. Janeway. Jr. .
`[et ai.].-- 5th ed.
`p. cm.
`includes bibliographical reierenoes and index.
`iSBN o-e153-ae42nx (pbk.)
`1. immunology. 2. immunity. l. Janeway. Charles. it. Title.
`
`.
`
`QR181 .I454 2001
`616.07'9-41021
`
`2001016039
`
`This book was produced using QuarkXpreas 4.11 and Adobe illustrator 9.0
`
`Published by Garland Publishing. a member of the Taylor 8. Francis Group,
`29 West 3511: Street, New York, NY 10001-2299.
`
`Printed in the United States of America.
`15141312111098785432
`
`Lassen — Exhibit 1039, p. 3
`
`Lassen - Exhibit 1039, p. 3
`
`

`

`
`
`[I
`
`CONTENTS
`
`PART I
`
`AN INTRODUCTION TO IMMUNOBIOLOGY AND INNATE IMMUNITY
`
`Chapter 1
`Chapter 2
`
`Basic Concepts in Immunology
`Innate Immunity
`
`THE RECOGNITION OF ANTIGEN
`
`Chapter 3
`
`Antigen Recognition by B-cell and T-cell Receptors
`
`Chapter 4
`
`The Generation of Lymphocyte Antigen Receptors
`
`Chapter 5
`
`Antigen Presentation to T Lymphocytes
`
`THE DEVELOPMENT OF MATURE LYMPHOCYTE RECEPTOR
`REPERTOIRES
`
`Chapter 6
`
`Signaling Through Immune System Receptors
`
`Chapter 7
`
`The Development and Survival of Lymphocytes
`
`
`_
`_
`__
`Chapters
`
`THE ADAPTIVE IMMUNE RESPONSE
`
`TCeII-Mediated Immunity
`
`Chapter 9
`
`The Humoral Immune Response
`
`Chapter 10 Adaptive Immunity to Infection
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Chapter 11
`
`Failures of Host Defense Mechanisms
`
`Chapter 12 Allergy and Hypersensitivity
`
`Chapter 13 Autoimmunity and Transplantation
`
`Chapter 14 Manipulation of the Immune Response
`
`Afterward Evolution 01 the Immune System: Past, Present, and Future,
`by Charles A. Janeway. Jr.
`
`Appendix I
`
`Immunologists’ Toolbox
`
`Appendix II
`
`CD Antigens
`
`Appendix III Cytokines and their Receptors
`
`Appendix IV Chemokines and their Receptors
`
`Appendix V Immunological Constants
`
`Biographies
`
`1
`35
`
`93
`
`123
`
`155
`
`187
`
`221
`
`295
`
`341
`
`381
`
`425
`
`471
`
`501
`
`553
`
`597
`
`613
`
`661
`
`677
`
`680
`
`681
`
`682
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glossary
`Index
`
`
`
`683
`
`Lassen — Exhibit 1039, p. 4
`
`Lassen - Exhibit 1039, p. 4
`
`

`

`F1
`
`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 structure of their antigen-binding site, 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-binding site,
`which is made up from the variable (V) regions of the receptor protein chains.
`In each chain the V region is linked to an invariant constant (C) region, which
`provides effector or signaling 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 encoded in full in the genome, as this
`would require more genes for antigen receptors than there are genes in the
`entire genome. Instead, we will see that the V regions of the receptor chains are
`encoded in 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. The selection of a gene segment of each type during gene rearrange-
`ment occurs at random, and the large number of possible different combi-
`nations accounts for much of the 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 immunoglobuiin and T-cell
`receptor genes. The basic mechanism is common to both B cells and T cells,
`and involves many if not all of the same enzymes. We will describe the details
`ol'the enzymology of this recombination process, the evolution of which was
`probably critical to the evolution of the vertebrate adaptive immune system.
`
`In B cells, but not T cells, the rearranged V region undergoes additional
`modification, known as somatic hypermutation. This does not occur until
`after B cells encounter and become activated by antigen. in 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
`0f B cells responding to antigen.
`
`In the third part of the chapter we consider the limited, but functionally
`important, diversity of immunoglobulin C regions. The C regions of T-cell
`receptors do not show such diversity as they function only as part of a
`membrane-bound antigen receptor. Their role is to anchor and support the
`
`Lassen — Exhibit 1039, p. 6
`
`Lassen - Exhibit 1039, p. 6
`
`

