The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90 -104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp 93 to Leu 94 occurs through the ␤-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiological conditions. These posttranslationally modified peptides may serve as an early immunologic stimulus in autoimmune disease.The immune system has evolved to be tolerant of self-proteins by the deletion of autoreactive cells in the thymus or bone marrow and by the establishment of B and T lymphocyte anergy in the peripheral circulation (1-6). These mechanisms are based on the presentation of a vast array of self-peptides to the lymphoid repertoire. Despite the efforts to instruct the immune system to ignore self-tissues, the appearance of various autoimmune diseases demonstrates that tolerance to self-antigens is not perfect. Flaws in the development of immune tolerance can be revealed by the immunization of animal models with a variety of self-peptides leading to B and T cell autoimmunity as well as autoimmune-mediated pathology (7-12).How tolerance is broken in the initiation of autoimmunity is not completely understood. The immunization of mice with a single self-peptide, the amino-terminal 11 amino acids of myelin basic protein (MBP) 1 in complete Freund's adjuvant can elicit pathology resembling that of human multiple sclerosis (7). The induction of disease requires a post-translationally acetylated form of MBP peptide 1-11. While this disease can be elicited with a single self-peptide or even with T cells of a single specificity, the autoimmune response diversifies to many sites on the MBP over the course of the disease. T cell responses originate with the dominant single self-peptide but rapidly evolve to include other cryptic peptide epitopes within MBP. Similar observations of determinant spreading have been made in murine models of diabetes and systemic lupus erythematosus (SLE), two diseases arising spontaneously in susceptible strains of mice (8,11,13).Antinuclear autoantibodies specific for double-stranded DNA and the U1/Sm ribonucleoprotein particle (snRNP) are diagnostic markers ...

Anti-Sm and anti-ssDNA transgenic (Tg) mice were generated using the VH-D-JH rearrangement of an anti-Sm hybridoma of MRL/Mp-lpr/lpr origin. B cells of each specificity account for 15%-35% of the splenic repertoire, but no circulating anti-Sm or anti-ssDNA antibodies are detected. Most autoreactive cells exhibit an immature B cell phenotype and have short half-lives equivalent to those of non-Tg immature B cells. However, at least some anti-Sm B cells are functional, because immunization with murine snRNPs induces anti-Sm secretion. We propose that anti-Sm and anti-ssDNA are eliminated during the transition to mature B cells and that this late stage of tolerance induction is consequential to their spontaneous activation in murine lupus.

Both B cells and dendritic cells (DCs) have been implicated as autoantigen-presenting cells in the activation of self-reactive T cells. However, most self-proteins are ubiquitously and/or developmentally expressed, making it difficult to determine the source and the exposure of autoantigens to APCs in a controlled manner. In this study, we have used an Ig transgenic mouse model to examine the mechanisms by which B cells and other APCs acquire and present lupus autoantigens in vivo. Targeting a lupus autoantigen, the small nuclear ribonucleoprotein particle D protein, to the BCR activates autoreactive T cells in the periphery. Our in vivo studies demonstrate that autoantigen-specific B cells, when present in the repertoire, are the first subset of APCs to capture and present self-proteins for activating T cells. Thereafter, DCs acquire self-Ag and become effective APCs for stimulating the same subsets of autoreactive T cells. This mechanism provides one explanation of how early steps in autoimmunity can focus responses, via BCR, at a small group of self-proteins among the total milieu of intracellular self-proteins. Subsequently, DCs and other professional APCs may then amplify and perpetuate the autoimmune T cell response.

The development of immune tolerance is dependent on the expression of self-peptides in the thymus and bone marrow during lymphocyte development. However, not all self-antigens are expressed in the thymus, particularly for proteins that become post-translationally modified during other biological processes in a cell. We have found that one such post-translational modification, the spontaneous conversion of an aspartic acid to isoaspartic acid (isoAsp), causes ignored self-antigens to become immunogenic. In order to determine the mechanism for this autoimmune response, pigeon cytochrome c peptide 88-104 (PCC p88-104) was synthesized with and without an isoaspartyl residue. Each form was digested with cathepsin D, an enzyme involved in antigen processing. The products of cathepsin digestion were dramatically different between the two forms of self-protein suggesting that cryptic self-peptides may be revealed to the immune system by natural modifications to self-proteins. This observation also held true if whole PCC protein contained isoaspartyl residues was digested with cathespsin D. Additionally, AND transgenic TCR T cells (recognizing PCC 88-104) proliferated to a greater extent in response to isoaspartyl PCC as compared to the normal form of PCC. These finding demonstrate the importance of post-translational modifications in shaping autoimmune responses in and the development of tolerance to self-proteins.

