Plant homeodomain finger protein 11 promotes class switch recombination to IgE in murine activated B cells.

BACKGROUND: Polymorphisms of the Plant homeodomain finger protein 11 (PHF11) are strongly associated with high serum IgE levels and clinical severity of atopic patients. However, the precise mechanism has not been fully elucidated. We investigated the role of Phf11 in class switch recombination (CSR) to IgE by activated B cells. METHODS: We generated Phf11 transgenic (Lckd-Phf11-Tg) mice that express the exogenous murine Phf11 in lymphocytes under the control of distal Lck promoter. We examined IL-4-induced CSR to IgE in activated Lckd-Phf11-Tg B cells in vitro. We analyzed production of ovalbumin (OVA)-specific IgE and nose-scratching symptoms in Lckd-Phf11-Tg mice using an OVA-induced allergic rhinitis model. RESULTS: The exogenous Phf11 promoted CSR to IgG1 and IgE in activated B cells with an increase in germ line transcript (GLT) γ1 and GLT ε expression. The exogenous Phf11 augmented transcriptional activity of the GLT γ1 and GLT ε promoters through permissive histone modifications and binding of NF-κB and STAT6. Furthermore, the exogenous Phf11 bound to the GLT ε promoter with increased binding of NF-κB. Silencing of the endogenous Phf11 reduced the frequency of CSR to IgE and GLT ε expression, but not to IgG1 or GLT γ1 expression, in activated B cells. In an allergic rhinitis model, Lckd-Phf11-Tg mice showed a significant increase in the production of OVA-specific IgE and the frequency of nose scratching. CONCLUSION: Phf11 accelerates CSR to IgE in activated B cells by increasing the transcriptional activity of GLT ε promoter and contributes to the exacerbation of allergic responses. These findings provide a novel therapeutic target for allergic diseases.

Over-expression of the LTC4 synthase gene in mice reproduces human aspirin-induced asthma.

BACKGROUND: The pathogenesis of aspirin-induced asthma (AIA) is presumed to involve the aspirin/non-steroidal anti-inflammatory drug (NSAID)-induced abnormal metabolism of arachidonic acid, resulting in an increase in 5-lipoxygenase (5-LO) metabolites, particularly leukotriene C(4) (LTC(4) ). However, the role of LTC(4) in the development of AIA has yet to be conclusively demonstrated. OBJECTIVE: The aim of this study was to evaluate the contribution of the lipid product LTC(4) secreted by the 5-LO pathway to the pathogenesis of AIA. METHODS: To evaluate antigen-induced airway inflammation, the concentrations of T-helper type 2 cytokine in bronchoalveolar lavage fluid (BALF) obtained from LTC(4) synthase-transgenic (Tg) and wild-type (WT) mice after challenge with ovalbumin were measured. Subsequently, the ex vivo and in vivo effects of the NSAID sulpyrine were investigated in these Tg and WT mice by measuring the secretion of LTC(4) from sulpyrine-treated BAL cells and the levels of LTC(4) in BALF following challenge with sulpyrine. Finally, the sulpyrine-induced airway response by the administration of pranlukast, an antagonist of the cysteinyl (cs)-LT1 receptor, was analysed. RESULTS: The concentrations of IL-4, -5, and -13 in BALF from Tg mice were significantly higher than those in WT mice. In addition, sulpyrine augmented the secretion of LTC(4) in BALF and by BAL cells in Tg mice, but not in WT mice. Additionally, the increased airway resistance induced by sulpyrine could be reduced by treatment with pranlukast. Furthermore, the secretion of LTC(4) from mast cells, eosinophils, and macrophages was increased in the allergen-stimulated LTC(4) synthase gene Tg mice, even in the absence of sulpyrine, as well as in BAL cells after sulpyrine. CONCLUSION AND CLINICAL RELEVANCE: The over-expression of the LTC(4) synthase in a mouse asthma model also replicates the key features of AIA. And our study supports that cys-LTs play a major role in the pathogenesis of AIA in patients with chronic asthma.

