Frequency and characterization of phenotypic Ig heavy chain allelically included IgM-expressing B cells in mice

Ig H chain (IgH) allelic exclusion remains a puzzling topic. Here, we address the following question: Do phenotypic IgH allelically included cells exist in normal mice and, if so, at what frequency? Sorted cells from heterozygous mice were evaluated for the expression of both IgM allotypes by double intracytoplasmic stainings. Dual expressors were found at a frequency of 1 in 104 splenic B cells. These data were confirmed by direct sequencing of IgH-rearranged alleles obtained after single cell (or clone) PCR on dual expressors. Typically, these cells have one rearranged J558 VH whereas, in the other allele, a D-proximal VH gene is used. Interestingly, dual expressors have rearranged IgH alleles with similar CDR3 lengths. These results show that, in contrast to the kappa L chain and the TCR beta-chain, IgH allelic exclusion is the result of an extremely stringent mechanism. We discuss two non-mutually exclusive scenarios for the origin of IgH dual expressors: 1) IgH allelically included cells arise when the first allele to rearrange productively is unable to form a pre-BCR; dual expressors could be a subset of this population in which, upon conventional L chain rearrangement, both IgH are expressed at the surface; and 2) synchronous rearrangement of the IgH alleles.     

The rearranged V(H) domain of a physiologically selected anti-single-stranded DNA antibody as a precursor for formation of IgM and IgG antibodies to diverse antigens

It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.     

Heavy chain revision in MRL mice: a potential mechanism for the development of autoreactive B cell precursors

Abs reactive to DNA and DNA/histone complexes are distinguished by the presence of positively charged amino acids, such as arginine, in the heavy chain complementarity-determining region 3. The presence of these amino acids partly results from atypical V(H)-D-J(H) rearrangements such as D-D fusions and D inversions. Previous results in our laboratory demonstrated that newborn autoimmune MRL/MpJ-+/+ mice undergo these unusual recombinations more frequently when compared with normal C3H/HeJ controls. In addition, the heavy chain junctions in newborn MRL mice demonstrated a preferred usage of V(H)-proximal D genes and distal J(H) genes suggestive of secondary gene rearrangements. In this study we explore the possibility that adult MRL B220(+)IgM(-) pre B cells, which have not yet undergone Ag selection, exhibit similar rearrangement patterns. Indeed, MRL pre-B cells possessed more atypical rearrangements (D-D fusions) than those of C3H/HeJ mice. However, the biased use of upstream D genes and downstream J(H) genes observed in the newborn MRL mice was not present in the pre-B cell library. These results suggest that the heavy chain rearrangement process persists later during B cell life in lupus-prone mice and lead us to propose a model of heavy chain receptor revision in the periphery of autoimmune mice.     

V gene rearrangement is required to fully activate the hypermutation mechanism in B cells

Nonproductively rearranged H and L chain loci of B cell hybridoma lines expressing heavily mutated antibodies were cloned and partially sequenced. The results confirm earlier data showing that somatic point mutations are as frequent in nonproductively rearranged loci containing a rearranged V gene as in productively rearranged loci. They establish in addition that in nonproductive H chain loci which bear a DJH rearrangement the frequency of somatic mutations is more than 10 times lower (0.2%) than in VDJH loci expressed by the same cells (2.5%). Thus, the hypermutation mechanism operating in B cell differentiation is targeted at V genes rearranged to the J locus and may require nucleotide sequences associated with both V and J elements in order to be fully activated. An inversion of the JH2 segment was detected in one DJH locus. This inversion appears to be the result of a secondary joining event occurring occasionally in the course of B cell development.     

Intraclonal diversity in the VH genes expressed by CD5- chronic lymphocytic leukemia-producing pathologic IgM rheumatoid factor.

