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In a variety of settings it is extremely helpful to be able to apply R functions through buttons, sliders and other types of graphical control. This is particularly true in plotting activities where immediate communication between such controls and a graphical display allows the user to interact with a plot in a very effective manner. The tcltk package provides extensive tools for this and the aim of the rpanel package is to provide simple and well documented functions which make these facilities as accessible as possible. In addition, the operations which form the basis of communication within tcltk are managed in a way which allows users to write functions with a more standard form of parameter passing. This paper describes the basic design of the software and illustrates it on a variety of examples of interactive control of graphics. The tkrplot system is used to allow plots to be integrated with controls into a single panel. An example of the use of a graphical image, and the ability to interact with this, is also discussed.

In a variety of settings it is extremely helpful to be able to apply R functions through buttons, sliders and other types of graphical control. This is particularly true in plotting activities where immediate communication between such controls and a graphical display allows the user to interact with a plot in a very effective manner. The tcltk package provides extensive tools for this and the aim of the rpanel package is to provide simple and well documented functions which make these facilities as accessible as possible. In addition, the operations which form the basis of communication within tcltk are managed in a way which allows users to write functions with a more standard form of parameter passing. This paper describes the basic design of the software and illustrates it on a variety of examples of interactive control of graphics. The tkrplot system is used to allow plots to be integrated with controls into a single panel. An example of the use of a graphical image, and the ability to interact with this, is also discussed.

Fcγ receptors (FcγRs) play key roles in the effector function of IgG, but their inappropriate activation plays a role in several disease etiologies. Therefore, it is critical to better understand how FcγRs are regulated. Numerous studies suggest that sialic acid-binding immunoglobulin-type lectins (Siglecs), a family of immunomodulatory receptors, modulate FcγR activity; however, it is unclear of the circumstances in which Siglecs can antagonize FcγRs and which Siglecs have this ability. Using liposomes displaying selective ligands to coengage FcγRs with a specific Siglec, we explore the ability of Siglec-3, Siglec-5, Siglec-7, and Siglec-9 to antagonize signaling downstream of FcγRs. We demonstrate that Siglec-3 and Siglec-9 can fully inhibit FcγR activation in U937 cells when coengaged with FcγRs. Cells expressing Siglec mutants reveal differential roles for the immunomodulatory tyrosine-based inhibitory motif (ITIM) and immunomodulatory tyrosine-based switch motif (ITSM) in this inhibition. Imaging flow cytometry enabled visualization of SHP-1 recruitment to Siglec-3 in an ITIM-dependent manner, while SHP-2 recruitment is more ITSM-dependent. Conversely, both cytosolic motifs of Siglec-9 contribute to SHP-1/2 recruitment. Siglec-7 poorly antagonizes FcγR activation for two reasons: masking by cis ligands and differences in its ITIM and ITSM. A chimera of the Siglec-3 extracellular domains and Siglec-5 cytosolic tail strongly inhibits FcγR when coengaged, providing evidence that Siglec-5 is more like Siglec-3 and Siglec-9 in its ability to antagonize FcγRs. Additionally, Siglec-3 and Siglec-9 inhibited FcγRs when coengaged by cells displaying ligands for both the Siglec and FcγRs. These results suggest a role for Siglecs in mediating FcγR inhibition in the context of an immunological synapse, which has important relevance to the effectiveness of immunotherapies., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Phospholipase D4 (PLD4) is an endolysosomal exonuclease of ssRNA and ssDNA, rather than a phospholipase as its name suggests. Human polymorphisms in the PLD4 gene have been linked by genome-wide association studies to systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. However, B6.129 Pld4-/- mice develop features of a distinct disease, macrophage activation syndrome, which is reversed in mice mutated in TLR9. In this article, we compare a Pld4 null mutant identified on the BALB/c background, Pld4thss/thss, which has distinct phenotypes: short stature, thin hair, and features of systemic lupus erythematosus. All phenotypes analyzed were largely normalized in Pld4thss/thssTlr9-/- mice. Thus, Pld4thss/thss represents a rare model in which mouse lupus etiology is TLR9 dependent. Compared with PLD4-deficient B6 mice, Pld4thss/thss mice had elevated levels of serum IgG, IgG anti-dsDNA autoantibodies, BAFF, and IFN-γ and elevated B cell numbers. Overall, the data suggest that PLD4 deficiency can lead to a diverse array of rheumatological abnormalities depending upon background-modifying genes, and that these diseases of PLD4 deficiency are largely driven by TLR9 recognition of ssDNA., (Copyright © 2023 The Authors.)