Bcl6 in pulmonary epithelium coordinately controls the expression of the CC-type chemokine genes and attenuates allergic airway inflammation.

BACKGROUND: There is synteny in the CC-type chemokine gene clusters between humans (CCL2/MCP-1, CCL7MCP-3, CCL11/eotaxin, CCL8/MCP-2, CCL13/MCP-4, and CCL1/I-309) and mice (CCL2, CCL7, CCL11, CCL12/MCP-5, CCL8, and CCL1). OBJECTIVE: As many putative Bcl6/STAT-binding sequences are observed in the clusters, we examined the roles of a transcriptional repressor Bcl6 and the regional histone modification in the expression of these chemokine genes in pulmonary epithelium. METHODS: We generated transgenic (Tg) mice carrying the Bcl6 or the dominant-negative (DN)-Bcl6 gene under the control of the surfactant protein C (SPC) promoter that induces the exogenous gene expression in the distal lung epithelium. For in vitro studies, A549, alveolar type II-like epithelial cell line transfected with the SPC-DN-Bcl6 gene were stimulated with IL-4+TNF-α, and Bcl6 or STAT6 binding to and histone modification of the cluster in the transfectants were analysed by chromatin immunoprecipitation assays. Tg mice sensitized with ovalbumin (OVA) were challenged with OVA inhalation. The amounts of mRNAs in each sample were analysed by quantitative RT-PCR. RESULTS: The amount of Bcl6 bound to the cluster decreased in A549 cells stimulated with IL-4 and TNF-α, whereas STAT6 binding increased in association with regional histone H3-K9/14 acetylation and H3-K4 methylation. The expression of all chemokine genes in the gene cluster was augmented in activated A549 cells transfected with the DN-Bcl6 gene. We also induced allergic airway inflammation in Tg mice. Expression of the chemokine genes and infiltrated cell numbers in the lungs of these Tg mice with allergic airway inflammation were inversely correlated with the amount of Bcl6 in the lungs. CONCLUSION AND CLINICAL RELEVANCE: Expression of the pulmonary epithelium-derived CC-type chemokine genes in the cluster is orchestrated by the conserved machinery related to Bcl6. Thus, Bcl6 in pulmonary epithelium may be a critical regulator for pathogenesis of various pulmonary inflammatory diseases.

Cellular consequences in the suppression of antibody response by the antigen-specific T-cell factor.

Cellular events mediated by antigen-specific soluble factor extracted from carrier-primed suppressor T cells (TsF) in the suppressive interaction was studied. Keyhole limpet hemocyanin (KLH)-specific TsF directly acts on KLH-primed, I-J positive, nylon-wool-adherent T cells that have an acceptor site for TsF. The nylon-wool-adherent T cells, after accepting TsF in the presence of specific antigen, generate new suppressor T cells acting as an actual effector cell type. Antigen-specificity and syngeneity at I-J between TsF and acceptor T cells are both required for the induction of new suppressor T cells. Newly induced suppressor T cells, however, suppress both syngeneic and allogeneic responses in an antigen-nonspecific fashion.

Two distinct allotypic determinants on the antigen-specific suppressor and enhancing T cell factors that are encoded by genes linked to the immunoglobulin heavy chain locus.

The alloantiserum was raised in BALB/c (H-2d, Igh-1a) mice hyperimmunized with spleen cells of Igh allotype congenic mice, CB-20 (H-2d, Igh-1b). It was found to define the new allotypic determinants (distinct from B cell Igh constant region determinants: Igh allotype) expressed only on a small population of T cells belonging to the Thy-1 dull-stained Lyt-2- or Lyt-2+ population of Igh-1b mice. Genes coding for the determinants were shown to be accommodated somewhere in the right side of the Igh variable region gene (Igh-V) cluster, as the antibody activity was completely absorbed with BAB-14 thymocytes. It was also demonstrated that the products detected by the antiserum represent the allotypic determinants (probably constant region determinants) on the antigen-binding moiety of the antigen-specific augmenting (TaF) and suppressor (TsF) T cell factors. Moreover, determinants on TsF were found to be distinct from those on TaF. Therefore, it can be suggested that the two genes coding for the T cell allodeterminants (distinct from those of the B cell Igh) are located in the right side of the B cell Igh-V on the 12th chromosome, and that both encode the antigen-recognition units of the functionally distinct T cell factors.

