Anti-Thy-1 monoclonal antibody-induced glomerulonephritis in Mongolian gerbils

Two novel murine monoclonal antibodies (mAbs) were produced to the Thy-1 molecule of Mongolian gerbils (Meriones unguiculatus). These mAbs, HUSM-M.g.27 of IgG1 isotype and HUSM-M.g.40 of IgG2a isotype, immunohistochemically reacted with the thymus, nervous system, and glomerular mesangium in partially different manners, suggesting that they recognize distinct epitopes, although they reacted with Thy-1 antigen, with apparent molecular weight of about 25 kDa, on gerbil thymocytes. Mild and severe forms of mesangioproliferative nephritis after glomerular deposition of the antibody was observed in gerbils administered mAbs HUSM-M.g.27 and HUSM-M.g.40, respectively, intraperitoneally, with or without guinea-pig serum as supplementary complement. Distinct pathogenicity and requirement of guinea pig serum for pathologic sequels are discussed as they relate to the rat model of anti-Thy-1-induced glomerulonephritis.     

TLR9 signaling in B cells determines class switch recombination to IgG2a

Although IgG2a is the most potent Ab isotype in the host response to viral and bacterial infections, the regulation of class switch recombination to IgG2a in vivo is not yet well understood. Recognition of pathogen-associated molecular patterns by dendritic cells expressing TLRs, like TLR7, recognizing ssRNA, or TLR9, recognizing DNA rich in nonmethylated CG motifs (CpG), favors induction of Th1 responses. It is generally assumed that these Th1 responses are responsible for the TLR-mediated induction of IgG2a. Using virus-like particles loaded with CpGs, we show here that TLR9 ligands can directly stimulate B cells to undergo isotype switching to IgG2a. Unexpectedly, TLR9 expression in non-B cells did not affect isotype switching in the Ab response against virus-like particles. Thus, TLR9 can regulate isotype switching to IgG2a directly by interacting with B cells rather than indirectly by inducing Th1 responses.     

Profiling the cellular immune response to multiple Brugia pahangi infections in a susceptible host

Human lymphatic filariasis is caused primarily by Brugia malayi and Wuchereria bancroffi. Unraveling this disease is complex, as people living in endemic areas exhibit a vast array of clinical states and immune responses. The Mongolian gerbil (Meriones unguiculatus)-B. pahangi model of human lymphatic filariasis has provided much information on immune parameters associated with filarial infection. Prior investigations in our laboratory have shown that gerbils closely mimic a subset of patients classified as microfilaremic but asymptomatic, a group that comprises the majority of people living in endemic areas. Worm recovery data suggest that gerbils carrying current B. pahangi infections do not show any resistance to subsequent subcutaneous B. pahangi infections. The aim of the present studies was to investigate the T cell cytokine response in gerbils receiving multiple infections of B. pahangi as a means of mimicking the conditions experienced by people in endemic areas. The T cell cytokine profile generated by multiply infected gerbils was not different from that previously generated by gerbils infected only once with B. pahangi. Gerbils infected multiple times with B. pahangi showed a transient increase in IL-5, which corresponded to the increased eosinophil levels previously reported from multiply infected gerbils. Chronically infected gerbils showed elevated IL-4 mRNA levels, as has been reported from gerbils infected only once with B. pahangi. Chronic infections were also associated with a state of immune hyporesponsiveness, as determined by the characterization of lymphatic thrombi and lymphoproliferation of spleen and renal lymph node cells to worm antigen.     

The immunoglobulin heavy chain constant region affects kinetic and thermodynamic parameters of antibody variable region interactions with antigen

A central dogma in immunology is that antibody specificity is a function of the variable (V) region. However serological analysis of IgG(1), IgG(2a), and IgG(2b) switch variants of murine monoclonal antibody (mAb) 3E5 IgG(3) with identical V domains revealed apparent specificity differences for Cryptococcus neoformans glucuronoxylomannan (GXM). Kinetic and thermodynamic binding properties of mAbs 3E5 to a 12-mer peptide mimetic of GXM revealed differences in the affinity of these mAbs for a monovalent ligand, a result that implied that the constant (C) region affects the secondary structure of the antigen binding site, thus accounting for variations in specificity. Structural models of mAbs 3E5 suggested that isotype-related differences in binding resulted from amino acid sequence polymorphisms in the C region. This study implies that isotype switching is another mechanism for generating diversity in antigen binding and that isotype restriction of certain antibody responses may reflect structural constraints imposed by C region on V region binding. Furthermore, isotype affected the polyreactivity of V region identical antibodies, implying a role for C region in determining self-reactivity.     

