Prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: We recently found that activation of the type III histone deacetylase sirtuin 1 suppresses T cell immune responses. Here we sought to determine the therapeutic potential of the sirtuin 1 activator resveratrol in the treatment of diabetes in the NOD mouse model of type 1 diabetes and the mechanisms underlying such potential. METHODS: NOD mice were fed or subcutaneously injected with resveratrol and evaluated for development of diabetes. Splenocytes from resveratrol-treated and control mice were analysed by gene array. The altered expression of inflammatory genes induced by resveratrol was validated and the role of changed gene expression in prevention of diabetes was determined. RESULTS: Resveratrol administration potently prevented and treated type 1 diabetes in NOD mice. Gene array analysis indicated a dramatic decrease in expression of Ccr6, which encodes chemokine (C-C motif) receptor (CCR) 6, in the splenocytes from resveratrol-treated mice. CCR6 abundance on IL-17-producing cells and CD11b(+)F4/80(hi) macrophages was inhibited by resveratrol treatment. Interestingly, CCR6(+) IL-17-producing cells and CD11b(+)F4/80(hi) macrophages accumulated in the spleens and pancreatic lymph nodes, but their presence in the pancreas was reduced, suggesting that resveratrol blocks their migration from peripheral lymphoid organs to the pancreas. Indeed, the migration of splenocytes toward media containing chemokine (C-C motif) ligand 20 (CCL20) was impaired by resveratrol treatment. CCL20 peptides, which block CCR6 binding to CCL20, inhibited development of type 1 diabetes. CONCLUSIONS/INTERPRETATION: Inhibition of CCR6-mediated migration of inflammatory cells by resveratrol may provide a powerful approach for treatment of type 1 diabetes and possibly of other inflammatory diseases.

Induction of a cross-reactive idiotype dextran-positive antibody response in two IgH-Cb mouse strains treated with anti-J558 cross-reactive idiotype antibodies.

The effect of IdX-specific rabbit and allogeneic antiidiotype antibodies (Ab2) was investigated in vivo in Igh-Cb mouse strains with respect to the induction of a cross-reactive idiotype (IdX)-positive anti-alpha (1-3) Dextran (Dex) response. These C.B20 and C57Bl/6 mice have an allotype-linked incapacity to respond with IdX-positive anti-alpha (1-3) Dex antibodies upon conventional immunization with Dex B1355. 7 d after the rabbit Ab2 injections, IdX-positive Ig (Ab3) and IdX-positive anti-alpha (1-3) Dex antibodies (Ab1') were detected in the sera of each tested mouse. The affinity-purified Ab1' were idiotypically indistinguishable from reference BALB/c IdX-positive myeloma proteins and BALB/c anti-alpha (1-3) Dex antibodies (Ab1) in a competitive inhibition radioimmunoassay, while Ab3 Ig appeared idiotypically deficient and did not bind to Dex. The response to the alpha (1-6) linkage of Dex was not affected in these mice. A large fraction of the Ab1' and Ab3 responses of both mouse strains were of the IgG1 class. The Ab1' antibodies differed from BALB/c Ab1 by lower relative binding to five of eight tested Dex, and by expressing the Igh4b allotype determinants on the IgG1 antibodies. This study identifies the products of a VHDex gene that appears to be under regulatory control in the Ighb mice. Its association with the b haplotype suggests that this gene may differ structurally from the BALB/c VHDex gene.

Neonatal exposure to a self-peptide-immunoglobulin chimera circumvents the use of adjuvant and confers resistance to autoimmune disease by a novel mechanism involving interleukin 4 lymph node deviation and interferon gamma-mediated splenic anergy.