Phospholipase D3 (PLD3) and PLD4 polymorphisms have been associated with several important inflammatory diseases. Here, we show that PLD3 and PLD4 digest ssRNA in addition to ssDNA as reported previously. Moreover, Pld3 -/- Pld4 -/- mice accumulate small ssRNAs and develop spontaneous fatal hemophagocytic lymphohistiocytosis (HLH) characterized by inflammatory liver damage and overproduction of Interferon (IFN)-γ. Pathology is rescued in Unc93b1 3d/3d Pld3 -/- Pld4 -/- mice, which lack all endosomal TLR signaling; genetic codeficiency or antibody blockade of TLR9 or TLR7 ameliorates disease less effectively, suggesting that both RNA and DNA sensing by TLRs contributes to inflammation. IFN-γ made a minor contribution to pathology. Elevated type I IFN and some other remaining perturbations in Unc93b1 3d/3d Pld3 -/- Pld4 -/- mice requires STING (Tmem173). Our results show that PLD3 and PLD4 regulate both endosomal TLR and cytoplasmic/STING nucleic acid sensing pathways and have implications for the treatment of nucleic acid-driven inflammatory disease., (© 2021. The Author(s).)

Soluble thrombomodulin plasma concentrations are elevated in steroid-resistant graft-versus-host disease (GVHD), implying endothelial hypofunctioning for thrombomodulin-dependent generation of activated protein C's (APC) anticoagulant, anti-inflammatory, and antiapoptotic functions. Recombinant thrombomodulin or APC administration decreases acute GVHD, manifested by intense inflammation and tissue destruction. Here, we administered recombinant murine wild-type (WT) APC to mice with established chronic GVHD (cGVHD), a less-inflammatory autoimmune-like disease. WT APC normalized bronchiolitis obliterans-induced pulmonary dysfunction. Signaling-selective APC variants (3A-APC [APC with lysine 191-193 replaced with 3 alanines] or 5A-APC [APC with lysine 191-193 replaced with 3 alanines and arginine 229/230 replaced with 2 alanines]) with normal cytoprotective properties, but greatly reduced anticoagulant activity, provided similar results. Mechanistically, WT APC and signaling-selective variants reduced T follicular helper cells, germinal center formation, immunoglobulin, and collagen deposition. WT APC can potentially cleave protease-activated receptor 1 (PAR1) at Arg41 or Arg46, the latter causing anti-inflammatory signaling. cGVHD was reduced in recipients of T cells from WT PAR1 or mutated Gln41-PAR1 donors but not from mutated Gln46-PAR1 donors. These data implicate donor T-cell APC-induced noncanonical cleavage at Arg46-PAR1, which is known to confer cytoprotective and anti-inflammatory activities. Together, these data indicate that APC anticoagulant activity is dispensable, whereas anti-inflammatory signaling and cytoprotective cell signaling by APC are essential. Because a phase 2 ischemic stroke clinical trial did not raise any safety issues for 3A-APC treatment, our studies provide a foundational platform for testing in clinical cGVHD therapy., (© 2019 by The American Society of Hematology.)

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

The ionic and electronic conductivity of orthorhombic LaMnO3 can be modified by introducing lower valence dopants at both the La and Mn sites. Alkaline earth doped perovskites, such as LaMnO3, have a variety of applications in catalysis, for nitrogen storage and reduction, and oxidation of volatile organic compounds, and as the oxygen electrode in solid oxide fuel cells. Here, we investigate doping with the divalent alkaline earth metals Mg, Ca, Sr and Ba, and the charge compensation mechanism. The energies of formation of isolated defects and clustered pairs were investigated at both La and Mn sites to establish the most probable site at which they will be introduced. The charge compensation mechanism for the introduction of alkaline earth dopants was examined by considering both ionic (formation of an oxygen vacancy for every two alkaline earth dopants introduced) and electronic compensation (a hole localised at the Mn site for each dopant introduced). Larger cations (Ca, Sr and Ba) were found to have lower defect formation energies when introduced at the La site, while the smaller Mg defect had lower formation energies when introduced to the Mn site. For all defects introduced, electronic compensation for the defect was found to be more energetically favourable, which will result in improved electronic conductivity of the material.

LaMnO 3 -based perovskites, which have been extensively studied as cathodes for high temperature solid oxide fuel cells (SOFCs), are also of interest for intermediate temperature SOFCs (T = 600-1000 K). Oxygen vacancy formation is required in LaMnO 3 for oxygen diffusion, therefore a low vacancy formation energy is preferable. The stability of the low index surfaces of orthorhombic LaMnO 3 has been investigated, with the {010} surface found to be the most stable. Surface stability was found to be affected by the La and Mn coordination, and the Mn-O bonds cleaved on surface formation. The crystal morphology has been predicted, in order to determine the most likely terminations to be present. The formation of oxygen vacancies in bulk LaMnO 3 and at all of its low index surfaces has been examined, and it has been found that formation of vacancies in the bulk has a high energy, while there is a large variation in formation energies at the low index surfaces, which is likely to lead to segregation of vacancies to the surface of orthorhombic LaMnO 3 .