Prostaglandin E2 promotes IL-4-induced IgE and IgG1 synthesis

PG of the E series are generally known to suppress immune responses, however, we have found that PGE synergizes with IL-4 to induce IgE and IgG1 production in LPS-stimulated murine B lymphocytes. PGE2 and PGE1 (10(-6) to 10(-8) M) significantly increase IgE and IgG1 production (up to 26-fold) at all concentrations of IL-4 tested. In addition to its effects on IgE and IgG1, PGE also causes a significant decrease in IgM and IgG3 synthesis, suggesting that PGE may promote IL-4-induced class switching. The specificity of the E series PG effect is demonstrated by the fact that PGF2 alpha (10(-6) M) does not alter production of any of these isotypes. Because PGE can mediate its effects through cAMP in some cases, we investigated the importance of cAMP levels in regulation of isotype expression. Other agents that increase intracellular cAMP levels (cholera toxin and dibutyryl cAMP) were assessed for their ability to regulate isotype differentiation. Cholera toxin (100 pg/ml) and dibutyryl cAMP (100 microM) significantly enhanced IgE and IgG1 production and diminished IgM and IgG3 synthesis. We also show that PGE and cholera toxin elevate intracellular cAMP in B lymphocytes in a dose-dependent manner. In contrast, PGF2 alpha (10(-6) M) and the B subunit of cholera toxin (100 pg/ml) did not increase cAMP and did not regulate the isotype of Ig produced, reiterating the importance of cAMP in enhancing isotype differentiation. Although PGE is known to inhibit a number of immune responses, our data show that it is not always inhibitory. PGE may play a role in atopy in vivo where PGE-secreting cells such as macrophages, follicular dendritic cells, and fibroblasts can promote IgE synthesis. This research emphasizes the importance of PGE in regulation of the humoral immune response and adds a new stimulatory action to the repertoire of known PGE effects.     

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell–tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c⁺ expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.     

Induction of enhanced immunity to intestinal nematodes using IL-9-producing dendritic cells

Dendritic cells can be considered natural adjuvants and are able to act as cellular vaccines to protect against disease. Adoptive transfer of Ag-pulsed bone marrow-derived dendritic cells (BMDCs) enhanced expulsion of the intestinal nematode, Trichinella spiralis, from the small intestine. IL 9 is a critical cytokine in protective immunity to intestinal nematode infection and is believed to enhance Th2 immune responses. Deriving dendritic cells from an IL-9 transgenic (IL-9t) mouse has enabled a detailed investigation of the importance of IL-9 during Ag presentation. Indeed, IL-9t dendritic cells significantly enhanced T cell proliferation and Th2 responses and, after adoptive transfer, enhanced parasite-specific IgG1 and intestinal mastocytosis in vivo, leading to accelerated expulsion of adult worms from the intestine. Overall, this paper demonstrates that dendritic cell vaccination can be used to successfully protect the host against intestinal nematode infection and suggests that IL-9 can act as a potent type 2 adjuvant during Ag presentation and the early stages of Th2 activation.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

