The majority of murine VH12-expressing B cells are excluded from the peripheral repertoire in adults

We have previously demonstrated that at birth most productive (P) V_H12 rearrangements in B10.H-2^aH-4^bp/Wts (2^a4^b) mice encode a ten-amino acid CDR3, and that a significant fraction of the expected repertoire is absent. We have now examined the adult V_H12 CDR3 repertoire involving all four J_H gene segments in both peritoneum and spleen. Of the 74 P V_H12 rearrangements from these tissues 67 encode a CDR3 of ten amino acids and include a Gly in the fourth position (designated 10/G4). Most of these rearrangements appear to derive from phosphatidylcholine (PtC)-specific B cells, which also have a 10/G4 V_HCDR3, since few 10/G4 P rearrangements were present in spleen cells depleted of PtC-specific B cells. Thus, the V_H12 B cell repertoire in adult mice is largely restricted to the use of a single CDR3 motif and to a single antigen specificity. This bias results from two selection events: (1) selective exclusion of most V_H12 B cells from the peripheral repertoire, and (2) clonal expansion in the periphery of V_H12 B cells that have a 10/G4 V_HCDR3 and bind PtC. Analysis of V_H12-J_H1 rearrangements in viable motheaten (me^vJme^v) mice, which have an abnormal B cell repertoire due to a defective phosphatase (Hcph) and have barely detectable numbers of PtC-specific B cells, indicates that selective exclusion of V_H12 B cells from the peripheral repertoire occurs normally, but that clonal expansion of 10/G4 V_H12 B cells is minimal. This is evidence that the selective exclusion of V_H12 B cells from the peripheral repertoire and the clonal expansion of V_H12 B cells with a 10/G4 CDR3 are due to independent signaling events.     

Autoreactivity and the positive selection of B cells

The diversity among B‐cell antigen receptor (BCR) specificities is generated by random rearrangement of gene segments during early B‐cell development. While such stochastic recombination of gene segments is important for diversity, it introduces the risk of producing self‐reactive BCRs which might lead to the development of autoimmune diseases. Therefore, it has been proposed that negative selection of autoreactive BCR specificities during early B‐cell development are required to establish tolerance towards self. In fact, transgenic mouse models have identified a number of “tolerance mechanisms” such as receptor editing, clonal deletion and anergy, all of which prevent the development of autoreactive B cells. Recent data, however, reveal that self‐recognition is crucial for the generation of B cells, and that the precursor‐BCR (pre‐BCR), which is essential for early B‐cell development, basically plays the role of an autoreactive BCR. Moreover, although it has become clear that autoreactive B cells are present in the periphery of healthy individuals, the role of autoantigen in their development, persistence and regulation is unclear. This review outlines the important role of autoreactivity in early B‐cell development and presents potential models for the regulation of the activation of peripheral B cells by different forms of self or foreign antigens.     

Anti-idiotypic antibody D12 and superantigen SPA both interact with human VH3-encoded antibodies on the external face of the heavy chain involving FR1, CDR2 and FR3

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V_H3 encoded antibodies. We used mutational analysis to determine the subregions of a V_H3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FR1, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V_H3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.     

Thymic stromal cells and positive selection

The intrathymic differentiation events leading to the development and export of mature T cells tolerant to self yet capable of responding to foreign peptide antigen in the context of self-MHC are clearly both dynamic and complex. The changing phenotype of the developing thymocyte as it migrates through and interacts with the heterogeneous thymic microenvironment and the intracellular signalling events associated with such interactions are being extensively studied, yet many aspects remain poorly defined, such as the precise relationship between stromal cells and thymic selection. Positive and negative selection are crucial events in the development of T cells, leading to a diverse yet non-autoreactive immune system. A breakdown in either of these processes could lead to either a reduced T cell repertoire or the escape into the periphery of autoreactive T cells - both clearly having deleterious consequences for the health of the individual. This review aims to summarise the current status of research in thymic positive selection with emphasis on the role of different cell types and peptides.     

