Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Mediated Calcium Signaling Is Active in Memory CD4+ T Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP), identified as one of the most potent calcium-mobilizing second messengers, has been studied in different eukaryotic cell types, including lymphocytes. Although aspects of NAADP-mediated calcium release in lymphocytes are still under debate, the organelles pertaining to NAADP-mediated calcium release are often characterized as acidic and related to lysosomes. Although NAADP-mediated calcium release in different subsets of T cells, including naïve, effector and natural regulatory T cells, has been studied, it has not been widely studied in memory CD4+ T cells, which show a different calcium flux profile. Using a pharmacological approach, the effect of Ned-19, an NAADP pathway antagonist, on the involvement of NAADP in TCR activation in murine memory CD4+ T cells and their downstream effector functions, such as proliferation and cytokine production, was studied. According to this study, Ned-19 inhibited TCR-mediated calcium flux and its downstream effector functions in primary memory CD4+ T cells. The study also revealed that both extracellular and intracellular calcium stores, including endoplasmic reticulum and lysosome-like acidic calcium stores, contribute to the TCR-mediated calcium flux in memory CD4+ T cells. NAADP-AM, a cell permeable analogue of NAADP, was shown to release calcium in memory CD4+ T cells and calcium flux was inhibited by Ned-19.

Synthesis and Evaluation of a Self-Adjuvanting Pseudomonal Vaccine Based on Major Outer Membrane Porin OprF Epitopes Formulated with Low-Toxicity QS-21-Containing Liposomes.

Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that the World Health Organization has ranked as a priority 1 (critical) threat. One potential prophylactic approach to preventing or reducing the incidence of PA would be development of a long sought-after vaccine. Both antibody and CD4+ T-cell responses have been noted as playing key roles in protection against infection. In these studies, we have designed a prototype vaccine consisting of several known linear B-cell epitopes derived from an outer membrane porin F (OprF). The resulting thiol-containing protein was conjugated to a version of the lipopeptide-based Toll-like receptor agonist Pam3CysSK4Mal (10) containing a maleimide moiety and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Mice immunized with the resulting vaccine generated antibodies that bound PA14 (serotype O10) in vitro and induced opsonization in the presence of rabbit complement and murine macrophage RAW264.7 cells. The liposome was optimized to contain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), Chol, Pam3CysSK4-OprF (12) and the Quillaja saponaria-derived saponin adjuvant QS-21. The resulting vaccine formulation produced significantly higher antibody titers, increased the IgG2a antibody isotype, and increased the number of IgG-producing B-cells as well as splenic primed T-cells. In summary, the liposomal vaccine platform was found highly useful for the generation of a robust and balanced TH1/TH2 response.

Synthesis and Immunological Evaluation of a Single Molecular Construct MUC1 Vaccine Containing l-Rhamnose Repeating Units.

A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.

Development of a Bioconjugate Platform for Modifying the Immune Response of Autoreactive Cytotoxic T Lymphocytes Involved in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disorder characterized by autoimmune cell mediated destruction of pancreatic beta cells. Pancreatic beta cells are the only source of insulin in the body. T1D patients then have to depend on insulin injections for their lifetime. Insulin injection can modulate the blood sugar levels, but insulin has little effect on the autoimmune process. Altered peptide ligands (APL) derived from known autoantigens in T1D are able to induce tolerance in autoreactive cells in T1D animal models, but are currently unable to elicit this protection in humans. There is a need to improve immunogenicity of the APLs, as these short peptides can be easily degraded by enzymes in the blood. GAD546-554 is a dominant epitope recognized by autoreactive T cells in the nonobese diabetic (NOD) mouse model that can cause destruction of beta cells. Alanine substitution at the eighth position of GAD546-554 peptide (APL9) induced tolerance in a GAD546-554 specific cytotoxic T lymphocyte clone. To improve the antigen presentation and endosomal escape of APL9, we developed a bioconjugate platform that consists of a liposome containing a bioconjugate of APL9 and toll-like receptor 2 ligand Pam3CysSK4 as well as an antibody against macrophage protein F4/80. APL9 bioconjugate liposome with F4/80 antibody was able to induce tolerance in a GAD 546-554 specific clone. Diabetic NOD splenocytes pretreated with APL9 bioconjugate were also not able to transfer diabetes into prediabetic NOD recipient mice. This work is beneficial to prevent T1D as an immunotherapy strategy to render autoreactive immune cells more tolerant of beta cells.