In the preceding paper it was suggested that the tumour localisation of 125I-labelled syngeneic rat monoclonal antibodies (mAbs) may be limited in immunocompetent hosts by the presence of competing endogenous serum antibodies. In syngeneic congenitally athymic (nu/nu) and cyclosporin-A-treated rats (both of which fail to mount immune responses to tumour antigens) increased uptake of mAbs in tumour tissue was obtained compared with that in immunocompetent animals. However, in the case of IgG2b and IgG1 mAbs, this appeared to be due primarily to enhanced "non-specific" localisation mediated by Fc binding, since it was abolished by the use of F(ab')2 fragments with two out of three mAbs tested. Normal tissue distribution was also influenced by host immune status: in nu/nu rats the uptake of IgG2b mAbs in the spleen was up to fivefold higher than that previously found in normal animals and the levels in liver were also increased. This effect was not seen in cyclosporin-A-treated hosts, suggesting that the reticuloendothelial system of congenitally athymic animals contains cells with enhanced IgG2b-FcR activity. This hypothesis was strengthened by the observation that splenic uptake was reduced by either the use of F(ab')2 fragments, or prior "blockade" of Fc receptors by "cold competition" with excess unlabelled IgG2b mAbs. This blockade could not be effected by mAbs of any other isotype or by IgG2b F(ab')2 fragments. The former manoeuvre resulted in higher tumour specificity ratios but usually at the expense of reduced levels of tumour associated radiolabelled mAb. The latter was found to increase "absolute" tumour localisation by up to 35%. In an attempt to characterise further and compare the Fc receptor activity of intratumour and intrasplenic host cells. The distribution of IgG2b mAbs was assayed in 3-week, 8-week and 12-week-old rats. We were able operationally to distinguish the activity of these two categories of cells, suggesting that they represent either different lineages or differentially activated subpopulations: the splenic IgG2b binding was fully expressed in weanling nu/nu rats whereas the FcR activity of cells infiltrating MC24 sarcoma was limited in 3-week-old compared with 8-12-week-old hosts. A further difference was apparent in the subclass "preference" of FcR binding: in immunodeprived rats both IgG1 and IgG2b mAbs were able to bind to tumour-infiltrating host cells, but uptake of IgG1 mAbs in the spleen was always low and not reduced further by the use of F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cutting edge: Langerin+ dendritic cells in the mesenteric lymph node set the stage for skin and gut immune system cross-talk

Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer's patch-null mice but abolished in the Peyer's patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after TCI. Unexpectedly, langerin(+)CD8alpha(-) dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin(+) cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin(+) dendritic cells in the MLN.     

In vivo allogeneic effects: shift in the isotype profile of primary TI-2 responses in mice undergoing graft-vs-host reaction

We showed previously that primary responses to T-dependent (TD) and T-independent type 2 (TI-2) antigens were differentially affected by allogeneic effects induced in vivo during a graft-vs-host reaction (GVH). TD responses were greater than or equal to 80% suppressed, whereas the TI-2 responses were greatly enhanced, particularly the IgG component, which normally is very low. We have analyzed the IgG subclass distribution in primary responses of normal and GVH F1 mice in order to determine whether the strong T cell signals that occur during GVH reactions also induce shifts in the isotype profile. The effect of GVH on responses to TI-2 antigens was of particular interest because they are usually dominated by IgM and IgG3 classes in normal mice. We found a threefold to 10-fold increase in the PFC numbers of all four IgG subclasses in the response to TI-2 antigens, with an apparent shift from the usual IgG3 dominance to IgG1 in GVH mice. This IgG1 dominance was not found in serum antibodies where IgG3, IgG1, and IgG2b were equally expressed, although total IgG was increased greater than 20-fold. No isotype shift was found in either the TNP-KLH response, which was greater than or equal to 75% suppressed (IgG1 dominance was retained), or in the TI-1 response to TNP-Ba. The latter response was reduced (25 to 50%) in GVH mice and continued to be dominated by IgG2b/2a and IgG3. Unlike the unique isotype patterns found in primary responses, TNP-KLH primed mice challenged with TD, TI-1, or TI-2 antigens gave memory responses with identical isotype profiles that were dominated by IgG1 PFC. The role of T cells in B cell differentiation and isotype expression is discussed.     

