REVIEW ARTICLE

Immunoglobulin V Regions and the B Cell

By A. Keith Stewart and Robert S. Schwartz

NVESTIGATIONS of normal and neoplastic B cells are being channeled into new directions by recent work on Ig variable region (V) genes. Fresh impetus to these studies comes from an informative directory of human germline V genes and an improved understanding of how B cells deploy those genes. But substantial questions about the molecular biology of V genes and B-cell maturation remain because of the unexpected finding that a limited group of Ig heavy chain (V,) genes predominates in the preimmune B-cell population. This peculiarity of the VHgene portfolio may hinge on unique properties of certain VH genes that predispose them to frequent rearrangement. It could equally reflect clonal selection in the bone marrow (BM), a process in which clones of immature B cells are selectively deleted (negative selection; clonal deletion) or singled out for preferential growth (positive selection). Emerging evidence suggests that the heavy chain of the B-cell antigen receptor engenders signals that mediate positive and negative selection. In this review we will discuss how the properties of surface Igs could result in clonal deletion or allow individual B cells to continue their development in the BM. Limitations on the V gene repertoire may seem to narrow the potential for antibody diversity, but the process of assembling heavy and light chain genes introduces enormous heterogeneity into the third complementarity determining regions (CDR3s) of Igs. For reasons that will become apparent below, variation in CDR3s is especially broad in heavy chains. Moreover, antigen-driven somatic mutations introduce another layer of permutations in antibody structures during the immune response. These diversifying mechanisms have interesting clinical applications. Our understanding of the clonal origins of malignant B cells has been improved by analysis of V gene structures in B-cell lymphomas and leukemias. Detection of very small populations of malignant clones within a larger population of normal B cells is now possible with the cell-specific markers furnished by the unique V gene sequences of the CDR3. In this report we will discuss the clinical utility of the genetic fingerprints of malignant B cells and comment on the limitations of the methods by which they are detected.
lg GENEREARRANGEMENT

identified, of which 83 are potentially functional.z'*2z As some of these genes are nearly identical, and may represent allelic variants, it is likely that 60 to 70 VH genes are available for rearrangement. These 60 to 70 genes belong to seven families (VHl-vH7) whose members have greater than 80% sequence homology.23 The presence of conserved nucleotide sequences in each family suggests evolutionary pressure to retain certain V gene structure^.^.'^ Less is known about the germline composition of light chain genes, although analysis of Southern blots suggests that VK and VX contain approximately 80 and 70 gene segments on chromosomes 2 and 22, respectively."." Recent compilations of sequence data identified only 75 known V K sequences, of which only 36 are potentially functi~nal'~ 36 known VX sequences of and which 24 were potentially functi0na1.I~The database of germline light chain sequences remains incomplete. Rearrangement of V gene segments9.10is dependent on the protein products of the recombinase activating genes RAG-l and RAG-2.l.' Although these genes are indispensable participants in V gene rearrangement,'.' the biochemical function of their gene products remains unknown.26Nevertheless, as all Ig V gene, and indeed T-cell receptor rearrangements, appear touse a common recombinase?' it follows that targeting of an individual gene for rearrangement may be modulated by the accessibility or exposure of the unrearranged gene to this recombinase.'.' Transcription of the Ig heavy chain locus is activated in pre-B cells and all rearranging loci in these cells are transcriptionally active at the time of their Consequently, it has been proposed that transcriptional activation by lymphoid specific enhancers and Ig promoter regions increases accessibility of V genes to rec~mbinase.~~.~' Hypomethylation of V genes appears characteristic of this transcriptionally active and thus accessible state.3zs33 imporThe tance of transcriptional activation has beenconfirmed in vitro; activation of germline gene transcription increases the frequency of V gene rearrangement' whereas deletion of an Ig-specific enhancer (Ep) dramatically inhibits germline V gene demethylation, transcription, and rearrange men^^^.^' Recombination of V genes starts in lymphoid progenitor (both T and B) cells,' within the IgH locus of either the

The mechanisms of Ig gene rearrangement have been reviewed in detail e1~ewhere.I.~ coding instructions for the The antibody V region arise from coordinated mechanisms of V gene rearrangement, joining and somatic m~dification.~-' Three distinct gene segments-the variable (VH),diversity (D), and joining region (JH) genes-encode the heavy chain of the variable region:" whereas two segments-variable (VK or VX) and joining (JK or JX) region genes-encode the light chains."-14 The Ig heavy chain (IgH) locus, on chromosome 14, contains an estimated 100 to 150 VHgenes, 30 D gene segments, and 6 J gene segment^.'^-^^ In a recent H census of VH genes compiled from a single individual and from the reported literature, 122 germline VH genes were
Blood, Vol 83,No 7 (April l), 1994 pp 1717-1730

