Minireview Vol. 264 No. 22 Issue of August 5, pp. 12745-12748,1989 0 19139 by The American Society for Biochemistry and Molecular Biology. Inc.

Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Human Immunoglobulin Heavy Chain Genes*

J. Donald Capra and Philip W. Tucker From the Department of Microbiology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235

Until recently, our understanding of the immunoglobulin heavy chain complex, especially the heavy chain variable (VH) genes in man, has lagged surprisingly behind similar information in the inbred mouse. The reasons for this are complicated but to some extent are related to the discovery of the myc oncogene. Thus, after genes encoding some of the constant region isotypes as well as the well defined “subgroups,” VHI, VHII, and VHIII, were cloned and sequenced using the B cell lymphoma lines then available (1-3), the discovery of myc translocations understandably diverted a considerable amount of attention to this first human oncogene implicated in B cell neopla- sia (4-6). Subsequently, as information accrued concerning the mu- rine IgH complex (7-10) a general hypothesis for the organization and expression of immunoglobulin variable genes developed, which has to some extent shaped our thinking about human VH genes. Some of these notions are being reexamined in the light of newer data that are rapidly emerging on the human IgH complex.

The IgH cluster has been mapped to chromosome 14 (11,12), band 14q32 (13, 14). The generally accepted gene order is shown in Fig. 1. The locus contains a t least 200 VH genes, over 20 diversity (D) segments, 6 joining ( J ) segments, and a constant region (CH) of 9 functional genes (p, 8 , y3, yl, a’, y2, y4, t, and a?) and 2 pseudogenes ($el and $7). The recombination of VH, D, and J H segments occurs early in B cell differentiation and determines the binding specificity of the antibody. The CH regions mediate effector functions, such as the ability to activate complement or cross the placenta. The total size of the locus has been estimated from pulsed-field gel measure- ment at 2500-3000 kb’ (15, 16). However, a precise map, particularly of the VH segments, with certain exceptions (see below) is lacking.

The Human Vn Genes From murine studies it became apparent that the approximately

1000 VH genes can be divided into approximately 10 families based on nucleotide and amino acid sequence data. In general, the murine VH families exist as clusters of related genes so that VH gene segments from each particular family are usually adjacent to other VH genes of the same family. While there are a number of exceptions, the pre- vailing wisdom is that the murine VH gene families are organized in such discrete clusters.

Immunologists interested in the human immune system have been at some disadvantage over those working in murine systems. Despite slow and steady progress, human hybridoma technology has lagged considerably behind murine accomplishments. While Epstein-Barr virus transformation of human peripheral blood lymphocytes is well known, this method of human B cell immortalization has rarely led to sufficient protein for structural analysis. New cDNA techniques have allowed, however, the rapid expansion of our understanding of human immunoglobulin VH gene expression through the synthesis of complementary DNA from only modestly expanded B cell lines derived from the peripheral blood of both normals and patients.

From these recent studies on the expressed human VH repertoire and laborious genomic work that is ongoing in several laboratories, we are beginning to appreciate the organization and method of expression of human VH genes (Fig. 1). These findings have led to a new paradigm suggesting that the organization and structure of the human VH locus (similar to the organization of the rabbit VH locus

AI 12127, CA 44016, Af18816, and AR 39169 and by Texas Higher Education This work was sup orted in part by National Institutes of Health Grants

Cyrdinating Board Grant 2812. The abbreviations used are: kb, kilobase pair($; bp, base pair(s).

(17)) are considerably different than those of the inbred mouse. There Are at Least Six Families of Immunoglobulin VH Genes-An

“immunoglobulin VH gene family” is a term that reflects nucleotide sequence similarity within the coding segments of immunoglobulin genes. Operationally it can be defined in one of two ways, both of which obviously reflect the nucleotide sequence. Under standard hybridization conditions, genes from one family do not cross-hybrid- ize well with genes from a second family. Thus, one can define an immunoglobulin VH family by setting hybridization conditions. An alternative way is to define a VH gene family at the nucleotide sequence level. Typically, immunoglobulin VH genes will differ from one another within a family by no more than 20% (80% nucleotide sequence identity). When comparing genes from different families, the homology is typically less than 70%. There is obviously an ambiguous area when between 70 and 80% nucleotide sequence sim- ilarity is the case, and hybridization conditions can be set such that a gene falling into this gray zone belongs to one or another family or represents a unique family.

