Jianan Wang

Active Tolerance Induction and Prevention of Autoimmune Diabetes by Immunogene Therapy Using Recombinant Adenoassociated Virus Expressing Glutamic Acid Decarboxylase 65 Peptide GAD500–585

Tolerance induction of autoreactive T cells against pancreatic β cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD500–585 could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD500–585 (3 × 1011 IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD500–585-responsive T lymphocytes, the enhanced GAD500–585-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-γ production; especially elevated anti-GAD500–585 IgG1 titer; and relatively unchanged anti-GAD500–585 IgG2b titer), the increased secretion of TGF-β, and the production of protective regulatory cells. Our studies also revealed that peptides 509–528, 570–585, and 554–546 in the region of GAD500–585 played important roles in rAAV/GAD500–585 immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD500–585, can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.

Interleukin-17-producing γδ + T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor-β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ+ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.

Complement C5a regulates IL‐17 by affecting the crosstalk between DC and γδ T cells in CLP‐induced sepsis

Complement 5a (C5a) and Interleukin‐17 (IL‐17) are two important inflammatory mediators in sepsis. Here we studied the mechanisms underlying regulation of IL‐17 by anaphylatoxin C5a. We found that C5a blockade increased the survival rate of mice following cecal ligation and puncture (CLP)‐induced sepsis and decreased IL‐17 expression in vivo. IL‐17 was secreted mainly by γδ T cells in this model. Importantly, our data suggest that C5a participates in the regulation of IL‐17 secretion by γδ T cells. Dendritic cells (DC) were found to act as a “bridge” between C5a and γδ T cells in a mechanism involving IL‐6 and transforming growth factor β (TGF‐β). These results imply that C5a affects the crosstalk between DC and γδ T cells during sepsis development, and this may result in a large production of inflammatory mediators such as IL‐17.

Blockade of complement activation product C5a activity using specific antibody attenuates intestinal damage in trinitrobenzene sulfonic acid induced model of colitis

Complement represents a chief component of innate immunity in host defense. However, excessive complement activation has been involved in the pathogenesis of inflammatory diseases. In this study, we investigated the contribution of complement to intestinal pathology of patients and rodents with inflammatory bowel disease. The expression of complement effectors (C3a and C3) was increased remarkably in inflamed colons of IBD patients compared with those of normal counterparts. In accordance with this, the sustained activation of complement in serum and colon (including elevated C3a and C5a levels, enhanced hemolytic activity, downregulated expression of C5a receptors) was observed, following the establishment of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, which peaked at 24 h. Mice pretreated with neutralizing anti-C5a antibodies (−2, 0, and 2 days after TNBS instillation) had significantly reduced weight loss and improved macroscopic/microscopic scores, comparable to the efficacy of prednisolone treatment. Strikingly, treatment with anti-C5a at 24 h after TNBS instillation showed remarkable therapeutic effects, whereas prednisolone did not. The efficacy of anti-C5a administration was associated with decreased release of proinflammatory chemokines and cytokines, inhibition of infiltration of neutrophils into colons, and enhanced Th2 response. These findings suggest a disease-promoting role of complement, particular C5a, in the pathology of TNBS-induced colitis in mice, indicating possible therapeutic potentials for C5a-specific antibody in IBD.

Essential roles of TGF-β in anti-CD3 antibody therapy: reversal of diabetes in nonobese diabetic mice independent of Foxp3+CD4+ regulatory T cells

Anti‐CD3 mAb have potentials to treat overt autoimmunity as reported recently. However, the underlying mechanisms remain unclear. In this report, using an animal model of type 1 diabetes, we found that TGF‐β1, an important immunoregulatory cytokine, plays a critical role in anti‐CD3‐mediated diabetes reversion and immune tolerance. Anti‐CD3 treatment increased the TGF‐β1 production, lasting for a long period of time, which contributed to maintaining peripheral tolerance by controlling pathogenic cells. Furthermore, we found that anti‐CD3 treatment did not increase the forkhead box p3+ (Foxp3+)CD4+ regulatory T cells (Tregs). When fractionated from anti‐CD3‐treated, remitting mice and cotransferred with splenic cells from diabetic NOD mice, these Tregs failed to inhibit diabetes development in NOD.scid mice. Moreover, we found that the depletion of these Tregs did not affect an anti‐CD3‐mediated, therapeutic effect and the level of TGF‐β1 production, which suggested that an increased level of TGF‐β1 may not derive from these Tregs. Thus, our data showed a dispensable role of Foxp3+CD4+ Tregs in anti‐CD3 antibody‐reversed diabetes in NOD mice. These findings may have an important implication for understanding the involved mechanisms responsible for immunomodulatory function of anti‐CD3 antibody on autoimmune diseases.

Regulation of IL-8 production by complement-activated product, C5a, in vitro and in vivo during sepsis.