Age-related impaired affinity maturation and differential D-JH gene usage in human VH6-expressing B lymphocytes from healthy individuals

To elucidate the basic molecular events underlying humoral immunity during ontogeny and senescence, we analyzed a panel of 179 polymerase chain reaction-derived VH6-D-JH rearrangements from cord blood, peripheral blood, and spleen. Nucleotide sequence analysis of the CDR3 region shows that there is a difference in D and JH gene usage in functional rearrangements between lymphocytes from peripheral blood and spleen. Analysis of the VH6 gene shows that the mutational frequencies rise from 0.81% in cord blood to 1.96% in peripheral blood lymphocytes derived from young adults, and decrease to 0.80% in samples from individuals older than 50 years. The number of rearrangements carrying mutations follows a similar pattern: 22% in cord blood, 73% in the age group 20–49 years, and 57% in the age group over 50 years. The mutational frequencies among the mutated genes are, however, similar for cord blood and young adults, 2.76% and 2.51%, respectively, and 1.3% in older adults. These data show an age-related impaired affinity maturation which might relate to the decrease in immunological responsiveness among the elderly.     

Available λ B cell repertoire in the mouse: Evidence of positive selection by environmental factors

We have recently shown that, from two BALB/c mice treated with rabbit anti-Cλ2/Cλ3 antibodies coupled to lipopolysaccharide, variable heavy chain (V_H) family repertoires associated with λ2 or λ3 light chains can differ from one λ. subtype to another and from one individual mouse to another. Indeed, 4 out of 6 λ2 (VxJ2) hybridomas from one mouse preferentially expressed the V_H10 family while 3 out of 8 λ2 (V2J2) and 5 out of 8 λ2 (VxJ2) hybridomas from a second mouse preferentially expressed the S107 and VGAM3.8 V_H families, respectively. In this report, we describe the structural basis of such preferential pairings by sequence analysis of the 12 λ2 hybridomas. The sequence comparison of their V_H regions show that each preferential association of a VH family to one Vλ region is restricted to the use of a single member or very closely related members inside a V_H family and that a great variability of CDR3 of heavy chain is observed. We, therefore, suggest that environmental factors can modify the available XλB cell repertoire through a positive selection of particular VH/Vλ pairings. Moreover, our data support that this selection does not require clonal expansion and punctual somatic mutation.     

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I-restricted T cell receptors (TcR) differentiate into CD8⁺ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8-null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I-restricted TcR. In all three instances CD8 was required for positive selection of MHC class I-restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I-restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I-restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.     

CDR3 regions in the preimmune VH B cell repertoire of lpr mice.

Previous studies have suggested that the CDR3 genetic element of the heavy chain variable region of autoantibodies is important in determining reactivity against self antigens, particularly against DNA. The lpr mutation was recently found to encode for a defective form of the fas protein, a molecule important for the transmission of the apoptotic signal into cells. Our aim was to determine whether CDR3 elements similar to those described for autoantibody-producing hybridomas derived from lupus-prone strains could be found in the preimmune repertoire of B cells in mice with the lpr mutation. The analysis of the junctions of the VH-C mu functional rearrangements derived by polymerase chain reaction (PCR) amplification of RNA obtained from splenic small, resting cells stimulated with lipopolysaccharide (LPS) from male lpr mice showed that a large proportion of them expressed D genes in the unusual reading frames 2 and 3. Two of the lpr joints were formed by D-D fusions. Similarly, nearly half of the lpr sequences had arginines, an amino acid which promotes binding to dsDNA and is seldom observed in normal junctions. Our results show that the preimmune repertoire of lpr animals has abnormal CDR3 elements which may result from a failure at different levels of selection. The antigen-dependent selection of such elements that leads to the expansion of specific, high-affinity anti-dsDNA antibody-producing clones might depend on other genetic factors not found in the C57B1/6-lpr strains but in the MRL-lpr.     

Differences in Vϰ gene utilization and VH CDR3 sequence among anti-DNA from C3H-lpr mice and lupus mice with nephritis

To investigate the molecular properties of anti-DNA from lpr mice that express high levels of anti-DNA without immune-mediated nephritis, the sequences of VH and Vϰ genes encoding 11 monoclonal anti-DNA antibodies derived from C3H-lpr/lpr (C3H-lpr) mice were studied. All of the C3H-lpr monoclonal anti-DNA bound single-stranded DNA while five also bound double-stranded DNA. Two of the hybridomas were clonally related as determined by Southern analysis and sequencing. Sequence analysis of C3H-lpr anti-DNA revealed the use of VH genes that encode anti-DNA from the MRL-lpr/lpr and (NZB X NZW)F1 mouse models of lupus, although differences occurred in the VH CDR3 amino acid content. In contrast, the Vϰ genes from C3H-lpr mice lacked significant identity with previously reported Vϰ genes for anti-DNA from lupus models. These results indicate that anti-DNA from C3H-lpr mice differ from anti-DNA from lupus mice with nephritis in patterns of V gene expression and suggest a molecular basis for the lack of pathogenicity of anti-DNA in these mice.     