It is clear that many factors can perturb T cell homeostasis that is critical in the maintenance of immune tolerance. Defects in the molecules that regulate homeostasis can lead to autoimmune pathology. This simple immunologic concept is complicated by the fact that many self-proteins undergo spontaneous posttranslational modifications that affect their biological functions. This is the case in the spontaneous conversion of aspartyl residues to isoaspartyl residues, a modification occurring at physiological pH and under conditions of cell stress and aging. We have examined the effect of isoaspartyl modifications on the effector functions of T lymphocytes in vivo using mice lacking the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT). PCMT−/− CD4+ T cells exhibit increased proliferation in response to mitogen and Ag receptor stimulation as compared with wild-type CD4+ T cells. Hyperproliferation is marked by increased phosphorylation of members of both the TCR and CD28 signaling pathways. Wild-type mice reconstituted with PCMT−/− bone marrow develop high titers of anti-DNA autoantibodies and kidney pathology typical of that found in systemic lupus erythematosus. These observations, coupled with the fact that humans have polymorphisms in the pcmt gene, suggest that isoaspartyl self-proteins may alter the maintenance of peripheral immune tolerance.

B cells play an active role in directing immunity against specific proteins in part because of their capacity to sequester antigen via B cell receptor (BCR). Our prior findings indicate that B cells can initiate an immune response in vivo to self proteins independent of other antigen‐presenting cells (APC). However, these studies also demonstrated that both dendritic cells and macrophages are important in the ongoing immune response. The present work illustrates a mechanism by which antigen acquired by B cells through BCR is specifically transferred to other APC, in particular, macrophages. The transfer of antigen is dependent on the specificity of BCR and requires direct contact between the cells, but does not require MHC compatibility between the cells and is independent of the activation state of macrophages. Antigen transfer is functional, in that macrophages, which received B cell derived‐antigen, can activate CD4 T cells. Overall, these results define a novel mechanism by which B cells can focus immunity toward a specific antigen and transfer the ability to activate CD4 T cells to other APC.

A hallmark of the immune system is the ability to ignore selfantigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of isoAsp TRP-2 peptide-immunized mice demonstrated that CD8 ؉ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8 ؉ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, posttranslational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens. Isoaspartyl (iso-Asp)3 post-translational protein modification occurs at physiological temperatures and pH values (1), and a wide variety of intracellular and extracellular proteins has been identified with iso-Asp modifications. Iso-Asp formation is prevalent in stressed (heat shock) or aged cells (2), and the presence of iso-Asp residues in proteins has been shown to decrease biological functions in some proteins (3). Iso-Asp residues can be repaired by the ubiquitous enzyme protein carboxyl methyltransferase (4). The importance of this enzyme to normal cellular biology is demonstrated in that mice with an insertional mutation in the protein carboxyl methyltransferase gene die at about 6 weeks of age (5).Another important, but under explored, consequence of isoaspartate formation is the altered immunogenicity of peptides/proteins. Our laboratory has shown that the spontaneous conversion of an aspartic acid to an isoaspartic acid (Fig. 1) induces both T and B cell immunity to model self-antigens (6). The B lymphocyte and antibody-mediated immune response is directed at not only the iso-Asp-modified form of self-antigen but also the native form of the self-antigen (6). Moreover, studies have shown that the post-translational modification of amino acids, such as phosphorylation, glycosylation, or citrullination, in self-antigens can induce an immune response to a self-antigen that was previously ignored by the immune system (7-9).In this study, we describe our efforts to apply this concept to developing immunity to tumor antigens. Many tumor-associated antigens are self-antigens that are often poorly recognized by the immune system. In an effort to make tumor-associated antigens more immunogenic, we introduced an isoaspartate into the peptide sequence of self-tumor proteins. We chose to look at the melanocyte differentiation antigen TRP-2 (tyrosinase-related protein-2) because melanoma continues to be an aggressive and difficult to treat cancer, with over 59,000 new cases d...

Modifications of both DNA and protein by methylation are key factors in normal T and B cell immune responses as well as in the development of autoimmune disease. For example, the failure to maintain the methylation status of CpG dinucleotides in DNA triggers T cell autoreactivity. Methylated proteins are known targets of autoimmunity, including the symmetrical dimethylarginine residues of SmD1 and SmD3 in SLE. Herein, we demonstrate that altering the metabolism of S-adenosylmethionine (SAM), the major methyl donor for transmethylation reactions, can suppress T cell immunity. A by-product of SAM metabolism, 5′-Deoxy-5′-methylthioadenosine (MTA), and an indirect inhibitor of methyltransferases, inhibits T cell responses including T cell activation markers, Th1/Th2 cytokines and TCR-related signaling events. Moreover, treatment of the lupus-prone MRL/lpr mouse with MTA markedly ameliorates splenomegaly, lymphadenopathy, autoantibody titers as well as IgG deposition and cellular infiltration in the kidney. Incubation of cells with SAM, which increases intracellular MTA levels, inhibits both TCR-mediated T cell proliferation and BCR (anti-IgM)-triggered B cell proliferation in a dose-dependent manner. These studies define the central role of MTA and SAM in immune responses and provide a simple approach to altering lymphocyte transmethylation and T cell mediated autoimmune syndromes.