Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PL×SJL F1 mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7–10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9–23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.

Recent studies have suggested the IL-4Rα expressed on lung epithelium is necessary for TH2-mediated goblet cell differentiation and mucus hypersecretion in a murine model of allergic lung disease. However, the IL-4Rα is expressed on numerous cell types that could contribute to the overall pathology and severity of asthma. The relative role of the receptor on these cells has not yet been conclusively delineated. To dissect the contribution of IL-4Rα in the development of pulmonary allergic responses, we generated murine radiation bone marrow (BM) chimeras. BM from IL-4Rα+ or IL-4Rα− mice was transferred into recipient mice that expressed or lacked IL-4Rα. In the absence of IL-4Rα in recipient mice, there was no goblet cell metaplasia or mucus hypersecretion in response to OVA, even in the presence of TH2 cells and substantial eosinophilic infiltration. More importantly, we found that expression of the IL-4Rα on a nonlymphoid, MHC class II+, BM-derived cell type contributes to the severity of inflammation and mucus production. These results suggest that IL-4 and IL-13 contribute to the development of allergic inflammation by stimulating a complex interaction between IL-4Rα+ cell types of both bone marrow and non-bone marrow origin.

A gene therapy model has been designed to induce tolerance to multiple epitopes expressed in-frame on a soluble IgG fusion protein scaffold. Tolerance to the λ repressor cI sequence p1-102 or its immunodominant epitopes (p12-26, p73-88) can be elicited when bone marrow (BM) or LPS blasts are transduced and injected into naive or even primed recipients. To explore the mechanism of tolerance, class II−/− (knockout, KO) BM cells were transduced with p1-102-IgG and transferred to irradiated recipients. These cells failed to induce tolerance to challenge with p1-102 epitopes, whereas transduced +/+ BM cells did. This supports the importance of class II MHC on the tolerogenic APC rather than secretion and representation in tolerogenesis. When BM cells from μMT KO mice were transfected with p12-26-IgG and injected into irradiated mice, these transduced BM cells also failed to induce tolerance to an immunodominant epitope. These results suggest the direct involvement of B cells in tolerance to p1-102 epitopes. IL-10 KO BM cells infected with a p12-26-IgG construct were still tolerogenic. Importantly, anti-CTLA-4 injections reversed tolerance in primed, but not in naive, recipients of transduced LPS blasts. These data emphasize the importance of MHC class II presentation, B cell involvement, and CTLA-4 engagement in induction and/or maintenance of tolerance.

IgG molecules can be highly tolerogenic carriers for associated antigens. Previously, we reported that recipients of bone marrow or lipopolysaccharide-stimulated B-cell blasts, both of which were retrovirally gene-transferred with an immunodominant peptide in-frame with the variable region of a murine IgG heavy chain, were rendered profoundly unresponsive to that epitope. To further investigate whether tolerance to larger molecules can be achieved via this approach and whether the IgG scaffold is important for induction and maintenance of immunological tolerance, we engineered two retroviral constructs encoding the cI repressor (MBAE-1-102 and MBAE-1-102-IgG) for gene transfer. Our results show that recipients of bone marrow or peripheral B cells, transduced with the MBAE-1-102-IgG recombinant, are hyporesponsive to p1-102. In addition, the self-IgG scaffold enhanced the induction and maintenance of such an immune hyporesponsiveness. Thus, our studies demonstrate that in vivo-expressed IgG heavy chain fusion protein can be processed and presented on the appropriate MHC class II, resulting in hyporesponsiveness to that antigen and offering an additional therapeutic approach to autoimmune diseases. Individuals normally develop tolerance to self-constituents during the development of the immune system. Tolerance induction, however, is a lifelong process and also must occur extrathymically (1). Moreover, the maintenance of this unresponsive state requires the persistence of antigen and continued induction in adults (2). The failure to discriminate between immunological self and nonself components leads to the clinical manifestations of autoimmunity. A number of experimental procedures have been proposed to induce tolerance to autoantigens and therefore to prevent and͞or reverse autoimmune diseases (3-5), although tolerance induction and maintenance in mature animals has proven difficult. Hence, novel methods need to be developed to promote tolerance induction in immunocompetent adults and to express the tolerogen in multipotential hematopoietic compartments for persistence of tolerogen and long-term maintenance of tolerance.Peptide fragments of multideterminant antigens can be divided into three main groups: dominant, subdominant, and cryptic epitopes (6-8). An immunodominant epitope is a peptide fragment specifically processed by antigen-presenting cells from a larger, multideterminant antigen and varies individually as a function of its MHC. Such an epitope is capable of binding to the MHC molecule, and this peptide͞MHC complex then is recognized by the T-cell repertoire (6, 7). Subdominant epitopes are the determinants that can stimulate native protein primed cells to proliferate, but less than dominant epitopes or the whole protein (7,8). In contrast, cryptic determinants are rarely revealed during antigen processing and therefore fail to activate T cells when the native antigen is used as immunogen (7,8). However, these hidden determinants might play a role in pathogenic autoimmune responses. One of the...