The first three immunoglobulin VH families, VHI, -11, and -111, were well known from the amino acid sequence studies of myeloma proteins. Some human VH gene segments were identified originally based on similarity to murine sequences while others were isolated from rearrangements in B cell malignancies. These first three human VH gene families were used by Kodaira et al. (18) to screen a cosmid library. They detected 120 recombinant clones and made two important observations. First, unlike the situation in the mouse, human VH genes from different families were adjacent to each other in the genome. Second, some of the cosmids contained VH genes that were not similar to any myeloma proteins previously sequenced. Lee et al. (19) defined the human VH IV family in this way. Using a probe isolated by a gene that was adjacent to a VHIII gene, six additional variable region gene segments were defined. They were more than 90% similar to each other and less than 65% similar to members of the three known VH families. They estimated that the human VHIV gene family had a minimum of nine members. Thus, the first of a number of “smaller” VH gene families became known just 2 years ago. This observation was rapidly followed by the description of a human VHV family by Shen et al. (20) and Humphries et al. (21). In analyzing JH rearrangements from leukemic lymphocytes of two siblings with chronic lymphocytic leukemia, they observed that the VH gene seg- ments were highly related (greater than 90% to each other) but unrelated (less than 60%) to previously published VH sequences. Southern filter hybridization of human liver DNA probed with either of the rearranged genes detected a limited set of similar sequences. Nucleotide sequence analysis of these genes defined a new VH family (VHV), and linkage of VH to J H using pulse field electrophoresis represented the first VH-DH-JH linkage in man.

The VHVI gene family was simultaneously described by Berman et al. (X) , Schroeder et al. (22), and Buluwela and Rabbitts (23). Each used different techniques, but each defined a new gene family that was less than 100 kb from JH that had less than 50% identity to any other previously sequenced VH gene. Taken with the DH-JH linkage of Matsuda et al. (16) and linkage distance between Cp and C8 (see below) the VHVI-D linkage can be estimated at 50 kb.

A Hind111 digest of human placental DNA probed with six human VH probes is illustrated in Fig. 2. It is estimated that approximately 200 human VH gene segments comprise the bulk of the human VH repertoire. While it is certainly possible that additional (probably small) gene families will be described, it is likely that these will be relatively few in number.

The Organization of Human VH Genes (Like the Rabbit) Involves Gene Segments from Several Families Which Are Interspersed-As mentioned above, when Kodaira et al. (18) examined 23 different cosmid clones containing VH gene segments they discovered that most of them contained several different genes. Usually, the different cosmids contained genes from several different families. Indeed, they described several clusters that contained members of all three VH gene families, and in retrospect with the definition of the VHIV family, some clusters had within a 100-kb stretch representatives of all four VH gene families. While this organization is similar to that described

12745

12746 Minireview: Human Immunoglobulin Heavy Chain Genes

of a Giemsa-stained human chromosome 14 is shown in A indicatmg the FIG. 1. The organization of Ig heavy chain genes. The banding pattern

position of the heavy chain gene cluster. The overall organization of the locus

in B. Estimated or known numbers of gene segments are indicated above the (without the fine exon structure) is shown relative to chromosomal orientation

below in kb. Pseudogenes are designated by +. map in parentheses. Known distances separating gene segments are denoted

FIG. 2. Human placental DNA dig- with restriction enzyme RindIII and probed with six human VE probes representative of the six VE families.

in the rabbit (17), it is decidedly different from the prevailing view of the murine VH gene organization. In the mouse, while there are certain clear exceptions, in general when phage (not cosmid) clones were examined, they invariably contained but a single VH germline gene, and when two genes were defined they invariably belonged to the same VH family. Recent examination of the 7183 family, however, suggests considerable interspersion with the Q52 family, and data from our own laboratory on the 5558 gene family suggest considerable interspersion with the 3609 family? However, in the human, this situation seems to be the rule rather than the exception. When the VHV family was described it was appreciated that a VHI gene was adjacent, and the VHVI gene family that has recently been described is within 20 kb of a VHV gene. Thus, the six VH gene families in the human are extensively interspersed as they are in human VK. Indeed, the original report for interspersion in immunoglobulin variable re- gion segments was in the human K locus (24).