From the Division of Hematology-Oncology, The Toronto Hospital, General Division, Toronto, Ontario, Canada; and the Division of Hematology-Oncology, New England Medical Center, Boston, MA. Submitted September 24, 1993; accepted December 27, 1993. Supported by grants from The Canadian Red CrosdCutter, The Medical Research Council of Canada, and National Institutes of Health Grant No. A128899. Address reprint requests to A. Keith Stewart, MD, Mulock-Larkin Wing, Room 2-024, Toronto Hospital-General Division, 200 Elizabeth St, Toronto, Ontario, M5G 2C4 Canada. 0 1994 by The American Society of Hematology. ooo6-4971/94/8307-0037$3.00/0
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STEWART AND SCHWARTZ

maternal or paternal chromosome 14.'"" Recombinase proteins must recognize andbind to conservedheptamer-spacernonamerrecombinationrecognition sequences flankingall V gene segments.36In B-lineage cells, rearrangementof one D gene to one JH gene followed by the addition of one of is the numerous VHgenesto the fused D-JH segment^.'^ If successful, resulting the VH-D-JH rearrangement is transcribed asa single unit intoRNA thatisspliced tothe constant region RNA (Cp) before translation into anIg heavy ( p ) chainin the pre-B ell.^.^ If the initial rearrangement yields a sequence that cannot be translated (nonproductive or abortive rearrangement) then rearrangement of the IgH locus proceeds on the other allele.37 The presence on the B-cell surface of a fully assembled p heavy chain inhibits further rearrangementsthatmight occurontheoppositeallele (allelic e x c l u ~ i o n ) . ~ Thus, ~~" each pre-B cellexpressesonlyone kind of heavy chain. However, if malignant transformation arrests B-celldevelopment before expression of the heavy chain on the cell surface, while RAG-l and RAG-2 are transcriptionally active, a new heavychain rearrangementmay occur. This phenomenon has been inferredfrom the ongoing VH gene replacement that occurs in clonallyrelated acute lymphoblastic leukemia (ALL) pre-B ~ e l l s ~and ~ ~ " - in the progenyof single transformed precursor B cells.44 RAG-l and RAG-2 expression is most prominent in early B cells and is limited or absent in later stages of d e v e l ~ p m e n t . ~Thus, the ability to un"~~ dergo Ig gene rearrangement decreases asB-cell maturation proceeds. After successful heavy chain gene rearrangement in preBcells, cytoplasmic p chain attachestotheendoplasmic reticulum predominantly in association with the Ig binding protein BiP.49Binding of newly formed p chain to surrogate light chain proteins (9 light chain),'" results in the displacement of small amounts of p from the endoplasmic reticulum and, in some latepre-Bcells, allows translocation of the resulting complex to the cell s u r f a ~ e . The~ ~ light chain ~~ * proteins are encoded by the Vpre.b A5114.1 genes, which and have significant homology with the Vh, Jh, and light chain constant region gene^.^"'^ The association of p, the light chain, and two associatedproteins, Ig-a and Ig-p, on the pre-B cell surface forms theB-cell antigen receptor comple~?~ which in many ways resembles its counterpart, the T-cell receptor complex. Although light chain assembly may occur in the absence of a functional heavy chain, the process is greatly enhanced by successful heavy chain rearrangement.2 Light chain rearrangement beginswhen one of an estimated70 kappa light chains (VK) rearranges to one five joining, JK, genes." first of If kappa light chain rearrangement is unsuccessful on both alleles, lambda light chains (VA and Jh) will subsequently rearrange. This hierarchical sequence of K before h may not be absolute, asstudies ofALL56 and fetal BM Bcells57 indicate that A light chains may occasionally rearrange first.

lacking functionalRAG-lSxand RAG-2 genes,"' together with the restoration of T-cell maturation in RAG-2-deficient mice by transgenic T-cell receptor variable region genes," constitute compelling evidencethat T cells and B cells must display antigen receptors on their surface if they are to complete differentiation. Moreover, B cells requirea surface heavy chain for completion of their development: targeted disruption of the p chain gene6' or deletionof the heavy chain joining region in mice6' arrests maturation at the preB cell stage. Era showed et that the appearance of a surface heavychain stimulates differentiation in immature interleukin-7(IL-7)-dependent mouse Bcells,whichbecome IL-7-independent when they express a surface light chain.The critical role oftheB-cellantigenreceptor in B-cell development is further emphasized by the dramatic reduction in early B-cell production shown in mice with a disabled As gene.64 How does the B-cell antigen receptor complex participate in B-cell development? A clue comes from the hereditary immunodeficiency disease X-linked agammaglobulinemia, in which a mutation in a gene belonging to the src family of signal transducing protein tyrosine kinases (the Ark gene) causes a block in B-cell e ~ e l o p m e n t . ~This~finding underd ',~ scores the necessity for signal transduction in B-cell maturation and raises the possibility that B-cell signals initiated by the B-cell antigen receptor complex may direct ongoing Bcell development at the pre-B cell stage. The p chain associated Ig-a0g-P heterodimer is both necessary and sufficient to mediate signal transduction by the Ig receptor and may well be one of the participants in the signaling required for B-cell mat~ration.'~
FORMATION OF CDR3

ANTIGEN RECEPTORS AND B-CELL DEVELOPMENT

The lack of mature B and T cells in mice that cannot rearrangevariableregion gene segments, suchassevere combined immunodeficiency disease (SCID) mice or mice

The Ig heavy and light chains each contain three hypervariable (complementarity determining regions or CDR) segments and four conserved framework areas.68The CDRs are the most diverse regions of the antibody molecule; all six associate to form the antigen-binding site,69 whose conformation is maintained by the framework sequence^.^" In the heavy chain, two CDRs (CDRl and CDR2) are encodedby the VH gene segment; collectively, they define 50 groups of germline VH genes.7' These groups may impose an upper 50 limit on the number of structurally different antigen binding sites in the naive Ig repertoire. The CDR3 is indirect contact with antigenand isthe most variable portion of the Ig molecule. Its extraordinary diversity-up to IOl4 different peptides are p o ~ s i b l e ' ~ - ~ ~ results from several mechanisms (Fig 1). The CDR3 encompasses the 3' end of VH, all of D, and the 5' end of JH. It often contains N nucleotides, which are randomly inserted at both the VH-D and D-JH junctions by the enzyme terminal deoxytransferase (TdT). The introduction of junctional nucleotides is developmentally regulated;insertions N are found in 68% of fetal B cells, 86% of neonatal B cells, and in 91% to100%of mature adultB ce11s.72~73.7s Another source of diversity in CDR3 is random deletion by nucleases of the terminalnucleotides of rearranging VH, D, and J genes. H Moreover, the D gene segmentsmay be rearranged in reverse orientation (3'to 5 ' ) fused withanotherD segment(D-