Induction of neonatal T cell tolerance to soluble antigens requires the use of incomplete Freund's adjuvant (IFA). The side effects that could be associated with IFA and the ill-defined mechanism underlying neonatal tolerance are setbacks for this otherwise attractive strategy for prevention of T cell-mediated autoimmune diseases. Presumably, IFA contributes a slow antigen release and induction of cytokines influential in T cell differentiation. Immunoglobulins (Igs) have long half-lives and could induce cytokine secretion by binding to Fc receptors on target cells. Our hypothesis was that peptide delivery by Igs may circumvent the use of IFA and induce neonatal tolerance that could confer resistance to autoimmunity. To address this issue we used the proteolipid protein (PLP) sequence 139-151 (hereafter referred to as PLP1), which is encephalitogenic and induces experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. PLP1 was expressed on an Ig, and the resulting Ig-PLP1 chimera when injected in saline into newborn mice confers resistance to EAE induction later in life. Mice injected with Ig-PLP1 at birth and challenged as adults with PLP1 developed T cell proliferation in the lymph node but not in the spleen, whereas control mice injected with Ig-W, the parental Ig not including PLP1, developed T cell responses in both lymphoid organs. The lymph node T cells from Ig-PLP1 recipient mice were deviated and produced interleukin (IL)-4 instead of IL-2, whereas the spleen cells, although nonproliferative, produced IL-2 but not interferon (IFN)-gamma. Exogenous IFN-gamma, as well as IL-12, restored splenic proliferation in an antigen specific manner. IL-12-rescued T cells continued to secrete IL-2 and regained the ability to produce IFN-gamma. In vivo, administration of anti-IL-4 antibody or IL-12 restored disease severity. Therefore, adjuvant-free induced neonatal tolerance prevents autoimmunity by an organ-specific regulation of T cells that involves both immune deviation and a new form of cytokine- dependent T cell anergy.

Presentation of a T cell receptor antagonist peptide by immunoglobulins ablates activation of T cells by a synthetic peptide or proteins requiring endocytic processing.

T cell receptor (TCR) antagonism is being considered for inactivation of aggressive T cells and reversal of T cell-mediated autoimmune diseases. TCR antagonist peptides silence aggressive T cells and reverse experimental allergic encephalomyelitis induced with free peptides. However, it is not clear whether free antagonist peptides could reverse natural disease where the antigen is presumably available for endocytic processing and peptides gain access to newly synthesized class II MHC molecules. Using an efficient endocytic presentation system, we demonstrate that a proteolipid protein (PLP) TCR antagonist peptide (PLP-LR) presented on an Ig molecule (Ig-PLP-LR) abrogates the activation of T cells stimulated with free encephalitogenic PLP peptide (PLP1), native PLP, or an Ig containing PLP1 peptide (Ig-PLP1). Free PLP-LR abolishes T cell activation when the stimulator is free PLP1 peptide, but has no measurable effect when the stimulator is the native PLP or Ig-PLP1. In vivo, Ig-PLP1 induces a T cell response to PLP1 peptide. However, when coadministered with Ig-PLP-LR, the response to PLP1 peptide is markedly reduced whereas the response to PLP-LR is normal. Free PLP-LR coadministered with Ig-PLP1 has no effect on the T cell response to PLP1. These findings indicate that endocytic presentation of an antagonist peptide by Ig outcompete both external and endocytic agonist peptides whereas free antagonist hinders external but not endocytic agonist peptide. Direct contact with antagonist ligand and/or trans-regulation by PLP-LR-specific T cells may be the operative mechanism for Ig-PLP-LR-mediated downregulation of PLP1-specific T cells in vivo. Efficient endocytic presentation of antagonist peptides, which is the fundamental event for either mechanism, may be critical for reversal of spontaneous T cell-mediated autoimmune diseases where incessant endocytic antigen processing could be responsible for T cell aggressivity.

Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus.

Several prior reports have identified peptides that are naturally associated with major histocompatibility complex (MHC) class II molecules on presenting cells. We have examined the delivery of a peptide from exogenous sources to MHC class II molecules. The peptide derives from the influenza virus hemagglutinin (HA) and activates a CD4+ T cell hybridoma. In functional assays of antigen presentation, this epitope is delivered effectively to T cells either in the context of influenza virus or chimeric immunoglobulin (Ig) molecules (Ig-HA) in which the peptide has replaced the CDR3 loop of the heavy chain. We find that the identical 11-mer peptide can be isolated from mouse MHC class II antigens whether the exogenous source of peptide is free HA peptide, the Ig-HA chimera, or ultraviolet-inactivated PR8 influenza virus. The Ig-HA chimera proves to be the most efficient vehicle for charging class II molecules via the exogenous route. Given the fact that self Igs represent natural long-lived carriers, we suggest that antigenized Igs have considerable potential for peptide delivery to MHC molecules in situ.