`

`
`
`IE Chapter 4: The Generation of Lymphocyte Antigen Receptors v.0
`
`;
`
`
`
`,
`'
`.
`'
`
`:
`-
`
`V regions at tlte 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 immunogiobttlina also serve these functions but in addition the
`C regions of the heavy chain are responsible for the effector functions of the
`secreted immunoglobttlins, or antibodies. made by activated B cells. These 1”
`regions come itt several different versions. or isotypes. each of which has a
`different effector function. In B cells that have become activated by antigen. the
`heavy-chain V region cart become associated with a different C” region by a fur.
`titer somatic recombination event. in tire process known as isotype switching
`This enables the different heavy-chain (J regions, each with a different i'unc.
`tion, to be represented among antibodies of the same antigen specificity.
`
`The generation of diversity in immanoglobulins.
`
`Virtually any substance cart elicit an antibody response. Furthermore, the
`response even to a sitttplc antigen bearing a single antigenic determinant is
`diverse, comprising many different antibody molecules each with a unique
`affinity, or binding strength, for the antigen anti :1 subtly different specificity.
`The total number of antibody specificities available to an individual is known
`as the antibody repertoire, or immunoglobulin repertoire, attd itt humans is
`at least 10”, perhaps tttany more. The ttumber of atttibody specificities pre-
`sent at any one time is, however, limited by the total number oill cells in an
`individual, as well as by each individttal's encounters with antigens.
`
`Before it was possible to examine the immunogloltulin genes directly. there
`were two main hypotheses for the origin of this diversity. The germllne theory
`held that there is a separate gene for each different itntttunoglobttliu chain
`and that the antibody repertoire is largely inherited. By contrast. somatic
`diversification theories proposed that the obscured repertoire is generated
`front a limited number of inherited V-region sequences that undergo alter»
`ation within B cells during the individual’s lifetime. Clotting of the
`immuttuglobulin genes revealed that the antibody repertoire is, in fact, gett-
`erated by DNA rearrangements dttring B/ceii development. As we will see itt
`this part of the chapter. a DNA sequence encoding a V region is 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. Thus tltc somatic diversification theory
`was essentially correct, although the concept of multiple germlitte genes
`embodied in the germlinc theory also proved true.
`
`4-1
`
`lmmunoglobulln genes are rearranged in antibody-producing cells.
`
`sequences. In gt'rmline DNA. front nottiympltoid cells. the V- and C-t'egion
`
`In nonlyrnphoid cells, the gene segments encoding the greater part of the V
`region of an inttttunoglobulin chain are some considerable distance away from
`the sequence encoding the C region. in mature B lymphocytes, however, the
`assembled V-region sequence lies ntuch nearer the C region, as a consequence
`ul'genc rearrangement. Rearrangement within the intntuttoglubulitt genes was
`originally discovered 25 years ago. when it first became possible to study Ill?-
`organization of the immunoglohulin genes in both B cells and ttoltlympitoid
`cells using restriction enzyme analysis and Southern blotting. In this procr
`dare, chromosomal DNA is first cut with a restriction enzyme, and the DNA
`fragments containing particular V- and C-region sequences are identified by
`hybridization with radiolabeled UNA probes specific for the tolerant DNA
`
`Lassen — Exhibit 1039, p. 7
`
`Lassen - Exhibit 1039, p. 7
`
`