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2 -/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2 -/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2 -/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Nucleotide-binding and oligomerization domain (NOD)-like receptors NOD1 and NOD2 are cytosolic innate immune receptors that recognize microbial peptidoglycans. Although studies have addressed the role of NOD proteins in innate immune responses, little attention has been given to their impact on the developing adaptive immune system. We have assessed the roles of NOD1 and NOD2 deficiency on T cell development in mice. Our results demonstrate that NOD1 and NOD2 promote the positive selection/maturation of CD8 single-positive thymocytes in a thymocyte-intrinsic manner. TCR-mediated ERK phosphorylation is significantly reduced in the absence of NOD proteins, but receptor-interacting protein 2 is not involved in CD8 single-positive thymocyte selection or ERK signaling. Commensal bacteria-free animals have thymocyte maturation defects, and exogenous NOD ligands can enhance thymocyte maturation in culture. These results raise the intriguing possibility that abnormal lymphocyte responses observed in NOD-dependent inflammatory diseases are not driven solely by microbial signals in the gut, but may also involve intrinsic lymphocyte defects resulting from impaired CD8 T cell thymic development., (Copyright © 2017 by The American Association of Immunologists, Inc.)

B cell activation leads to proliferation and Ab production that can protect from pathogens or promote autoimmunity. Regulation of cell metabolism is essential to support the demands of lymphocyte growth and effector function and may regulate tolerance. In this study, we tested the regulation and role of glucose uptake and metabolism in the proliferation and Ab production of control, anergic, and autoimmune-prone B cells. Control B cells had a balanced increase in lactate production and oxygen consumption following activation, with proportionally increased glucose transporter Glut1 expression and mitochondrial mass upon either LPS or BCR stimulation. This contrasted with metabolic reprogramming of T cells, which had lower glycolytic flux when resting but disproportionately increased this pathway upon activation. Importantly, tolerance greatly affected B cell metabolic reprogramming. Anergic B cells remained metabolically quiescent, with only a modest increase in glycolysis and oxygen consumption with LPS stimulation. B cells chronically stimulated with elevated BAFF, however, rapidly increased glycolysis and Ab production upon stimulation. Induction of glycolysis was critical for Ab production, as glycolytic inhibition with the pyruvate dehydrogenase kinase inhibitor dichloroacetate sharply suppressed B cell proliferation and Ab secretion in vitro and in vivo. Furthermore, B cell-specific deletion of Glut1 led to reduced B cell numbers and impaired Ab production in vivo. Together, these data show that activated B cells require Glut1-dependent metabolic reprogramming to support proliferation and Ab production that is distinct from T cells and that this glycolytic reprogramming is regulated in tolerance.

Protein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη-/- T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation. In contrast, only PKCη-deficient T cells showed defective homeostatic proliferation, which requires self-antigen recognition. PKCη was dispensable for thymocyte development; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor development, indicating some redundancy between the PKC isoforms. Deficiency in PKCη or PKCθ had opposing effects on the relative numbers of CD4+ and CD8+ T cells. PKCη-/- mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ-/- mice had a lower ratio. Mice deficient in both isoforms exhibited normal cell ratios. Together, these data suggest that PKCη shares some redundant roles with PKCθ in T cell biology and also performs nonredundant functions that are required for T cell homeostasis and activation.

To analyze B lymphocyte central tolerance in a polyclonal immune system, mice were engineered to express a superantigen reactive to IgM of allotype b (IgM(b)). IgM(b/b) mice carrying superantigen were severely B cell lymphopenic, but small numbers of B cells matured. Their sera contained low levels of IgG and occasionally high levels of IgA. In bone marrow, immature B cells were normal in number, but internalized IgM and had a unique gene expression profile, compared with those expressing high levels of surface IgM, including elevated recombinase activator gene expression. A comparable B cell population was defined in wild-type bone marrows, with an abundance suggesting that at steady state ∼20% of normal developing B cells are constantly encountering autoantigens in situ. In superantigen-expressing mice, as well as in mice carrying the 3H9 anti-DNA IgH transgene, or 3H9 H along with mutation in the murine κ-deleting element RS, IgM internalization was correlated with CD19 downmodulation. CD19(low) bone marrow cells from 3H9;RS(-/-) mice were enriched in L chains that promote DNA binding. Our results suggest that central tolerance and attendant L chain receptor editing affect a large fraction of normal developing B cells. IgH(a/b) mice carrying the superantigen had a ∼50% loss in follicular B cell numbers, suggesting that escape from central tolerance by receptor editing from one IgH allele to another was not a major mechanism. IgM(b) superantigen hosts reconstituted with experimental bone marrow were demonstrated to be useful in revealing pathways involved in central tolerance.