V REGIONS AND B CELLS

1719

VH

JH

REARRANGEMENT

CDR1 CDR2

LIGATION

v]
n n n n n n n m mnnnn Fig 1. Formation of CDRB. Duringgene V rearrangement variable deletionsof the ends of V, D, and JH, combined with nonternplated nucleotide (nl inat sertions tionr contribute to the CDRB. During antibody maturation,somaticmutations (x) furtherdiversify the CDR fingerprint.

1 CHAIN

CDR3

+
ANTIGEN DRIVEN
SOMATIC MUTATION

and V-D

D), and transcribed in any of three potential reading frames depending on the VH-D junctional ~equence.~.~~ At junctional sites in which no nucleotide deletions occur there may be templated p nucleotide^.^^.^^ These sequences, usually only one or two bases long, are complementary copies of the last nucleotides of the coding region they abut. They may be used to repair asymmetrical breaks in the hairpin ends of V genes undergoing rec~mbination.~ Exceptionally long stretches of p nucleotides have been found in thymocytes from mice with SCID, a finding that implicates abnormal DNA repair in the pathogenesis of this disease.79 All of these variables contribute to the CDR3 antibody fingerprint even before the somatic mutations that further distinguish and individualize V They result in a CDR3 sequence that is unique to each rearrangement, and that therefore identifies individual B cells or clonal B-cell expansions. Indeed, it has been calculated that only 5 in 1 0O O circulating B cells share the same CDR3.72 0, O
ANTIGEN SELECTION OF

B CELLS

Although rearrangement stops after the loss of recombinase activity in the BM, further changes in V genes occur during the immune response. The adaptive response to an immunogen occurs in the germinal centers of lymphoid follicles. B cells whose antigen receptors bind to the immuno-

genn3,n4 react by proliferating. By thus acquiring a growth advantage, they become the targets of clonal ~election.~ Under the influence of T-helper cells,86the clonal progeny of these antigen-stimulated B cells mobilize a mechanism that causes somatic mutations (nucleotide substitutions) of rearranged V genes. The mutations occur only in the coding regions of the Ig genes; they may be silent or they may result in amino acid replacements that change the affinity of the antibodys binding site for the antigen. If the mutation causes a loss of the receptors affinity for the antigen, the B cell loses its growth advantage. If the mutation causes a gain in the receptors affinity, the B cell gains a proliferative ad~antage.8~-~ Because mutations in CDRs are the most likely to affect the antibodys binding site, a hallmark of an antigen-stimulated B cell is the clustering of replacement mutations in the C D R S . ~ ~Thus, the B-cell response to -~ antigen is a microcosm of Darwinian evolution in which the environment (antigen) and V gene mutation collaborate to select B-cell clones whose surface Igs best fit the antigenic challenge. The resulting increase in antibody affinity for antigen is the hallmark of a maturing antibody response. Because V gene mutations are unique to each B cell and its progeny, and because they occur onlyin B cells that have engaged in an immune response, the V gene nucleotide sequence can show the biography of a B-cell clone. As will

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STEWART AND SCHWARTZ

CMOKINES STROMA

? SELF ANTIGEN

FOREIGN ANTIGEN FOLLICULAR LYMPHOMA NHL MYELOMA

ALL

BURKITS LYMPHOMA

CLL

PRO-B

PRE-B

MATURE B

ANTIGEN SELECTION

r
CLONAL

A N T I B O D Y

EXPAN-

PRODucTlON

LYMPHOID MARROW BONE CIRCULATION

BONE MARROW

T CELL INDEPENDENT GM UNMUTATED V GENES ALLELIC EXCLUSION

T CELL DEPENDENT ISOTYPE SWITCH MUTATED V GENES

Fig 2. 6-cell development is represented schematically. Features characteristic of V gene structures at each p h a r of 541development are highlighted. Althoughthe early development of B-lineage cells is generally consideredto be independent of the influonce of antigen, we propose that recognition of self antigens is important in B c l clonal selection (see text). $ represents the surrogate lg t chain; p the Ig - el ih heavy chain; S, IgD; and 7, IgG. TdT expression islimited to the earliest stages of B-cell development; expression 9 ir limited to Iate-stoge of pre-B cells. It should be noted that only 10% to 20% of late pre-B calls expross $. Expression of RAG 1 and RAG2 occursat high level8in eerly preB cells and decreases dramatically as 6 cells mature. normal The counterparts of 6-41 malignancies are The majority of circulating B cells are CD5 negative and the expression of CD5 is used here to show the maturetion stage in 6-cell differentiation at which CLL arises.