`

`
`
`The generation of diversity in immunoglobullns 125
`
`Fig. 4.1 lmmunoglobulln genes are
`rearranged In B cells. The two photo-
`graphs on the left (garmline DNA) show
`a Southern blot ot a restriction enzyme
`digest of DNA from nonlymphoid cells
`from a normal person. The locations oi
`lmmunoglobulin DNA sequences are
`identified by hybridization with V- and
`c—region probes. The V and C regions
`are found in distinct DNA fragments in
`the nonlymphoid DNA. The two photo-
`graphs on the right (B-cali DNA) are of
`the same restriction digest of DNA from
`peripheral blood lymphocytes irom a
`patient with chronic lymphocytic
`leukemia (see Chapter 7), in which an
`single clone at 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 predominance in 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 irom
`either the C- or the V-region germline
`fragments. Although not shown in this
`figure, a population of normal B lympho-
`cytes has many different rearranged
`genes, so they yield a smear of DNA
`fragment sizes, which are not visible as
`a crisp band. Photograph courtesy of
`S. Wagner and L. Luzzatto.
`
`
`
`sequences identified 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
`occurred. A typical experiment using human DNA is shown in Fig. 4.1.
`
`This simple experiment showed that segments of genomic DNA within the
`immunoglobulin genes are rearranged in cells of the B-lymphocyte lineage.
`but not in other cells. This process of rearrangement is known as somatic
`recombination, to distinguish it from the meiotic recombination that takes
`place during the production of 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 immunoglobuiin heavy or light chain is
`encoded by more than one gene segment. For the light chain. the V domain
`is encoded by two separate DNA segments. The first segment encodes the
`first 95-10] amino acids of the light chain and is termed a V gene segment
`because it encodes most of the V domain. The second segment encodes the
`remainder of the V domain (up to 13 amino acids) and is termed a joining or
`1 gene segment.
`
`The rearrangements that lead to the production oln complete intmttnoglobttlin
`light-chain gene are shown in Fig. 4.2 (center panel). The joining ofa V and a
`J gone segment creates a continuous exon that encodes the whole ot'the light-
`chaln V region. in the ttnrcarranged DNA. the V gene segments are located
`relatively far away from the C region. The 1 gene segments are located close
`to the C region. however, and joining of a V segment to a J gene segment also
`brings the V gene close to a C—rcgion sequence. The I gene segment of the
`rearranged V region is separated frotn a C-rogton sequence only by an intron.
`in the experiment shown in Fig. 4 . I. the gonnline DNA fragment identified by
`tho ‘V-region probe' contains the V gene segment. and that identified by the
`'C-rcgion probc' actually contains both the J gent- segment and the C-rcglon
`sequence. To make a complete immunoglobulin light-chain messenger RNA,
`the V~region exon is joined to thc C-region sequence by RNA splicing after
`transcription (see Fig. 4.2).
`
`A heavy-chain V region is encoded in three gent.- segments. In addition to the
`V and i gene segments (denoted V" and in to distinguish them from the light-
`Cltnin VI. and it}. than: is a third gene segment called the diversity or D" gene
`Segment, which lies between the V“ and in ltenc segments. The process of
`teetnnhlnation that generates a complete heavy-chain V region is shown in
`
`Lassen — Exhibit 1039, p. 8
`
`Lassen - Exhibit 1039, p. 8
`
`

`

`g
`a
`
`D—J )oined
`rearranged DNA
`Somatic
`recombination
`li-JorV—DJ -
`
`rearranged D"———‘
`
`-
`
`Transcription
`
`Primary
`transcript RNA
`"-—-‘-"'
`
`1116 ll Chapter4: The Generation of Lymphocyte Antigen Receptors
`
`Gerrnline DNA
`
`Somatic
`recombination
`
`SPH‘W
`
`Fig. 4.2 V-region genes are constructed from gene
`segments. Lightscheln V-reglon genes are constructed lrom two
`segments (center panel). A variable (V) and a )oining (J) gene
`segment in the genomic DNA are joined to term a complete
`light-chain V-region exon. lmrnunoglobulin chains are extra-
`cellular proteins and the V gene segment is preceded by an
`axon encoding a leader peptide (L), which directs the protein
`into the cell's secretory pathways and is then cleaved, The light-
`chain C region is encoded in a separate exon and is joined to
`the V-region exon by splicing oi the light-chain FINA to remove
`
`the L-to-V and the J-to-C introns. Heevy-oheln V regions are
`constructed from three gene segments (right panel). First. the
`diversity (D) and J gene segments join, then the V gene
`segment joins to the combined DJ sequence, lorrning a complete
`VH axon. A heavy-chain C-reglon gene is encoded by several
`axons. The C-reglon axons, together with the leader sequence.
`are spliced to the V-dornein sequence during processing oi the
`heavy-chain FtNA transcript. The leader sequence is removed
`alter translation and the disullide bonds that link the polypeptide
`chains are formed. The hinge region is shown in purple.
`
`Fig. 4.2 (right panel), and occurs in two separate stages. In the first. a I)” gene
`segment is joined to n in gene segment: then a V“ gene segment rearranges
`to DJ” to make a complete Vlrrcgion own. As with the light-chain genes,
`RNA splicing joins the assembled V-region sequence in the neighboring
`C-region gene.
`
`4-3
`
`There are multiple ditterent V-reglon gene segments.
`
`For simplicity, we have so far discussed the formation of a complete
`immunoglobuiin V-region sequence as though there were only a single copy
`of each gene segment. In fact, there are multiple copies of all of the gene seg-
`ments in germiine 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
`V regions among immunoglobulins. The numbers of functional gene segments
`
`Lassen — Exhibit 1039, p. 9
`
`Lassen - Exhibit 1039, p. 9
`
`

