Murine Valpha14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Valpha14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-gamma upon in vivo stimulation with a specific ligand, alpha-galactosylceramide (alpha-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Valpha14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Valpha14 NKT-deficient mice that Valpha14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with alpha-GalCer, Valpha14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Valpha14 NKT- or IFN-gamma-deficient mice. Consistent with these results, we also found that ligand-activated Valpha14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Valpha14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, our studies have revealed that IL-4 produced by Valpha14 NKT cells has only a minor effect on Th2 cell differentiation.

Conserved differences between the transmembrane and cytoplasmic domains of membrane immunoglobulin (Ig)M and IgG may alter the function of antigen receptors on naive versus memory B cells. Here, we compare the ability of these domains to signal B cell allelic exclusion and maturation in transgenic mice. A lysozyme-binding antibody was expressed in parallel sets of mice as IgM, IgG1, or a chimeric receptor with IgM extracellular domains and transmembrane/cytoplasmic domains of IgG1. Like IgM, the IgG1 or chimeric IgM/G receptors triggered heavy chain allelic exclusion and supported development of mature CD21(+) B cells. Many of the IgG or IgM/G B cells became CD21(high) and downregulated their IgG and IgM/G receptors spontaneously, resembling memory B cells and B cells with mutations that exaggerate B cell antigen receptor signaling. Unlike IgM-transgenic mice, "edited" B cells that carry non-hen egg lysozyme binding receptors preferentially accumulated in IgG and IgM/G mice. This was most extreme in lines with the highest transgene copy number and diminished in variant offspring with fewer copies. The sensitivity of B cell maturation to transgene copy number conferred by the IgG transmembrane and cytoplasmic domains may explain the diverse phenotypes found in other IgG-transgenic mouse strains and may reflect exaggerated signaling.

The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

Immature bone marrow-derived myeloid dendritic cells (BMDCs) are induced to undergo phenotypic maturation and secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-12, and IL-10 when pulsed in vitro with intact Streptococcus pneumoniae. After transfer to naive mice, pulsed BMDCs induce immunoglobulin (Ig) isotype responses specific for both protein and polysaccharide pneumococcal antigens, having in common the requirement for viable BMDCs, T cells, and B7-dependent costimulation in the recipient mice. Whereas primary Ig isotype responses to bacterial proteins uniformly require BMDC expression of major histocompatibility complex class II, CD40, and B7, and the secretion of IL-6, but not IL-12, similar requirements for antipolysaccharide Ig responses were only observed for the IgG1 isotype.

Contact-mediated interactions between CD4+ T cells and B cells are considered crucial for T cell-dependent B cell responses. To investigate the ability of activated CD4+ T cells to drive in vivo B cell responses in the absence of key cognate T-B interactions, we constructed radiation bone marrow chimeras in which CD4+ T cells would be activated by wild-type (WT) dendritic cells, but would interact with B cells that lacked expression of either major histocompatibility complex class II (MHC II) or CD40. B cell responses were assessed after influenza virus infection of the respiratory tract, which elicits a vigorous, CD4+ T cell-dependent antibody response in WT mice. The influenza-specific antibody response was strongly reduced in MHC II knockout and CD40 knockout mice. MHC II-deficient and CD40-deficient B cells in the chimera environment also produced little virus-specific immunoglobulin (Ig)M and IgG, but generated a strong virus-specific IgA response with virus-neutralizing activity. The IgA response was entirely influenza specific, in contrast to the IgG2a response, which had a substantial nonvirus-specific component. Our study demonstrates a CD4+ T cell-dependent, antiviral IgA response that is generated in the absence of B cell signaling via MHC II or CD40, and is restricted exclusively to virus-specific B cells.