be seen below, the presence or absence of V gene mutations in B-cell malignancies can pinpoint the developmental stage of a neoplastic cell (Fig 2). Besides somatic mutation, other molecular signs of antigen selection include the repetitive use of particular V genes to encode antibodies of a given specificity and the. IgM to IgG isotype switch. Up to 25% of circulating B cells with surface IgM (Cb) may show molecular evidence of a previous antigen c ~ n t a c t ,but somatic mutation predominates ~~.~~ in the Cy,89-92 Ca,93and populations of B cells. A relevant example of these principles is a study of 14 V genes used by anti-Rh blood group antib0dies.9~ Both low-affinity IgM and high-affinity IgG anti-Rh antibodies were shown to originate from a limited pool of V genes. The four IgM antibodies were encoded by two minimally mutated VH genes, V&-21 and hv3019b9 (a vH3 family member). Of the IgG antibodies, 6/10 were somatically mutated variants of those same two genes. High affinity in these anti-Rh antibodies correlated with frequent replacement mutations. The J H gene (rearranged in 18% of normal human circulating ~ B cells8), was used by 79% (1 1/14) of the antibodies. This very high frequency may reflect structural requirements for binding to the Rh antigen, as 9/11 CDR3 segments encoded by JH6result in the protein sequence tyr-x-met-glu-Val. In another study, marked variations in the representation of individual V genes in populations of naive Cp and experi-

enced CyB cells were found over time. These population shifts presumably resulted from accumulated antigenic experience; they suggest that antigen selection has a major role in shaping the V gene repertoire in humans.g0
lg V GENE REPERTOIRE

The B-cell repertoire may be divided into a naive (preimmune) repertoire, consisting of virginal B cells that have not been exposed to external antigen, and the immune repertoire of mature B cells in peripheral blood and lymphatic tissues. If each functional germline gene segment has an equal chance of undergoing rearrangement to form a VH-D-JH transcription unit, then the V gene repertoire (ie, the distribution of V segments in the population of B-cell clones) of naive B cells should contain all germline V genes in equal frequency. For instance, if there are 83 potentially functional germline VHgene segments, the frequency of each VHgene in a representative population of preimmune B cells should be 1.2%, and all functional VH genes should be present in rearranged form. However, the actual finding is a pronounced over-representation of certain V The naive repertoire has been studied in B cells from fetal lymphoid or cord and in pre-B cells of ALL.103.w NormalBM pre-B cells have been studiedin mice but not yet in humans. Cp+ B cells from each of these sources probably rearrange their V genes independently of

V REGIONS AND B CELLS

1721

rearrangements in the pre-B cells of ALLLo3 and in the aborted K light chain rearrangements of A light chain-expressing B cells in CLL.'" Therefore, bias in V gene distribution can be introduced into preimmune B-cell populations before expression of the surface Ig, and thus before any possibility of influence by antigen. A clear-cut example has been found for the case of the VK gene humkv325. The expected frequency of rearrangement of h u h 3 2 5 is 1.4% (1/70), yet 14% of nonproductive VK rearrangements in A light chain-expressing CLL contained this gene.'" Thus, at least in CLL, humkv325 has an inherent tendency to rearrange, ie, its high frequency in the repertoire is independent of antigen selection. Of interest, biased utilization of T-cell receptor variable region genes has also been noted to occur in the absence of any possibility of antigen election."^ Repertoire bias that appears before the expression of a surface Ig receptor could be a consequence of the number of copies of individual V genes in the germline, advantageous accessibility of the V gene to recombinase, the presence of V gene specific promoter sequences, or enhanced V gene transcriptional activation. Indeed, one of the frequently expressed VH genes (VH26) is present at two unique chromosomal sitesLz6 and appears to be flanked by sequences that may function as promoters or enhancers of tran~cription.'