Epitope-specific regulation. I. Carrier-specific induction of suppression for IgG anti-hapten antibody responses.

The epitope-specific regulatory system selectively controls IgG antibody production to the individual (haptenic) determinants on a complex antigen. This system can be specifically induced to suppress primary and secondary IgG antibody responses to dinitrophenyl hapten (DNP) without interfering with antibody responses to epitopes on the carrier molecule on which the DNP is presented. Furthermore, once induced, it will specifically suppress responses to DNP presented on unrelated carrier molecules. Results summarized here obtained using widely different immunization conditions, and a variety of haptens and carrier molecules indicate that this regulatory system controls antibody production in most T-dependent antibody responses. Carrier-specific suppressor T cells (CTs) that arise shortly after priming with a carrier molecule such as keyhole limpet hemocyaninin (KLH) induce the epitope-specific system to suppress in situ and adoptive antibody responses to epitopes (e.g., DNP) presented subsequently on the priming carrier. These well-known regulatory T cells are commonly believed to regulate antibody production by interfering with carrier-specific help; however, by repeating the original CTs transfer experiments with additional controls that define the specificity of the mechanism mediating suppression in CTs recipients, we show that KLH-specific CTs regulate responses by inducing typical isotope- specific suppression for anti-DNP responses when the recipients are immunized with DNP-KLH. Thus, whether KLH-primed animals are immunized directly with DNP-KLH (KLH/DNP-KLH immunization sequence) or whether T cells from these animals are challenged with DNP-KLH in (nonirradiated)recipients, anti-DNP responses are persistently suppressed while anti-carrier responses proceed normally. The aqueous KLH-priming protocols usually used to generate CTs are marginally more effective in priming for in situ suppression-induction than the alum KLH-priming protocols commonly used to generate KLH-specific helper T cells and used here in KLH/DNP-KLH immunizations. Thus, studies presented show that priming with an antigenic (carrier) molecule simultaneously prepares the animal for the production of typical secondary (anamnestic) antibody responses to epitopes on the priming antigen and for the induction of epitope-specific suppression for antibody production to determinants presented subsequently on the same antigenic molecule. We discuss the mechanism(s) responsible for this duality and its significance for antibody responses in an accompanying publication that describes the bistable regulatory capabilities of the epitope-specific system.

Properties of the antigen-specific suppressive T-cell factor in the regulation of antibody response of the mouse. III. Dual gene control of the T-cell-mediated suppression of the antibody response.

The antigen-specific suppressive T-cell factor of mice, which had previously been shown to be an I region gene product, could effectively suppress the in vitro secondary antibody response of spleen cells from syngeneic or H-2 compatible mouse strains but not that of H-2 incompatible strains. The identities among genes in the left side half (K, I-A, and I-B) of the H-2 complex between the donor and recipient strains were found to be both necessary and sufficient for the induction of suppression. This suggests that the acceptor site for the suppressive T-cell factor is also determined by the gene present in the left side half of the H-2 complex. The cell type which expresses the acceptor site was found to be a subset of T cell. In general, the suppressive T-cell factor obtained from F1 mice could suppress the responses of both parental strains, and the parental factors could suppress the response of F1 mice. The results indicate that both suppressor and acceptor molecules are codominantly expressed on F1 T cells. There were found two types of defects in the expression of suppressor and acceptor molecules among mouse strains: A/J mice could not produce the suppressive T-cell factor despite that they could accept the factor produced by other H-2 compatible mouse strains. In contrast, all the B10 congeneic lines could produce the T-cell factor, but could not accept the factor produced by syngeneic and H-2 compatible non-B10 congeneic lines. The F1 hybrid of A/J and B10. A could both produce and accept the T-cell factor, and thus the expressions of suppressor and acceptor molecules were found to be dominant traits. These results indicate that the antigen-specific T-cell-mediated suppression is regulated by at least two genes both present in the H-2 complex, and that the complementation of these two genes is required for the induction of suppression.