Coupling of peripheral tolerance to endogenous interleukin 10 promotes effective modulation of myelin-activated T cells and ameliorates experimental allergic encephalomyelitis.

Several immune-based approaches are being considered for modulation of inflammatory T cells and amelioration of autoimmune diseases. The most recent strategies include simulation of peripheral self-tolerance by injection of adjuvant free antigen, local delivery of cytokines by genetically altered T cells, and interference with the function of costimulatory molecules. Although promising results have been obtained from these studies that define mechanisms of T cell modulation, efficacy, practicality, and toxicity, concerns remain unsolved, thereby justifying further investigations to define alternatives for effective downregulation of aggressive T cells. In prior studies, we demonstrated that an immunoglobulin (Ig) chimera carrying the encephalitogenic proteolipid protein (PLP)1 peptide corresponding to amino acid sequence 139-151 of PLP, Ig-PLP1, is presented to T cells approximately 100-fold better than free PLP1. Here, we demonstrate that aggregation endows Ig-PLP1 with an additional feature, namely, induction of interleukin (IL)-10 production by macrophages and dendritic cells, both of which are antigen-presenting cells (APCs). These functions synergize in vivo and drive effective modulation of autoimmunity. Indeed, it is shown that animals with ongoing active experimental allergic encephalomyelitis dramatically reduce the severity of their paralysis when treated with adjuvant free aggregated Ig-PLP1. Moreover, IL-10 displays bystander antagonism on unrelated autoreactive T cells, allowing for reversal of disease involving multiple epitopes. Therefore, aggregated Ig-PLP1 likely brings together a peripheral T cell tolerance mechanism emanating from peptide presentation by APCs expressing suboptimal costimulatory molecules and IL-10 bystander suppression to drive a dual-modal T cell modulation system effective for reversal of autoimmunity involving several epitopes and diverse T cell specificities.

High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire.

The autoreactive T cells that escape central tolerance and form the peripheral self-reactive repertoire determine both susceptibility to autoimmune disease and the epitope dominance of a specific autoantigen. SJL (H-2(s)) mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP). The two major encephalitogenic epitopes of PLP (PLP 139-151 and PLP 178-191) bind to IA(s) with similar affinity; however, the immune response to the PLP 139-151 epitope is always dominant. The immunodominance of the PLP 139-151 epitope in SJL mice appears to be due to the presence of expanded numbers of T cells (frequency of 1/20,000 CD4(+) cells) reactive to PLP 139-151 in the peripheral repertoire of naive mice. Neither the PLP autoantigen nor infectious environmental agents appear to be responsible for this expanded repertoire, as endogenous PLP 139-151 reactivity is found in both PLP-deficient and germ-free mice. The high frequency of PLP 139-151-reactive T cells in SJL mice is partly due to lack of thymic deletion to PLP 139-151, as the DM20 isoform of PLP (which lacks residues 116-150) is more abundantly expressed in the thymus than full-length PLP. Reexpression of PLP 139-151 in the embryonic thymus results in a significant reduction of PLP 139-151-reactive precursors in naive mice. Thus, escape from central tolerance, combined with peripheral expansion by cross-reactive antigen(s), appears to be responsible for the high frequency of PLP 139-151-reactive T cells.

Presentation of a viral T cell epitope expressed in the CDR3 region of a self immunoglobulin molecule.

Synthetic peptides corresponding to microbial epitopes stimulate T cell immunity but their immunogenicity is poor and their half-lives are short. A viral epitope inserted into the complementarity-determining region 3 (CDR3) loop of the heavy chain of a self immunoglobulin (Ig) molecule was generated from the Ig context and was presented by I-Ed class II molecules to virus-specific, CD4+ T cells. Chimeric Ig-peptide was presented 100 to 1000 times more efficiently than free synthetic peptide and was able to prime virus-specific T cells in vivo. These features suggest that antigenized Ig can provide an improved and safe vaccine for the presentation of microbial and other peptides.