Excessive complement-activated product complement 5a (C5a) has been implicated in the pathogenesis of sepsis development. Herein, we employed in vitro and in vivo models of sepsis to investigate the functional relationship between overtly produced C5a and IL-8. Our data revealed that C5a could strongly amplify IL-8 expression from human whole blood cells induced by LPS and other types of TLR agonists. ERK1/2 and p38, but not JNK, were mainly participated in signaling pathways for IL-8 production. In the whole blood stimulated by Escherichiacoli, C5a levels were quickly elevated and blockage of C5a significantly decreased E. coli-elicited IL-8 production. In the mouse model of sepsis induced by cecal ligation and puncture (CLP), the markedly increased keratinocyte-derived cytokine (KC) could be strongly suppressed by blockage of C5a. These data suggest that excessive C5a functions as a critical inflammatory mediator to enhance IL-8 production mainly through MAPK signaling pathways.

CD8+ regulatory T cells are responsible for GAD‐IgG gene‐transferred tolerance induction in NOD mice

Our previous studies demonstrated that lipopolysaccharide (LPS)‐stimulated splenocytes, retrovirally transduced with a glutamate decarboxylate 65 (GAD) and immunoglobulin G (IgG) fusion construct, can protect non‐obese diabetic (NOD) mice from diabetes by inducing GAD‐specific tolerance, and also that there are increased numbers of CD4+ regulatory T cells (Tregs) in GAD‐IgG‐treated NOD mice. However, little is known about the role of CD8+ Tregs in GAD‐IgG gene‐transferred tolerance induction in NOD mice. Here, we found that GAD‐IgG‐transduced splenocytes induced an increase in the number of CD8+ Foxp3+ Tregs in vitro. Using a T‐cell depletion assay, we found that, compared with undepleted groups, NOD recipients transfused with CD8– or CD8– CD25– GAD‐IgG‐transduced splenocytes showed a decrease in the percentage of CD8+ Foxp3+ T cells, a high incidence of diabetes, serious insulitis, GAD‐specific hyperresponsiveness at both the cellular and humoral levels, and changes in cytokine expression. These results indicate that CD8+ Tregs, which were induced in vitro by GAD‐IgG‐transduced splenocytes, were also responsible for GAD‐IgG gene‐transferred tolerance induction in NOD mice.

Glutamic Acid Decarboxylase-Derived Epitopes with Specific Domains Expand CD4+CD25+ Regulatory T Cells

Background CD4+CD25+ regulatory T cell (Treg)-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used. Methodology/Principal Findings Splenic lymphocytes from nonobese diabetic (NOD) mice were stimulated with different glutamic acid decarboxylase (GAD)-derived epitopes for 7–10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524–538)-expanded CD4+CD25+ T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509–528)- or p530 (GAD530–543)-expanded CD4+CD25+ T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes) between the above-mentioned epitopes and MHC class II I-Ag7 were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-Ag7, while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-Ag7. Furthermore, p524 bound to I-Ag7 more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570–585, p570), which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4+CD25+ T cells suppressed the onset of diabetes in NOD mice. Conclusions/Significance These data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of protective epitopes to expand functional Tregs.

cDNA cloning and function analysis of two novel erythroid differentiation related genes

Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen λ-gtll human cDNA expression library of fetal liver in order to obtain the relevant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins’ functions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigated the expression pattern ofEDRF1 andEDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid development, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

GAD-IgG-inducing CD4+Foxp3+Treg cells suppressing diabetes are involved in the increasing ratio of CD80+:CD86+ CELLS in NOD mice.

BACKGROUND Our previous studies have demonstrated that GAD-IgG-transduced splenocytes protect non-obese diabetic (NOD) mice from diabetes by in vitro-inducing CD4(+)Foxp3(+)Treg cells. However, the underlying mechanisms by which CD4(+)Foxp3(+)Treg cells suppress diabetes remain unclear. METHODS Seven-week-old female NOD mice were intravenously injected with GAD-IgG-transduced splenocytes. The ratio of CD80(+):CD86(+) cells in splenocytes was analyzed by flow cytometry. The effect of the ratio of CD80(+):CD86(+) cells on tolerance, diabetes prevention, and Foxp3 expression in GAD-IgG-treated NOD mice was tested by in vitro proliferation, in vivo antibody block, and semi-quantified RT-PCR, respectively. RESULTS We found that the ratio of CD80(+):CD86(+) cells increased in GAD-IgG-treated NOD mice. After CD4(+)Treg cells were depleted from GAD-IgG-transduced splenocytes before transfer, the ratio of CD80(+):CD86(+) cells decreased in NOD mice recipients. The increasing ratio of CD80(+):CD86(+) cells was positively associated with tolerance, diabetes prevention, and the high level of Foxp3 in GAD-IgG-treated NOD mice. CONCLUSIONS These findings suggest that the high ratio of CD80(+):CD86(+) cells is required for the suppressive function of GAD-IgG-inducing CD4(+)Foxp3(+)Treg cells in NOD mice.