~' The VH26gene (also called 30P1, 18/2, and 3-23),"' one Transcriptional activation greatly increases the probability of at least 29 potentially functional germline vH3 family of V gene therefore, it has been postumembers:' illustrates the point. This VHgene was found to lated that transcription may make V genes more accessible be expressed in 24% of fetal B-cell cDNA clone^,'^"^' 15% to recombinase.l,2However, no evidence has directly linked of cord blood cDNA clone^^^*'^' and 6% to 10% of unsegene duplication or preferential transcription to V gene seleclected adult peripheral blood B cells.''' It also shows up in tion bias. The position of VHgenes within the IgH locus (5' 8% of published sequences of autoantibodies with a wide or 3') influences the fetal mouse repertoire,'" perhaps range of specificities,%including antibodies against platelets, cardiolipin, myelin, and thyroglobulin."4"'8 At last count, through accessibility to recombinases, but VHgene position has no role in the formation of the human B-cell repertoire. 23% of published anti-DNA antibody sequences were enIndeed, VHgenes that rearrange frequently in human fetal B coded by this one VH gene.%."""8 Furthermore, VH26also encodes antipathogen antibodies, including antibodies to racells are not only dispersed over 890 kb of germline DNA bies?' herpes simplex,Io8 hemophilus B,'w*"oand human imbut also interspersed with other VHgenes that are not develmunodeficiency virus (HIV),"' often in a somatically muopmentally sele~ted.''~"'~ There is good experimental evidence that V gene selection tated form. Thus, the predominance of VH26extends from the fetal liver B-cell to adult germinal center B cells engaged occurs in fetal mice during the processes of recombination in the secondary immune response. VHrestriction is not conand ligation that join D to J H and VHto D-JH Short identifined to VH26.On the contrary, a detailed analysis of the cal nucleotide sequences at the ends of VH,D, and J H appear expressed VHgene repertoire of two normal subjects showed to promote rearrangement; this results in limited junctional that one third of over 100 C p cDNA clones used only 6 of diversity, with a predominance of particular coding sethe 83 available VH genes. Moreover, only 23 VH genes quences at the sites of h o m ~ l o g y . ~ The translated prod~'~'~~ encoded 90% of all the C p clones in unselected adult periphucts of these rearrangements predominate in the expressed eral blood B cells?' V gene restriction is not unique to heavy fetal r e ~ e r t 0 i r e .This form of directed rearrangement may l~~ chain genes; for example, a K light chain gene, humkv325, also result in the pairing of homologous segments of VH which can be identified by the idiotype it encodes, has been and D genes in a nonproductive reading frame."5 Such a shown in 4% of tonsillar B cells,"' in 25% of light-chain junctional "mistake," if it is the rule rather than the excepbearing B cells in chronic lymphocytic leukemia (CLL),'20,121 tion, can explain the relative under-representation of certain and frequently in IgM paraproteins with rheumatoid factor V genes among the productively rearranged V genes of the activity.'22.'2' fetal mouse re~ert0ire.l'~ mice deficient in the enzyme In TdT, homology-directed joining is a prominent feature of FORMATION OF THE V GENE REPERTOIRE early V gene rearrangement, prompting speculation that the How can bias in the rearranged V gene repertoire be expresence of TdT obscures or inhibits this important mechaplained? There are two main possibilities: inherent properties nism of nonrandom V gene a~sociation."~*"~ of the overrepresented genes themselves or B-cell selection. Evidence for clonal selection o V genes. In contrast to f Germline determinants of V gene rearrangement. Rethe mouse, there is no evidence of directed V gene recombistricted use of V genes has been found in nonproductive nations in embryonic muscovy ducks."' Onthe contrary, the influence of external antigen, an assumption supported by the absence of somatic mutation in V gene coding sequences derived from the C p cDNA of these cells. A consistent pattern of dominance of certain individual VH,D, and J H genes has emerged from these studies. In the naive V gene repertoire, the use of JH genes is biased. The JA gene segment, which is one of the six potentially available J H genes, occurs not in the predicted frequency of 17% (1/6) but in 32%of rearranged Ig cDNA clones from fetal liver:83w in 42% of pre-B cell ALL clones,'" and in 50% of cord blood cDNA c l ~ n e s . ' ' ~ ~ ~ ~is also bias in the use of D There gene segments; HQ52, is found in 45% of fetal liver B cells and Dnl, with an expected prevalence of 3.3%,was actually found in 15% of pre-B ALL cells. A small group of individual germline VHgenes, to be mentioned presently, also predominate in fetal liver, cord blood, and in pre-B cell ALL. Studies in the mouse suggest a similar V gene restriction in normal pre-B cells.lo5 The small set of recurrent V gene segments that fetal liver B cells rearrange are also ubiquitous in unselected populations of adult peripheral blood B cell^,^^'"^^^^ B cells that produce specific antibodies against bacterial or viral pathogens,89,91.1"-' I ' and B cells that produce autoantibodies."'

