Frequency of λ light chain subtypes in mouse antibodies to the 2,4-dinitrophenyl (DNP) group

Of the three λ chain subtypes made by inbred mice, chains of the λ1 subtype are much more frequent than those of the other subtypes (λ2,λ3) in antibodies (Ab) to those few antigenic structures that are known to elicit responses, in which λ chains are the predominant type of light chain [(4-hydroxy-3-nitrophenyl)acetyl (NP) and dextran]. The reasons for the frequency differences are not understood, and the large difference between the λ1 and λ3 frequencies is particularly puzzling, because in nearly all (about 95%) chains of these subtypes the N-terminal 97 or 98 amino acids are endoded by the same Vλ-gene segment. In an effort to identify an Ab response that has different λ subtype frequencies, we analyzed the light chains of the Ab made by BALB/c and B6 mice in response to 2,4-dinitrophenylated chicken gamma globulin (DNP-CGG). We found that approximately 40% of the elicited anti-DNP molecules had λ chains and of these approximately 40% were of the λ2 or λ3 subtype. Polyacrylamide gel electrophoresis indicated that the λ2 and λ3 chains were about equally abundant. Similar λ subtype frequencies were found in the anti-DNP Ab produced by the hybridomas made with spleen cells from the same immunized mice. In the anti-DNP Ab elicited by DNP-CGG and in the anti-NP Ab elicited by NP-CGG the different λ subtype frequencies (λ1/λ2 + λ3 = ca. 1.0-1.5 in anti-DNP and ca. 30 in anti-NP) were unaffected by immunizing mice with each of these antigens alone or with a mixture of the two. This finding, though preliminary, suggests that isotype-specific regulatory T cells are not responsible for the markedly different λ subtype frequencies in anti-DNP and anti-NP Ab.     

T cell receptor gamma chain variable gene rearrangements in acute lymphoblastic leukemias of T and B lineage

The status of immunoglobulin and T cell receptor genes in acute lymphoblastic leukemia (ALL) of T and B lineage has been studied. Our data indicate that illegitimate gene rearrangements at immunoglobulin heavy chain (in T cell ALL), and T cell receptor beta chain (in pre-B ALL) genes are only rarely found (2 out of 30 patients). In contrast, T cell receptor gamma chain gene rearrangements, characteristically found in T-ALL, are also present in 7 of 18 patients with pre-B ALL. Several features distinguish these illegitimate T cell receptor gamma chain gene rearrangements from those in normal and leukemic T cells. V gamma genes located far upstream of the J gamma/C gamma complexes (V gamma 2, V gamma 3, V gamma 4, V gamma 5) appear to be preferentially used in normal adult peripheral blood T cells. In contrast, V gamma genes located immediately 5' to the J gamma/C gamma complexes (V gamma 8, V gamma 9, V gamma 10, V gamma 11) predominate in V gamma -J gamma recombinations observed in T-ALL and pre-B ALL. Whereas the J gamma 2 region is primarily used in T cell receptor gamma gene rearrangements observed in T-ALL, those in pre-B ALL are confined mostly to the J gamma 1 region. These data suggest a limited accessibility of the T cell receptor gamma chain gene locus for recombination processes in early stages of B cell differentiation.     

Nucleotide deletion of T cell receptor V gamma 2 and J gamma 2 coding sequences at V gamma 2-J gamma 2 junctions in immature B cell lines.

Immature B cell lines transformed with a temperature-sensitive mutant of Abelson murine leukaemia virus undergo preferentially V gamma 2 to J gamma 2 joinings during culture at a non-permissive temperature (39 degrees C). Here we examined nucleotide addition and deletion at the V gamma 2-J gamma 2 junctions in these V gamma 2 to J gamma 2 joinings, in comparison with the V gamma 2-J gamma 2 junctional sequences reported previously in T cells. Forty-eight V gamma 2-J gamma 2 junctions were PCR-amplified and sequenced. Only three of 48 V gamma 2-J gamma 2 junctions had nucleotide addition. The average nucleotide deletion of V gamma 2 coding sequences at the V gamma 2-J gamma 2 junctions was 6.9 nucleotides in immature B cells and 5.1 nucleotides in T cells (p less than 0.05). Also, the average nucleotide deletion of J gamma 2 coding sequences at the V gamma 2-J gamma 2 junctions was 3.1 nucleotides in immature B cells and 1.9 nucleotides in T cells (p less than 0.01). The average of total number of the deleted nucleotides at the V gamma 2-J gamma 2 junctions was 10.0 nucleotides in immature B cells and 7.0 nucleotides in T cells (p less than 0.01). No correlation was found between the extent of the nucleotide deletion of the V gamma 2 coding sequence and that of the J gamma 2 coding sequence at each V gamma 2-J gamma 2 junction in immature B and T cells. These results demonstrated that nucleotide deletion at the V gamma 2-J gamma 2 junctions was significantly wider in immature B cells than in T cells.     