Specific enrichment of the suppressor T cell bearing I-J determinants: parallel functional and serological characterizations.

A simple procedure to enrich the antigen (keyhole limpet hemocyanin, KLH)-specific suppressor T cell was described. The suppressor T cell from KLH)-immunized mice specifically bound to the KLH-coated Sephadex G-200 column at 37 degrees C, and was eluted from the column by cold (0-4 degrees C) medium. The helper T cell did not bind to the column under the identical condition. The suppressor T cell thus obtained had 100 times as potent suppressor activity as the original spleen cells in in vivo and in vitro secondary antibody responses against a hapten coupled to KLH. This procedure also enriched the cells bearing I-J determinants and Lyt-2,3 alloantigens, allowing us to study the phenotypic expressions on the suppressor T cell by direct serological procedures as well as by the use of the fluorescence activated cell sorter. Parallel functional and serological analyses indicated that the antigen-specific suppressor T cell belongs to a population of I-J+, Lyt-2+,3+ and Fc R- T cells.

Monoclonal alloantibodies specific for the constant region of T cell antigen receptors.

We established three distinct monoclonal antibodies (7C5, 7D1; IgM and 6A4; IgG1) by the fusion of P3U1 and BALB/c (Igh-1a) spleen cells hyperimmunized with T cell blasts from the immunoglobulin heavy chain (Igh) allotype congenic CB-20 (Igh-1b) mice. The 7C5 or 6A4 antibody reacts with the constant region determinants on the antigen-binding molecule (Ct) of the antigen-specific suppressor T cell factor (TsF) or augmenting T cell factor (TaF), respectively. The 7D1 antibody, however, recognizes the shared determinants on the Ct molecules of TsF and TaF. Genetic studies of determinants recognized by these monoclonal antibodies have also suggested that the distinct Ct molecules of TsF and TaF are encoded by two discrete genes linked to the Igh-1b genes, which are located on the right side of the variable genes of Igh on the 12th chromosome. By using the immunoadsorbent columns of 6A4 antibody and anti-I-Ab, TaF, in a manner similar to TsF, was demonstrated to be composed of two chains, i.e., the Ct molecules and the I-A-encoded products. Furthermore, the Ct-bearing molecules were shown to possess the antigen-binding moiety.

Presence of interchain disulfide bonds between two gene products that compose the secreted form of an antigen-specific suppressor factor.

The secreted form of the suppressor T cell factor specific for keyhole limpet hemocyanin derived from the hybridoma 34S-704 was found to consist of the two distinct polypeptide chains, i.e., the antigen-binding and the I-J-encoded chains. They were linked in covalent association with disulfide bonds. The two chains were cleaved by the reduction with dithiothreitol and were easy to reconstitute the active form of TsF. The association of the two distinct chains was suggested to be essential for the expression of the TsF activity.

Functional roles of two polypeptide chains that compose an antigen-specific suppressor T cell factor.

The functional roles of the two polypeptide chains that compose the T cell suppressor factor (TsF) that mediates the antigen-specific and genetically restricted suppressor function were studied by using the heavy or light chains isolated from the conventional TsF or the 11S and 13S mRNA translation products of TsF. Either the heavy or the light chain of mRNA translation products reconstitutes the active TsF that suppresses the antibody response in an antigen-specific and genetically restricted manner when it is combined with the isolated heavy or light chain from the conventional TsF. As a consequence, the antigen-binding heavy chain mediates the antigen specificity of TsF. On the other hand, the I-J-positive light chain works as an element to determine the genetic restriction specificity. Thus, the identity of the histocompatibility between the I-J haplotypes on the light chain and the responding cell is essential for the functional expression of TsF. No genetic preference, however, was observed, in the association of the heavy and light chains of TsF.

Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors.