The organization of the VH gene families, particularly their prox- imity to DH and JH, is thought to be crucial in gene expression. Again, while it remains somewhat controversial, the paradigm that has developed in the murine system is that D and JH proximal genes are

Tucker, and J. D. Capra, EMBO J., submitted for publication. *K. M. Meek, G. Rathbun, L. Reininger, J.4. Jaton, R. Kofler, P. W.

more commonly rearranged early in ontogeny than more distal genes (25-29). While antibody diversity results largely from random com- binatorial and mutational mechanisms, there is a growing consensus that the acquisition of antibody specificities is developmentally pro- grammed. While a careful analysis of this program is not within the scope of this review, one theory which might explain the develop- mentally ordered appearance of antibody specificities is relaced to the proximity of the rearranging gene segment. This would suggest that chromosomal order and the timing of gene rearrangements might be related. In the mouse there is a great deal of evidence both for and against this concept, and thus it is important to establish the order of such genes in man. While these kinds of studies are still being reported it appears that the relatively small gene families (VHV and VHVI) are quite D proximal. Whether or not such developmentally programmed responses relate to the gene orders in man is yet to be determined since few expressed VHIV, VHV, or VHVI antibodies of defined specificity have been described (30,31). Schroeder et d. (22) in an analysis of cDNA clones from a 160-day fetal human liver found no VHV and only a single VHVI representative (22). This might suggest that such proximity to DH may not be as important in the human system as it may be in the mouse. However, Fougereau and his colleagues3 have recently examined by in situ hybridization Epstein- Barr virus lines derived from a 7-week-old human fetus and found exclusively VHV and VHVI genes expressed.

Some Human VH Genes Display Surprisingly Little Polymor- phism-The conventional wisdom is that in man, as an outbred species, the structure of variable region genes differs between individ- uals. This view is supported by studies with restriction fragment length polymorphism and, indeed, early human gene sequences which suggested considerable variation particularly in germline genes en- coding VHI, VHII, and VHIII gene families (1-3,32,33).

A large body of experimental evidence in the mouse suggests that, whether large or small, gene families are quite polymorphic when one compares the VH genes from one strain to another. Indeed, few murine VH genes isolated from one strain are identical to those from another strain. In the human, we rapidly were led to the conclusion that at least some human VH genes must be remarkably preserved in evolu- tion. Sanz et d. (34) described an anti-Sm antibody that had an identical VHsequence to two cDNA clones isolated from fetal liver by Perlmutter's group (22). The VHV anti-insulin antibody, also se- quenced by Sanz et d. (30), had a structure remarkably similar to the VHV sequence V ~ 2 5 1 (20, 21). Similarly, polyreactive antibodies sequenced by Sanz et d. (30) demonstrated remarkable similarities among expressed VHIV and VHIII genes to those previously described. We suggested that a reasonable explanation for cross-reactive idi- otypes in several human diseases and among several human antibod- ies might derive from the fact that the anti-idiotypic antibodies were directed toward framework structures on these small VH families (35).

The Human DaJRComplex The number and organization of diversity (D) segments is un-

known, but as shown in Fig. 1, over 20 have been described. Four D segments were originally shown to be clustered near the J segments (36). Later a fifth functional D segment (D5) was described (37), and recently Ichihara et ~ l . (38) have reported up to a total of 24 new germline DH genes. Curiously, these human germline D segments are found in very few of the known expressed sequences. This raises the possibility that additional functional D segments will be located. The human JH locus consists of six functional segments, clustered in -3 kb about 6 kb upstream of the p gene (39).

