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.     

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.     

Differences in kappa to lambda (κ:λ) ratios of serum and urinary free light chains

Free light chains (FLC) are a natural product of B lymphocytes and, as such, represent a quantifiable biomarker of cellular proliferation. Accurate measurement of the concentrations of these components in serum and urine provides a unique means of ascertaining B cell immunoglobulin synthesis during physiologic and, especially, pathologic states, where such information has important diagnostic and therapeutic implications. Previously, use of such quantitative assays has been limited due to the lack of potent serologic reagents specific for these components. We have immunized mice with κ- and λ-type monoclonal human light chains (Bence Jones proteins (BJP)) and have obtained monoclonal antibodies (MoAbs) that differentiate between unbound and bound light chains. These highly specific MoAbs were used to measure by ELISA the concentrations of FLC in the serum of 22 normal individuals and in urine from 16 of these subjects. The mean serum κ and λ FLC concentrations were found to be 16.6 ± 6.1 μg/ml and 33.8 ± 14.8 μg/ml, respectively. In contrast, the values for urinary κ and λ FLC were 2.96 ± 1.84 μg/ml and 1.07 ± 0.69 μg/ml, respectively. In each case studied, the serum κ:λ ratio was consistently less than that of urine (mean values, serum ≈ 1:2; urine ≈ 3:1). That the rate of synthesis of λ-type FLC exceeded that of κ was evidenced in assays of culture fluid supernatants of unstimulated normal peripheral blood mononuclear cells (PBMC), where the mean κ:λ ratio was determined to be 1:1.4. Metabolic studies in which mice were injected with pools of κ- and λ-type BJP prepared in ratios of 1:1, 1:2 and 1:4 demonstrated that, regardless of the proportion, κ FLC were preferentially excreted. Our studies provide the first evidence that λ FLC are secreted by normal PBMC at a greater rate than are κ FLC, as evidenced in biosynthetic studies and by measurement of their serum concentrations. Further, we posit that quaternary structural differences between the two light-chain isotypes may account for the predominance of κversusλ components in urine.     

Molecular analysis of human immunoglobulin Vλ germline genes: Subgroups VλIII and VλIV

Three human immunoglobulin Vλ germline genes have been isolated: two from the VλIV subgroup and one from the VλIII subgroup. The VλIII gene and one of the VλIV genes appear to be functional (each being utilized in at least two expressed Vλ genes), despite deviations from the reported consensus sequences in their promoter TATA-box and recombination signal sequence elements. The other VλIV gene is a pseudogene. Of the 20 human Vλ germline genes characterized to date, 45% are pseudogenes or vestigial genes.     

Secretory immune system of the duck ( Anas platyrhynchos ). Identification and expression of the genes encoding IgA and IgM heavy chains

IgA has not previously been identified in waterfowl. Studies instead revealed physical and antigenic similarities between duck bile immunoglobulin (Ig) and serum IgM. Here, a differential screening approach was used to clone, from a duck spleen library, the cDNA encoding the heavy (H) chains of IgM and the Ig, identified here as IgA, occurring in duck secretions. Phylogenetic comparisons of inferred amino acid sequences of entire H chain constant (C) regions and of individual domains revealed that the duck μ chain was closest to chicken μ (54 % overall identity), and duck α was closest to chicken α (50 % identity). Comparison of the μ and α C regions revealed areas of up to 65 % amino acid similarity within the C4 domains, accounting for the previously noted antigenic overlap of duck IgM and IgA. Messages for α and μ were detected in duck lymphoid organs but the α message was most abundant in the respiratory, alimentary and reproductive tracts. The α message first appeared around 14 days of age and reached adult levels of expression only at 35 – 50 days. The results indicate that the duck has a mucosal immune system which utilizes IgA; however, the delayed expression and secretion of duck IgA explains the susceptibility of ducklings to mucosal pathogens. Since the waterfowl are among the most primitive extant birds, the recognition of IgA in the duck supports the conclusion that IgA occurs throughout the class Aves and also existed in the common ancestors of birds and mammals.     

