B cell maturation in the chicken Harderian gland

We have characterized maturation of B lymphocytes in the chicken Harderian gland. Expression of Ig genes was studied by using lambda L and mu H chain-specific DNA probes. In unstimulated chickens, the concentration of mu H chain and lambda L chain mRNA in the Harderian gland was observed to be greater than 8 times higher than in the bursa of Fabricius or spleen. By using in situ hybridization, the plasma cells expressing mu mRNA were located in central area of the gland packed around the tubules. Antibodies produced by the Harderian plasma cells were measured from the tears before and after antigenic stimulation. In unstimulated chickens high levels of total IgM, IgA, and IgG were observed. After ocular stimulation with tetanus toxoid, specific antitetanus IgG and IgA antibodies appeared in the tears but IgM antibodies were barely detectable. These results indicate that after antigenic stimulation the Harderian B cells rapidly mature through IgM secretion to the production of IgG or IgA. Southern blot analysis of the Harderian total genomic DNA showed strong rearrangement in the lambda L chain locus. In contrast, the band indicating major rearrangement in the mu H chain locus gave a very poor hybridization signal, indicating deletion of C mu genes in the Harderian gland DNA. As a conclusion, our present data indicate for the Harderian gland a role in terminal B cell differentiation and Ig class switch.     

IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of T15i knockin mice.

T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the J(H) region of chromosome 12. This T15H chain combines with a kappa22-33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id-. Analysis of L chains expressed in these T15-Id-, PC-specific B cells revealed that two L chains, kappa8-28 and kappa19-15, were expressed in this population. The V(kappa) region of these L chains was recombined to J(kappa)5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id- B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id- B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:kappa8-28L and T15H:kappa19-15L Abs revealed L chain differences from the kappa22-33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:kappa22-33L Ab.     

Influence of the isotype of the light chain on the properties of IgG.

It is widely appreciated that the isotype of the H chain of the Ab molecule influences its functional properties. We have now investigated the contribution of the isotype of the L chain to the structural and functional properties of the Ab molecule. In these studies, the L chain variable region of a murine anti-dansyl Ab was joined to either human kappa or lambda constant region domains and expressed with mouse-human chimeric H chains of the four human IgG isotypes. The resulting Abs were secreted as fully assembled molecules although, as has been previously observed, IgG4 with either kappa or lambda L chains was also secreted as HL half-molecules. However, the isotype of the L chain can influence the kinetics of intracellular assembly with IgG1lambda, IgG2lambda, and IgG4lambda assembling more slowly than their kappa counterparts. The isotype of the L chain also influenced the susceptibility of the interchain disulfide bonds to attack by reducing agents with variable effects, depending on the isotype of the H chains. For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain influenced the rate of clearance in mice, with IgG2lambda having a shorter in vivo half-life than IgG2kappa. Only slight differences were also observed between lambda and kappa molecules in their kinetics of binding to and dissociation from the hapten dansyl. These studies demonstrate that the isotype of the L chain has only a slight impact on the structural and functional properties of variable region identical Abs.     

Gene targeting in the Ig kappa locus: efficient generation of lambda chain-expressing B cells, independent of gene rearrangements in Ig kappa.

The production of lambda chain-expressing B cells was studied in mice in which either the gene encoding the constant region of the kappa chain (C kappa) or the intron enhancer in the Ig kappa locus was inactivated by insertion of a neomycin resistance gene. The two mutants have similar phenotypes: in heterozygous mutant mice the fraction of lambda chain-bearing B cells is twice that in the wildtype. Homozygous mutants produce approximately 7 times more lambda-expressing B cells (and about 2.3 times fewer total B cells) in the bone marrow than their normal counterparts, suggesting that B cell progenitors can differentiate into either kappa- or lambda-producing cells and do the latter in the mutants. Whereas gene rearrangements in the Ig kappa locus are blocked in the case of enhancer inactivation, they still occur in that of the C kappa mutant, although in this mutant RS rearrangement is lower than in the wildtype. This indicates that gene rearrangements in the Ig lambda locus can occur in the absence of a putative positive signal resulting from gene rearrangements in Ig kappa, including RS recombination. Complementing these results, we also present data indicating that in normal B cell development kappa chain rearrangement can be preceded by lambda chain rearrangement and that the frequency of kappa/lambda double producers is small and insufficient to explain the massive production of lambda chain-expressing B cells in the mutants.     

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.     

Gene transfer of immunoglobulin light chain restores heavy chain secretion

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.     

B cell abnormalities induced by a mu Ig transgene extend to L chain isotype usage

We have analyzed the phenotype of B cell populations from mice transgenic for a rearranged Ig mu H chain gene. We find a decrease in the number of B cells in the spleens of these mice. Transgenic B cells have decreased surface levels of both IgM and IgD. The circulating IgM in these mice is 3- to 10-fold enriched in lambda L chains, compared with that in non-transgenic mice. Analysis of IgM-producing hybridomas, from transgenic mice that express the transgene at high levels, demonstrates that this higher lambda frequency is observed in transgene-nonexpressing as well as transgene-expressing hybridomas. A partial loss of L chain isotype exclusion is also noted in these hybridomas, and a significant proportion of primary B cells expressing both kappa and lambda L chains on their surface can be demonstrated. These findings suggest an ability of the transgenic Ig H chain to affect events in B cell ontogeny beyond the H chain locus. Our results support a quantitative model of exclusion for both the H chain alleles and the L chain isotypes.     