Monoclonal antibodies against oncofetal mucin M1 antigens associated with precancerous colonic mucosae.

We obtained seven monoclonal antibodies (MAb) against a gastric mucin of an ALeb patient. By immunoperoxidase on normal gastric mucosae, two MAbs (3-3A and 2-25 LE) reacted exclusively with the A and Lewis-positive individuals, respectively; the five other MAbs (1-13 M1, 2-11 M1, 2-12 M1, 9-13 M1, and 58 M1) stained the mucus cells of surface gastric epithelium independently of ABO or Lewis status. They did not stain normal colonic mucosae, but did stain fetal and precancerous colonic mucosae. Using serial sections, each anti-M1 MAb stained the same goblet cells in fetal and precancerous colon. Extensive search of other normal tissues showed that M1 antigens were restricted to the epithelium embryologically derived from the foregut (gastric and bronchial epithelium) and from Müllerian ducts (mucus cells of endocervix and prostatic utriculus). Some differences in the reactivities of the various anti-M1 MAb were observed in subesophageal, subtracheal, and endocervical mucus cells, suggesting that each anti-M1 MAb characterized a different M1 epitope. A mixture of these five anti-M1 MAbs allowed the estimation of M1 mucus modification in the precancerous colonic mucosae with a sensitivity near to that obtained with polyclonal anti-M1 antibodies. Papain and mercaptoethanol treatments destroyed the M1 epitopes, at variance with the A- or Lewis-related antigens. Our results therefore suggest that the expression of M1 epitopes in precancerous colonic mucosae cannot be due exclusively to alterations in mucin glycosylation but may be related to the reexpression of antigens associated with native gastric mucin which is normally produced by the fetal colon during the sixth month of gestation.

Regulation of the response to alpha(1-3) dextran: an anti-dextran associated idiotope of BALB/c mice is also expressed on A/J anti-NIP antibodies.

The functional properties of a mAb reactive to the B5 idiotope were analyzed in BALB/c, A/J and C.B20 mice. the B5 idiotope was specifically associated with the alpha(1-3) DEX response in a strain-specific manner in BALB/c mice (responder strain). It was serologically distinct from the previously described IdX idiotype and overlapped on part of the IdX+ and IdX- BALB/c anti-alpha(1-3) DEX response. While treatment of adult BALB/c mice with mAb B5 enhanced the serum anti-alpha(1-3) DEX antibody synthesis and induced de novo antibody synthesis in non-responder C.B20 mice, A/J non-responder mice remained unresponsive, despite elevated levels of serum B5+ dextran non-binding Ab3 immunoglobulins. However, in some of these A/J mice, we detected an enhancement of anti-NIP antibody synthesis. This anti-NIP antibody component was (1) specifically induced by mAb B5, (2) found in A/J, but not in BALB/c or C.B20 mice, and (3) specific towards the NIP hapten. These A/J anti-NIP antibodies bore either the lambda 1 or the kappa light chain and some of them also expressed the B5 idiotope. The B5 id thus appears as a typical id determinant shared by antibodies having different binding specificities, and it may have a role in the regulation of these responses.

A sensitive method to detect defined peptide among those eluted from murine MHC class II molecules.

We developed a sensitive competitive inhibition radioimmunoassay able to trace pmoles of a defined peptide eluted from major histocompatibility complex (MHC) class II molecules that were subsequently fractionated by RP-HPLC. In this assay we used a model synthetic peptide corresponding to amino acid residues 110-120 from the hemagglutinin (HA) of PR8 influenza virus, and affinity purified rabbit antibodies specific for this peptide. The HA110-120 peptide binds to I-Ed class II molecules on the surface of APCs and is recognized by specific CD4+ T helper cells. 2PK3 B lymphoma cells (H-2d) were pulsed with HA110-120 peptide or PR8 virus, lysed, the MHC class II molecules extracted, and bound peptides eluted. After separation by RP-HPLC, the fractions were tested for inhibition of the binding of rabbit anti-HA110-120 antibodies to peptide coated microtiter plates. A significant inhibitory activity was observed with one peak when the cells were pulsed with HA110-120 peptide and two peaks when pulsed with PR8 virus. The inhibitory activity was correlated with the presence of HA110-120 peptide as demonstrated by peptide sequencing. The assay is reproducible and sensitive to 1 pmol of antigenic peptide. This assay can be useful to identify microbial peptides with defined structure and antigenicity among the multiple peptides bound to class II molecules.