`

`
`
`Thcgeneretion oirllverslty in immunoglobullns l 127
`
`of each type in the human genome. as determined by gene cloning and
`sequencing, are shown in Fig. 4.3. Not all the gene segments discovered are
`functional, as a proportion have accumulated mutations that prevent them
`from encoding a functional protein. These are termed 'pseudogenes.’
`Because there are many V. D, and ] gene segments in germlinc DNA, no
`single one is essential. This reduces the evolutionary pressure on each gene
`segment to remain intact, and has resulted in a telatively large number of
`pseudogenes. Since some of 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 loci—the x, it, 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 the 1 light- chain locus. located on chromosome 22, a cluster of V1
`gene segments is followed by four sets of I). gene segments each linked to a
`single Ct gene. In the K light-chain locus, on chromosome 2, the cluster of V“-
`gene segments is followed by a cluster of In gene segments, and then by a
`single CK gene. The organization of the heavy-chain locus, on chromosome
`14. resembles that of the K locus. with separate clusters ot‘Vu, D. I, and in gene
`segments and of Cu genes. The heavy-chain locus differs in one important
`way: instead of a single C-region, it contains a series of (1 regions arrayed one
`after the other, each of which corresponds to a different isorype. Generally. a
`cell expresses only one at a time, beginning with lgM. The expression of other
`isolypes, such as lgG, 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
`member shares 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 V;~ gene seg-
`ments. The families can be grouped into clans, made up of families that are
`
`
`
`Fig. 4.3 The numbers of functional
`gene segments for the V regions of
`human heavy and light chains. These
`numbers are derived lrom exhaustive
`cloning and sequencing of DNA from
`one individual and exclude all pseudo-
`genes (mutated and nonfunctional
`versions ot a gene sequence). Owing
`to genetic polymorphism, the numbers
`will not be the same for all people.
`
`A llght-chalnlocus
`L1 V11
`
`L2 V72
`
`K light-chain locus
`L1
`vt1
`L2 v,.2
`
`L3 vta
`
`L v..-- to
`
`J.1--5
`
`Heavy--chaln locus
`
`
`
`
`
`
`
`
`
`
`L2 VH2L1 Uri! La Vitaml lflvflufifi } [
`
`
`Dul--2'a"
`
`1
`
`.ifli--fi
`
`Fig. 4.4 The gormllno organization of the lmmunoglobulin
`heavy- and light-chain loci In the human genome. The
`genetic locus for the it light chain (chromosome 22) has about
`30 lunotional Vt, gene segments and tour pairs at functional
`J}. gene segments and Ca. genes. The K locus (chromosome 2)
`Is organized in a similar way, with about 40 lunctional V» gene
`segments accompanied by a cluster oi live J,t gene segments
`but with a single Ct gene. In approximately 50% oi individuals.
`the entire cluster of r: V gene segments has undergone an
`increase by duplication (not shown for simplicity). The heavy-
`chain locus (chromosome 14) has about as functional VH gene
`
`segments and a cluster of around 27 D segments lying between
`these VH gene segments and six JH gene segments. The
`heavy-chain locus also contains a large cluster of CH genes that
`are described in Fig. 4.18. For simplicity we have shown only a
`single CH gene in this diagram without illustrating its separate
`axons. have omitted pseudogenes. and have show all v gene
`segments in the same orientation. L. leader sequence. This
`diagram is not to scale: the total length at the heavy-chain locus
`is over 2 megabases (2 million bases). whereas some oi the
`D 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 such clans. All or the V“ gene segments identified
`from amphibians, reptiles, anti mammals also fall into the same three clans,
`suggesting that these clans existed in a common ancestor of these modern
`animal groups.
`
`4-4
`
`Rearrangement ow, D, and J gene segments is 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] gene segment coding regions. In
`addition. joins must be regulated such that a V gene segment joins to a D or i
`and not to another V. DNA rearrangements are 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'—which is always contiguous
`with the coding sequence. followed by a nonconservcd region known as the
`spacer. which is either [2 or 23 nucleotides long. This is followed by a second
`conserved block of nine nucleotides—the nonnnter 5‘ACAAAAACC3' (Fig.
`4.5). The spacer varies in sequence but its conserved length corresponds to
`one or two turns of the DNA double helix. This brings the heptamer and non-
`amer sequences to the same side of the DNA helix. where they can be bound