Allergic diseases, including asthma, represent a major threat to human health. Over the three last decades, their incidence has risen in western countries. Aspirin treatment has been shown to improve allergic diseases, especially asthma, and the decreased use of aspirin has been hypothesized to contribute to the increase in childhood asthma. Because salicylate compounds suppress a number of enzymatic activities, and signaling through IL-4R participates in the development of allergic responses, we tested the effect of salicylates on IL-4 signal transduction. We found that treatment of cell lines and primary cells with aspirin and salicylates, but not acetaminophen, inhibited the activation of STAT6 by IL-4 and IL-13. This effect correlated with the inhibition of IL-4-induced CD23 expression. Although salicylates inhibited the in vivo activation of Janus kinases, their kinase activity was not affected in vitro by salicylates, suggesting that other kinases were involved in IL-4-induced STAT6 activation. Furthermore, we found that an Src kinase was involved in STAT6 activation because 1) Src kinase activity was induced by IL-4, 2) Src kinase activity, but not Janus kinase, was inhibited by salicylates in vitro, 3) cells expressing viral Src had constitutive STAT6 phosphorylation, and 4) cells lacking Src showed low STAT6 phosphorylation in response to IL-4. Because STAT6 activation by IL-4 and IL-13 participates in the development of allergic diseases, our results provide a mechanism to explain the beneficial effects of aspirin and salicylate treatment of these diseases.

No abstract

I-A(g7) is a unique class II MHC molecule that is clearly associated with autoimmune diabetes in non-obese diabetic (NOD) mice. To determine if I-A(g7) is defective in its ability to deliver tolerogenic signals in vivo, H-2(g7) mice were nasally pretreated with antigen, prior to immunization, to induce antigen-specific regulation. Nasally pretreated NOR (H-2(g7)) and (NON).NOD (H-2(g7)) congenic mice showed responses similar to those of NON (H-2(nb1)), BALB/c (H-2(d)) and B10.PL (H-2(u)) mice-a reduced recall response and a deviated T(h) cytokine profile. However, we found that NOD (H-2(g7)) mice are comparatively resistant to immunological tolerance induced by nasal pretreatment, such that at the usually effective dose no significant reduction was seen in the proliferative recall responses to nominal antigen after immunization. (NOD x BALB/c)F(1) (H-2(g7/d)) and (NOD x NOR)F(1) (H-2(g7)) mice were similarly resistant to nasal-induced tolerance, although significantly higher nasal doses of antigen were able to overcome the resistance in NOD and F(1) mice. Interestingly, activated NOD T cells were resistant to cell death induced by re-stimulation with plate-bound anti-CD3. These results demonstrate that activated T cells in NOD mice are defective in their ability to respond to regulatory signals delivered in vivo or in vitro. Furthermore, NOD T cells have an increased resistance to tolerance induced by I-A(g7)-dependent (antigen) or I-A(g7)-independent (anti-CD3) mechanisms. Thus, while I-A(g7) may contribute to insulin-dependent diabetes mellitus by selecting a particular repertoire of self-reactive T cell clones, additional defects in the peripheral T cells themselves are required to allow the expansion of diabetogenic clones and the development of autoimmune disease.

Tolerance induction would be an ideal way to treat autoimmune diseases, especially if achievable in primed individuals. Moreover, specific tolerance would preclude the need for immunosuppressive treatment with its side effects. In this review, we will revisit the historical concepts of tolerance, and introduce a novel approach to tolerance via gene therapy. Our model system is based on the tolerogenicity of IgG carriers and B-cell antigen presentation. Results with this model demonstrate that it is simple and effective even in primed recipients, and has proven efficacy in three autoimmune models. We discuss the mechanisms of tolerance with fusion IgG's and analyze how our model of gene therapy approached can be utilized to fit in the future treatment of autoimmune conditions.