Synthesis of λ1, λ2, and λ3 light chains by mouse spleen B cells

To determine whether the infrequency of immunoglobulins with λ3 light chains is due to a corresponding scarcity of λ3 B cells, the production of the various λ chain subtypes (λ 1, λ2, and λ3) by normal spleen cells was compared. The results showed that λ1, λ2, and λ3 chains are produced in a ratio of about 1.0 :0.7 : 0.3, respectively. The argument is made that λ1, λ2, and λ3 B cells exist in the same ratio. Results obtained with neonatal and nude mouse spleen cells suggest that these small differences are not due to stimulatory effects of environmental antigens or regulatory T cells. The much greater disparity in the abundance of λ subtypes in various antibody responses and serum Ig suggests that λ1 B cells may be more likely than λ2 or λ3 B cells to differentiate into antibody-secreting plasma cells.     

The kappa gene repertoire of human neonatal B cells

The kappa chain repertoire of individual IgD(+) human neonatal B cells was analyzed using a single cell PCR technique. A total of 104 productive and 90 non-productive VkappaJkappa rearrangements from three cord blood B cell samples were sequenced and compared to the adult IgM(+) peripheral B cell VkappaJkappa repertoire. All six Vkappa families were present in neonatal B cells, but the distribution was not random. In the non-productive repertoire Vkappa2 and Vkappa6 families were less frequent, Vkappa1 and Vkappa3 families were as frequent, and Vkappa4 and Vkappa5 families were more frequent than expected from random chance. Notably, the Vkappa2 family was negatively selected into the productive repertoire. In contrast, the Vkappa1 family was positively selected because of positive selection of three specific genes, O12/O2, L12a and L9. B3 (Vkappa4) and B2 (Vkappa5) were over-represented in the non-productive repertoire and then were expressed less frequently in the productive repertoire. In contrast, the Vkappa3 family gene, A27, was also over-represented in the non-productive repertoire but not further selected into the productive repertoire. Compared to the adult repertoire, junctional diversity was less marked because of a diminished influence of TdT activity, whereas the mean CDR3 length was comparable to that of normal adult B cells. Comparison of the distribution of Vkappa and Jkappa genes with those found in normal adult subjects suggested that there was less receptor editing in neonatal B cells. When neonatal CD5(+) B cells were compared with CD5(-) IgD(+) B cells, it was noted that the Vkappa gene A30 was used only in CD5(+) B cells in both the productive and non-productive repertoires. The results indicate that the usage of Vkappa genes by neonatal B cells is biased by both intrinsic molecular processes and selection. The evidence of selection indicates that the Vkappa repertoire is shaped by self antigens, since exposure to exogenous antigens is limited at the time of birth.     

Involvement of SLP-65 and Btk in tumor suppression and malignant transformation of pre-B cells

Signals from the precursor-B cell receptor (pre-BCR) are essential for selection and clonal expansion of pre-B cells that have performed productive immunoglobulin heavy chain V(D)J recombination. In the mouse, the downstream signaling molecules SLP-65 and Btk cooperate to limit proliferation and induce differentiation of pre-B cells, thereby acting as tumor suppressors to prevent pre-B cell leukemia. In contrast, recent observations in human BCR-ABL1(+) pre-B lymphoblastic leukemia cells demonstrate that Btk is constitutively phosphorylated and activated by the BCR-ABL1 fusion protein. As a result, activated Btk transmits survival signals that are essential for the transforming activity of oncogenic Abl tyrosine kinase.     

Isolation of peritoneal precursors of B-1 cells in the adult mouse

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220⁺IgM^?lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of λ5, V_preB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220⁺IgM^? cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220⁺IgM^? cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.     