Little is know about the nature of peripheral B cell tolerance or how it may vary in distinct lineages. Although autoantibody transgenic studies indicate that anergy and apoptosis are involved, some studies claim that receptor editing occurs. To model peripheral B cell tolerance in a normal, polyclonal immune system, we generated transgenic mice expressing an Igκ-light chain-reactive superantigen targeted to the plasma membrane of hepatocytes (pAlb mice). In contrast to mice expressing κ superantigen ubiquitously, in which κ cells edit efficiently to λ, in pAlb mice, κ B cells underwent clonal deletion. Their κ cells failed to populate lymph nodes, and the remaining splenic κ cells were anergic, arrested at a semi-mature stage without undergoing receptor editing. In the liver, κ cells recognized superantigen, down-regulated surface Ig, and expressed active caspase 3, suggesting ongoing apoptosis at the site of B cell receptor ligand expression. Some, apparently mature, κ B1 and follicular B cells persisted in the peritoneum. BAFF (B cell-activating factor belonging to the tumor necrosis factor family) overexpression rescued splenic κ B cell maturation and allowed κ cells to populate lymph nodes. Our model facilitates analysis of tissue-specific autoimmunity, tolerance, and apoptosis in a polyclonal B cell population. The results suggest that deletion, not editing, is the major irreversible pathway of tolerance induction among peripheral B cells.

It is not fully understood how the expression level of autoantigens in beta cells impacts autoimmune diabetes (T1D) development. Earlier studies using ovalbumin and also insulin had shown that secreted antigens could enhance diabetes development through facilitated presentation by antigen presenting cells. Here we sought to determine how the expression level of a membrane bound, non-secreted or cross-presented neo-antigen, the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), would influence T1D. We found that an RIP-LCMV transgenic mouse line exhibiting higher levels of beta cell GP expression developed more severe diabetes after LCMV infection or transfer of high numbers of activated autoreactive T cells. Importantly, all beta cells were lost and a significant increase in morbidity and mortality from T1D was noted. Insulitis and accumulation of autoaggressive CD8 cells was more profound in the RIP-LCMV-GP high-expressor line. Interestingly, the additional introduction of neo-antigen-specific CD4(+) helper or regulatory T cells was able to influence diabetogenesis positively or negatively. We conclude that a higher degree of autoantigen expression results in increased diabetes susceptibility. Therefore, autoantigens such as insulin that are expressed at higher levels in beta cells might have a more profound impact on diabetes pathogenesis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The TNF-family cytokine BAFF (BLyS) promotes B lymphocyte survival and is overexpressed in individuals with systemic lupus erythematosus and Sjögren's Syndrome. BAFF can rescue anergic autoreactive B cells from death, but only when competition from nonautoreactive B cells is lacking. Yet, high BAFF levels promote autoantibody formation in individuals possessing diverse B cells. To better understand how excess BAFF promotes autoimmunity in a polyclonal immune system, Ig L chain usage was analyzed in 3H9 site-directed IgH chain transgenic mice, whose B cells recognize DNA and chromatin when they express certain endogenous L chains. BAFF levels were manipulated in 3H9 mice by introducing transgenes expressing either BAFF or its natural inhibitor ΔBAFF. B cells in BAFF/3H9 mice were elevated in number, used a broad L chain repertoire, including L chains generating high-affinity autoreactivity, and produced abundant autoantibodies. Comparison of spleen and lymph node B cells suggested that highly autoreactive B cells were expanded. By contrast, ΔBAFF/3H9 mice had reduced B cell numbers with a repertoire similar to that of 3H9 mice, but lacking usage of a subset of Vκ genes. The results show that limiting BAFF signaling only slightly selects against higher affinity autoreactive B cells, whereas its overexpression leads to broad tolerance escape and positive selection of autoreactive cells. The results have positive implications for the clinical use of BAFF-depleting therapy.

During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered gamma2a-reactive superantigen (gamma2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag. In normal mice, self-reactive IgG2a-switched B cells were deleted, leading to the selective absence of IgG2a memory responses. These findings identify a novel negative selection mechanism for deleting mature B cells that acquire reactivity to self-Ag. This process was only partly dependent on the Bcl-2 pathway, but markedly inefficient in MRL-Fas(lpr) lupus mice, suggesting that defective apoptosis of isotype-switched autoreactive B cells is central to Fas mutation-associated systemic autoimmunity.

Transitional B cells turn over rapidly in vivo and are sensitive to apoptosis upon BCR ligation in vitro. However, little direct evidence addresses their tolerance sensitivity in vivo. A key marker used to distinguish these cells is IgD, which, through alternative RNA splicing of H chain transcripts, begins to be coexpressed with IgM at this stage. IgD is also expressed at high levels on naive follicular (B-2) and at lower levels on marginal zone and B-1 B cells. In this study, mice were generated to ubiquitously express a membrane-bound IgD-superantigen. These mice supported virtually no B-2 development, a greatly reduced marginal zone B cell population, but a relatively normal B-1 compartment. B cell development in the spleen abruptly halted at the transitional B cell population 1 to 2 stage, a block that could not be rescued by either Bcl-2 or BAFF overexpression. The developmentally arrested B cells appeared less mature and turned over more rapidly than nontransgenic T2 cells, exhibiting neither conventional features of anergy nor appreciable receptor editing. Paradoxically, type-2 T-independent responses were more robust in the transgenic mice, although T-dependent responses were reduced and had skewed IgL and IgH isotype usages. Nevertheless, an augmented memory response to secondary challenge was evident. The transgenic mice also had increased serum IgM, but diminished IgG, levels mirrored by the increased numbers of IgM(+) plasma cells. This model should facilitate studies of peripheral B cell tolerance, with the advantages of allowing analysis of polyclonal populations, and of B cells naturally lacking IgD.