1722

STEWART AND SCHWARTZ

Table 1. Molecular Events That Result in Unique V Gene Structures initial rearrangements occur randomly, whereas later in emand a Diverse Antibody Population are Contrasted With Those bryonic developmentthere is a progressive positive selection Features of V Gene Rearrangement That Place Structural for individual p chains. This selection favors particular V,Limitations on Antibody Formation D and D-JH junctionalsequencesandmaybedriven by Restriction Diverslty Antibody Antlbody the protein product of the CDR3. Presumably, the antigenbinding characteristics of the favored CDR3 aresome way in 1. Variable associations of 1. Fixed germline V gene pool advantageous to the animal's developing immune system.In 2. Preferentialrearrangement VH,D, and JH VL and JL humans,selection forthe proteinproduct of CDR3 may Gene copy number Recombinase Heavy and light chains explain the predominance of recurrent amino acids in the accessibility 2. Hypervariable CDR 3 junctional sequencesof some germline-encoded cold agglutiTranscriptional "P" templated nucleotides n i n ~ ' ~ ' anti-DNA antibodiest4' and the predominance and of enhancers "N" junctional nucleotides polyreactive autoantibodies with particular binding properHomology directed Deletions at ends of VH, D, ties produced by the malignant B cells of CLL.I4' joining and JH Sequence conservationwithin humanVH families also 3. Nonrandom V gene D gene fusion (D-D) suggests a selective pressure to retain structural features of association D gene inversion (3'-5') theheavychain p ~ l y p e p t i d e .Some frequentlyexpressed ~~ 4. Clonal deletion Gene conversion VH genes,such as VH26 and V&, areconserved between 5. Receptor editing Three potential reading frames 6. Structural limitations species, exhibit little polymorphism, and occurin up to 98% 3. Somatic mutation of the population.126,'4'.'44Thus,advantageous features of antigen-binding sites encoded by conserved V, genes may contribute totheir over-representation in the preimmune repertoire. from the endogenous mouse Ig locus. This "receptor editNegative selection (clonal deletion) can influence the V ing," if successful, offers the developing B cell an opportugene repertoire of earlyB cells in n ~ r m a l ' ~ 'and transgenic .'~~ nity to avoid d e l e t i ~ n . ~ ~ ~ * ~ ~ ~ mice.'47 Clonal deletionof B cells has been shown unequivoContinuous positive and negative selection of pre-B cells cally in cases where the B cells express transgenic V genes may explain the death of the vast majority of B cells before of potentially harmful a ~ t o a n t i b o d i e s . ' ~For'example, a ~~ ~' they leavethe BMI6'; thenumerousautoreactivesurface dramatic reduction in splenic and lymph node B cells was IgM' B cells in normal blood; and the restricted heavy and found in mice that were transgenic for the VH andV Kgenes light chain V gene repertoire.Aparallelmodel of T-cell of ahemolyticanti-redblood cell (RBC) a~toantibody.'~' differentiationinthe thymusalso requiresT-cellreceptor Furthermore,peritonealB cells that evadedcontact with recognition of self antigen^.'^'"^^ The molecular events that RBC autoantigens were rapidly destroyed after intraperitoresultin diverse antibody structures and impose limitson neal injection of syngeneic RBCS.'"~"' The V gene reperthe immune repertoire are summarized in Table 1. toire of transgenic mice bearingV genes for self-reactive antibodies is further limited by light chain "receptor editing," 152-154 during which autoreactive B cells try to escape CLINICALRELEVANCE OF V GENESELECTION deletion by replacing a pathogenic light chain with a harmCold agglutinins. Adramatic example of VHgene reless light chain. striction has emerged from molecular studiescold agglutiof Autoreactive antibodies and clonal selection. The manins. The first anti-I cold agglutinin to be sequenced was jority of polyreactive IgM autoantibodies (natural autoantiproduced by aB-cell lymphoma thatuseda VH4 family bodies) that are presentinall humansand m i ~ e ~ ' ~are ' genesegment, VJ-21.166 This V, genewas subsequently "~ encoded by unmutated (germline) V genes.'58.t5y The physioobserved in high frequency in normal B cells and in other logic role, if any, of these autoantibodies is unknown. It has autoantibodies.'" Analysis of 10 additional monoclonal cold been proposed that the B-cell surface heavy chain counteragglutinins showed an absolute V J - 2 1 gene restriction, irreparts of natural autoantibodies participate in clonal selection spective of the anti-i or anti-I specificity of the antibody,14~.t67,i68 of early B cells.'60 In this model, the newly formed surface That heavy chain structures encoded by V&heavy chain, by reacting with a self antigen, initiates signal21 itself are essential for coldagglutinin binding was brought ing pathways that promote B-cell differentiation. Affinity of out by the unique CDR3 sequences of each cold agglutinin the surface heavy chain for self antigens would determine heavy chain and the lack of association with any particular K light chain. The V&-21 genes of the anti-iagglutinins whether the pre-B cell is deleted (high affinity) or allowed were unmutated; in the anti-I antibodies they had few or no to differentiate (low affinity). According to this scheme, the mutations. In contrast, the cold agglutinin light chain genes entire preimmune repertoire should be weakly autoreactive. contained many nonrandom somatic mutations of the CDRs. The role of self antigen in clonal selection is supported by As VJ-21 also predominates in other anti-RBC antibodrecent experimental evidence which shows that strongly auies," it has been hypothesized that it encodes a polyreactive toreactiveB cellsmay modifytheir surface Ig toescape heavy chain with the capacity to bind weakly to a variety clonal d e l e t i ~ n . ' In ~ ~ ' ~ ~ mice, developing B cells ~ transgenic of RBC carbohydrates. In the case of cold agglutinins, fine expressing a surface light chain with high-affinity anti-DNA specificity and high avidity binding appear to be conferred activity are destined for deletionunless the pathogenic light on the autoantibody by the VH CDR3 and by somatic mutachain isremovedandreplaced with alight chain derived

V REGIONS AND B CELLS

1723

tion of the light chain V region gene segments. These latter features signify an antigen-driven immune response. The restriction in the V-D junction of these antibodies (a glycine residue at this site occurred in 6/10 cold agglutinin sequences) probably represents a structural requirement for cold agglutinin binding. It is important to keep in mind that many of these studies on monoclonal cold agglutinins dealt with the Ig products of malignant B cells. The results we have just summarized imply that the neoplasms which produced the monoclonal cold agglutinins originated from antigen-stimulated progenitor B cells. We will take up below other examples of malignancies that appear to arise from antigen-stimulated B cells. Although pathogenic monoclonal cold agglutinins show an absolute restriction to V&-21, the natural cold agglutinins in normal serum do not.169 cells from two normal subjects B were transformed by Epstein-Barr virus (EBV) and screened for production of anti-i and anti-I cold agglutinins; two antiI clones and one anti-i clone were found. The anti-I clones usedvH3 family genes, whereas the anti-i clone used the V d . 2 1 gene. Additional studies confirmed that most serum cold agglutinins from normal subjects were not related to the V A . 2 1 gene segment, as in monoclonal cold agglutinins. Thus, it appears that the tight correlation between cold agglutinin activity and V d . 2 1 holds only for B-cell neoplasms. CDR3 as a clonal marker. The remarkable diversity of VH CDR3 has been exploited to identify clonal populations of B cell^.'^@'^^ The polymerase chain reaction (PCR) is advantageous for this purpose because it amplifies the CDR3 sequence in question thousands of times. To amplify around the CDR3 segment and flanking sequences, primers corresponding to the conserved sequences of the framework l or framework 3 regions of VH and the 5' end of J have been H used,170-173With such PCR primers it is possible to detect