The lambda gene immunoglobulin repertoire of human neonatal B cells

The dynamics of immunoglobulin rearrangements and selection, which depend on age, antigen exposure and tolerance functions, are only partly understood. Thus, we analyzed and compared the lambda chain immunoglobulin repertoire of individual IgD+ human neonatal B cells with the adult peripheral B cell VlambdaJlambda repertoire. Some Vlambda genes, 4C, 2A2, 2B2, 5A, 1G and 4B, were overexpressed in the non-productive neonatal repertoire, whereas other Vlambda genes (2E, 2A2, 3H, 2B2, 1C and 1G) were overexpressed in the productive repertoire. The adult B cell repertoire revealed nearly the same predominance of genes in the non-productive and productive repertoire. A comparison of the non-productive and productive repertoire indicated that the genes 3H and 1C were positively selected, whereas the genes 4C, 2A1, 3I, 5A, 9A, 4A and 4B were negatively selected. All four functional Jlambda genes were used in both repertoires. Jlambda2/3 was used mainly. Insertions of non-templated nucleotides at the VlambdaJlambda-junction by the enzyme TdT were less frequent as compared to the adult, but the CDR3 length was the same. Comparison of CD5+IgD+ and CD5-IgD+ B cells revealed no differences between neonatal productive rearrangements. However, the genes 1C and 1G were used more often in the non-productive repertoire of CD5+ B cells, whereas gene 4B was used significantly more frequent in CD5- B cells. These data provide evidence that the primary usage and subsequent selection of Vlambda genes in the neonate are surprisingly comparable with the adult. This suggests that selection into the productive Vlambda repertoire in principal might be driven mainly by autoantigens in the newborn, as well as in adulthood, since newborns have not been exposed to exogenous antigens.     

Contribution of secondary Igkappa rearrangement to primary immunoglobulin repertoire diversification

Abs reactive to DNA and DNA/histone complexes are a distinguished characteristic of primary immunoglobulin repertoires in autoimmune B6.MRL-Fas^lpr and MRL/MpJ-Fas^lpr mice. These mice are defective in Fas receptor, which is critical for the apoptosis of autoreactive B cells by an extrinsic pathway. In the present study, we explored the possibility that bone marrow small pre-B and immature B cells from adult B6.MRL-Fas^lpr mice and MRL/MpJ-Fas^lpr mice respectively, which contain autoreactive B-cell antigen receptors (BCR) and manifest autoimmune syndromes, exhibit enhanced receptor editing patterns. Indeed, FAS^lpr pre B and immature B cells were shown to possess more ongoing replacements of non-productive (nP) than productive (P) primary VκJκ rearrangements. Significantly, the P vs nP ratios of these replaced primary rearrangements were 1:2, thus indicating that κ light-chain production appears not to inhibit secondary rearrangements. In addition, we identified multiple atypical rearrangements, such as Vκ cRS (cryptic recombination signals) cleavages. These results suggest that the onset of light chain secondary rearrangements persists similarly as a non-selected mode and independent of BCR autoreactivity during certain developmental windows of bone marrow B cells in lupus-prone mice and control, and leads us to propose the function of secondary, de novo Igκ rearrangements to increase BCR diversity.     

The efficiency of B cell receptor (BCR) editing is dependent on BCR light chain rearrangement status

Anti-DNA knock-in mice serve as models for studying B cell tolerance mechanisms to a ubiquitous antigen. We have constructed six strains of double transgenic (C57BL/6xBALB/c)F1 mice, each expressing an unmutated or somatically mutated anti-DNA heavy (H) chain, combined with one of three different light (L) chains, namely V(kappa)1-J(kappa)1, V(kappa)4-J(kappa)4 and V(kappa)8-J(kappa)5. In vitro analysis of the various Ig H/L chain combinations showed that all had a similar specificity for single-stranded DNA and double-stranded DNA, but that antibodies encoded by the mutated H chain had higher affinities for the autoantigen. None of the targeted mouse strains exhibited significant levels of serum anti-DNA activity. However, while B cells from mice carrying the V(kappa)1-J(kappa)1 transgenic L chains were tolerized almost exclusively by L chain receptor editing in an affinity-independent manner, the mice expressing V(kappa)8-J(kappa)5 L chains have utilized affinity-dependent clonal anergy as their sole mechanism of B cell tolerance. V(kappa)4-J(kappa)4 targeted mice exhibited an intermediate phenotype with respect to these two mechanisms of B cell tolerance. Our results suggest that receptor editing is the preferred mechanism of B cell tolerance and that the efficiency of L chain editing is directly related to the number of available J(kappa) segments on the expressed V(kappa) allele.     

