Renelle J. Gee

Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins.

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 Asp93 to Leu94 occurs through the β-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 cpeptide 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 post-translationally modified peptides may serve as an early immunologic stimulus in autoimmune disease.

Autoreactive B Cell Regulation: Peripheral Induction of Developmental Arrest by Lupus-Associated Autoantigens

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.

B Cells Drive Early T Cell Autoimmunity In Vivo prior to Dendritic Cell-Mediated Autoantigen Presentation

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.

A failure to repair self-proteins leads to T cell hyperproliferation and autoantibody production.

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.

Altered immunogenicity of isoaspartate containing proteins.

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.

Isoaspartyl Post-translational Modification Triggers Anti-tumor T and B Lymphocyte Immunity

A hallmark of the immune system is the ability to ignore self-antigens. 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 iso-Asp 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, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens.

B lymphocytes as autoantigen-presenting cells in the amplification of autoimmunity.

The exact role of B cells in antigen presentation to naive T cells in vivo is presently not known. Here, we demonstrate the ability of a B cell subset consisting of B7-2pos-B cells to prime autoreactive T cells in B cell-deficient mice. In contrast, B cell-deficient mice are unable to mount a similar initiation and expansion of the autoimmune response. The expression of the B7-2 costimulatory molecule as well as the specificity to a self-antigen, either murine cytochrome c or murine ribonucleoproteins (the target of autoimmunity in SLE), enabled B cells as antigen-presenting cells to induce naive lymph node T cells to proliferate and to express IFN-gamma, IL-4, IL-5, and IL-10 cytokine mRNAs. In contrast, neither adoptively transferred B7-2neg-B cells nor nonspecific B7-2pos-B cells were able to activate naive T cells. In addition, anti-B7-2 treatment prevented the in vivo expression of the IL-4, IL-5, and IFN-gamma cytokine mRNA responses. Our results suggest a major role of autoantigen-specific B7-2pos-B cells in breaking T cell tolerance to self-antigen.

Intracellular protein modification associated with altered T cell functions in autoimmunity

Posttranslational protein modifications influence a number of immunologic responses ranging from intracellular signaling to protein processing and presentation. One such modification, termed isoaspartyl (isoAsp), is the spontaneous nonenzymatic modification of aspartic acid residues occurring at physiologic pH and temperature. In this study, we have examined the intracellular levels of isoAsp residues in self-proteins from MRL+/+, MRL/lpr, and NZB/W F1 mouse strains compared with nonautoimmune B10.BR mice. In contrast to control B10.BR or NZB/W mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and T lymphocytes. Moreover, T cells that hyperproliferate to antigenic stimulation in MRL mice also have elevated intracellular isoAsp protein content. Protein l-isoaspartate O-methyltransferase activity, a repair enzyme for isoAsp residues in vivo, remains stable with age in all strains of mice. These studies demonstrate a role for the accumulation of intracellular isoAsp proteins associated with T cell proliferative defects of MRL autoimmune mice.

Autoantigens: Novel forms and presentation to the immune system

Abstract It is clear that lupus autoimmunity is marked by a variety of abnormalities, including those found at a macroscopic scale, cells and tissues, as well as more microenvironmental influences, originating at the individual cell surface through to the nucleus. The convergence of genetic, epigenetic, and perhaps environmental influences all lead to the overt clinical expression of disease, reflected by the presences of autoantibodies and tissue pathology. This review will address several specific areas that fall among the non-genetic factors that contribute to lupus autoimmunity and related syndromes. In particular, we will discuss the importance of understanding various protein post-translational modifications (PTMs), mechanisms that mediate the ability of “modified self” to trigger autoimmunity, and how these PTMs influence lupus diagnosis. Finally, we will discuss altered pathways of autoantigen presentation that may contribute to the perpetuation of chronic autoimmune disease.

Lupus autoimmunity altered by cellular methylation metabolism

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.