Members of the Fgd (faciogenital dysplasia) gene family encode a group of critical guanine nucleotide exchange factors (GEFs), which, by specifically activating Cdc42, control cytoskeleton-dependent membrane rearrangements. In its first characterization, we find that FGD2 is expressed in antigen-presenting cells, including B lymphocytes, macrophages, and dendritic cells. In the B lymphocyte lineage, FGD2 levels change with developmental stage. In both mature splenic B cells and immature bone marrow B cells, FGD2 expression is suppressed upon activation through the B cell antigen receptor. FGD2 has a complex intracellular localization, with concentrations found in membrane ruffles and early endosomes. Although endosomal localization of FGD2 is dependent on a conserved FYVE domain, its C-terminal pleckstrin homology domain mediates recruitment to membrane ruffles. FGD2 overexpression promotes the activation of Cdc42 and leads to elevated JNK1 activity in a Cdc42- but not Rac1-dependent fashion. These findings are consistent with a role of FGD2 in leukocyte signaling and vesicle trafficking in cells specialized to present antigen in the immune system.

Marginal zone (MZ) B cells resemble fetally derived B1 B cells in their innate-like rapid responses to bacterial pathogens, but the basis for this is unknown. We report that the MZ is enriched in "fetal-type" B cell receptors lacking N regions (N(-)). Mixed bone marrow (BM) chimeras, made with adult terminal deoxynucleotidyl transferase (TdT)(+/+) and TdT(-/-) donor cells, demonstrate preferential repertoire-based selection of N(-) B cells into the MZ. Reconstitution of irradiated mice with adult TdT(+/+) BM reveals that the MZ can replenish N(-) B cells in adult life via repertoire-based selection and suggest the possibility of a TdT-deficient precursor population in the adult BM. The mixed chimera data also suggest repertoire-based bifurcations into distinct BM and splenic maturation pathways, with mature "recirculating" BM B cells showing a very strong preference for N(+) complementarity-determining region (CDR) 3 compared with follicular B cells. Because the T1 and MZ compartments are both the most enriched for N(-) H-CDR3, we propose a novel direct T1-->MZ pathway and identify a potential T1-MZ precursor intermediate. We demonstrate progressive but discontinuous repertoire-based selection throughout B cell development supporting multiple branchpoints and pathways in B cell development. Multiple differentiation routes leading to MZ development may contribute to the reported functional heterogeneity of the MZ compartment.

Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell-dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.

Ligand-dependent aggregation of FcgammaRIIa initiates multiple biochemical processes including the translocation to detergent resistant membrane domains (DRMs) and receptor tyrosine phosphorylation. Palmitoylation of cysteine residues is considered to be one process that assists in the localisation of proteins to DRMs. Within the juxtamembrane region of FcgammaRIIa there is cysteine residue (C208) that we show to be palmitoylated. Mutation of this cysteine residue results in the disruption of FcgammaRIIa translocation to DRMs as empirically defined by insolubility at high Triton X-100 concentrations. This study also demonstrates that the lack of lipid raft association diminishes FcgammaRIIa signaling as measured by receptor phosphorylation and calcium mobilisation functions suggesting that FcgammaRIIa signaling is partially dependent on lipid rafts.

In mice carrying a synthetic Igkappa-reactive superantigen ("kappa macroself antigen"), low level expression induced split peripheral B cell tolerance in the sIgkappa+ compartment, with striking reductions in follicular and marginal zone (MZ) B cells and the retention of significant numbers of sIgkappa+ B-1a but not B-1b cells in the peritoneum. Here, we characterize the transgenic line pKkappa with this split tolerance phenotype and assess the effects of B cell competition and the survival cytokine BAFF (B cell activating factor belonging to the TNF family) on peripheral tolerance. In pKkappa mice the surviving peritoneal and splenic kappa+ B cells were largely lost in mice carrying one copy of the human Ckappa exon in place of the mouse version, a maneuver that generates additional antigen non-reactive competitor B cells in this model. Furthermore, overexpression of BAFF suppressed kappa-macroself antigen-induced deletion and promoted production of both IgM,kappa and IgA,kappa antibodies in mice with normal Igkappa alleles but not in mice carrying one copy of the human Ckappa allele. These findings suggest that BAFF overexpression has minimal effects on the survival of autoreactive B cells in a polyclonal immune system and that B cell:B cell competition plays a potent role in suppressing the survival of B-1 and splenic B cells with excessive autoreactivity.

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.

Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and DeltaBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and DeltaBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and DeltaBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and DeltaBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.