Regulation of Abortion by γλ T Cells

PROBLEM: T cells are present at the feto-maternal interface, but their function during pregnancy has not been fully elucidated. T cells bearing γλ T-cell receptor (TCR) may be particularly important, as some subsets can react to trophoblast cells by producing cytokines, such as interleukin-2 (IL-2). METHOD: We depleted T cells bearing the γλ receptor by injecting monoclonal antibodies (mAB) into females of the abortion-prone animal model CBA x DBA/2. We investigated the percentage and number of γλ T-cell receptor positive (TCR)⁺ cells in decidua and spleen during pregnancy in control and γλ-depleted female mice. Pregnant females were also exposed to ultrasonic sound stress to boost the abortion rate. RESULTS: Stress failed to increase the abortion rate in the γλ TCR-depleted mice. FACScan analysis show that the ratio of cells bearing the γλ TCR dramatically decreased after injection of mAB to the γλ TCR in spleen and decidua, these cells recovered six days after depletion, showing a change in cytokine pattern. Levels of TNF-α in decidual γλ T cells decreased; similar effects of decreasing Th₁ cytokines could be observed in splenic γλ T cells. We further identified increased levels of intracellular TNF-α in the Vλ4 subset in the decidua, compared to spleen. CONCLUSIONS: Trophoblast recognition by the Vλ4 T-cell subset in the decidua may cause the release of abortogenic cytokines such as TNF-α. Depletion of such γλ TCR T cells during early pregnancy may promote successful pregnancy outcome in normal pregnancy and prevent stress-induced abortions.     

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.     

Primary localized amyloidosis of the eyelid: two cases of immunoglobulin light chain-derived proteins, subtype λV respectively λVI

Primary localized amyloidosis has been described in many different organs in the body. Studies by immunohistochemical techniques have suggested an immunoglobulin light chain origin of the amyloid material. Only in a limited number of cases has the amyloid protein been characterized by amino acid sequence analysis as subtypes of immunoglobulin light chain or heavy chain. In this report, two cases of primary localized amyloidosis of the eyelid are presented. The amyloid substance has been extracted and a major fibril protein subjected to amino acid sequence analysis. Both amyloid proteins were part of the variable region of immunoglobulin light chains, subtype λV and subtype λVI, respectively. While λVI has been shown to be a common subtype in systemic immunoglobulin light chain-amyloidosis, it has never been demonstrated in localized amyloid. Very few λV immunoglobulin light chains have been characterized and the subgroup has never been found in amyloid before.     

Influence of nucleotide excision repair on N-hydroxy-2-acetylaminofluorene-induced mutagenesis studied in λlacZ-transgenic mice

To study the influence of nucleotide excision repair (NER) on mutagenesis in vivo, ERCC1+/−, XP−/−, and wild-type (ERCC1+/+ and XP+/+, respectively) λlacZ-transgenic mice were treated i.p. with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and lacZ mutant frequencies were determined in liver. No significant effect of the treatment on the mutant frequency in wild-type or ERCC1-heterozygous mice was observed. The liver mutant frequency appeared to be significantly increased in treated XP−/− mice only. To distinguish N-OH-AAF-induced from spontaneous mutations, lacZ mutants derived from treated XP−/− mice were subjected to DNA-sequence analysis and the spectrum obtained was compared to that established for lacZ mutants in liver of PBS-treated λlacZ-transgenic mice of the parentstrain 40.6. The N-OH-AAF-induced mutation spectrum appeared to be significantly different from the spontaneous mutation spectrum: the former consisted of mainly (19/22) single bp substitutionstargeted at G, of which the majority (12/19) were G:C → T:A transversions, suggesting that N-(deoxyguanosin-8-yl)-2-aminofluorene [dG-C8-A], the major DNA adduct in N-OH-AAF-treated mice, is the premutagenic lesion. After analysis of 21 spontaneous mutants, only ten single bp substitutions targeted at G were found, of which five were G:C → T:A transversions. This study with XP−/− λlacZ-transgenic mice shows that one of the components of NER, that is, the XPA protein, suppresses mutagenesis in vivo. Environ. Mol. Mutagen. 31:41–47, 1998. © 1998 Wiley-Liss, Inc.     

