Peter H. Brodeur

Molecular cloning of the primary IgH repertoire: a quantitative analysis of VH gene usage in adult mice.

The generation of the primary antibody repertoire requires the somatic rearrangement of germline gene segments. It is not known, however, whether all functional V and J gene segments have an equal probability of contributing to this initial set of antibody specificities. To address this issue, we have examined the relative utilization of VH and JH gene segments of the mouse. We have constructed VH cDNA phage libraries from C mu transcripts obtained from polyclonally activated spleen cells of the BALB/c and C57BL/6 strains. We show that probes specific for either one, two or three functional VH gene segments hybridize to cDNAs at frequencies directly proportional to the number of functional germline VH genes detected by each probe. In contrast, the representation of 10 VH gene families within each library indicates that certain families are under‐represented relative to their estimated germline gene number. These families must either have extraordinary proportions of nonfunctional genes or are influenced by as yet unidentified regulatory mechanisms or constraints on rearrangement.

Accessibility changes across the mouse Igh-V locus during B cell development

During their development, B and T lymphocytes are thought to undergo several cycles of chromatin remodeling at their antigen receptor loci that serve to regulate access of a common V(D)J recombinase to particular gene segments. We used germ‐line transcription and susceptibility to DNaseI as markers to examine tissue and stage‐specific changes in chromatin structure surrounding genes of the VH J558, VH10, and VH S107 families, whose mem bers are located at discreet subregions of the locus. Germ‐line VH transcripts from all three families were detectable at pro‐ and pre‐B cell stages. Transcripts from the VH10 and VH S107 families, but not VH J558, remained detectable at the immature and mature B cell stages. Unexpectedly, none of the germ‐line VH loci examined were markedly nuclease sensitive, regardless of cell type or transcriptional activity. A modest degree of nuclease sensitivity was noted at the VH J558 loci of pro‐B and pre‐B cells, however. Our data suggest that the entire Igh‐V locus becomes accessible at early B cell stages, and returns thereafter to an inaccessible state. However, the timing of these accessibility changes does not occur uniformly across the VH array. These results imply that multiple long‐range elements are involved in targeting VH genes for rearrangement.

Identification of a candidate regulatory element within the 5' flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A.

The Igh locus is controlled by cis-acting elements, including Eμ and the 3′ IgH regulatory region which flank the C region genes within the well-studied 3′ part of the locus. Although the presence of additional control elements has been postulated to regulate rearrangements of the VH gene array that extends to the 5′ end of the locus, the 5′ border of Igh and its flanking region have not been characterized. To facilitate the analysis of this unexplored region and to identify potential novel control elements, we physically mapped the most D-distal VH segments and scanned 46 kb of the immediate 5′ flanking region for DNase I hypersensitive sites. Our studies revealed a cluster of hypersensitive sites 30 kb upstream of the most 5′ VH gene. Detection of one site, HS1, is restricted to pro-B cell lines and HS1 is accessible to restriction enzyme digestion exclusively in normal pro-B cells, the stage defined by actively rearranging Igh-V loci. Sequence motifs within HS1 for PU.1, Pax5, and E2A bind these proteins in vitro and these factors are recruited to HS1 sequence only in pro-B cells. Transient transfection assays indicate that the Pax5 binding site is required for the repression of transcriptional activity of HS1-containing constructs. Thus, our characterization of the region 5′ of the VH gene cluster demonstrated the presence of a single cluster of DNase I hypersensitive sites within the 5′ flanking region, and identified a candidate Igh regulatory region defined by pro-B cell-specific hypersensitivity and interaction with factors implicated in regulating V(D)J recombination.