DeltaBAFF is a novel splicing isoform of the regulator B cell-activating factor (BAFF, BLyS), a TNF family protein with powerful immunoregulatory effects. Overexpression of BAFF leads to excessive B cell accumulation, activation, autoantibodies, and lupus-like disease, whereas an absence of BAFF causes peripheral B cell immunodeficiency. Based on the ability of DeltaBAFF to multimerize with full-length BAFF and to limit BAFF proteolytic shedding from the cell surface, we previously proposed a role for DeltaBAFF in restraining the effects of BAFF and in regulating B lymphocyte homeostasis. To test these ideas we generated mice transgenic for DeltaBAFF under the control of human CD68 regulatory elements, which target expression to myeloid and dendritic cells. We also generated in parallel BAFF transgenic mice using the same expression elements. Analysis of the transgenic mice revealed that DeltaBAFF and BAFF had opposing effects on B cell survival and marginal zone B cell numbers. DeltaBAFF transgenic mice had reduced B cell numbers and T cell-dependent Ab responses, but normal preimmune serum Ig levels. In contrast, BAFF transgenic mice had extraordinarily elevated Ig levels and increases in subsets of B cells. Unexpectedly, both BAFF and DeltaBAFF appeared to modulate the numbers of B-1 phenotype B cells.

The tumor necrosis family member BAFF is limiting for the survival of follicular B lymphocytes, but excessive BAFF signaling can lead to autoimmunity, suggesting that its activity must be tightly regulated. We have identified a conserved alternate splice isoform of BAFF, called deltaBAFF, which lacks 57 nt encoding the A-A1 loop and is co-expressed with BAFF in many mouse and human myeloid cells. Mouse deltaBAFF appears on the plasma membrane, but unlike BAFF it is inefficiently released by proteolysis. DeltaBAFF can associate with BAFF in heteromultimers and diminish BAFF bioactivity and release. Thus, alternative splicing of the BAFF gene suppresses BAFF B cell stimulatory function in several ways, and deltaBAFF may promote other functions as well.

The mouse Fc gamma RI is one of the most fundamentally important FcRs. It participates in different stages of immunity, being a low affinity receptor for T-independent IgG3 and yet a high affinity receptor for IgG2a, the product of a Th1 immune response. However, analysis of this receptor has been difficult due largely to the failure to generate specific Abs to this FcR. We have made use of the polymorphic differences between BALB/c and NOD/Lt mice to generate mAb specific for the Fc gamma RI of BALB/c and the majority of in-bred mouse strains. Three different mAb were obtained that detected Fc gamma RI encoded by the more common Fcgr1(a) and Fcgr1(b) alleles, and although they identified different epitopes, none inhibited the binding of IgG to Fc gamma RI. When bound to Fc gamma RI, these mAb induced calcium mobilization upon cross-linking. Several novel observations were made of the cellular distribution of Fc gamma RI. Resting and IFN-gamma-induced macrophages expressed Fc gamma RI as well as mast cell lines. Both bone marrow-derived and freshly isolated dendritic cells from spleen and lymph nodes expressed Fc gamma RI. A class of DC, uniquely found in s.c. lymph nodes, expressed the highest level of Fc gamma RI and also high levels of MHC class II, DEC205, CD40, and CD86, with a low level of CD8 alpha, corresponding to the phenotype for Langerhans-derived DC, which are highly active in Ag processing. Thus, in addition to any role in effector functions, Fc gamma RI on APC may act as a link between innate and adaptive immunities by binding and mediating the uptake of T-independent immune complexes for presentation, thereby assisting in the development of T-dependent immune responses.

Introduction: This study pilots a method of measuring health outcomes in a general practice population of patients with type 2 diabetes., Method: The Diabetic Register of the Perth and Osborne Divisions of General Practice was linked to the Western Australian Health Services Research Linked Database., Results: Of the 487 patients in the study, 332 (68%) had been admitted before their diagnosis of diabetes (40% with a diabetes related condition), and 56% were admitted postdiagnosis (55% with a diabetes related condition). The admission rate increased with age and duration of diabetes., Discussion: The data show that a large proportion of diabetic patients suffer from serious comorbidity both pre- and post-diagnosis and demonstrate that their hospital admission rate is higher than that in the general population., Conclusion: The project demonstrates that linked hospital morbidity data can be used to monitor health outcomes in a general practice population of diabetic patients.

The inactivation of the mouse high-affinity IgG Fc receptor FcgammaRI resulted in a wide range of defects in antibody Fc-dependent functions. These studies showed the primary importance of FcgammaRI in endocytosis of monomeric IgG, kinetics, and extent of phagocytosis of immune complexes, in macrophage-based ADCC, and in immune complex-dependent antigen presentation to primed T cells. In the absence of FcgammaRI, antibody responses were elevated, implying the removal of a control point by the deletion of FcgammaRI. In addition, FcR-gamma chain-deficient mice were found to express partially functional FcgammaRI. Thus, FcgammaRI is an early participant in Fc-dependent cell activation and in the development of immune responses.