The Human Constant Region Locus Enhancer sequences required for transcription of heavy chains are

conserved among mice and men in the J H - C ~ intron (40, 41). The enhancer includes an element similar to the 72-bp repeat of SV40 (42), although unlike the virus, the IgH enhancer has only been found active in lymphoid cells. Another of the enhancer motifs, ATGCAAAT, is also found upstream of all VH segment promoters (43,44), and together they provide the B cell-specific component of heavy chain expression. No enhancers have yet been identified within or flanking other CH genes.

All CH genes of the human locus have yet to be physically linked. The relatively compact JH-Cp-Cb region is followed by two major nonoverlapping CH gene clusters which lie an unknown distance further downstream (39). The gene organization of the two groups of

M. Fougereau, personal communication.

Minireview: Human Immunoglobulin Heavy Chain Genes 12747

intron structure of the nine functional and two pseudo ( J . ) heavy chain constant FIG. 3. Structure of the heavy chain constant region genes. The exon-

region genes that are resent on chromosome 14 is shown. Repetitive sequences are shown b htche8bozes designated S. Globular domain encoding exons are designated &A. Exons encoding hinge region sequences are indicated by H H , and those encoding transmembrane and cyto lasmic tail sequences (including a short connecting sequence extending beyonfthe membrane) are identified by MI and M2. The known 3’-untranslated regions and polyadenylation RNA cleavage recopition sites for each gene are shown as lowered boxes and dots, respectively. equences that remain uncharacterized are indicated by dashed lines or open circles.

constant regions was determined by cosmid cloning (45). The two clusters most likely arose by a duplication event because the first cluster consists of Cy3, Cyl, a pseudo (I)) Ce, and Cal, whereas the second cluster encodes Cy2, Cy4, Ce, and Ca2 (Fig. 2). A processed I)Ce gene (i.e. without introns) has been located on chromosome 9 (46, 47), and a $Cy has been mapped by linkage disequilibrium (48) and deletion analysis (49, 50) somewhere between the two constant region clusters on chromosome 14.

Fine Structure of Human CHGenes-The constant region genes are quite varied in their structure (Fig. 3). All Cy genes have highly homologous domain-encoding exons but differ in the binge, a segment proposed to engender flexibility to the molecule. The C-yl, C72, and Cy4 genes each have a single exon encoding the binge region (51-54). Cy3 utilizes four small hinge exons (52,53) postulated to have arisen by unequal crossing over between the $Cy and Cy1 genes. The C6 hinge is encoded by two exons (55) , whereas the Cal and Ca2 genes encode their hinge regions continuously with the 5’ end of the second constant region (56). The Cp and Ct genes do not contain hinge regions; rather, four globular domains are encoded by separate exons, and $Ct retains only the 3’ domain exons 3 and 4 (57).

Each of the functional CH genes is preceded by a “switch sequence” that undergoes recombination in the later stages of B cell differentia- tion (reviewed in Ref. 58). The consequence is a shift of Ig class produced (e.g. IgM to IgG) through deletion of intervening DNA between the switch region of Cp and the switch region of the particular CH gene expressed. With the exception of C6, switch sequences are approximately 3 kb in length and consist of tandem repeats with overall similarity of, at best, 60% with the “master” switch sequence 5‘ of Cp. The human Cp switch sequence is similar to that in the mouse, composed primarily of pentamer repeats (GAGCT, GGGGT) (59). However, in the Cy, Ce, and Ca switch regions, repeat units are not pentamer-dominated and have different sequence composition than the corresponding murine sequences.‘ C6 has evolved a quite different mode of deleting Cp. A 443-bp block of unique sequence

‘ F. Mills and D. Camarini, personal communication; A. Richardson and P. Tucker, unpublished results.

located some 200 bp 3’ of the enhancer (60) is duplicated almost perfectly (96% similarity) in the Cp-C6 intervening sequence (61,621. Recombination between these homologous regions5 appears respon- sible for the increased level of secreted 6 chain mRNA in human relative to rodents (63).