Germline diversity of the expressed BALB/c VhJ558 gene family

Although the mouse immunoglobulin heavy chain (Igh) locus contains 15 heavy chain V (Vh) gene families, at least half of the Vh gene segments are members of the VhJ558 family. This large Vh gene family represents the least characterized germline coding regions of any of the mouse antigen receptor loci and the contribution of individual VhJ558 genes to the preimmune repertoire is poorly understood. In fact, relatively few germline VhJ558 sequences have been reported for BALB/c, the foundation strain for mouse immunoglobulin genetics and the prototypic strain of the Igh(a) haplotype. Here we present a database consisting of 66 sequences estimated to represent one-half of the total number of functional BALB/c VhJ558 genes. Our results indicate that a subset of the VhJ558 genes is highly expressed in the preimmune repertoire, with just nine Vh sequences accounting for nearly 50% of the VhJ558 heavy chains expressed by splenic B cells. We show that this disparity in the expressed Vh gene repertoire is not due to the position of the Vh genes relative to the Dh cluster or to multiple germline copies of the highly expressed VhJ558 genes. Together, these data constitute the first detailed analysis of functional BALB/c VhJ558 genes, demonstrate a striking bias in the use of particular VhJ558 genes in the preimmune repertoire, and provide sufficient information to study the regulation of the Dh-distal region of the Igh-V locus at the level of individual genes.

Polymorphism and Evolution of Igh-V Gene Families

The immunoglobulin heavy chain locus (Igh) is located on chromosome 12 of the mouse. It is a complex locus, containing over one hundred variable region gene segments (Igh-V or Vh), approximately 12 diversity (Dh), 4 joining (Jh) and 8 constant region (Igh-C or Ch) gene segments. Although extensive diversity of antibody structures is generated in the assembly of active variable region genes by fusion of all combinations of V, D and J, it is apparent that there is as well a very high degree of genetic polymorphism at this locus. Examination of a panel of eighteen inbred strains selected to represent a variety of constant region allotypes (Igh-C alleles) revealed correlated allelism of the entire array of Vh and Dh gene segments (Brodeur and Riblet 1984; Brodeur et al. 1984). The extent of this genetic diversity parallels that of the MHC (Klein 1975) consistent with the parallel importance of this locus in protecting an individual from a variable environment. In this study we present further examination of the extent of this polymorphism and some aspects of the evolution of the locus.

Cutting Edge: Stat5 Mediates the IL-7-Induced Accessibility of a Representative D-Distal VH Gene

During B cell development, discrete domains within the Igh locus are activated independently for recombination. The D-distal VH genes are uniquely dependent on IL-7R signaling, which is thought to establish local chromatin accessibility through an unknown mechanism. To dissect this mechanism, we used a murine B cell line that responds to IL-7 by specifically inducing accessibility of a representative D-distal gene (A1) but not a D-proximal gene (V11). We demonstrate that IL-7-activated Stat5 is recruited rapidly to the A1 gene, with a concomitant increase in germline transcription and H4 acetylation. Furthermore, retroviral transduction of dominant negative or constitutively active Stat5 demonstrated that Stat5 activation is both necessary and sufficient for the IL-7-induced A1 germline transcription. Lastly, as with all known Stat5 target genes, A1 germline transcription requires a deacetylase activity. These results demonstrate that in response to IL-7 signaling, Stat5 is recruited to the D-distal A1 gene and induces accessibility.

Isolation and immunological characterization of a group of new B lymphomas from CBA mice

We have isolated and characterized a new series of B lymphoma which occurred spontaneously in a group of CBA/N mice that were transferred with spleen or lymph node cells from 24-month-old CBA/Ca mice. Tumor cell lines from six CBA/N mice that received spleen cells were rescued and designated as BKS-2, BKS-3, BKS-4, BKS-5, BKS-6, and BKS-7. Also, tumor cells from a recipient of lymph node cells were rescued and the resulting cell line was designated BKL. These tumor cells expressed membrane immunoglobulin (mu, kappa), major histocompatibility complex Class I and Class II molecules, B220, Lyb8, Fc receptors, J11d, interleukin 2 receptors, and Ly1. All of the tumors did not express the T cell specific markers Thy 1.2, L3T4, and Lyt2.1. They appeared to be clonal in origin, since they exhibited common rearrangements at both heavy and light chain immunoglobulin loci. Phenotypically, these lymphomas appeared to be analogous to immature B cells. Also, these lymphomas displayed different functional reactivities when treated with various B cell mitogens and growth factors in vitro. Anti-mu antibodies which normally induce B cell growth inhibited the proliferation of these lymphoma cells in vitro, whereas they responded to lipopolysaccharide, T cell-derived growth factors, and interleukin 5 by enhanced proliferation. These tumor cells expressed constitutively high levels of c-myc mRNA.