Cell- and nuclear-penetrating anti-dsDNA autoantibodies have multiple arginines in CDR3 of VH and increase cellular level of pERK and Bcl-2 in mesangial cells

Investigation of characteristics of cell- and nuclear-penetrating anti-double stranded (ds)DNA autoantibodies (autoAbs) is important to understand pathogenesis of lupus nephritis, but has not been clearly explored. The present study reports that three anti-dsDNA monoclonal autoAbs, which contain more than two arginine residues in their CDR3s of variable heavy domain (VH), penetrated into living murine mesangial cells and the cell nuclei. However, an anti-dsDNA monoclonal Ab (mAb) having only one arginine in the CDR3-VH did not penetrate cells. To assess the contribution of antigen-binding sites, especially the VH, in cell- and nuclear-penetration, we evaluated the characteristics of recombinant single chain Fv(scFv), VH, and variable light domain (VL) of a penetrating mAb. The scFv and VH domain, containing arginine in CDR3-VH maintained the ability to penetrate cells and the cell nuclei, whereas the VL domain, having no arginine in CDR3, did not penetrate cells. The penetratingm Abs, scFv, and VH activated ERK and increased cellular protein levels of Bcl-2, whereas the non-penetrating Ab and VL did not. The cell survival was decreased by the penetrating mAbs, scFv and VH, not by the non-penetrating mAb and VL. The data indicate that an antigen-binding site is required for cell-penetration and that positively-charged arginine residues in CDR3-VH contribute to the cell- and nuclear-penetrating ability of a subset of anti-dsDNA autoAbs. Furthermore,the nuclear-penetrating anti-dsDNA autoAbs could possibly function as a pathogenic factor for lupus nephritis by up-regulating ERK activation and Bcl-2 production in mesangial cells. The cell- and nuclear-penetrating VH domain may be exploited as a vehicle for the intra cellular delivery of various useful molecules.     

Progressive decrease in VH3 gene family expression in plasma cells of HIV-infected patients

We have analysed the expression of V_H gene families In totalperipheral plasma cells of µ and isotypes from a groupof HIV-infected patients. CD19^– and CD20^– cellswere separated from B lymphocytes, and anchored RT-PCR productswere hybridized with specific V_H gene family probes and witha consensus J_H region probe. The V_H/J_H hybridization Intensityratios were taken as a parameter to measure V_H expression. Invivo V_H3 gene family expression is reduced In plasma cells ofall HIV-infected patients compared with adult healthy donors.This decrease is maximal in patients with AIDS: >90%. Thisis true for both studied Isotypes. Conversely, the two othermain V_H gene families, V_H1 and V_H4, show no significant variationIn expression. We and others have previously shown that V_H3gene family expression is first expanded and then decreasedIn peripheral B lymphocytes of HIV-infected patients. The presentresults extend these observations and show that V_H3 gene familyexpression is also affected In plasma cells. The existence ofa B cell superantlgen In HIV Infection may explain these data.This pronounced reduction In Vh3 family expression may participateIn the Impaired humoral responses against bacterial agents foundIn HIV-infected patients.     

Biased VH gene expression in murine CD5 B cells results from age-dependent cellular selection.

Flow cytometry-purified, peritoneal and splenic CD5+ and CD5- B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and Vx gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain-treated mouse red blood cells (BrMRBC), single-stranded DNA, trimethyl ammonium and bovine gamma-globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D-proximal VH preferences that are lost with age. Biased VH11 expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti-BrMRBC reactivity, which is considerably higher in peritoneum than in spleen. No particular Vx bias was observed in any of the populations studied with the possible exception of Vx22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age-dependent cellular selection mechanisms.     