The leukemic cells from 95% of patients with B cell chronic lymphocytic leukemia (CLL) coexpress B cell differentiation antigens and the pan-T lymphocyte surface antigen CD5 (Leu 1). As such, CLL generally may be considered a malignancy of the CD5 B cell, a minor B cell subpopulation implicated in the production of autoantibodies. Recent data indicate that CD5+ leukemic cells may express autoantibody-associated V region genes with little or no somatic mutation. We examined the Heavy chain V genes expressed by an unusual CLL that secretes rheumatoid factor (RF) autoantibodies but does not express the CD5 surface Ag. Nucleic acid sequence analyses of the rearranged VH genes of three independent rDNA clones demonstrated intraclonal diversity not apparent in previously studied cases of CD5+ CLL. Comparison of the rearranged VH genes reveals that they belong to the VH4 gene subgroup and share highest homology with a rearranged VH gene (Ab44) that encodes a polyreactive autoantibody. That these productively rearranged VH genes encode the RF generated by this unusual CLL population is demonstrated by immunoblotting of the RF paraprotein using primary sequence dependent antipeptide antisera. These results indicate that CD5- CLL, like their CD5+ counterparts, may produce RF. However, unlike CD5+ CLL examined to date, CD5- CLL may have intraclonal diversity in their expressed Ig genes.     

The multiple shark Ig H chain genes rearrange and hypermutate autonomously

Sharks and skates are representatives of the earliest vertebrates with an immune system based on V(D)J rearrangement. They possess a unique Ig gene organization consisting of 15 to >50 individual IgM loci, each with one VH, two DH, one JH, and one set of constant region exons. The present study attempts to understand how multiple Ig genes are regulated with respect to rearrangement initiation and to targeting during somatic hypermutation. The linkage of three single-copy IgH genes was determined, and single-cell genomic PCR studies in a neonatal animal were used to examine any relationship between relative gene position and likelihood of rearrangement. Our results show that one to three IgH genes are activated independently of linkage or allelic position and the data best fit with a probability model based on the hypothesis that V(D)J rearrangement occurs as a sequence of trials within the B cell. In the neonatal cell set, two closely related IgH, G2A, and G2B, rearranged at similar frequencies, and their membrane forms were expressed at similar levels, like in other young animals. However, older animals displayed a bias in favor of the G2A isotype, which suggests that although rearrangement at G2A and G2B was randomly initiated during primary repertoire generation, the two very similar IgM sequences appear to be differentially expressed with age and exposure to Ag. We performed genomic single-cell PCR on B cells from an immunized individual to study activation-induced cytidine deaminase targeting and found that hypermutation, like V(D)J rearrangement, occurred independently among the many shark IgH.     

Analyses of recombinant stereotypic IGHV3-21-encoded antibodies expressed in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells that use IgH encoded by IGHV3-21 and that have a particular stereotypic third CDR (HCDR3), DANGMDV (motif-1), almost invariably express Ig L chains (IgL) encoded by IGLV3-21, whereas CLL that use IGHV3-21-encoded IgH with another stereotypic HCDR3, DPSFYSSSWTLFDY (motif-2), invariably express κ-IgL encoded by IGKV3-20. This nonstochastic pairing could reflect steric factors that preclude these IgH from pairing with other IgL or selection for an Ig with a particular Ag-binding activity. We generated rIg with IGHV3-21-encoded IgH with HCDR3 motif-1 or -2 and IgL encoded by IGKV3-20 or IGLV3-21. Each IgH paired equally well with matched or mismatched κ- or λ-IgL to form functional Ig, which we screened for binding to an array of different Ags. Ig with IGLV3-21-encoded λ-IgL could bind with an affinity of ～ 2 × 10(-6) M to protein L, a cell-wall protein of Peptostreptococcus magnus, independent of the IgH, indicating that protein L is a superantigen for IGLV3-21-encoded λ-IgL. We also detected Ig binding to cofilin, a highly conserved actin-binding protein. However, cofilin binding was independent of native pairing of IgH and IgL and was not specific for Ig with IgH encoded by IGHV3-21. We conclude that steric factors or the binding activity for protein L or cofilin cannot account for the nonstochastic pairing of IgH and IgL observed for the stereotypic Ig made by CLL cells that express IGHV3-21.     