`by the complex of proteins
`that
`catalyzes
`recombination. The
`heptamer—spacer—nonamer is 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 segment flanked
`by a RSS with a lZ-base pair tbp) spacer can be joined to one flanked by a 23
`bp spacer RSS. This is known as the 12/23 rule. Thus. for the heavy chain. a
`I)” gene segment can be joined to a In gene segment and a Vn gene segment
`to n D; 1 gene segment. but V” gene segments cannot be joined to in gene seg-
`ments directly, as both V" and In gene segments are flanked by 23 bp spuccrs
`and the DH gene segments have 12 bp spacers on both sides (see Fig. 4.5).
`
`it is now apparent. however. that, even though it violates the 12/23 rule. direct
`joining of one D gene segment to another can occur in most species. in humans.
`D—D fusion is 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 CDRBs and unusual amino acid combinations,
`these DFD fusions add further to the diversity of the antibody repertoire.
`
`The mechanism of DNA rearrangement is similar for the heavy- and light-
`chain loci, although only one joining event is 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
`
`Fig. 4.5 Conserved hoptamsr and
`nonemer sequences flank the gene
`segments encoding the V regions of
`heavy (H) and light (it and w.) chains.
`The spacer (white) between the
`heptamer (orange) and nonamer
`(purple) sequences is always either
`approximately 12 Do or approximately
`23 bp. and Joining almost always
`involves a 12 bp and a 25 bp
`
`GACAGTG
`GTGTCAC
`
`
`
`recombination signal sequence.
`
`_ GGTTITI'GT
`CCAMMCA
`
`CACTGTG _
`GTGACAC
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`Lassen — Exhibit 1039, p. 11
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`Lassen - Exhibit 1039, p. 11
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`The generation of diversity in immunogiobuilns
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`129
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`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 same orienta-
`tion in the DNA. A second mode of recombination can occur between two
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`gene segments that have opposite transcriptional orientations. This mode of
`recombination is less common, although such rearrangements account for
`about half of all Vt.- to it; joins; the transcriptional orientation of half of the
`human VK gene segments is opposite to that of the 1,; gene segments. The
`mechanism of recombination is essentially the same, but the DNA that lies
`between the two gene segments meets a differentiate (Fig. 4.6, right panels).
`When the R855 in such cases are brought together and recombination takes
`place. the intervening DNA is not lost from the chromosome but is retained
`in an inverted orientation.
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`4-5
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`The reaction that recombines V, D, and .l gene segments involves
`both lymphocyte-specific and ubiquitous DNA-modifying enzymes.
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`The molecular mechanism ofV-reglon DNA rearrangement, or V(D)l recombi-
`nation, is illustrated in Fig. 4.7. The 12 bp spaced and 23 bp spaced R859 are
`brought together by interactions between proteins that specifically recognize
`the length of spacer and thus enforce the 12123 rule for recombination. The
`DNA molecule is then broken in two pieces and rejoined in a dii'ferent
`cOi‘itiguration. The ends of the heptamer sequences are joined precisely in a
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`Fig. 4.6 V-region gene segments are
`joined by recombination. In every
`V-region recombination event. the
`signals flanking the gene segments are
`brought together to allow recombination
`to take piece. For simplicity. the
`recombination of a light-chain gene is
`Illustrated: for the heavy-chain gene.
`two separate recombination events are
`required to generate a functional
`V region. In some cases. as shown in
`the left panels, the V and J gene
`segments have the same transcriptional
`orientation. Juxtapoeition of the
`recombination signal sequences results
`in the looping out at the intervening
`DNA. Heptamers are shown In orange,
`nonamers in purple, and the arrows
`represent the directions of the heptamsr
`and nonamer recombination signals
`(see Fig. 4.5). Fiecombinailon occurs at
`the ends of the heptamer sequences.
`creating a signal joint and releasing the
`intervening DNA in the form of a closed