The following evidence, mainly presented here, suggests that IgD receptors play a crucial role in determining the potential for affinity maturation in memory B cell populations. IgD receptors are present on the first memory B cells to appear after priming. These memory cells give rise to more-mature memory cells that have lost their IgD receptors. The proportions of early (IgD(+)) and mature (IgD(-)) memory cells found in individual donors vary with time, priming conditions, and the availability of T cell help, and both populations frequently coexist for long periods of time. IgD(+) and IgD(-) memory cells carry IgG receptors and give rise to IgG responses with identical isotype representation in adoptive recipients. IgD(+) memory cells, however, always give rise to predominantly low-affinity antibody responses, whereas IgD(-) memory cells consistently generate responses of substantially higher average affinity. This affinity differential is maintained between early and mature memory populations in the same donor and does not appear to be a result of selective differentiation of higher-affinity IgD(+) memory cells into the IgD(-) memory pool. Thus, the selective forces responsible for affinity maturation appear to operate mainly in mature memory cell populations that have already lost IgD receptors; or, stated conversely, little or no selection towards high-affinity memory appears to occur among memory cells that retain IgD receptors. In discussing these findings, we suggest that the IgD receptors themselves are responsible for maintaining early memory populations at a lower average affinity than IgD(-) populations in the same animal. The IgD receptors, we argue, serve to increase the antigen-binding capacity of lower-affinity memory cells so that these cells can survive, expand, and differentiate (to IgD(-)) at antigen concentrations that select against expansion of low- affinity memory cells no longer carrying IgD receptors. Thus, when antigen is limiting, IgD(-) memory populations will be selectively expanded to higher average affinities, whereas coexisting IgD(+) populations will retain their initial affinity profile. This hypothesis suggests that mechanisms that regulate expression and loss of IgD receptors are central to the adaptability of the immune system in its response to invading pathogens. Two related roles can be envisioned for the IgD receptors in this regard. First, they extend the lower boundary of the affinity range of early memory cell populations induced by a given antigenic stimulus and therefore broaden the diversity of responses obtainable from these populations. Secondly, they support the persistence of low-affinity memory populations under conditions where antigen becomes limiting and eventually disappears. These persisting populations then serve as a diversely reactive reservoir from which mature memory populations can be drawn with higher affinities either for the original antigen or, more importantly, for related antigens that the animal may subsequently encounter. Thus the existence of IgD receptors on early memory cells maintains the full range of response diversity despite ongoing selective expansion of (mature) memory populations to produce antibodies with high combining affinities for individual antigens. The flexibility inherent in such an organizational system, we believe, could be expected to account for the evolutionary development of IgD receptors and the regulatory capabilities that support operation of the system.

Monoclonal antibody to an IgD allotype induces a new type of allotype suppression in the mouse.

Studies presented here show that perinatal exposure to anti-IgD allotype antibodies induces a persistant IgG-allotype suppression in the mouse that differs markedly from either the short-term or chronic allotype suppressions induced by antibodies to IgG or IgM allotypes. This novel form of allotype suppression induced by injecting neonatal BALB/c x SJL mice with monoclonal antibody to the paternal Igh-5b (IgD) allotype drastically reduces paternal allotype production during the first 6 mo of the affected animal's life and simultaneously stimulates compensatory production of maternal allotype IgG. In addition, it interferes with the development of B cells carrying the paternal IgD allotype and impairs the development of memory B cells destined to give rise to paternal allotype IgG-producing cells. Thus, its properties make it more like allotype suppression as described in the rabbit than like the known forms of allotype suppression in the mouse. As shown here, Igh-5b-bearing (5b+) B cells are completely depleted from the neonate after anti-5b exposure and only gradually appear as the animal ages. The recovery of the 5b+ population to near normal size (by approximately 14 wk of age) substantially preceeds recovery of the ability to generate normal-size memory B cell populations. Paternal allotype levels in serum remain well below normal until the anti-5b-exposed animals reach approximately 6 mo of age and then climb rapidly, finally stabilizing at levels comparable to levels in controls of the same age. The elevated maternal allotype levels characteristic of the suppression period began falling somewhat earlier and are clearly stabilized within the normal range in 6-mo-old animals. Thus, perinatal exposure to anti-5b compromises B cell development and IgG production throughout early adulthood but has little apparent effect in older animals. Perinatal exposure to antibody to the paternal IgG2a allotype (Igh-1b) or IgM allotype (Igh-6b), in contrast, induces a chronic allotype suppression that has relatively little affect on IgG production in young adults but severely suppresses allotype production in older animals. Furthermore, this type of (chronic) suppression does not influence maternal allotype production and does not interfere with memory B cell development. These differences, illustrated here by data from parallel sets of animals exposed either toi anti-5b or anti-1b, raise a series of intriguing questions concerning the mechanisms regulating B cell development and expression and the nature of the neonatal (B) cell populations with which the suppression-inducing antibodies react.