TLR signaling fine-tunes anti-influenza B cell responses without regulating effector T cell responses

Influenza is a ssRNA virus that has been responsible for widespread morbidity and mortality; however, the innate immunological mechanisms that drive the adaptive anti-influenza immune response in vivo are yet to be fully elucidated. TLRs are pattern recognition receptors that bind evolutionarily conserved pathogen-associated molecular patterns, induce dendritic cell maturation, and consequently aid the development of effective immune responses. We have examined the role of TLRs in driving effective T and B cell responses against influenza virus. We found TLR3 and its associated adapter molecule, Toll/IL-R domain-containing adaptor-inducing IFN-beta, did not play a role in the development of CD4(+) or CD8(+) T cell responses against influenza virus, nor did they influence influenza-specific B cell responses. Surprisingly, TLR7 and MyD88 also played negligible roles in T cell activation and effector function upon infection with influenza virus; however, their signaling was critical for regulating anti-influenza B cell Ab isotype switching. The induction of appropriate anti-influenza humoral responses involved stimulation of TLRs on B cells directly and TLR-induced production of IFN-alpha, which acted to reduce IgG1 and increase IgG2a/c class switching. Notably, direct TLR signaling on B cells or T cell help through the CD40-CD40L interaction was sufficient to support B cell proliferation and IgG1 production, whereas IFN-alpha was critical for fine-tuning the nature of the isotype switch. Taken together, these data reveal that TLR signaling is not required for anti-influenza T cell responses, but through both direct and indirect means orchestrates appropriate anti-influenza B cell responses.     

Cross-linking surface Ig delays CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for independent regulation of B cell proliferation and differentiation

T cells stimulate B cells to divide and differentiate by providing activating signals in the form of inducible membrane-bound molecules and secreted cytokines. Provision of these signals in vitro reproduces many of the consequences of T-B collaboration in the absence of any form of Ag stimulation. Although clearly not obligatory, Ag signals appear to play an important regulatory role in numerous aspects of the B cell response. To examine directly the effect of an Ag signal, naive B cells were stimulated in the presence of rCD40 ligand, with or without IL-4 in the presence or absence of different anti-Ig mAbs. Anti-Ig mAbs exerted variable effects on the B cell division rate, from enhancement to no effect to inhibition. In contrast, all anti-Ig mAbs tested inhibited division-linked isotype switching to IgG1 and IgE. Thus, B cell Ag receptor ligands could modify the rates of B cell expansion and class switching independently. The ability of anti-Ig reagents to modify class switching suggests the B cell Ag receptor may play an important role in the selection of Ig isotypes during T cell-dependent humoral immune responses to Ags of different physical structure.     

Determinants of autoantibody induction by conjugated papillomavirus virus-like particles

Immunization of mice with self-Ag arrayed on the surface of papillomavirus-like particles induces long-lasting high-titer IgG production by autoreactive B cells. In contrast, immunization with disorganized self-Ag linked to foreign Th epitopes induces weak autoantibody responses that are predominantly of the IgM isotype. In this study, we evaluated the structural correlates of autoantibody induction to determine the basis of these disparate observations, using a system in which mice were vaccinated with a fusion protein containing self (TNF-alpha) and foreign (streptavidin) components, conjugated to biotinylated virus-like particles (VLPs). Similar titers of autoantibodies to TNF-alpha were elicited using conjugated polyomavirus VLPs and papillomavirus VLPs, indicating that acute activation of dendritic cells by the Ag is not required. Strong autoantibody responses were also induced by conjugated papillomavirus capsid pentamers, indicating that a higher order particulate structure is also not required. However, a reduction of self-Ag density on VLP surfaces dramatically reduced the efficiency of IgG autoantibody induction. In contrast, the negative effects of reductions in foreign Ag density were limited and could be overcome by dosage and adjuvant. These data suggest that the immune system has evolved to differentially recognize closely spaced repetitive Ags and that the signals generated upon interactions with high-density self-Ags can overwhelm the normal mechanisms for B cell tolerance.     

Immune response of the Mongolian gerbil (Meriones unguiculatus) to sheep red blood cells.

The immune responses of Mongolian gerbils, Meriones unguiculatus, to sheep red blood cells (SRBC) were studied as compared to those of mice. After a single injection of SRBC, hemagglutinin titers in gerbils were significantly lower and hemolytic plaque-forming cells (PFC) in the spleen were less in number as compared to the response of mice. In gerbils the PFC response to a higher dose of bacterial lipopolysaccharide (LPS) was rather higher than in mice. The delayed-type hypersensitivity (DTH) assay on the foot-pad revealed that the responsiveness was considerably lower in gerbils than in mice.     