of lo5 normal B cells.'79Evaluations of allele-specific PCR to detect minimal residual disease are ongoing; in one early report this technology was unable to predict relapse in ALL."' This result is not surprising because oligoclonal Bcell populations that differ in the CDR3,'8'*'82 and ongoing VHgene rearrangements that disrupt the CDR3 of the malignant are frequent in ALL. Similar problems are likely to be encountered in follicular lymphomas, which have a high CDR3 somatic mutation rate.IE3 Conversely, the more stable V genes in the B-cell clones of intermediate-grade non-Hodgkin's lymphoma(NHL),Is4 multiple myeloma,'85 and CLLl86-188are likely to lend themselves more readily to study with PCR. ALL. Nucleotide N regions are less frequent in fetal B cells than in neonatal and adult B Therefore, the absence of N nucleotides can serve as a relatively specific temporal marker of B-cell development. Wasserman et showed that 39% of sequenced V genes in childhood ALL lacked N insertions, a frequency similar to that of fetal B cells. The lack of N regions was more frequent in leukemias in younger children; 87.5% of the ALL V genes from children under the age of 3 lacked N regions in CDR3, whereas only 11% of leukemic clones from children older than age 3 lacked N insertions. This finding may imply that the transforming event in the younger patients occurred in utero, and that the latency period for development of leukemia was less than 3 years. Other possibilities include low-level TdT expression in a subset of neonatal B cells or more frequent loss of TdT activity in leukemic clones arising before the age of 3. In 98% of ALLs, clonal IgH rearrangements can be identified, whereas only 48% 23% and show K and A rearrangements, respective1y.l8' There isno correlation between immunophenotype and Ig rearrange~nent.'~~with As normal B-cell populations, over-representation of individual clonal populations that make up only 1% to 5% of the total VH,D, and J genes occurs in B-cell ALL.".'" For example, H pop~lation.'~' However, because the primers are not clone when ALL clones with rearranged vH1 family genes were specific, the PCR, if allowed enough cycles, will eventually examined, five of nine rearranged VHgenes used the VH20P3 amplify all CDR3 regions in the sample. Thus, detection of gene that was originally identified in fetal liver." Although B-cell clonality requires a limited number of PCR cycles to differentiate clonal from polyclonal populations of B ~ e i I s . ' ~ * exceptions have been published4*the majority of V genes in ALL are unmutated. Therefore, blind reliance on the PCR as an indicator of BMultiple clonal Ig rearrangements have been foundin 45% cell malignancy is to be discouraged. In up to 15% of reactive of patients with ALL.'" After the exclusion of extra copies B-cell proliferations in lymph nodes or spleen, B-cell clonof chromosome 14 as a contributing factor, 27%of ALL ality has been detected by the PCR reaction.'74Furthermore, cases were biclonal and 13% oligoclonal. Oligoclonal leukelimitations of the consensus primers result in the failure to mic populations share the same karyotypic abnormality; amplify clonal sequences in 20% of B-cell malignancies thus, the Ig rearrangements must have occurred after neoeven when the abnormal clone is dominant. This phenomeplastic transformation.ls2Secondary V gene rearrangements non reflects loss of primer annealing because of 3' VHor 5' that result in oligoclonal populations reflect ongoing recomJ nucleotide deletions'73or aberrant V gene rearrangements H binase activity in the immature leukemic c l ~ n e ~ ~ ,are and ' ~ such as those noted in Burkitt's 1ymph0rna.l~~ most often characterized by identical D-JH rearrangements Thus, the sensitivity of PCR for identifying B-cell clones joined to unique VH Thus, the oligoclonality of is little better than that of Southern analysis. To search for B-cell ALL is caused by ongoing VHgene rearrangement or smaller populations of B cells, it is necessary to design tumor-specific primers. This method requires sequencing the VH gene replacement. The presence of clonally related yet clonal CDR3 PCR products and designing PCR primers diverse minor populations of leukemic blasts is clinically complimentary to the unique VH-D-JH junction (allele-spesignificant as these clones may with time predominate and cific primer^)."^"^^ Allele-specific primers amplify only remay compromise the ability to detect minimal residual disarranged Ig DNA from the particular malignant B cells under ease at the molecular level.'" study and can detect one malignant B cell in a population Burkitt's lymphoma. The V genes encoding surface Ig