The mouse Fcgr1 gene encoding the high-affinity IgG receptor (FcgammaRI) exists as two known alleles, FcgammaRI-BALB and FcgammaRI-NOD, and these alleles exhibit functional differences. To determine whether other alleles exist in mouse strains, Fcgr1 coding regions from 35 strains of mice were sequenced and a further five alleles were identified. The FcgammaRI-BALB and NOD alleles are now designated the "a" and "d" alleles, respectively. Analysis of the five new alleles revealed that although no polymorphisms were observed in the two leader exons, nucleotide and subsequent amino acid changes were observed in the exons encoding the extracellular domains, and transmembrane and cytoplasmic tail. The cDNA of the seven alleles (a-g) were isolated and transiently transfected into COS cells, and IgG-binding studies were performed. Receptors encoded by four of the five new alleles (b, c, f, g) bound IgG2a with high affinity, displaying IgG binding characteristics similar to the a allele (previously FcgammaRI-BALB). The d allele (previously FcgammaRI-NOD) and the e allele [derived from Mus spretus (SPRET/Ei)] encoded receptors which showed broader specificity by binding monomeric IgG2a, IgG2b, and IgG3.

Recombinant Congenic Strains (RCS) are useful for dissecting complex polygenic traits. Here, we describe genetic and phenotypic characterization of six new RCS generated from outcrosses between NOD/Shi and CBA/LsLt, followed by sib mating of first backcross progeny (to CBA) for 20 generations, whereupon genetic and phenotypic analysis commenced. Four of the RCS were selected on the basis of residual heterozygosity present at F20 in one of the three original RCS. Contrary to expectations for RCS developed at first backcross, all derived at least 50% of the polymorphic markers typed from the NOD parental strain. Development of autoimmune insulin-dependent diabetes mellitus (IDDM) in NOD is a strain-specific characteristic. The major genetic component predisposing NOD mice to IDDM, their H2(g7) haplotype, was present in all RCS. Nevertheless, the presence of variable amounts of CBA genome at non-MHC loci conferred complete resistance in all RCS to spontaneous IDDM development, and rendered them strongly resistant to cyclophosphamide-induced IDDM. Although the RCS more resemble NOD in regard to certain strain-specific characteristics, such as prolificacy, an immunologic phenotype that was significantly reduced when compared to both parental strains was the number of peripheral CD8(+) T cells. Given the genetic characterization presented, these new RCS should prove valuable to investigators interested in studying genes controlling differential susceptibilities distinguishing the NOD and CBA inbred strain backgrounds.

It has been postulated that, during evolution of the Ig superfamily, modifications of the function of individual receptors might occur by acquisition of exons and their subsequent modification, though evidence of this is lacking. Here we have analysed the interaction of mouse IgG subclasses with high-affinity FcgammaRI (CD64) which contains three Ig-like domains and is important in innate and adaptive immunity. This analysis has identified a mechanism by which the postulated modification of newly acquired exons provides gains in function. Thus, the most widely distributed FcgammaRI allele in mice (e.g. BALB/c), bound only a single IgG subclass, IgG2a, with high affinity. However, non-obese diabetic (NOD) mice expressed a unique allele that exhibits broader specificity and, in addition to binding IgG2a, FcgammaRI-NOD bound monomeric IgG3 and bound IgG2b with high affinity, an IgG subclass not bound by FcgammaRI of other mouse strains, either as monomer or multivalent immune complexes. Analysis of mutants of FcgammaRI wherein segments of the interdomain junctions were exchanged between FcgammaRI-BALB and FcgammaRI-NOD identified these regions as having major influence in 'gain-of-function' by the NOD form of FcgammaRI. Nucleotide sequence analysis of intron/exon boundaries encoding the interdomain junctions of the FcgammaRI alleles showed these to have arisen by mutation to alter existing or create new mRNA splice donor/acceptor sites, resulting in generation of modified junctions.

Mouse IgG3 appears early in immune responses independently of T cell help and, as such, is an early effector molecule of the immune system. Yet, a specific IgG3 cellular receptor remains undefined. In transfection experiments, mouse Fc gammaRI was clearly able to bind immune complexes of IgG3, whereas mouse Fc gammaRII could not. Furthermore, macrophages from mice expressing Fc gammaRII and Fc gammaRIII but lacking Fc gammaRI were unable to phagocytose IgG3 immune complexes, thus identifying mouse Fc gammaRI as the sole receptor for IgG3 immune complexes. Competition studies demonstrated that monomeric mouse IgG3 could inhibit IgG2a binding to mouse Fc gammaRI with an ID50 approximately 10(-7) M (fivefold lower than IgG2a). The identification of mouse Fc gammaRI as the IgG3 receptor establishes Fc gammaRI as a participant in events at the interface between innate and adaptive immunity, implying a greater role for this receptor in the development of normal and pathologic immune responses than previously recognized.