Liposomal Fc Domain Conjugated to a Cancer Vaccine Enhances Both Humoral and Cellular Immunity.

Targeted delivery of antigens to antigen-presenting cells (APCs) by utilizing natural anticarbohydrate antibodies is a promising approach for selective uptake and enhanced antigen presentation. Previously, we reported that in the presence of a natural antibody, anti-rhamnose antibody (anti-Rha), the bacterial sugar rhamnose conjugated with liposomal cancer antigen MUC1-Tn enhances antigen presentation by APCs such as dendritic cells by targeting Fc gamma receptors. The idea was to utilize the natural human anti-Rha antibodies present in human serum for targeted delivery of cancer-specific antigens. Recently, we found that the IgG3 antibody isotype was the most prevalent anti-Rha antibody generated in mice immunized with rhamnose-Ficoll (Rha-Ficoll) antigen. In this manuscript, we have conjugated the murine IgG3-Fc with a MUC1-containing cancer vaccine and compared the humoral and cellular immune response to this vaccine with one targeted via the human anti-Rha antibody and to the MUC1 vaccine alone. This Fc approach enhanced antibody production and T-cell proliferation almost to the same level as using the anti-Rha antibody. These results suggest that targeting Fc directly to dendritic cells can be an alternative approach to human anti-Rha for generating effective antigen-primed T-cells.

Use of Dendritic Cell Receptors as Targets for Enhancing Anti-Cancer Immune Responses.

A successful anti-cancer vaccine construct depends on its ability to induce humoral and cellular immunity against a specific antigen. Targeting receptors of dendritic cells to promote the loading of cancer antigen through an antibody-mediated antigen uptake mechanism is a promising strategy in cancer immunotherapy. Researchers have been targeting different dendritic cell receptors such as Fc receptors (FcR), various C-type lectin-like receptors such as dendritic and thymic epithelial cell-205 (DEC-205), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), and Dectin-1 to enhance the uptake process and subsequent presentation of antigen to T cells through major histocompatibility complex (MHC) molecules. In this review, we compare different subtypes of dendritic cells, current knowledge on some important receptors of dendritic cells, and recent articles on targeting those receptors for anti-cancer immune responses in mouse models.

Augmenting Vaccine Immunogenicity through the Use of Natural Human Anti-rhamnose Antibodies.

Utilizing natural antibodies to augment vaccine immunogenicity is a promising approach toward cancer immunotherapy. Anti-rhamnose (anti-Rha) antibodies are some of the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. It was shown previously that anti-Rha antibody generated in mice binds effectively with Rha-conjugated vaccine and is picked up by antigen presenting cells (APCs) through stimulatory Fc receptors. This leads to the effective uptake and processing of antigen and eventually presentation by major histocompatibility complex (MHC) molecules. In this article, we show that natural human anti-Rha antibodies can also be used in a similar mechanism and immunogenicity can be enhanced by targeting Rha-conjugated antigens. In doing so, we have purified human anti-Rha antibodies from human serum using a rhamnose affinity column. In vitro, human anti-Rha antibodies are shown to enhance the uptake of a model antigen, Rha-ovalbumin (Rha-Ova), by APCs. In vivo, they improved the priming of CD4+ T cells to Rha-Ova in comparison to non-anti-Rha human antibodies. Additionally, increased priming of both CD4+ and CD8+ T cells toward the cancer antigen MUC1-Tn was observed in mice that received human anti-Rha antibodies prior to vaccination with a rhamnose-modified MUC1-Tn cancer vaccine. The vaccine conjugate contained Pam3CysSK4, a Toll-like receptor (TLR) agonist linked via copper-free cycloaddition chemistry to a 20-amino-acid glycopeptide derived from the tumor marker MUC-1 containing the tumor-associated carbohydrate antigen α- N-acetyl galactosamine (GalNAc). The primed CD8+ T cells released IFN-γ and killed tumor cells. Therefore, we have confirmed that human anti-Rha antibodies can be effectively utilized as a targeting moiety for making an effective vaccine.

Immunological Evaluation of Recent MUC1 Glycopeptide Cancer Vaccines.