The pre-B cell receptor and its function during B cell development

The process of B cell development in the bone marrow occurs by the stepwise rearrangements of the V, D, and J segments of the Ig H and L chain gene loci. During early B cell genesis, productive Ig H chain gene rearrangement leads to assembly of the pre-B cell receptor (pre-BCR), which acts as an important checkpoint at the pro-B/preB transitional stage. The pre-BCR, transiently expressed by developing precursor B cells, comprises the Ig muH chain, surrogate light (SL) chains VpreB and lambda5, as well as the signal-transducing heterodimer Ig alpha/Ig beta. Signaling through the pre-BCR regulates allelic exclusion at the Ig H locus, stimulates cell proliferation, and induces differentiation to small post-mitotic pre-B cells that further undergo the rearrangement of the Ig L chain genes. Recent advances in elucidating the key roles of pre-BCR in B cell development have provided a better understanding of normal B lymphopoiesis and its dysregulated state leading to B cell neoplasia.     

Pairing of VH gene families with the λ light chain: Evidence for a non-stochastic association

The frequencies at which four V_H gene families pair with the λ1 light (L) chain were determined by sequential hybridization of V_H- and λ1-specific DNA probes to mitogen-induced colonies of B cells. Analysis of pair frequencies indicates that the repertoire of λ L chain antibodies is generated by the stochastic pairing of smaller 3′-to-mid-locusV_H gene families (X-24, S107, Q52). However, the large 5′ V_H J558 family appeared to associate with the λ1L chain non-stochastically; the frequency of VhJ558/λ1⁺ colonies among all λ1⁺ colonies was significantly lower than the frequency of J558 expression among all (Cμ⁺) B cell colonies. This difference suggests that selection, either intrinsic at the level of rearrangement or heavy and L chain pairing, or extrinsic following surface immunoglobulin expression, may operate to shape the λ antibody repertoire prior to the introduction of exogenous antigen.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

Subtle differences in antibody responses and hypermutation of λ light chains in mice with a disrupted χ constant region

Analysis of λ light chain use in normal mice is made difficult by the dominant x light chain repertoire. We produced mice rendered deficient in x light chain expression by gene targeting and focused on questions concerned with the generation of λ light chain diversity. Whilst these mice compensate the x deficiency with increased λ titers, and their Ig level is therefore not significantly reduced, they show major differences in immunization titers, germinal center (GC) development and somatic hypermutation. After immunization, using antigens that elicit a restricted IgL response in normal mice, we obtained in the x^?/? mice elevated primary antibody titers but a subsequent lack in titer increase after repeated antigen challenge. Analysis of the Peyer's patches (PP) revealed a dramatically reduced cell content with rather small but highly active GC. Flow cytometric analysis showed different cell populations in the PP with enriched peanut agglutinin (PNA)^hi/CD45R(B220)⁺ B cells, implying that the apparent compensation for the lack of x light chain expression involves the GC microenvironment in cell selection, the initiation of hypermutation and high affinity expansion. The three Vλ genes, V1, V2 and Vx, are mutated in the GC B cells, but show no junctional diversity. In contrast, a reduced rate of Vλ hypermutation is found in the hybridoma antibodies, which appears to reflect a selection bias rather than structural constraints. However, mechanisms of somatic mutation and specificity selection can operate with equal efficiency on the few Vλ genes.     

The transient expression of pre-B cell receptors governs B cell development

Only a subpopulation of relatively large pre-B cells express pre-B cell receptors (preBCR) that can be seen with very sensitive immunofluorescence methods. Inefficient assembly of the multicomponent preBCR coupled with their ligand-induced endocytosis may account for the remarkably low in vivo levels of preBCR expression. Signaling initiated via the preBCR promotes cellular proliferation and RAG-1 and RAG-2 downregulation to interrupt the immunoglobulin V(D)J gene rearrangement process. Silencing of the surrogate light chain genes, VpreB and lambda5, then terminates preBCR expression to permit cell cycle exit, recombinase gene upregulation, and VJ(L) rearrangement by small pre-B cells destined to become B cells.