`circle. Subsequently. the joining oi 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 ol the DNA. Joining the ends of
`the two heptamer sequences new
`results in the inversion and integration
`of the intervening DNA. Again. the
`joining of the V and J segments creates
`a functional eregion axon.
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`Lassen — Exhibit 1039, p. 12
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`Lassen - Exhibit 1039, p. 12
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`FEB—Quill Chapter 4: The Generation oi Lymphocyte Antigen Receptors
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`Fig. 4.7 Enzymatic steps In the
`rearrangement of immunoglobulln
`gene segments. Rearrangement begins
`with the binding of RAG-1, RAG-2. and
`high mobility group (HMG) proteins
`(not shown). These HAG-tthAG-Z
`complexes (domes. colored green or
`purple tor clarity although they are
`identical at each recombination site)
`recognize the recombination signal
`sequences (arrows) flanking the coding
`sequences to be joined (red and yellow
`rectangles). These are then brought
`together (second panel). toilowing which
`the RAG complex is activated to cut one
`strand oi 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 oi 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:KuBO (indicated
`in blue) that join the complex (fourth
`panel) along with the RAG proteins. the
`DNA hairpin is cleaved at a random site
`to yield a single-stranded DNA end. This
`and is then modified by the action of
`TdT and exonuciease (indicated in pink.
`titth panel). which randomly creates
`diverse. imprecise ends. Finally (sixth
`panel) the two heptamer sequences,
`which are not modiiied. are ligated to
`form the precise signal joint. while the
`coding joint is also ligated, both by the
`action of DNA iigase iV.
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`head-to-head fashion to form a signal joint in a circular piece of extra-
`chromosomal DNA, which is lost from the genome when the cell divides. The
`V and I 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.
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`The complex of enzymes that act in concert to effect somatic V(D)I recombi-
`nation is termed the V(D)] recombinaee. The products of the two genes
`RAG-1 and HAG-2 (recombination-activating genes) comprise the lymphoid—
`specific components of 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)I recombination. Indeed, these genes. when expressed together, are
`sufficient to confer on nonlymphoid cells such as fibroblasts the capacity to
`rearrange exogenous segments of DNA that contain appropriate RSSs; this is
`how RAG-1 and RAG-2 were initially discovered.
`
`Although the RAG proteins are required for ViDii recombination, they are not
`the only enzymes in 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 DNA iigase W. the enzyme DNA-dependent
`protein kinase (DNA-PK). and Ku, a well-known autoantigen. which is a
`heterodimer (Ku 70:i<u 80) that associates tightly with DNA-PK.
`
`VtDii recombination is a multistep enzymatic process in which the first
`reaction is an endonucleoiytlc 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
`11885 that are guiding the join (see Fig. 4.7). RAG-l is thought to specifically
`recognize the nonarncr of the 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 at sites 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 other strand. sealing the end of the double-stranded DNA
`to create a DNA ‘hairpin' out of the gene segment coding region. This process
`simultaneously creates a flush double-stranded break at the ends of the two
`heptamer signal sequences. The DNA ends do not float apart, however. but
`are held tightly in a complex by the RAG proteins and other associated DNA
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`Lassen — Exhibit 1039, p. 13
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`Lassen - Exhibit 1039, p. 13
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`The generation of diversity In lmmunoglobuilns 131}
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`repair enzymes until the join is completed. The two 11885 are precisely joined
`to form the signal joint. Coding joint formation is more complex. First, the
`DNA hairpin is nicked open by a single-stranded break. again by the RAG
`proteins. The tricking can happen at various points along the hairpin. which
`leads to sequence variability in the eventual joint. The DNA repair enzymes
`in the complex then modify the opened hairpins by removing nucleotides (by
`exonuclease activity) and by randomly adding nucleotides (by terminal
`deoxynucl