Aberrantly glycosylated mucin 1 (MUC1) is a recognized tumor-specific antigen on epithelial cell tumors. A wide variety of MUC1 glycopeptide anti-cancer vaccines have been formulated by many research groups. Some researchers have used MUC1 alone as an immunogen whereas other groups used different antigenic carrier proteins such as bovine serum albumin or keyhole limpet hemocyanin for conjugation with MUC1 glycopeptide. A variety of adjuvants have been used with MUC1 glycopeptides to improve their immunogenicity. Fully synthetic multicomponent vaccines have been synthesized by incorporating different T helper cell epitopes and Toll-like receptor agonists. Some vaccine formulations utilized liposomes or nanoparticles as vaccine delivery systems. In this review, we discuss the immunological evaluation of different conjugate or synthetic MUC1 glycopeptide vaccines in different tumor or mouse models that have been published since 2012.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.

Synthesis of a Liposomal MUC1 Glycopeptide-Based Immunotherapeutic and Evaluation of the Effect of l-Rhamnose Targeting on Cellular Immune Responses.

Generation of a CD8(+) response to extracellular antigen requires processing of the antigen by antigen presenting cells (APC) and cross-presentation to CD8(+) T cell receptors via MHC class I molecules. Cross-presentation is facilitated by efficient antigen uptake followed by immune-complex-mediated maturation of the APCs. We hypothesize that improved antigen uptake of a glycopeptide sequence containing a CD8(+) T cell epitope could be achieved by delivering it on a liposome surface decorated with an immune complex-targeting ligand, an l-Rhamnose (Rha) epitope. We synthesized a 20-amino-acid glycopeptide TSAPDT(GalNAc)RPAPGSTAPPAHGV from the variable number tandem repeat region of the tumor marker MUC1 containing an N-terminal azido moiety and a tumor-associated α-N-acetyl galactosamine (GalNAc) at the immunogenic DTR motif. The MUC1 antigen was attached to Pam3Cys, a Toll-like receptor-2 ligand via copper(I)-catalyzed azido-alkyne cycloaddition (CuAAc) chemistry. The Rha-decorated liposomal Pam3Cys-MUC1-Tn 4 vaccine was evaluated in groups of C57BL/6 mice. Some groups were previously immunized to generate anti-Rha antibodies. Anti-Rha antibody expressing mice that received the Rha liposomal vaccine showed higher cellular immunogenicity compared to the control group while maintaining a strong humoral response.

Synthesis of α-L-rhamnosyl ceramide and evaluation of its binding with anti-rhamnose antibodies.

An α-L-rhamnosyl ceramide (1, α-L-RhaCer) has been prepared that was recognized by anti-L-rhamnose (anti-Rha) antibodies. During these studies we explored the use of an α-L-rhamnosyl thioglycoside and a trichloroacetimidate as a glycosyl donors. Subsequently, the acceptors desired for glycosylation, 3-O-benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from D-xylose. The thioglycoside donor, 2,3,4-tri-O-acetyl-1-(4-tolyl)thio-α-L-rhamnopyranoside, and the trichloroacetimidate donor, 2,3,4-tri-O-acetyl-1-(2,2,2-trichloroethanimidate)-α-L-rhamnopyranoside, were synthesized in 50% and 78% yield overall, respectively. The synthesis of the glycosylation acceptor employed an addition-fragmentation olefination that was successfully carried out in 53% yield. With the successful synthesis of key intermediates, α-L-RhaCer (1) was prepared without any insurmountable obstacles. Anti-Rha antibodies were prepared in BALB/c mice by immunizing them with rhamnose-ovalbumin (Rha-Ova) with Sigma Adjuvant System (SAS) and the anti-L-Rha antibodies were isolated from the blood sera. Liposomes and EL4 tumor cells were used as model systems to demonstrate the ability of 1 to insert into a lipid bilayer. The interaction of the liposomes or the EL4 cells with α-L-RhaCer (1) and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to confirm the ability of glycolipid 1 to be displayed on the tumor cell surface as well as the ability to be recognized by anti-Rha antibodies.

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: difference in specificity between human and sea urchin NAADP receptors.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure-activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel "caged" 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca2+ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.

Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.

Synthesis of a single-molecule L-rhamnose-containing three-component vaccine and evaluation of antigenicity in the presence of anti-L-rhamnose antibodies.