Adjuvanticity of stearyl tyrosine on the antibody response to peptide 503-535 from HIV gp160.

In this present report we compare the humoral immune response induced by immunization with an HIV-1 gp160 peptide corresponding to amino acid sequence 503-535 complexed with different adjuvants. Specifically, the antipeptide, anti-HIV-1 gp160 and neutralizing antibody responses were measured in groups of mice and baboons that received peptide 503-535 conjugated to a carrier protein in either saline, alum, or stearyl tyrosine. The highest antibody responses were induced when mice and baboons were immunized with peptide adsorbed on stearyl tyrosine. These data indicate that stearyl tyrosine represents a potent candidate as a nontoxic adjuvant not only for subunit viral vaccines, but also for HIV peptides.

Multi-modal antigen specific therapy for autoimmunity.

Peripheral tolerance, represents an attractive strategy to down-regulate previously activated T cells and suppress an ongoing disease. Herein, immunoglobulins (Igs) were used to deliver self and altered self peptides for efficient peptide presentation without costimulation to test for modulation of experimental allergic encephalomyelitis (EAE). Accordingly, the encephalitogenic proteolipid protein (PLP) sequence 139-151 (referred to as PLP1) and an altered form of PLP1 known as PLP-LR were genetically expressed on Igs and the resulting Ig-PLP1 and Ig-PLP-LR were tested for efficient presentation of the peptides and for amelioration of ongoing EAE. Evidence is presented indicating that Ig-PLP1 as well as Ig-PLP-LR given in saline to mice with ongoing clinical EAE suppresses subsequent relapses. However, aggregation of both chimeras allows crosslinking of Fcgamma receptors (FcgammaRs) and induction of IL-10 production by APCs but does not promote the up-regulation of costimulatory molecules. Consequently, IL-10 displays bystander suppression and synergizes with presentation without costimulation to drive effective modulation of EAE. As Ig-PLP1 is more potent than Ig-PLP-LR in the down-regulation of T cells, we conclude that peptide affinity plays a critical role in this multi-modal approach of T cell modulation.

Engineered immunoglobulin molecules as vehicles for T cell epitopes.

The variable regions (V) of immunoglobulins (Ig) bear antigenic determinants that can stimulate both humoral and cellular immune responses subsequent to hetero, allo or iso-immunization. The expression of these determinants by Igs usually correlates with the presence of specific amino acid residues within the CDR loops of the V regions. Since the CDR loops varies in length, we reasoned that they would represent permissive sites to insert foreign peptides and create antigenized Igs expressing selected determinants. Taking advantage of these properties and the fact that Igs are self and long-lived molecules, we expressed a CTL and a helper epitope of influenza virus nucleoprotein and hemagglutinin respectively, within the heavy chain CDR3 loop of an anti-arsonate antibody. We found that foreign peptides comprised of 11 to 15 amino acid residues can be expressed within the V region of the heavy chain without alteration of pairing with the light chain. More striking, the cellular processing machinery is able to generate the peptides from the Ig context which were then recognized by specific T cells. Furthermore, the engineered Igs are able to induce T cell responses specific for the inserted peptide and for influenza virus. The use of engineered Ig molecules as vehicles for T and B cell peptides might represent a valuable strategy to generate safe, long lived reagents able to stimulate strong specific immune responses. This would then overcome the short half life of synthetic peptides based vaccines and the side effects seen in case of recombinant viral proteins or inactivated viruses based vaccines.

Neonatal tolerant immunity for vaccination against autoimmunity.