Epitope-specific regulation. II. A bistable, Igh-restricted regulatory mechanism central to immunologic memory.

Antibody responses to commonly used antigens are regulated by an epitope- specific system composed of Igh-restricted elements responsible for controlling the isotype and allotype responses mounted to each of the epitopes on the antigen. Because these elements can be independently induced to either suppress or support antibody production, this system as a whole provides an effector mechanism capable of selectively controlling the amount, affinity, isotype representation, and epitope-specificity of an antibody response. Sequential immunizations with a carrier molecule and a hapten conjugated to that carrier (carrier/hapten-carrier immunization) induce suppression for IgG responses to the hapten. IgG(2a), IgG(2b), and IgG(3) responses are easily suppressed, whereas IgG(1) responses tend to be more resistant. Once induced, suppression tends to be maintained; however, repeated stimulation with the hapten (on any carrier) eventually induces antibody production, first for IgG(1) and later for the more suppressible isotypes (IgG(2a), IgG(2b), IgG(3)). Antibody production, once initiated, also tends to be maintained. Ongoing IgG antihapten responses in animals primed with a hapten-carrier conjugate can be suppressed by subsequent carrier/hapten-carrier immunization (using a different carrier molecule); however, the suppression induced under these circumstances is substantially weaker, i.e., it mainly affects the more suppressible isotypes and is only strong enough to detect clearly in about one-half the immunized animals. Thus, the initiation of antibody production impairs the subsequent induction of suppression, and the initial induction of suppression tends to prevent subsequent initiation of antibody production. This reciprocal relationship defines a bistable regulatory mechanism, i.e., one that tends to maintain its initially induced state but is capable of shifting to the alternate state when stimulatory conditions so dictate. The operation of such a mechanism permits conditions surrounding the first immunization with an epitope (hapten) to strongly influence but not absolutely determine which and how many of the anti-epitope memory B cells generated by that immunization will subsequently be expressed. Thus, epitope- specific regulation, although subordinate to mechanisms that control memory B cell development (as opposed to expression), plays a key role in determining the magnitude, affinity, and isotype representation of anamnestic (memory) responses produced in response to previously encountered epitopes.

Two distinct types of helper T cells involved in the secondary antibody response: independent and synergistic effects of Ia- and Ia+ helper T cells.

We have described here two distinct types of carrier-specific helper T cells which act independently and synergistically to augment the B-cell response to a hapten. They are separable by passage through a nylon wool column. The first type of helper T cell, which we designate as Th1, is nylon nonadherent, and can help the response of hapten-primed B cells only if the haptenic and carrier determinants are present on a single molecule (cognate interaction). The second type of helper T cell, Th2, adheres to the nylon wool column, and can help the B-cell response to a hapten coupled to a heterologous carrier upon stimulation with unconjugated relevant carrier (polyclonal interaction). The addition of a small number of Th2 to the mixture of Th1 and B cells significantly augmented the net response to the hapten carrier conjugate. Both Th1 and Th2 cells belong to the Lyt-1+,2-,3- subclass. Th1 has no detectable Ia antigen, whereas Th2 is killed by certain anti-Ia antisera and complement. The Ia antigen detected on Th2 was found to be controlled by a locus in the I-J subregion. The results clearly established the fact that there are two distinct pathways in the T- and B-cell collaboration, which involves two different subsets of carrier-specific helper T cells.

