Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.

Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA-transfected Epstein-Barr virus-immortalized autologous B cells.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

B cells are not essential for peripheral T-cell tolerance.

Some self-reactive T cells avoid thymic tolerance and become mature peripheral cells. Nevertheless, these cells do not usually attack their hosts because T cells can be inactivated or killed, even after they are mature, by various means. The details of these processes are not fully understood; however, a number of experiments have suggested that peripheral tolerance may be induced in mature mouse T cells by exposure to antigen on resting B cells, cells that can express antigen bound to major histocompatibility complex proteins but that lack critical costimulatory molecules such as B7-1 and B7-2. Conversely, previous experiments have indicated that mature T cells can be stimulated by exposure to antigen on cells such as dendritic cells, cells that are thought to express the essential costimulatory molecules. We tested this idea in vivo by using mice that lack B cells. Unexpectedly, T-cell tolerance and antigen-induced T-cell death occurred normally in mice free of B cells. On the other hand, antigen-specific T-cell expansion in the spleens of such mice was impaired. Finally, we have recently shown that T-cell death in mice can be prevented by exposure to antigen and an inflammatory agent such as bacterial lipopolysaccharide. This was also true in mice that lacked B cells. Overall, these data show that mature T cells can be tolerized and rescued from tolerance in the absence of B cells.

Differentiation of B cells in the nonlymphoid tissue of the synovial membrane of patients with rheumatoid arthritis.

In patients with rheumatoid arthritis the synovial membrane of the affected joint is infiltrated with lymphoid cells which may be arranged in structures resembling germinal centers. We have directly isolated such infiltrates to determine whether B-cell clones within them are selected and expanded in a process analogous to that which normally takes place in the germinal centers in secondary lymphoid organs. The data suggest that an antigen-driven process leads to the accumulation of B cells in the synovial membrane. The finding of identical sequences in consecutive sections suggests that under conditions of chronic stimulation, memory B cells may enter a stage of differentiation in which they proliferate without further accumulation of somatic mutations. Further we see intraclonal diversity which underlines the germinal center-like character of these infiltrates and demonstrates that a microenvironment is built up in this nonlymphoid tissue which supports antigen-dependent differentiation of B cells. This is the first demonstration, to our knowledge, of a germinal center-like reaction outside lymphoid tissue.

CD27-CD70 interactions regulate B-cell activation by T cells.

CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis.

Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Epitope-specific tolerance induction with an engineered immunoglobulin.

Isologous and heterologous immunoglobulins have been shown to be extremely effective as tolerogenic carriers for nearly 30 years. The efficacy of these proteins is due in part to their long half-life in vivo, as well as their ability to crosslink surface IgM with Fc receptors. The concept of using IgG as a carrier molecule to induce unresponsiveness in the adult immune system has been exploited for simple haptens, such as nucleosides, as well as for peptides. To further evaluate the in vivo potential of these molecules for inducing tolerance to a defined epitope, we have engineered a fusion protein of mouse IgG1 with the immunodominant epitope 12-26 from bacteriophage lambda cI repressor protein. This 15-mer, which contains both a B-cell and T-cell epitope, has been fused in-frame to the N terminus of a mouse heavy chain IgG1 construct, thus creating a "genetic hapten-carrier" system. We describe a novel in vitro and in vivo experimental system for studying the feasibility of engineered tolerogens, consisting of a recombinant flagellin challenge antigen and a murine IgG1 tolerogen, both expressing the lambda repressor epitope 12-26. Herein, we show that peptide-grafted IgG molecules injected i.v., or expressed by transfected, autologous B cells, can efficiently modulate the cellular and humoral immune responses to immunodominant epitopes. This model displays the feasibility of "tailor-designing" immune responses to whole antigens by selecting epitopes for either tolerance or immunity.

Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains.

Bacterial lipopolysaccharide (LPS) is a potent stimulator of B-cell activation, proliferation, and differentiation. We examined the genetic response of B-lineage cells to LPS via trapping of expressed genes with a gene-trap retrovirus. This analysis showed that expression of only a small fraction of genes is altered during LPS stimulation of B-lineage cells. Isolation of the cellular portion of the trapped LPS-response genes via 5' RACE (rapid amplification of cDNA ends) cloning identified novel genes for all the cloned loci. These novel LPS-response genes were also found to have differentiation stage-restricted expression within the B-lymphoid lineage. That LPS-response genes in B cells also have differentiation stage-restricted expression suggests that these genes may be involved in the control of B-cell function and differentiation, since the known members of this class of genes have frequently been found to play a role in the function and differentiation of B-lineage cells. The isolation of novel members of this class of genes, including a gene that contains a putative SH2 domain, will further increase our understanding of the molecular events involved in the control of B-cell differentiation and function.

Concerted repression of an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2).

The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus-controlling region, is highly responsive to any of several participating factors.

The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c.

Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags.

Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma.

The BCL6 gene encodes a zinc-finger transcription factor and is altered by chromosomal arrangements in its 5' noncoding region in approximately 30% of diffuse large-cell lymphoma (DLCL). We report here that, in 22/30 (73%) DLCL and 7/15 (47%) follicular lymphoma (FL), but not in other tumor types, the BCL6 gene is also altered by multiple (1.4 x 10(-3) -1.6 x 10(-2) per bp), often biallelic, mutations clustering in its 5' noncoding region. These mutations are of somatic origin and are found in cases displaying either normal or rearranged BLC6 alleles indicating their independence from chromosomal rearrangements and linkage to immunoglobulin genes. These alterations identify a mechanism of genetic instability in malignant B cells and may have been selected during lymphomagenesis for their role in altering BCL6 expression.

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.