1724

STEWART AND SCHWARTZ

Serial study of the V genes of follicular lymphomas that in Burkitt's lymphoma are germline (unmutated), but in contrast toALL there isno evidence of ongoing junctional progress to diffuse lymphoma has shown numerous shared changes.'" This difference presumably reflects the low levels mutations in both tumors, indicating the origin of the diffuse of RAG-l and RAG-2 mRNA in surface IgM positive Burlymphoma from a single follicular lymphoma cell."' Friedkitt's lymphoma cells.lgOStructural abnormalities of Ig V man et a1'" showed that a minor clone in a transforming genes have been reported in cell lines derived from patients low-grade lymphoma emerged over time to predominate and with Burkitt's l y r n p h ~ m a . ~ ~ ' . 'these lines the VHheavy In ~ ~ eventually dominate the diffuse NHL.In contrast, de novo chain sequence was truncated and the residual VH (frameintermediate-grade NHLs show no evidence for ongoing somatic m u t a t i ~ n . A similar lack of mutation was noted in '~~ work1)was directly rearranged to the p constant region a case of gastrointestinal mucosa associated lymphoid tissue with no intervening VHCDR1-D-JH. The site at which many of these breaks occurred may be a potential alternative splice (MALT) lymphoma associated with Sjogren's syndrome that site."' These kinds of truncated p chain products are typical was studied over a period of 13 years."' Thus, it would appear that the machinery for somatic mutation is active in of heavy chain disease'94; they have also been shown in EBV-transformed B-cell lines. The absence of light chain follicular B-cell lymphomas, butnot in diffuse or MALTproduction in malignant B cells with truncated p chains associated NHL. provides further support for the necessity of a functional Multiple myeloma. Asmightbe expected, themajority heavy chain during B-cell d e v e l ~ p m e n t . ' ~ ~ ~ ' ~ ~ of V genes in multiple myeloma appear heavily mutated, in CLL. V gene usein CLL is highly r e s t r i ~ t e d " " . ' ~ ~ ~ ' ~ 'keeping withthemature ~~" B-cell derivation of this maligand frequent pairing of the same germline VHand VK genes nancy.2'8 However, these results are not definitive because the V gene sequences in the myeloma cells were not sought has been noted.'" The V genes in this disease are usually in the patient's germline. In humans, V gene mutations can unmutated"6-1'8and expression of surface IgM is weak. The be distinguished from polymorphic variants or unknown V absence of somatic mutation places the CD5+ (B-l) B cell of CLL in the preimmune repertoire, or at an early stage in genes only by comparisons withunmutated germline sequences. Both idiotypic2" and sequence analysis2'*suggest the immune response."' However, isotype switching in CLL that the V gene repertoire of this mature B-cell malignancy cells has been noted:" albeit infrequently, and unusual variis restricted, whereas analysis of tumor-derived V genes indiants of CLL may have features of the more differentiated cates that there is little intraclonal variation or ongoing soB-cell malignancies. Consistent with this observation scatmatic mutation,"' although isotype switching may occur.220 tered examples of CLL harbor V genes with somatic mutations202.204., thus, they resemble the malignant cells of nonThe use of CDR3 as a clonal marker has been rewarding in myeloma. Circulating malignant cells can be identified in Hodgkin's lymphoma. Consistent with clinical patterns, V the majority of patients at a frequency of 0.001% to 1.0%.22' gene sequences in Richter's transformation indicate a comMoreover, circulating Cp B cells have beenfoundtobe mon clonal origin of the developing high-grade lymphoma clonally related to, and perhaps precursors of, the plasma and the preceeding CLL.'"' cells that secrete C y or Ca paraproteins.222.223 These results NHLs. V gene restriction has been found in follicular ~ ~ ~ . 2 0 6 - 2 0 8 In eight cases that had rearranged a VH4family implythat multiple myeloma may arise from a germinal center Cp+ B cell which, under the influence of an immunoAs gene, three used the V&-21 gene and three VH71-4.208 genic stimulus and T-cell help, makesan isotype switch, with other B-cell malignancies, the restricted use of V genes mutates its V genes, and returns to the BM for clonal expanin follicular lymphoma is unlikely to be disease specific. sion. Unlike malignancies arising from early B cells, follicular Analysis of somatic mutations may provide clues to the lymphomas characteristically have heavily mutated V pathogenic nature of some Igs in this disease. This is illusgenes.2flR-2'fl influence of an antigenic stimulus is sugThe trated by the structural analysis of a V gene encoding a light gested by prominent replacement mutations in the CDR rechain antibody associated with light chain nephr~pathy."~ gions.'" By contrast, the majority of mutations in the frameThis light chain V gene showed mutations that created new work regions are silent. Thus, in follicular NHL the forces potential N-glycosylation sites. Indeed, the K light chain proof clonal selection may preserve the structural framework tein eluted from diseased kidneywas abnormally heavily of the Ig while continuing to modify its CDRs."' glycosylated, a finding that suggests a role for aberrant glyV genes in follicular lymphoma B cells can also exhibit cosylation in light chain nephropathy. In nonsecretory myintraclonal diversity."' Analysis of the V genes sequences eloma, alterations in the coding sequence of thevariable from a single lymphoma strongly suggested that theyderived region result in a failure of VH transcription and rapid intrafrom a common precursor V gene thatwasmodified by cellular degradation of the truncated y chain.*" somatic mutation, a process that resulted in an oligoclonal population of neoplastic B cells.213The ongoing somatic SUMMARY mutation in follicular lymphoma suggests that antigen stimuThere is now substantial evidence that a small group of lation notonly perpetuates the malignant clone but also V genes predominates in the Ig repertoire of preimmune B causes the waxing and waning course of the disease.'" Socells. This phenomenon of V gene restriction may reflect matic mutation in low-grade lymphoma has further clinical preferential accessibility of these genes to recombinase, horelevance because V gene mutationmay result in escape mology-directed V gene rearrangement, promoters and enfrom anti-idiotype sensitivity and thus limit the efficacy of hancers of V gene transcription, or positive and negative monoclonal antibody therapy.''4.''s

V REGIONS AND B CELLS

1725

selection mediated by theanti-self binding propertiesof the B cells surface Ig. Thesemechanismsmayoperatealone or in combination to influence V gene rearrangementand populations of immature B cells. Althoughconstraintson the pool of rearranged V genes may seem disadvantageous to theimmune system, themechanismsthatgeneratethe CDR3s of heavy and light chains ensure extensive diversity inthe pre-B-cell population.Inmature B cells, somatic mutation of V genes adds further diversity. CDR3 sequences and somaticmutationsnotonlyprovidepotentiallyuseful clonal markers but also help to identify the normal counterparts of malignant B cells.
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