Isolation of pure functionally active CD8 T cells positive selection with monoclonal antibodies directly conjugated to monosized magnetic microspheres

A monoclonal antibody of the IgM isotype, ITI-5C2, which binds with high affinity to CD8 molecules, was directly conjugated to the monosized magnetic microspheres M-450. This permits selective removal of the CD8⁺ T cell subset (T8) from peripheral blood mononuclear cell suspensions in a rapid one-step procedure. With a low ratio of microspheres to cells (2:1), functionally active T8 cells can be recovered. In vitro experiments involving such positively selected T8 cells or recombinations of isolated T8 and T4 subsets, demonstrate that the presence of M-450 microspheres coated with ITI-5C2 do not interfere with the immunological functions of the positively selected cells. The method has possible application in the isolation of all cell populations where high avidity mAbs of appropriate specificity are available.     

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4⁺CD8^la intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class 1.     

Biased VH family usage in primary gastric B cell lymphoma of mucosa-associated lymphoid tissue-type and the selected Vβ expression of T cells infiltrating into the lymphoma cells

Seven cases of primary gastric low-grade B cell lymphoma of mucosa-assoclated lymphoid tissue (MALT) type, two cases of high-grade B cell lymphoma with a low-grade component and three cases of pure high-grade lymphoma were selected for the current study. The Ig VH gene use of lymphoma cells and the Vβ repertoires of infiltrating T cells were Investigated. The VH gene analysis showed multiple VH family usage In 12 cases, but the MALT-type lymphoma cell usage was found to be biased for the families that have a low number of VH genes (VHIV and V). Another analysis of lymphoma-lnfiltrating T cells showed restricted expressions of the Vβ repertoire in all seven low-grade cases and three high-grade cases. In those 10 cases, a considerable number of CD4-postttve T cells Infiltrated Into lymphoma cells and RAG-1 was also prominently expressed. Based on these findings, It was thus assumed that the normal counterpart of gastric B cell lymphoma of MALT type is different from the conventional B cell lymphoma, and the restricted expression of Vβ repertoires Is therefore considered to be a characteristic finding in low-grade B cell lymphomas of MALT type as well as in a proportion of high-grade lymphomas (the so called 'high-grade lymphoma of MALT type').     

The influence of invariant chain on the positive selection of single T cell receptor specificities

The appearance of peptide-loaded major histocompatibility complex (MHC) class II molecules at the cell surface depends critically on the invariant chain (Ii). We have studied the influence of Ii on the positive selection of CD4⁺ T cells, mediated by class II molecules expressed on thymic stromal cells. Invariant chain-deficient mice (Ii°) were crossed with different T cell receptor (TcR) transgenic strains and the emergence of mature CD4 single-positive thymocytes measured in Ii°/TcR transgenic offspring. Positive selection was nearly absent in Ii°/2B4 mice, which display receptors specific for a moth cytochrome c (MCC) peptide in the context of E^k. In addition, no T cell response was elicited when nontransgenic Ii° animals were injected with this peptide, even though antigenpresenting cells (APC) from such mice were perfectly capable of presenting it, suggesting that selection of the entire anti-MCC 88-103 repertoire depends on Ii. Positive selection also appeared strongly reduced in another line of Ii°/TcR transgenic mice (Ii°/BDC2.5). However, in sharp contrast, a third line (Ii°/3A9) exhibited almost normal positive selection of thymocytes displaying the transgene-encoded receptor. These thymocytes were exported to the periphery; peripheral T cells could respond normally to the appropriate peptide in vitro. The most likely interpretation of these findings is that selection of most CD4⁺ T cells depends on MHC class II complexes loaded with peptide in an Ii-dependent pathway, but some can be selected on class II complexes that are either loaded along an alternative, Ii-independent, route or are empty. This is consistent with the involvement of peptide in positive selection of CD4⁺ T cells, for which there exists little prior evidence.     

Pertussis toxin can replace T cell receptor signals that induce positive selection of CD8 T cells