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.     

The purinergic P2×7 receptor is expressed on monocytes in Behçet's disease and is modulated by TNF‐α

The P2×7 receptor (P2×7r) is expressed in innate immune cells (e.g. monocyte/macrophages), playing a key role in IL‐1β release. Since innate immune activation and IL‐1β release seem to be implicated in Behçet's disease (BD), a systemic immune‐inflammatory disorder of unknown origin, we hypothesized that P2×7r is involved in the pathogenesis of the disease. Monocytes were isolated from 18 BD patients and 17 healthy matched controls. In BD monocytes, an increased P2×7r expression and Ca²⁺ permeability induced by the selective P2×7r agonist 2′‐3′‐O‐(4‐benzoylbenzoyl)ATP (BzATP) was observed. Moreover, IL‐1β release from LPS‐primed monocytes stimulated with BzATP was markedly higher in BD patients than in controls. TNF‐α‐incubated monocytes from healthy subjects almost reproduced the findings observed in BD patients, as demonstrated by the increase in P2×7r expression and BzATP‐induced Ca²⁺ intake. Our results provide evidence that in BD monocytes both the expression and function of the P2×7r are increased compared with healthy controls, as the possible result, at least in part, of a positive modulating effect of TNF‐α on the receptor. These data indicate P2×7r as a new potential therapeutic target for the control of BD, further supporting the rationale for the use of anti‐TNF‐α drugs in the treatment of the disease.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Cloning and sequencing of human immunoglobulin λ. gene segments

To provide the building blocks for making synthetic antibody fragments we have used the polymerase chain reaction (PCR) to clone human variable (V) gene segments of λ light chains. The PCR primers were based on the sequences of known human Vλ segments, and were used to isolate 14 new Vλ segments (including 4 pseudogenes) from a single individual. We have compiled a sequence directory from this data and other sources to include all known human Vλ segments with open reading frames and we have identified a new Vx family (Vλ IX). Almost all of the segments (22/24) have different sequences in the complementarity-determining regions, setting a lower limit to the structural diversity of the antigen binding sites encoded by human Vλ genes in the human population.     

Analysis of individual immunoglobulin λ light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenviroment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse λ light chain genes which arose in response to a specific antigen. Laboratory mice possess three Vλ subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single λ light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V_λ1 genes from these single cells for mutational analysis. None of the 32 V_λ1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other Vλ subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.     

TAK1 maintains the survival of immunoglobulin λ‐chain‐positive B cells

TAK1 (MAP3K7) mediation of the IκB kinase (IKK) complex?nuclear factor‐κB (NF‐κB) pathway is crucial for the activation of immune response and to perpetuate inflammation. Although progress has been made to understand TAK1 function in the B‐cell receptor (BCR) signaling, the physiological roles of TAK1 in B‐cell development, particularly in the bone marrow (BM), remain elusive. Previous studies suggested that the IKK complex is required for the development of immunoglobulin light chain λ‐positive B cells, but not for receptor editing. In contrast, NF‐κB activity is suggested to be involved in the regulation of receptor editing. Thus, NF‐κB signaling in early B‐cell development is yet to be fully characterized. Therefore, we addressed the role of TAK1 in early B‐cell development. TAK1‐deficient mice showed significant reduction of BM Igλ‐positive B‐cell numbers without any alteration in the BCR editing. Furthermore, the expression of survival factor Bcl‐2 was reduced in TAK1‐deficient BM B cells as assessed by microarray and quantitative PCR analyses. Ex vivo over‐expression of exogenous Bcl‐2 enhanced the survival of TAK1‐deficient Igλ‐positive B cells. TAK1–IKK–NF‐κB signaling contributes to the survival of λ‐chain‐positive B cells through NF‐κB‐dependent anti‐apoptotic Bcl‐2 expression.