IgH V-region sequence does not predict the survival fate of human germinal center B cells

Germinal center (GC) B cell survival fate is governed in part by the outcome of successful/failed BCR-mediated interactions with accessory cells. However, the extent to which the BCR primary sequence influences such interactions is not fully understood. Over 1000 IgV(H)4 family cDNAs were sequenced from living (annexin V(-)) and apoptotic (annexin V(+) or from within tingible body macrophages) GC B cell fractions from seven tonsils. Results surprisingly demonstrate that living and dying GC B cells do not significantly differ in IgV(H), D, or J(H) gene segment use; HCDR3 length or positive charge; or mutation frequency. Additionally, equivalent IgH cDNA sequences were identified in both fractions, suggesting that BCR sequence alone is an unreliable predictor of GC B cell survival.     

B lymphoblastoid cell lines with rearranged T cell receptor beta-chain genes retain conventional B cell surface antigen and Ig expression

Transformation of peripheral blood lymphocytes by co-incubation with EBV produces B lymphoblastoid cell lines, but rearrangement of TCR beta-chain genes was observed in three different cell lines derived from two individuals. Because rearrangement of TCR genes in B lymphocytes is considered a rare event, these B lymphoblastoid cell lines with rearranged TCR beta-genes were examined in detail to determine whether there were any additional characteristics to distinguish them from B lymphoblastoid cell lines with germ-line TCR beta-genes. All B lymphoblastoid cell lines contained rearranged Ig H and kappa L chain genes, secreted Ig, and expressed B and not T cell surface markers. Cell lines with rearranged TCR beta-genes had rearranged both IgH genes and had rearranged and subsequently deleted both kappa C region genes. Furthermore all three B lymphoblastoid cell lines with rearranged TCR beta-genes produced small amounts of Ig with lambda-L chains. Although the cellular mechanisms maintaining lineage-specific rearrangement events remain unknown, extensive Ig gene rearrangement and inefficient Ig production by B cells may be indicators of a cellular status where normally stringent lineage-specific control elements fail to function efficiently.     

Cutting edge: TCR delta gene is frequently rearranged in adult B lymphocytes

TCR gene rearrangement generates diversity of T lymphocytes by V(D)J recombination. Ig genes are rearranged in B cells using the same enzyme machinery. Physiologically, TCR gene is postulated to rearrange exclusively in T lineage, but malignant B precursor lymphoblasts contain rearranged TCR genes in most patients. Several mechanisms by which malignant cells break the regulation of V(D)J recombination have been proposed. In this study we show that incomplete TCR delta rearrangements V2-D3 and D2-D3 occur each in up to 16% alleles in B lymphocytes of all healthy donors studied, but complete VDJ rearrangement was negative at the sensitivity limit of 1%. Data are based on real-time quantitative PCR validated by PAGE and sequencing of the cloned products. Therefore, TCR genes rearrange not exclusively in T lineage. This study opens up further questions regarding the exact extent of the "cross-lineage" TCR or Ig rearrangements in normal lymphocytes, specific subsets in which the cross-lineage rearrangements occur, and the physiological importance of these rearrangements.     

Similarities and differences between the light and heavy chain Ig variable region gene repertoires in chronic lymphocytic leukemia

Analyses of Ig V(H)DJ(H) rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the V(L) and J(L) gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express V(L) genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific V(L) and J(L) segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the V(H) repertoire, V(L) gene use was not significantly different than that of normal B-lymphocytes. In addition, Vkappa genes that lie more upstream on the germline locus were less frequently mutated than those at the 3' end of the locus; this was not the case for Vlambda genes and is not for V(H) genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.     

V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer.