Detailed analysis of the public idiotype of anti-hen egg-white lysozyme antibodies.

For several years we have studied the idiotypy of the immune response to hen egg-white lysozyme (HEL). We have shown that almost all anti-HEL antibodies, late in the primary response and after secondary immunization, are characterized by the presence of a predominant idiotype, IdXL. This idiotype dominates the anti-HEL response in all animals so far tested: many strains of inbred mice, outbred mice, rats, deermice, and rabbits.’ IdXL, however, is largely absent on early primary anti-HEL and on monoclonal anti-HEL generated by the hybridoma technique from B cells of mice given a primary immunization nine days previously.* IdXL is also absent on antibody raised against human lysozyme (HUL) except for those rare molecules showing cross-reactivity with hen egg-white lysozyme.’ On the other hand, monoclonal anti-HEL antibodies produced by hybridomas generated from B cells of mice given a secondary immunization 3 to 5 days previously, universally show the presence of IdXL.2 These “late” monoclonal antibodies have allowed us to begin an examination of the relation of epitypic specificity and the presence of IdXL. We were able to show that two monoclonal anti-HEL of clearly different specificity (for different regions of HEL) were each reactive with anti-IdXL rabbit sera. Strikingly, each of these antibodies when immobilized on a column was able to completely absorb all anti-IdXL from rabbit sera.3 In this report, we more extensively examine the range of epitope specificity of a collection of IdXL positive secondary monoclonal anti-hen egg-white lysozyme. Also examined are the heavy chain variable region gene rearrangements present in hybridomas producing these antibodies.

Analysis of mice lacking DNaseI hypersensitive sites at the 5' end of the IgH locus.

The 5′ end of the IgH locus contains a cluster of DNaseI hypersensitive sites, one of which (HS1) was shown to be pro-B cell specific and to contain binding sites for the transcription factors PU.1, E2A, and Pax5. These data as well as the location of the hypersensitive sites at the 5′ border of the IgH locus suggested a possible regulatory function for these elements with respect to the IgH locus. To test this notion, we generated mice carrying targeted deletions of either the pro-B cell specific site HS1 or the whole cluster of DNaseI hypersensitive sites. Lymphocytes carrying these deletions appear to undergo normal development, and mutant B cells do not exhibit any obvious defects in V(D)J recombination, allelic exclusion, or class switch recombination. We conclude that deletion of these DNaseI hypersensitive sites does not have an obvious impact on the IgH locus or B cell development.

Expansion of CD4 T cells expressing a highly restricted TCR structure specific for a single parasite epitope correlates with high pathology in murine schistosomiasis.

The hepatic immunopathology in schistosomiasis mansoni is mediated by CD4 T cells specific for egg antigens and varies considerably among mouse strains. Previous studies in high pathology C3H mice suggested that a strong T cell response was due to the recognition of an immunodominant epitope within the major egg antigen Sm‐p40 (Sm‐p40234–246). Using a panel of T cell hybridomas, we have now examined the egg antigen‐specific TCR repertoire in two high pathology strains, C3H and CBA. We found that nearly half of the hybridomas responded to the Sm‐p40234–246 epitope and, of these, nearly all expressed Vα11.3 associated with Vβ8. Furthermore, in response to egg antigen stimulation, transcript levels of Vα11.3J36 (the most prevalent rearrangement expressed by Sm‐p40234–246‐specific hybridomas), increased in high pathology (CBA) but not in low pathology (BALB/c) strains. Our findings suggest that exacerbated schistosome egg‐induced immunopathology can be driven by T cells expressing a highly restricted TCR structure specific for a single parasite epitope.