The non-obese diabetic mouse (NOD) expresses a unique form of the high affinity receptor for IgG (FcgammaRI), containing multiple mutations that result in substitutions and insertions of amino acids and a truncated cytoplasmic tail. As a result of these major changes, receptor affinity for IgG increases 10-fold over that of wild-type FcgammaRI from BALB/c mice, while the specificity for ligand is retained. Kinetic analysis revealed that while the association rate of IgG with FcgammaRI from NOD mice (FcgammaRI-NOD) and FcgammaRI from BALB/c mice (FcgammaRI-BALB) is similar, IgG bound much more tightly to FcgammaRI-NOD as revealed by a profoundly diminished dissociation rate. Transfection studies demonstrated that FcgammaRI-NOD was expressed at one-tenth of the level of FcgammaRI-BALB. Although mouse FcgammaRI was previously not known to associate with the FcepsilonRI gamma-subunit, transfection of COS-7 cells demonstrates that like human FcgammaRI, mouse FcgammaRI is also able to associate with this signaling subunit. Furthermore, expression levels of FcgammaRI-NOD were not restored by the presence of the FcepsilonRI gamma-subunit. The difference in the levels of expression was mapped to mutations in the extracellular region of FcgammaRI-NOD as replacement of the extracellular domains with those of human FcgammaRI or FcgammaRI-BALB restored expression to that of human FcgammaRI or FcgammaRI-BALB.

Control of IgG immune complex formation and deposition is important in determining the nature and extent of subsequent immune effector responses, and appears to be aberrant in some autoimmune diseases. In this study we demonstrate that recombinant soluble Fc gamma RII (rsFc gamma RII) is an effective modulator of immune complex formation, delaying immune precipitation in a manner which is dose-dependent, and can be specifically inhibited by anti-Fc gamma RII MoAb Fab' fragments. This inhibitory role in immune precipitation also provides a possible mechanistic explanation for our previous demonstration of the efficacy of rsFc gamma RII as an inhibitor of immune complex-induced inflammation in the Arthus reaction in vivo. RsFc gamma RII inhibited immune complex precipitation in two different experimental systems. First, rsFc gamma RII inhibited the precipitation of 125I-bovine serum albumin (BSA)-anti-BSA complexes in a dose-dependent manner, while an irrelevant protein (soybean trypsin inhibitor) had no effect on the precipitation of the immune complexes. Moreover, rsFc gamma RII inhibited the precipitation of ovalbumin (OVA)-anti-OVA complexes as determined by turbidimetric analysis, where the inhibition of immune complex precipitation by rsFc gamma RII was dose-dependent and was specifically blocked by prior incubation with Fab' fragments of a blocking MoAb to Fc gamma RII. RsFc gamma RII could inhibit the precipitation of BSA-anti-BSA complexes in the presence of excess bystander IgG and did not inhibit complement-mediated prevention of immune precipitation, demonstrating that rsFc gamma RII did not block C1 binding to the BSA-anti-BSA complex. Unlike complement, rsFc gamma RII could not cause re-solubilization of pre-formed precipitated BSA-anti-BSA complexes. Soluble Fc gamma Rs have been detected in biological fluids of normal and inflammatory disease patients, yet the role of sFc gamma R is still unclear. However, they now play a potential role in the modulation of immune complex solubility.

Recombinant soluble IgE Fc receptors (rsFc epsilon RI) are potent inhibitors of type I hypersensitivity reactions tested in a local inflammatory setting. However, the fate of these receptors in vivo is dependent on the cellular source of the rsFc epsilon RI. We have produced these by transiently transfecting Cos-7 cells with a cDNA encoding the extracellular domains of human Fc epsilon RI alpha-chain. Following affinity purification, the rsFc epsilon RI was characterized as 58,000 MW, which was reduced to 23,000 MW following endoglycosidase F treatment. The purified rsFc epsilon RI could inhibit mouse IgE binding to Fc epsilon RI+ transfected CHO-K1 cells in vitro, bind sIgE+ B lymphoma cells in vitro, and inhibit the passive cutaneous anaphylaxis model in vivo in Sprague-Dawley rats. Pharmacokinetic studies in vivo involving intravenous injection of radiolabelled rsFc epsilon RI in mice revealed the receptor to have a rapid initial blood clearance (t1/2 early phase of 15 min) and to accumulate in the liver before being detected in urine. The localization of rsFc epsilon RI in the liver could be blocked by administration of mannose glycosylated ovalbumin and mannan, demonstrating that liver uptake involved the mannose receptor that is expressed on liver sinusoid cells and Kupffer cells. The production of rsFc epsilon RI using a stable expression system in CHO-K1 cells produced functional receptor of the same molecular weight as the Cos-7 system by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). However, biodistribution studies demonstrated differences; the CHO-K1 cell-produced material did not localize to the liver in comparison to the Cos-7-produced rsFc epsilon RI.