Ordered Utilization of CH Genes in Preimmune and Immune De- uelopment-The nine functional immunoglobulin constant region genes are not used randomly but in an ordered process. After func- tional VH-DH-JH joining occurs, transcription and translation of the proximal Cp locus results in a complete p heavy chain protein. Therefore, exclusive usage of Cp is observed in “preB” cell develop- ment (implying heavy chain but not light chain rearrangement). Extrapolating from murine data on the basis of gestation age equiv- alence (64), this corresponds to the 7th and 8th week of the human fetus. Subsequent rearrangement of light chain gene segments allows production of complete IgM heterodimers to assemble on B cell membranes by week 9 (65, 66). At this point the transcription blockade 3’ to Cp is relaxed allowing progression through the adjacent C6 gene some 8 kb further downstream (63). Alternative splicing of this large pre-mRNA results in simultaneous utilization of Cp and C6 with the identical VDJ segment (63). As B lymphocytes develop, immunoglobulin receptors are evident on their cell surface. Surface positive IgM and IgD lymphocytes are detectable by week 12 of gestation (67) and represent the major transitional phenotype be- tween fetal and neonatal life. Although some antibody responses can be demonstrated at this time (68), immunocompetence to broad antigenic challenge develops slowly over several months. The IgM’IgD+ cells either develop directly into IgM secreting plasma cells or undergo class switching to CH genes 3’ of C6 and then proceed to the plasma cell stage secreting IgG, IgE, or IgA of the original antigen specificity around week 20 (69). The temporal order of expression of Cp, then C6, then downstream CH genes is a feature conserved across mammalian evolution and relies on the linear ordering of tho CH locus.

This logic may be at least partially applicable to Cy gene prefer- ential usage. The T-cell-independent response to complex carbohy- drates including B cell mitogens often, but not always, utilizes the most J H proximal CH gene, Cy3 (70, 71). These observations along with analysis of hybridomas (72, 73) and spontaneous “switching” murine B cell lines (74) have led to the concept that progeny of a single B cell clone can undergo successive switchings in an order compatible with the linear arrangement of CH genes. Conversely a position-independent bias is observed in most T cell-dependent im- mune responses, correlating most significantly with the variety of T cell growth/differentiation factors (lymphokines) elaborated. For ex- ample, the lymphokine IL-4 induces biased utilization of Ct and Cy4 genes (reviewed in Ref. 75), perhaps by rendering them more acces- sible to the enzymes promoting switch recombination. An equally strong bias is exerted by the anatomical site where the antigenic challenge is encountered. For example, secretory responses in gut and other epithelia are highly biased toward IgA. Again this specificity is likely engendered by T cells.

DNA Sequence Polymorphism in the CH Locus-Interests in the IgH region vary from genetic to anthropological to medical (reviewed in Ref. 76). Thus, considerable efforts have been undertaken to study variation in CH genes using both serological and molecular hybridi- zation approaches. Nonrandom association of alleles (linkage dis- equilibrium) for most IgH genes has resulted in preferred combina- tions or “haplotypes.” The number of haplotypes present in the Caucasian population has reached 82. This level of heterogeneity rivals that found for hemoglobin (77) or for mitochondrial DNA (78). However, there are only two major haplotypes from which the other rare haplotypes seem to have been derived primarily through deletions of multiple constant regions. It is probable that the primary event leading to separation of the two main Caucasian haplotypes occurred early in evolution and has been maintained by heterozygous advan- tage. Based on the time of disappearance of Neanderthal in southwest Europe, the date has been estimated between 30,000 and 35,000 years ago (79).

Acknowledgments-We acknowledge the skilled technical assistance of Carol Williams. We are grateful to Drs. Kathy Meek, Inaki Sanz, Charlotte Word, Bill Kuziel, and Andrea Richardson for many helpful discussions and permis- sion to cite some of their unpublished work. We thank Margaret Wright, Angela Houston, and Marilyn Gardner for skilled secretarial assistance.

Note Added in Proof-The organization of the human constant region has

M. White, F. Blattner, and P. Tucker, manuscript in preparation.

12748 Minireview: Human Immui been extended (80). Cy3 maps 60 kb 3' to Cd; Cy2 maps 80 kb 3' to C d ; $Cy maps 35 kb 3' of Cnl and is in the same orientation as the other genes.

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