We have assessed the importance of the immunoglobulin heavy chain (IgH) intron enhancer for recombination of variable gene segments (V, D and J) during B cell development. We generated chimeric mice with embryonic stem cells lacking the intron enhancer from one of their IgH loci. The IgH intron enhancer was substituted by a short oligonucleotide through homologous recombination using the 'Hit and Run' procedure. V(D)J recombination occurred less frequently on mutant alleles, but was not blocked completely. Quantitative polymerase chain reaction analyses demonstrated that 15-30% of the mutated loci in mature B cells were unrearranged, in striking contrast to the wild-type alleles. The remainder of the mutated loci underwent D-J (65-80%) as well as V-DJ rearrangements, although the latter were less frequent (3-6%). These results are in line with previous data which showed that the V(D)J recombination machinery is modulated through cis-regulatory elements within the intron enhancer. However, our data predict the existence of additional cis-regulatory element(s) which, together with the intron enhancer, are required to activate the V(D)J recombination machinery fully. Such cis-regulatory element(s) might function as an enhancer of recombination or as a locus control region regulating the accessibility of the IgH locus.     

Immunoglobulin gene rearrangements in normal mouse B cells.

We have analyzed the structure of rearranged mu heavy-chain genes obtained from the genomic DNA of normal BALB/c mouse spleen cells expressing surface immunoglobulin M. Examples were found of two types of nonproductive rearrangements, which may be responsible for allelic exclusion in normal B cells. In one of these rearrangements, a germ line D gene segment has joined to the JH4 gene segment but no V/D joining has occurred. We present evidence that D gene segments lie as a cluster between V and J gene segments in the germ line. A comparison of conserved sequences in V and D gene segments suggests that the D gene segments, which are found only in the heavy-chain gene family, may have evolved from V gene segments similar to the Vk family.     

Cutting edge: B cell linker protein is dispensable for the allelic exclusion of immunoglobulin heavy chain locus but required for the persistence of CD5+ B cells

The pre-B cell receptor (pre-BCR) and the BCR are required for B lymphopoiesis and for the allelic exclusion of Ig genes. Mice lacking B cell linker (BLNK) protein that is a component of the BCR signaling pathway have impaired B cell development. In this report, we show that allelic exclusion is intact in BLNK(-/-) mice harboring a V(H)12 transgene. This differs from mice lacking the tyrosine kinase Syk that is upstream of BLNK in BCR signaling and contrasts with mice lacking SLP-76 that is the equivalent adaptor molecule in TCR-signal transduction. We also show that, whereas most wild-type V(H)12-expressing B cells are CD5(+), the majority of the splenic V(H)12-expressing BLNK(-/-) B cells are CD5(-). A small population of V(H)12-expressing, BLNK(-/-) CD5(+) B cells is detectable in the peritoneal cavity of younger but not older mice. This suggests that BLNK deficiency affects not only the generation but also the persistence of B-1 cells.     

A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. Definition of the VH11 family

By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).     

Neonatal and adult primary B cells use the same germ-line VH and V kappa genes in their (T,G)-A-L-specific repertoire

Although there is a nonrandom usage of VH gene families by primary B cells early in ontogeny, at issue is whether the preferential rearrangement of 3' germ-line VH genes, e.g., VH7183 and VHQ52 family genes, influences the neonatal B cell repertoire that can be expressed in response to Ag. In order to address this issue, and to determine whether neonatal B cells can use the same germ-line VH and V kappa genes as adult B cells in their primary response, we have analyzed at the molecular level the neonatal antibody response to (T,G)-A-L and compared it with the adult primary response. Among the TGB5 Id+, GT+ antibodies, which dominate the neonatal response to (T,G)-A-L, two VH gene families were used: J558 (high frequency) and 36-60 (low frequency). The majority of Id+ neonatal hybridomas used the same germ-line VH gene (H10, from the VHJ558 family), but with enormous diversity in the D region, and one of two germ-line V kappa 1 genes (V kappa 1A, V kappa 1C). These are the same germ-line V-genes used by most primary adult Id+ hybridomas, and the frequency of expression of this germ-line V-gene combination appears equivalent in the neonatal and adult primary repertoires. Therefore, it is clear from this study that as early as day 5, neonatal B cells can use the same germ-line V-genes as adult primary B cells in their Ag-specific repertoire.