Human B cells secrete predominantly lambda L chains in the absence of H chain expression

Ig H and L chains are independently assembled in B cells and then secreted together as a functional protein. H chains cannot be secreted without assembly to L chains; however, L chains can be secreted in the absence of H chains by both mice and human cells. To examine the influence of H chain expression on human L chain isotype selection (kappa or lambda), we compared the kappa/lambda ratio of L chains unassociated with H chains (free L chains) to the kappa/lambda ratio of L chains associated with H chains. Culture supernatants of human splenocytes were assayed for kappa and lambda L chains. Free L chains were the predominant form of L chains detected in unstimulated cultures, accounting for 68 to 70% of the total. This was in contrast to the minor proportion that free L chains represented (less than 20%) in cultures stimulated with PWM or LPS (p less than 0.01). Furthermore, the kappa/lambda ratio of light chains detected in unstimulated cultures was 0.5 as compared to 1.3 for PWM stimulated cultures (p = 0.0001). To demonstrate that the decreased kappa/lambda ratio of L chains in the supernatants of cultures of unstimulated B cells was due to free L chains, we measured the kappa/lambda ratio of IgG and IgM-associated L chains. In both the stimulated and unstimulated cultures, the kappa/lambda ratio of L chains associated with H chains was greater than the ratio determined for free L chains. Free L chains were shown to be predominantly lambda as compared to the predominantly kappa phenotype of L chains associated with H chains. Thus absence of H chain expression affects selection of L chain isotypes secreted by human B cells.     

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.     

Antigen receptor editing in anti-DNA transitional B cells deficient for surface IgM

In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM(-/low)). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM(-/low) anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vlambdax) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgM(low) transitional splenic B cells of wild-type mice.     

Analysis of VpreB expression during B lineage differentiation in lambda5-deficient mice

The VpreB/lambda5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of lambda5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in lambda5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.     

Clonal diversity in human B cell lymphoma. I. Idiotypic and genetic analysis of lymphoma heterohybrids

Secretory heterohybrid clones from seven pristine human B cell lymphomas of diverse histologic types were established to investigate the question of tumor clonal diversity. We found that in six tumors, heterohybrid-derived Ig showed similar band patterns in IEF; families of anti-Id prepared from tumor Ig reacted uniformly with individual heterohybrids and original tumor; and the V gene loci displayed little variation on Southern analysis. In one patient who was followed with serial multiple site biopsies over a 14-mo period, clonal Id was preserved until the final stage of his disease, in spite of cytotoxic treatment. In a single follicular tumor (J.M.), each of the anti-Id reacted uniformly with the parent tumor and the individual heterohybrids, except that three of six clones failed to react with a single anti-Id family member. A Southern analysis of the VH gene locus revealed an identical gene rearrangement that was shared by the parent tumor and each heterohybrid. However, there was considerable heterogeneity of J.M. heterohybrid Ig in IEF gels, and we demonstrated the production of variant lambda L chains by the heterohybrid clones. One type of lambda L chain had a normal mobility in SDS-PAGE gels but larger lambda variants were produced by four of six heterohybrids. A Southern analysis of the VL gene displayed considerable variation in the type of lambda rearrangement present in the various heterohybrids, suggesting extensive diversity at the VL gene locus. In a second tumor (S.C.) that exhibited uniform anti-Id tumor reactivity we were also able to demonstrate the presence of a second minor tumor cell population (a biclonal tumor). Our data suggest that intraclonal VH variation may vary considerably with lymphoma subtype and mutagenic exposure and that an additional mechanism for generating spontaneous intraclonal heterogeneity is genetic variation at the VL locus.     

Multiple levels of selection responsive to immunoglobulin light chain and heavy chain structures impede the development of Dmu-expressing B cells

The truncated/V(H)-less mouse H chain Dmu forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dmu-lambda1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dmu's unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent lambda1 L chains could associate with both full-length H chains and Dmu, whereas secretion-incompetent lambda1 L chains could only do so with full-length H chains. In contrast, Dmu could not form receptors with a panel of kappa L chains irrespective of their secretion properties. This was due to an incompatibility of Dmu with the kappa-joining and constant regions. Finally, the Dmu-lambda1 receptor was less active than the full-length mouse mu-lambda1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dmu H chain alleles to the repertoire.     

Murine lambda gene rearrangements: the stochastic model prevails over the ordered model.

The ontogeny of the immunoglobulin (Ig) gene rearrangement in mammalian B cells seems to be ordered. Heavy chain gene segments rearrange first, followed by light chain gene segments, kappa before lambda. The genomic organization of murine lambda locus does not preclude the simultaneous expression of two subtypes from the same chromosome. In order to distinguish between an ordered and a stochastic model of rearrangement, a panel of 67 B cell hybridomas secreting either lambda 1, lambda 2, lambda 3 or lambda x (recently described) were analysed for V lambda J lambda rearrangements. The results show that in 97% of cases, a single rearrangement occurred, favouring the stochastic model over the ordered one. Strikingly, the possibility of having a productive rearrangement if the first try results in an aberrant one is rare. We propose therefore, that the lambda Ig is not necessarily required to ensure allelic and subtypic exclusion mechanisms. Moreover, in 97% of the cases, at least one kappa allele is rearranged. Furthermore, the RS recombination has been detected in 77% of the cases. This suggests that, although the stimulation of kappa precedes that of lambda locus, the RS recombination acts as a transacting albeit dispensable lambda activator.     

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.