Carbohydrates are generally considered to be poorly immunogenic. Therefore, new approaches for enhancing their immunogenicity are important for the development of carbohydrates as vaccine components. We hypothesized that conjugation of an l-rhamnose (Rha) moiety to a carbohydrate antigen would enhance the antigenicity of the antigen in mice possessing anti-Rha antibodies via an antibody-dependent antigen uptake mechanism. To explore this hypothesis, we synthesized a single-molecule three-component vaccine containing the GalNAc-O-Thr (Tn) tumor-specific antigen, a 20 amino acid helper T-cell epitope (YAF) derived from an outer-membrane protein of Neisseria meningitides, and a Rha moiety. The vaccine was synthesized by automated Fmoc-based solid-phase peptide synthesis and deacetylated by brief treatment with NaOMe. Groups of female BALB/c mice were immunized and boosted with Rha-ovalbumin (Rha-OVA) formulated with either TiterMax Gold or Sigma Adjuvant System for a period of 35 days in order to determine optimal conditions for generating anti-Rha titers in mice. Anti-Rha antibody titers were >100 fold higher in groups of mice immunized with Rha-OVA than in the control groups. Mice producing anti-Rha were challenged with Rha-YAF-Tn or YAF-Tn. Sera collected from the groups initially immunized with Rha-OVA and later challenged with Rha-YAF-Tn showed a 2-fold increase in anti-Tn titer at 1/100 serum dilution relative to mice not immunized with Rha-OVA. An in vitro T-cell proliferation study using cells primed with either Rha-YAF-Tn or YAF-Tn was done to examine possible differences in antigen uptake and presentation due to anti-Rha antibody and chemical modification. Proliferation of T cells was stimulated by a 10-fold lower antigen concentration in the presence of Rha antibodies. The results strongly suggest that T cells present in the spleen were presented with higher concentrations of Rha-YAF-Tn as a result of the presence of the anti-Rha antibodies.

Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines.

Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on different carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glycoconjugate was then injected into mice and pre- and post-immune antibody levels in the mice sera were measured by enzyme-linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glycoconjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multidrug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines.

Recall immune memory: a new tool for generating late onset autoimmune myasthenia gravis.

Most patients with autoimmune myasthenia gravis (MG) produce autoantibodies against their muscle acetylcholine receptors (AChR), causing debilitating muscle weakness. Approximately 60% of MG patients first exhibit myasthenic symptoms after the age of 40. Yet, in the C57BL/6 mouse model of MG, older mice are resistant to induction of myasthenia gravis. To understand the immunological basis for this resistance, the effects of age on the B-cell responses to AChR from Torpedo californica, the inducing antigen, were addressed. As expected, the primary B-cell response was lower in 20-month-old mice than in 2-month-old mice; the isotype profile was not altered by age. When mice were re-immunized, the anti-T-AChR titers increased in both young and old animals, suggesting that a memory response was elicited. Importantly, memory B-cells activated in young animals were largely resistant to the age-associated loss of immune function and the recall memory response was vigorous. Furthermore, the antibodies produced in re-stimulated older mice were functional, as evidenced by the appearance of MG symptoms in some of these animals. Thus, by eliciting a recall memory response, the first examples of late onset MG in mice have been generated. By analogy, late onset MG in humans may be due to re-activation of B-cell responses initiated at a younger age.

Split tolerance in a novel transgenic model of autoimmune myasthenia gravis.

Because it is one of the few autoimmune disorders in which the target autoantigen has been definitively identified, myasthenia gravis (MG) provides a unique opportunity for testing basic concepts of immune tolerance. In most MG patients, Abs against the acetylcholine receptors (AChR) at the neuromuscular junction can be readily identified and have been directly shown to cause muscle weakness. T cells have also been implicated and appear to play a role in regulating the pathogenic B cells. A murine MG model, generated by immunizing mice with heterologous AChR from the electric fish Torpedo californica, has been used extensively. In these animals, Abs cross-react with murine AChR; however, the T cells do not. Thus, to study tolerance to AChR, a transgenic mouse model was generated in which the immunodominant Torpedo AChR (T-AChR) alpha subunit is expressed in appropriate tissues. Upon immunization, these mice showed greatly reduced T cell responses to T-AChR and the immunodominant alpha-chain peptide. Limiting dilution assays suggest the likely mechanism of tolerance is deletion or anergy. Despite this tolerance, immunization with intact T-AChR induced anti-AChR Abs, including Abs against the alpha subunit, and the incidence of MG-like symptoms was similar to that of wild-type animals. Furthermore, evidence suggests that this B cell response to the alpha-chain receives help from T cells directed against the other AChR polypeptides (beta, gamma, or delta). This model offers a novel opportunity to elucidate mechanisms of tolerance regulation to muscle AChR and to clarify the role of T cells in MG.