Development of monoclonal antibodies that specifically interact with necrotic lymphoma cells

The immune system has evolved mechanisms to sense not only microbes, but also necrotic cells. The pattern-recognition receptors in macrophages/dendritic cells that stimulate the acquired immune system are closely associated with danger signaling. In this study monoclonal antibodies (mAbs) that specifically interact with necrotic cells were developed. One IgG1 and two IgM mAbs were established, and they recognized a 80 kDa protein expressed in necrotic, but not live or apoptotic, cells. These mAbs, which serve as a probe for necrosis, facilitate analyses of the role of the immune complex that consists of necrotic cells and Ab and contributes to the formation of the inflammatory milieu induced by necrotic cell death.     

Local and systemic antibody responses in gerbils following vaccination with irradiated or non-irradiated Trichostrongylus colubriformis larvae

Groups of 10 Mongolian gerbils, Meriones unguiculatus, were vaccinated with 1,500 gamma-irradiated Trichostrongylus colubriformis infective larvae (L3) or with non-irradiated larvae. 25 days later five gerbils from each group were necropsied and the remaining gerbils challenged with 1,500 non-irradiated T. colubriformis infective larvae. Systemic, local intestinal and coproantibody levels were compared in each group of gerbils 25 days after vaccination and 26 days after challenge. Strong local intestinal and faecal antibody responses were detected. Coproantibodies reflected antibody levels in the intestinal contents and in mucosal extracts. The results gave further support to the view that coproantibody measurements provide a sensitive index of immunity at mucosal surfaces to intestinal parasites.     

Variable-region-identical antibodies differing in isotype demonstrate differences in fine specificity and idiotype

A central tenet of the current understanding of the relationship between Ab structure and function is that the variable region domain is solely responsible for Ag specificity. However, this view was recently challenged by the observation that families of mouse-human chimeric Abs with identical V regions demonstrate differences in fine specificity and by reports of changes in Ab Id structure with isotype switching. Here we revisited this question by evaluating the reactivity of two families of murine IgG switch variants that differed in V region usage for Cryptococcus neoformans glucuronoxylomannan, glucuronoxylomannan peptide mimetics, and anti-Id mAbs. The results reveal isotype-related differences in fine specificities and Id for two mAb isotype switched families, thus establishing the validity of this observation with sets of homologous Abs. The results suggest that the C region affects V region protein conformation, leading to differences in fine specificity and Id. The finding that isotype can affect fine specificity has major implications for current concepts of the generation of secondary responses, idiotypic network regulation, and isotype function. Given that isotype class switching and Ig gene somatic hypermutation share molecular mechanisms, these observations unify these processes in the sense that both can alter specificity and affinity.     

Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.     

Mechanism for the isotype dependence of antibody-mediated toxicity in Cryptococcus neoformans-infected mice

Ab-based therapies have undergone a renaissance in recent years, but infusion-related reactions are a significant clinical problem. Administration of certain mAbs to Swiss Webster mice infected with Cryptococcus neoformans can result in acute lethal toxicity (ALT) characterized by cardiovascular collapse. The ability of a mAb to produce ALT is isotype dependent and occurs with IgG1 but not IgG3. To investigate this phenomenon, we measured spleen and liver cytokine responses and platelet-activating factor (PAF) content in mice given C. neoformans glucuronoxylomannan (GXM) followed by specific Ab of IgG1 or IgG3 isotype. We found no evidence to suggest that the differences in IgG1 and IgG3 toxicity were due to differences in chemokine or cytokine response. In contrast, liver and spleen tissue PAF content was significantly greater in mice IgG1. Furthermore, our results show differences in the response to IgG1- and IgG3-GXM complexes regarding: 1) macrophage-inflammatory protein-1alpha and monocyte chemoattractant protein-1 regulation, 2) splenic and hepatic PAF content, and 3) hepatic PAF content in infected mice. IgG1-associated ALT appears to be the result of greater production of PAF in response to IgG1-GXM complex formation. The results are consistent with the view that IgG1 and IgG3 interact with different Fc receptors. Our findings strongly suggest that the mechanism for Ab-mediated ALT is different from the cytokine release syndrome described after administration of other therapeutic mAbs.