CD4⁺ helper T lymphocytes and CD8⁺ killer T lymphocytes are both generated in the thymus from common precursor cells expressing CD4 and CD8. The development of immature CD4⁺ CD8⁺ thymocytes into mature ‘single-positive’ T cells requires T cell antigen-receptor (TCR)-mediated positive selection signals. Although it is known that the recognition specificity of TCR expressed by CD4⁺ CD8⁺ thymocytes determines their fate to become either CD4⁺ or CD8⁺ T cells, the molecular signals that direct precursor thymocytes to become CD4⁺ and CD8⁺ T cells are unclear. By using ZAP-70^? mutant thymus organ cultures in which T cell development is arrested at the CD4⁺ CD8⁺ thymocyte stage, the present study shows that distinct biochemical treatments can selectively restore the generation of mature CD4⁺ and CD8⁺ T cells, bypassing TCR-induced positive selection signals. The combination of phorbol ester and ionomycin selectively restores the generation of CD4⁺CD8^? TCR^high cells consistent with previous results. On the other hand, we find that the generation of CD4^? CD8⁺ TCR^high cells is selectively induced by pertussis toxin. Interestingly, the signals generated by pertussis toxin, which increase Notch expression, can dominate the signals by phorbol ester and ionomycin, steering thymocyte development to CD8 lineage. These results indicate that distinct biochemical signals replace TCR signals that selectively induce positive selection of CD4⁺ and CD8⁺ T cells, and that biochemical treatment can manipulate the development and choice of CD4⁺ and CD8⁺ T cells.     

小鼠白细胞介素12基因转染及其在黑色素瘤细胞B16F10中的表达

目的:探索小鼠白细胞介素12(mIL-12)基因在小鼠黑色素瘤B16F10细胞中的表达。方法:应用DNA重组技术将mIL-12基因插入pcDNA3.1真核表达载体中, 通过电穿孔转染B16F10细胞, 筛选出阳性细胞克隆后, 应用PCR、RT-PCR及Westernblot技术检测mIL-12基因在B16F10细胞中的整合及表达。结果:在DNA、mRNA及蛋白质3个水平均证实mIL-12基因已转染到B16F10细胞中并表达。结论:mIL-12基因可成功地转染体外培养的B16F10细胞并表达, 为进一步研究IL-12基因修饰的肿瘤细胞的基因瘤苗奠定了基础。     

HMGB1 promotes CXCL12-dependent egress of murine B cells from Peyer's patches in homeostasis

High mobility group box-1 protein (HMGB1) is an alarmin that, once released, promotes inflammatory responses, alone and as a complex with the chemokine CXCL12. Here, we report that the HMGB1–CXCL12 complex plays an essential role also in homeostasis by controlling the migration of B lymphocytes. We show that extracellular HMGB1 is critical for the CXCL12-dependent egress of B cells from the Peyer's patches (PP). This promigratory function of the complex was restricted to the PPs, since HMGB1 was not required for B-cell migratory processes in other locations. Accordingly, we detected higher constitutive levels of the HMGB1–CXCL12 complex in PPs than in other lymphoid organs. HMGB1–CXCL12 in vivo inhibition was associated with a reduced basal IgA production in the gut. Collectively, our results demonstrate a role for the HMGB1–CXCL12 complex in orchestrating B-cell trafficking in homeostasis, and provide a novel target to control lymphocyte migration in mucosal immunity.     

Inhibition of murine B1 lymphocytes by interleukin-12

B1 cells are a subset of B lymphocytes found in many spectes and are implicated in the development of autoimmunity. B1 cells have previously been shown to be suppressed by the T helper (Th)1 cytokine interferon (IFN)-γ, and to be stimulated by the Th2 cytokines interleukin (IL)-2, IL-4, IL-5 and IL-10. To examine further the interactions of B1 cells and Th1 cells, we have now tested the effects of the Th1 cell-inducing cytokine IL-12 on murine B1 cells. BALB/c mice were immunized with phosphorylcholine conjugated to keyhole limpet hemocyanin (PC-KLH) and simultaneously treated with 1 μg recombinant murine IL-12 for 3 consecutive days. In addition to altering the isotype and idiotype distribution of anti-PC antibodies, IL-12 treatment was found to cause a loss of peritoneal, but not splenic B lymphocytes in immunized mice. B cell depletion required exposure to IL-12 plus antigenic stimulation. Levels of peritoneal B lymphocytes were fully restored by day 45, but the majority of these cells belonged to the B2 subset. Additionally, proliferation of B1 cells in vitro induced by IL-5 was substantially inhibited by IL-12. IL-12 itself had no effect on viable cell recovery of peritoneal cells (PeC) cultured in vitro, but viable cell recovery was significantly decreased in PeC cultured with IL-5 plus IL-12. These results show that IL-12 causes the loss of murine peritoneal B1 cells and suggest that treatment with this cytokine may be useful for disease conditions that involve B1 cell dysfunction.