Autoimmunity arises when the immune system no longer tolerates self and precipitates lymphocyte reactivity against our own antigens. Although the developing T cell repertoire is constantly purging, self-recognition events do exist when such tight control is evaded and autoreactive lymphocytes escape the thymus (the sites of T cell development) and migrate to the periphery. Upon activation these autoreactive cells may exert aggressive behavior toward one's own tissues and organs leading to autoimmune disease. Multiple sclerosis, Rheumatoid arthritis, and type I diabetes are autoimmune diseases mediated by autoreactive T cells. A logical approach to prevent such autoimmunity would be to reprogram those lymphocytes to tolerate the self antigen. Injection of antigen at the neonatal stage promotes a state of tolerance such that successive encounter with antigen does not precipitate aggressive reactions. The mechanism underlying neonatal tolerance involves priming of T cells whose effector functions do not cause inflammatory reactions upon recognition of antigen but rather induce protective immunity. This form of tolerant immunity provides an attractive strategy for vaccination against autoimmunity. Herein, it is shown that neonatal exposure to a self-peptide-immunoglobulin chimera drives a tolerant immunity toward the self-peptide and protects against the autoimmune disease, experimental allergic encephalomyelitis.

The LY-1 gene expression in murine hybridomas producing autoantibodies.

Studies presented here demonstrate the expression of the Ly-1 gene and the detection of the Ly-1 cytodifferentiation antigen in murine hybridomas producing autoantibodies. We examined the transcription of the Ly-1 gene in thymocytes and 140 hybridomas producing autoantibodies of various specificities which were obtained from normal and autoimmune disease prone mouse strains. As previously demonstrated thymocytes stain brightly for Ly-1 by immunofluorescence and express Ly-1 transcripts. In our panel of hybridomas producing autoantibodies Ly-1 transcripts were detected in 31 (45%) out of 69 NZB hybridomas and 7 (88%) out of 8 viable motheaten hybridomas. S1 nuclease protection experiments showed that Ly-1 transcripts detected in thymocytes and B cells are the product of the same gene. The B cell transcripts are functional since immunofluorescence and Western data presented here detected the Ly-1 protein in hybridomas cells which were found to transcribe the Ly-1 gene. Interestingly a polymorphic transcription of the Ly-1 gene was observed in B cells and B cell hybridomas as compared to thymocytes. Our results obtained in the hybridoma system firmly establish a major contribution of the Ly-1 B cell subset to the production of DNA specific autoantibodies and a smaller contribution to the production of rheumatoid factors and "natural", multispecific autoantibodies.

Purification of antigenized immunoglobulins derivatized with monomethoxypolyethylene glycol.

Genetically engineered immunoglobulins (Igs) carrying viral B or T cell peptides in the CDR3 loop, function as efficient delivery system of the defined viral epitopes. Two of these antigenized Igs (AIgs) were derivatized with 2-O-monomethoxypolyethylene glycol-4,6-dichloro-s-triazine (mPEG). Herein, we describe a two-step strategy to purify mPEG-derivatized AIgs (AIgs-mPEG). Unreacted mPEG polymers were removed by size-exclusion chromatography using ammonium hydrogencarbonate as a buffer system. Mildly PEGylated AIgs were isolated from free and highly derivatized AIgs by anion-exchange chromatography. Electrophoretic analysis indicated that the AIgs-mPEG preparation contained less than 4 x 10(-4) M unreacted mPEG. This strategy may be applied to other mPEG-derivatized monoclonal antibodies.

The significance of idiotype-anti-idiotype interactions in the activation of self-reactive clones.

Autoantibodies against a wide range of self antigens have been found in the serum of normal mice and healthy humans. It is now accepted that the production of self reactive clones is not an aberration and that the immune system does not purge itself of the previously called "horror" clones. Recently it has been shown that the majority of autoantibodies are polyspecific and able to react with both self and foreign antigens. Following these observations, it was speculated that the activation of self reactive clones may result from common shared epitopes between self and foreign (bacterial, viral and parasitic) antigens. In this report, we show on the one hand that autoantibodies specific for Sm antigen express an idiotype originally found on antibodies specific for bacterial levan. On the other hand, using an anti-idiotypic antibody bearing the internal image of the glutamic-tyrosine polymer, we succeed in inducing polyspecific antibodies reacting with GT and self antigens in both normal and autoimmune disease prone mice. These findings may reflect one side of the mechanism(s) behind the activation of self reactive clones. The significance of idiotype-anti-idiotype interactions in the activation of self reactive clones which may be responsible for autoimmune diseases is discussed.