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a peculiar T cell lymphoma, as expanding B cell clones are often present besides the malignant T cell clones. In addition, large numbers of Epstein-Barr virus (EBV)-infected B cells are frequently observed. To analyze the differentiation status and clonal composition of EBV-harboring B cells in AILD, single EBV-infected cells were micromanipulated from lymph nodes of six patients with frequent EBV(+) cells and their rearranged immunoglobulin (Ig) genes analyzed. Most EBV-infected B cells carried mutated Ig genes, indicating that in AILD, EBV preferentially resides in memory and/or germinal center B cells. EBV(+) B cell clones observed in all six cases ranged from small polyclonal to large monoclonal expansions and often showed ongoing somatic hypermutation while EBV(-) B cells showed little tendency for clonal expansion. Surprisingly, many members of expanding B cell clones had acquired destructive mutations in originally functional V gene rearrangements and showed an unfavorable high load of replacement mutations in the framework regions, indicating that they accumulated mutations over repeated rounds of mutation and division while not being selected through their antigen receptor. This sustained selection-free accumulation of somatic mutations is unique to AILD. Moreover, the survival and clonal expansion of "forbidden" (i.e., Ig-deficient) B cells has not been observed before in vivo and thus represents a novel type of viral latency in the B cell compartment. It is likely the interplay between the microenvironment in AILD lymph nodes and the viral transformation that leads to the survival and clonal expansion of Ig-less B cells.

T cell receptors on CD4(+) lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcgammaRIIb2 and FcgammaRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcgammaRIIb2 or FcgammaRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcgammaRIIb2 and FcgammaRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcgammaRIII, but not through FcgammaRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcgammaRIIb2 by addition of the FcgammaRIII-associated gamma chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the gamma chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens.

The biological functions of immunoglobulin (Ig)A antibodies depend primarily on their interaction with cell surface receptors. Four IgA receptors are presently characterized. The FcalphaRI (CD89) expressed by myeloid cells selectively binds IgA1 and IgA2 antibodies, whereas the poly-IgR, Fcalpha/muR, and asialoglycoprotein receptors bind other ligands in addition to IgA. IgA binding by mesangial cells, epithelial cells, and proliferating lymphocytes is also well documented, but the nature of the IgA receptors on these cells remains elusive. A monoclonal antibody (A24) is described here that specifically blocks IgA binding to epithelial and B lymphocyte cell lines. Both the A24 antibody and IgA1 myelomas bind a cell surface protein that is identified as the transferrin receptor (CD71). The transferrin receptor selectively binds IgA1 antibodies, monomeric better than polymeric forms, and the IgA1 binding is inhibitable by transferrin. Transferrin receptor expression is upregulated on cultured mesangial cells as well as on glomerular mesangial cells in patients with IgA nephropathy. The characterization of transferrin receptor as a novel IgA1 receptor on renal mesangial cells suggests its potential involvement in the pathogenesis of IgA nephropathy.

Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2.

Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2- activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor-associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-gamma/delta+ T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6.

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

In both humans and animals, immunoglobulin (Ig)G autoantibodies are less frequent but more pathogenic than IgM autoantibodies, suggesting that controls over Ig isotype switching are required to reinforce B cell self-tolerance. We have used gene targeting to produce mice in which hen egg lysozyme (HEL)-specific B cells can switch to all Ig isotypes (SWHEL mice). When crossed with soluble HEL transgenic (Tg) mice, self-reactive SWHEL B cells became anergic. However, in contrast to anergic B cells from the original nonswitching anti-HEL x soluble HEL double Tg model, self-reactive SWHEL B cells also displayed an immature phenotype, reduced lifespan, and exclusion from the splenic follicle. These differences were not related to their ability to Ig class switch, but instead to competition with non-HEL-binding B cells generated by VH gene replacement in SWHEL mice. When activated in vitro with B cell receptor (BCR)-independent stimuli such as anti-CD40 monoclonal antibody plus interleukin 4 or lipopolysaccharide (LPS), anergic SWHEL double Tg B cells proliferated and produced IgG anti-HEL antibodies as efficiently as naive HEL-binding B cells from SWHEL Ig Tg mice. These results demonstrate that no intrinsic constraints to isotype switching exist in anergic self-reactive B cells. Instead, production of IgG autoantibodies is prevented by separate controls that reduce the likelihood of anergic B cells encountering BCR-independent stimuli. That bacteria-derived LPS could circumvent these controls may explain the well-known association between autoantibody-mediated diseases and episodes of systemic infection.