Blockade of transforming growth factor beta/Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway inflammation and airway reactivity.

Transforming growth factor (TGF)-beta has been implicated in immunosuppression. However, it remains obscure whether regulation of T cells by TGF-beta contributes to the immunosuppression in vivo. To address this issue, we developed transgenic mice expressing Smad7, an intracellular antagonist of TGF-beta/Smad signaling, selectively in mature T cells using a plasmid construct coding a promoter element (the distal lck promoter) that directs high expression in peripheral T cells. Peripheral T cells were not growth inhibited by TGF-beta in Smad7 transgenic mice. Although Smad7 transgenic mice did not spontaneously show a specific phenotype, antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice associated with high production of both T helper cell type 1 (Th1) and Th2 cytokines. Thus, blockade of TGF-beta/Smad signaling in mature T cells by expression of Smad7 enhanced airway inflammation and airway reactivity, suggesting that regulation of T cells by TGF-beta was crucial for negative regulation of the inflammatory (immune) response. Our findings also implicated TGF-beta/Smad signaling in mature T cells as a regulatory component of allergic asthma.

Monoclonal antibodies that recognize the product controlled by a gene in the I-J subregion of the mouse H-2 complex.

The B cell hybridomas producing monoclonal antibodies (E10, D7, F4, H6, and D4) were established by the fusion of P3U1 or NS-1 murine myeloma cell lines and spleen cells of B10.A(5R) mice hyperimmunized with mitomycin C-treated B10.A(3R) spleen and thymus cells. Two types of monoclonal antibodies specific for the products controlled by a gene in the I-Jb subregion of the H-2 complex were characterized: one specific for the private type of I-Jb determinant, the other recognizing the cross-reactive determinant between the I-Jb and I-Jd products. By using these monoclonal reagents, the I-J-encoded product on the antigen-specific suppressor T cells was found to be expressed on their soluble suppressor factors. Furthermore, the I-Jb products were successfully detected not only on the T cell hybridoma with suppressor activity specific for keyhole limpet hemocyanin (KLH), but also on KLH-primed suppressor T cells enriched by antigen-coated petri dishes and concanavalin A-induced thymocyte blasts of C57BL/6 mice by complement-dependent cytotoxic assays and membrane fluorescence techniques.

Disruption of the Bcl6 gene results in an impaired germinal center formation.

The Bcl6 gene has been identified from the chromosomal translocation breakpoint in B cell lymphomas, and its products are expressed highly in germinal center (GC) B cells. To investigate the function of Bcl6 in lymphocytes, we have generated RAG1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient mice (Bcl6(-/-)RM). Lymphogenesis in primary lymphoid tissues of Bcl6(-/-)RM is normal, and Bcl6(-/-)RM produced control levels of primary IgG1 antibodies specific to T cell-dependent antigens. However, GCs were not found in these mice. This defect was mainly due to the abnormalities of B cells. Therefore, Bcl6 is essential for the differentiation of GC B cells.

Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

A novel pathogenesis of megacolon in Ncx/Hox11L.1 deficient mice.

The Ncx/Hox11L.1 gene, a member of the Hox11 homeobox gene family, is mainly expressed in neural crest-derived tissues. To elucidate the role of Ncx/Hox11L.1, the gene has been inactivated in embryonic stem cells by homologous recombination. The homozygous mutant mice were viable. These mice developed megacolon with enteric ganglia by age 3-5 wk. Histochemical analysis of the ganglia revealed that the enteric neurons hyperinnervated in the narrow segment of megacolon. Some of these neuronal cells degenerated and neuronal cell death occurred in later stages. We propose that Ncx/Hox11L.1 is required for maintenance of proper functions of the enteric nervous system. These mutant mice can be used to elucidate a novel pathogenesis for human neuronal intestinal dysplasia.