T-dependent B cell responses in the spleen are initiated in the outer periarteriolar lymphoid sheath (PALS) and culminate in the generation of proliferative foci and germinal center reactions. By pulsing anti-hen egg lysozyme (HEL) immunoglobulin transgenic (IgTg) B cells with various concentrations of HEL in vitro before adoptive transfer into normal recipients, it was shown that a critical number of B cell receptors (BCRs) must be ligated for B cells to undergo arrest in the outer PALS. T cell help was manipulated independently of the BCR stimulus by incubating B cells expressing the appropriate major histocompatibility complex class II antigen with a peptide recognized by CD4(+) TCR Tg T cells. B cells which either failed to arrest in the outer PALS due to a subthreshold BCR stimulus, or arrested only transiently due to the brevity of the BCR stimulus, underwent an abortive response within the follicles when provided with T cell help. In contrast, naive B cells stimulated by a sustained, suprathreshold concentration of either foreign or self-antigen and given T cell help, proliferated in the outer PALS and then differentiated. Outer PALS arrest was not influenced by the nature of the B cells occupying the follicle, but appeared to be determined solely by the magnitude of BCR stimulation. Thus antigen-pulsed B cells arrested in the outer PALS in an identical manner irrespective of whether the follicles comprised a population of normal B cells with multiple specificities, a monoclonal naive population, or a monoclonal population of tolerant B cells. In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding. Similarly, when anti-HEL Tg mice were crossed with a second HEL Tg strain expressing a higher concentration of soluble HEL, the tolerant anti-HEL Tg B cells were located constitutively in the outer PALS. Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen. A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.

To determine the function of immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, we generated mice in which Igalpha ITAM tyrosines were replaced by phenylalanines (Igalpha(FF/FF)). Igalpha(FF/FF) mice had a specific reduction of B1 and marginal zone B cells, whereas B2 cell development appeared to be normal, except that lambda1 light chain usage was increased. The mutants responded less efficiently to T cell-dependent antigens, whereas T cell-independent responses were unaffected. Upon B cell receptor ligation, the cells exhibited heightened calcium flux, weaker Lyn and Syk tyrosine phosphorylation, and phosphorylation of Igalpha non-ITAM tyrosines. Strikingly, when the Igalpha ITAM mutation was combined with a truncation of Igbeta, B cell development was completely blocked at the pro-B cell stage, indicating a crucial role of ITAM phosphorylation in B cell development.

The pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN), the most prevalent form of glomerulonephritis worldwide, involves circulating macromolecular IgA1 complexes. However, the molecular mechanism(s) of the disease remain poorly understood. We report here the presence of circulating soluble FcalphaR (CD89)-IgA complexes in patients with IgAN. Soluble CD89 was identified as a glycoprotein with a 24-kD backbone that corresponds to the expected size of CD89 extracellular domains. To demonstrate their pathogenic role, we generated transgenic (Tg) mice expressing human CD89 on macrophage/monocytes, as no CD89 homologue is found in mice. These mice spontaneously developed massive mesangial IgA deposition, glomerular and interstitial macrophage infiltration, mesangial matrix expansion, hematuria, and mild proteinuria. The molecular mechanism was shown to involve soluble CD89 released after interaction with IgA. This release was independent of CD89 association with the FcRgamma chain. The disease was induced in recombination activating gene (RAG)2(-/-) mice by injection of serum from Tg mice, and in severe combined immunodeficiency (SCID)-Tg mice by injection of patients' IgA. Depletion of soluble CD89 from serum abolished this effect. These results reveal the key role of soluble CD89 in the pathogenesis of IgAN and provide an in vivo model that will be useful for developing new treatments.

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcepsilon receptor I (FcepsilonRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the Fc epsilonRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of Fc epsilonRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch mu region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgamma2a and Cgamma1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon gamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.

Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-gamma were biologically active, as supernatants from anti-CD28-treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release. These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines.