Features of Systemic Autoimmune Disease

The systemic autoimmune diseases are a complex group of disorders, which include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune myopathies, Sjögren's syndrome (SS), vasculitis, and scleroderma. The diseases manifest in a wide range of clinical phenotypes, often united by chronic inflammation and involvement of multiple organ systems. A dominant feature of these diseases is the elaboration of high titer, high affinity autoantibodies, which are strongly associated with phenotype.1, 2, 3 Although the clinical presentations of the individual systemic autoimmune diseases are often quite distinct, the presence of overlapping features in different diseases suggest the involvement of common effector pathways and shared disease mechanisms.4 This includes autoimmune myopathies occurring in myositis, SLE, and scleroderma, or the sicca complex (dry eyes/mouth) in many of the systemic autoimmune diseases. Autoantigens in systemic autoimmune diseases appear to be ubiquitously expressed molecules and may be central to the disease process, but the mechanisms underlying their selection remain unclear. Defining the features unifying these autoantigens, which may impact their selection by the immune response, is an important priority.

One striking feature of autoimmune rheumatic diseases is their self-sustaining nature, and their striking capacity for autoamplification (exemplified by disease flares). The autoantibody response in active disease is typically of high affinity and titer, and seems to be driven by ongoing antigen release. This amplification strongly suggests the presence of a feed-forward cycle in disease propagation consisting of two key components: active immune effector pathways and immunogenic self-antigens. Immune effector pathways drive the generation/release of autoantigens from target tissue, which in turn provide fuel to propagate the immune response. Autoantigens are not passive players in this process, but rather have a direct function in shaping the autoimmune response. Identifying such amplifying pathways may have important therapeutic implications.

There is a growing awareness that structural changes of autoantigens during cytotoxic lymphocyte granule-mediated cell death are a frequent feature of systemic autoimmune diseases. Although this review focuses on structural changes of intracellular autoantigens induced by the granule component granzyme B (GrB), it is important to note that there are several other mechanisms by which cytotoxic granules may contribute to the self-sustaining cycle of tissue damage in autoimmune disorders. First, structural modification of an overlapping yet distinct set of autoantigens has been shown for other members of the granzyme family including granzyme A (GrA)5, 6 and granzyme H (GrH).7 Second, granzymes can mediate direct extracellular effects on target tissue. Neurotoxicity is induced after incubation of neurons with GrB through G-coupled protein receptor signaling and may have a function in the pathogenesis of neuroinflammatory diseases.8 In addition, degradation of extracellular matrix proteins by GrB is thought to have a function in cytotoxicity of smooth muscle cells in inflammatory vascular disease.9, 10 Finally, a function for granzymes as independent pro-inflammatory modulators of antigen-presenting cell (APC) function has been suggested by the finding that mouse macrophages treated with GrA secrete pro-inflammatory cytokines IL-1β, TNFα, and IL-6.11 This review discusses the GrB-mediated changes in autoantigen structure and mechanisms whereby these changes may contribute to the initiation and propagation of autoimmunity.

Granzymes Modify the Structure of Autoantigens during Cytolytic Granule-Mediated Cell Death

During lymphocyte-induced cytotoxicity, granzymes and the pore-forming protein perforin act in concert to provide protease access to the target cell cytosol. Granzymes induce cell death by several processes, including activation of the caspase cascade, as well as direct cleavage of various intracellular substrates that participate in nuclear fragmentation and cell death.12 Significant data exists to suggest an important function for the cytotoxic lymphocyte granule pathway in the pathogenesis of systemic autoimmunity. Activated cytotoxic lymphocytes are enriched in the lung in scleroderma,13, 14 the salivary gland in SS,15 and the synovium in RA.16 In the muscle of individuals with polymyositis (PM), T cells in direct contact with muscle fibers have been shown to orient components of the cytolytic granule pathway toward the site of cell–cell contact.17 Cytolytic CD8+ T cells invading muscle fibers of PM patients exhibit a restricted T-cell receptor repertoire suggesting clonal expansion of autoantigenic T cells in the inflamed target tissue.18 Furthermore, increased circulating levels of cytolytic cells and components of the granule pathway correlate with disease activity. For example, the number of circulating activated cytolytic CD8+ T cells correlates with disease activity in SLE,19 whereas the levels of GrB in the serum and synovial fluid of RA patients is strikingly associated with the severity of erosive joint disease.20, 21

Multiple studies have shown that the majority of autoantigens targeted in systemic autoimmune diseases are substrates for granzymes, particularly GrB5, 6, 22, 23, 24 (Table 1). The association between GrB cleavability and autoantigen status is striking as, until recently, there have been few GrB substrates identified which are not autoantigens. A recent comprehensive proteomic analysis by Van Damme et al.25 revealed hundreds of novel GrB targets, and may suggest a pool of earlier undefined autoantigens.

Table 1 Autoantigens cleaved by granzyme B have diverse functions and subcellular localizations
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The tetrapeptide substrate specificity of human GrB has been determined. In addition to the requirement for aspartic acid in the P1 position, P4 is specific for isoleucine (Ile), valine (Val), and leucine (Leu), whereas P3 and P2 are able to accommodate a wider range of amino acids26, 27 (Figure 1a). Although there are many tetrapeptides satisfying the GrB consensus sequence within molecules, most of these sites are not cleaved by GrB.28 Indeed, GrB typically cleaves autoantigen substrates at a limited number of sites.22 Interestingly, although GrB has similar tetrapeptide substrate specificity to the upstream group III caspases (which process effector caspases 3 and 7),27 distinct sites are used generating unique fragments of autoantigens not seen during caspase-mediated cell death.22

Figure 1
figure 1

Cleavage sites in autoantigens are enriched in amino acids specifically preferred by GrB over group III caspases. (a) Specificities of GrB and group III caspases are similar, but distinct. A comparison of the P2, P3, and P4 position requirements for a group III caspase (caspase-9) and GrB reveals differences in preferred amino acids (modified from Thornberry et al26). (b) Cleavage sites of autoantigens known to be GrB substrates are enriched in amino acids that are preferred by GrB in the P2 and P3 positions over caspase-9. Amino acids preferred by GrB are shown in green for the P2 and in blue for the P3 positions. Amino acids are indicated as follows: A, alanine; R, arginine; N, asparagine; D, aspartic acid; Q, glutamine; E, glutamic acid; G, glycine; H, histidine; I, isoleucine; L, leucine; M, methionine; K, lysine; P, proline; S, serine; T, threonine; V, valine

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The GrB cleavage sites defined within autoantigens reveal several interesting features (Figure 1b). First, all have Ile, Leu, or Val in P4 and are cleaved after the P1 aspartic acid.22 Second, amino acids in the P2 and P3 positions in autoantigens are almost universally favored by human GrB, but not tolerated by caspases (e.g. proline in P2).26 Finally, the native three-dimensional antigen structure may determine GrB cleavage site accessibility. For example, although GrB is able to cleave an in vitro generated autoantigen (SS transmembrane autoantigen, muscarinic receptor 3) at two distinct sites, only one site is used during granule-induced killing of intact human salivary gland target cells.23 These features reinforce the strong relationship between autoantigen status and this unique cleavage susceptibility.

Not unexpectedly, granzyme cleavage of antigens frequently has consequences in terms of function,5, 24 including redistribution of nuclear antigens to the cytosol during cytotoxic granule-induced cytotoxicity.6, 29 The SS nuclear autoantigen La/SSB is a substrate for both GrB and GrH, and undergoes dramatic redistribution during granule-mediated cell death after uncoupling the C-terminal nuclear-localization signal (NLS).29 Similarly, cleavage of SLE and autoimmune hepatitis antigen lamin B by GrA and B causes disruption of the nuclear lamina, by uncoupling lamin B from its NLS.6 The granule pathway has also been shown to trigger the release of autoantigens from target cells. Blanco et al.19 showed that cytolytic CD8+ T cells from patients with active, but not quiescent, SLE were able to stimulate the release of nucleosomes from target cells, together with cleavage of nuclear autoantigens (e.g. U1-70 kDa). It is conceivable that cytoplasmically redistributed granzyme-cleaved antigens are released during granule-induced death, potentially influencing pathways of antigen presentation.

The ability of GrB to modify the structure of self-antigens is not limited to systemic autoimmunity. There are examples of tissue specific28, 30 and cancer autoantigens being GrB substrates.31 Myasthenia gravis (MG) is a tissue-specific autoimmune disease mediated by the antibody-dependent reduction of acetylcholine receptors (AchR) at the neuromuscular junction.32 Both GrB and its substrate (AchR ɛ subunit) are aberrantly expressed in thymuses from patients with MG, suggesting that an immune response to AchR may propagate in the thymus.30 In medullary carcinoma of the breast (MCB), GrB-expressing cytotoxic T cells are found in large numbers, often nearby apoptotic tumor cells. Autoantibodies to β-actin have been identified in these patients and are able to recognize actin that has been redistributed to the surface of the apoptotic tumor cells. GrB cleavage fragments of β-actin have been detected in MCB tumor lysates, suggesting that CTL-mediated death of MCB tumor cells and actin redistribution may have a function in the generation of autoimmunity to β-actin.31 Interestingly, patients with MCB have a significantly higher survival rate than patients with non-MCB breast cancer, suggesting that autoimmunity generated against tumor self-antigens may contribute to the favorable prognosis.33, 34

These data show that a group of molecules, which share little in common in terms of structure, function, or distribution, are unified by their susceptibility to cleavage by a highly specific and fastidious protease that has a major function in immune effector pathways. To place in context the possible relevance of GrB-mediated cleavage of autoantigens to initiation and propagation of autoimmunity, it is necessary to review the concepts of immunodominance and the ability of single, early proteolytic events to influence epitope selection.

Protein Structure and Early Proteolytic Events Influence Epitope Selection during Antigen Presentation

Structural modification of autoantigens including protein–protein interactions, post-translational modifications, mutation of key residues, and differential sensitivity to proteases can lead to striking changes in the peptides that are ultimately presented to T cells.35 Molecules that are found in, or gain access to the extracellular space (including those derived from dying cells), are phagocytosed by professional APCs and gain access to the major histocompatibility complex (MHC) class II antigen processing pathway. On entry, antigens are exposed to a variety of proteases in the endosomal compartment, which process the native antigen into peptide fragments, available for loading onto class II molecules. Interestingly, although a large number of potential peptides can be generated, only a few peptides are successfully presented on the surface of the APC. These peptides are termed as ‘immunodominant’.36

There is accumulating evidence to suggest a function for protein conformation and MHC-guided processing as one mechanism underlying immunodominance.37 During antigen processing, exposed structural determinants of native antigens can bind to MHC class II molecules and be protected from proteolysis by endosomal cathepsins, whereas the rest of the molecule is degraded. Alterations in antigen structure could, therefore, have profound effects on the regions of the protein available for binding to, and protection by, class II molecules. During development of immune tolerance, T cells with high affinity for dominant peptides derived from self-proteins are often deleted or rendered non-responsive.38 Those peptides that are not presented on class II molecules at significant levels are termed as ‘cryptic’, and T cells recognizing such cryptic determinants are not tolerized and persist. Differences in antigen processing of self-proteins occurring as a result of structural modification could enhance cryptic epitope presentation, leading to activation of self-reactive T cells.36 Activation of T cells recognizing cryptic epitopes has been proposed to be an important mechanism underlying the development of autoimmunity.39

Recent studies showing the importance of early and highly specific proteolytic events in presentation of immunodominant and cryptic epitopes are highly relevant to the observation that autoantigens are proteolyzed in a novel way by granzymes. For example, asparaginyl endopeptidase (AEP) is a cysteine protease involved early in MHC class II antigen processing. Cleavage of intact endocytosed protein by AEP occurs at a limited number of asparagine residues, yet has profound effects on downstream processing by lysosomal proteases.40 Thus, cleavage of tetanus toxoid C fragment (TTCF) at a single asparagine residue by AEP is critical for presentation of multiple peptide epitopes derived from other regions of the protein. Mutation of the TTCF AEP cleavage site alters the pattern of fragments generated during subsequent processing and dramatically reduces activation of several antigen-specific T-cell hybridomas.41

Processing by AEP has been implicated in the generation of autoimmunity. For example, a major disease-associated epitope in the multiple sclerosis autoantigen myelin basic protein (MBP) contained within amino-acids 84–102 is normally cryptic, because of proteolysis at Asn94 by AEP. Inhibition of AEP activity in APCs enhances presentation of this epitope, which is a dominant epitope when autoimmunity to MBP is subsequently initiated.42 In addition, proteolytic activity of AEP is impaired when asparagine residues are N-glycosylated, suggesting a way in which post-translational modifications can affect susceptibility to proteolysis and subsequent antigen presentation.40

In this regard, it is notable that antigen proteolysis and epitope selection is also influenced by subtle changes in antigen structure and conformation in several models of autoimmunity. For example, the post-translational conversion of aspartic acid to isoaspartyl residues is an isomerization that occurs naturally during cell stress, aging, and activation. The presence of isoaspartyl residues in full-length pigeon cytochrome C alters processing by cathepsin D and stimulates T-cell proliferation. However, the aspartyl form is not stimulatory, suggesting that accumulation of a naturally occurring amino-acid modification can render self-antigens immunogenic.43 Immune responses to isoaspartyl, but not aspartyl, forms of the SLE autoantigen U1/Sm ribonucleoprotein have also been observed. Interestingly, activated B and T cells contain increased levels of isoaspartyl residues and may act as sources of autoantigen to drive the self-sustaining loop of autoimmunity.44 Furthermore, modification of single amino-acid residues within endogenous proteins can generate autoimmunity to cryptic epitopes. For example, dopachrome tautomerase (Dct) is an antigen normally expressed in melanocytes. When randomly mutated constructs of Dct are expressed in mice, autoimmunity to Dct is induced, resulting in skin hypopigmentation and activation of autoreactive T cells recognizing cryptic Dct epitopes. Autoreactive T cells and hypopigmentation are also observed when the Dct constructs encoding truncated versions of the molecule, not generated during normal processing of the full-length protein, are expressed.45

Taken together, these studies show that relatively minor changes in autoantigen structure, including single proteolytic events, can have striking effects on epitope selection during antigen processing and presentation on MHC class II molecules. Autoimmunity may arise when normally dominant epitopes of self-antigens are destroyed or cryptic epitopes are revealed.

Conformational State of Antigens Affects Susceptibility to GrB Cleavage

As noted above, proteolysis and epitope selection can be affected by minor changes in antigen structure, including post-translational modifications,40 isomerizations,43 and early cleavage events.41 Several studies have shown that the tissue-specific expression46, 47, 48 or conformation49, 50 of proteins can affect their susceptibility to cleavage by GrB. For example, GrB cleavage of extracellular matrix proteins (vitronectin and fibronectin) and the clotting mediator, von Willebrand factor, only occurs when the proteins are in distinct matrix-associated conformations.49, 50 These studies suggest that autoantigens may be susceptible to GrB cleavage only under very specific circumstances associated with unique activation, differentiation, or mitotic states of the cell (Figure 2).

Figure 2
figure 2

Cleavage of GrB-sensitive forms of autoantigens may reveal cryptic epitopes in disease-relevant target tissue. GrB-sensitive forms of autoantigens may be present in the disease-relevant target tissue because of unique intermolecular interactions or post-translational modifications (*) exposing GrB cleavage sites (red star). Seemingly ubiquitously expressed autoantigens may be susceptible to GrB cleavage (black triangle) only when in tissue-specific immunogenic conformations and may drive the generation of autoimmunity to cryptic epitopes (yellow)

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Granzyme-sensitive forms of autoantigens have been observed in the disease-relevant target tissue in myositis and scleroderma. For example, expression of a GrB-sensitive form of histidyl-transfer RNA synthetase (HRS/Jo-1), a target antigen in patients with interstitial lung disease associated with myositis,51 has also been noted exclusively in lung tissue as compared with other tissues including placenta, liver, muscle, ovary, and colon. The exclusive expression of granzyme-sensitive HRS/Jo-1 in the lung (which is the target of immune-mediated damage in this disease) is of interest. It is consistent with the proposal that GrB-mediated cleavage has a function in antigen selection, and that expression of cleavable autoantigens in specific target tissues may focus amplifying injury at that site.46 A similar tissue-specific expression of a granzyme-sensitive form of the nucleolar autoantigen nucleophosmin (B23) has been observed in scleroderma. Autoantibodies to B23 are associated with the development of pulmonary hypertension in which vascular smooth muscle cell damage is believed to have a function in pathogenesis.52 Interestingly, B23 from differentiated, but not undifferentiated, vascular smooth muscle cells is efficiently cleaved by GrB. The restricted expression of cleavable autoantigens to particular cells in specific differentiation states within the target tissue suggests that either the property of cleavability or cleavage events themselves may be relevant to immune-mediated damage.47

B23 is also a target antigen in patients with hepatocellular carcinoma (HCC).53 Unlike normal regions of liver tissue, tumorous areas of liver also express a highly cleavable form of B23, which exists primarily as an SDS-stable oligomer. This cleavable form of B23 is preferentially recognized in HCC tumors by an antibody specifically raised to a peptide within the GrB cleavage site, suggesting that HCC liver expresses a form of B23 in which the GrB cleavage site is exposed, rendering it more sensitive to cleavage.48

Distinct or aberrant post-translational processing of autoantigens may also affect susceptibility to cleavage by GrB (Figure 2). The glutamate receptor subunit 3 (GluR3) is an autoantigen in the tissue-specific autoimmune disease Rasmussen's encephalitis.54 The glycosylation state of GluR3 seems to be critical in determining susceptibility to GrB cleavage. Although the N-glycosylated granzyme-insensitive form predominates in neuronal tissue, a percentage is not glycosylated and is, therefore, susceptible to cleavage by GrB. It has been proposed that the balance of the glycosylated versus unglycosylated forms is modified during inflammation, potentially providing an opportunity for GrB-mediated cleavage only under very limited circumstances.28

Together, these data highlight that changes in protein conformation affect susceptibility to GrB cleavage in target tissues in autoimmunity as well as cancer. The association between status as an autoantigen and susceptibility to cleavage by GrB is striking, as are the clear examples in which the cleavable conformation of an autoantigen is enriched or expressed exclusively within the target tissue associated with an immune response to that antigen. Taken together, the data implicate novel cleavage events directed by immune effector pathways, occurring at specific tissue sites, as components of the pathways selecting self-antigens in autoimmune rheumatic diseases. It must be noted, however, that there is to date no direct evidence that GrB cleavage of autoantigens alters the immune response to that antigen, or participates in disease propagation. It is possible that cleavable forms of antigens exist in disease-relevant target tissues and represent an altered, immunogenic conformation that can drive the generation of autoimmunity even in the absence of GrB cleavage.

Unfortunately, both the tetrapeptide and the macromolecular specificities of human GrB differ significantly from mouse GrB, and the unique unifying features around GrB cleavage sites in human autoantigens are largely unshared by mouse antigens.55, 56, 57 Defining the function of GrB in human systemic autoimmune diseases, therefore, depends on addressing the effects of GrB cleavage events on antigen immunogenicity in the human model – which constitutes a significant challenge. Possible ways around this barrier include: (i) examining effects of cleavage with human GrB (or fragments resulting from such cleavage) on immunogenicity of defined human autoantigens in mice; (ii) studying initiation and propagation of various forms of systemic autoimmunity in a GrB knock-out mouse or in a human GrB knock-in mouse; and (iii) directly testing effects of GrB on autoantigen immunogenicity and epitope selection in peripheral blood from human beings with autoimmune diseases in which GrB substrates are targeted.

Limited studies have been performed in this regard and suggest conflicting functions for GrB cleavage of antigens in the generation of autoimmunity. Mouse experiments have been performed, which suggest that fragments of antigens elicit more robust immune responses than intact protein.45, 58 For example, T cells from transgenic mice expressing human MHC class II molecules (DRB1*0301/DQB1*0201) associated with the development of immune responses to SLE autoantigen La exhibit enhanced T-cell proliferation on peptide re-challenge after priming with a truncated form of the protein. This naturally occurring frame-shift mutant (amino-acids 1-204) is very similar in length to the N-terminal fragment of La generated after GrB cleavage (amino-acids 1–220) suggesting that fragmentation of La may enhance antigen processing and release of cryptic epitopes.58 A study in GrB knock-out mice shows that GrB is not required for the development of pristane-induced SLE and may even have a protective effect. GrB knock-out mice injected with pristane exhibited features of SLE, including autoantibody production, but seemed to have increased mortality after pristane treatment, compared with wild-type mice.59 Experiments in human GrB knock-in mice may provide insights into the effect of autoantigen cleavage in a mouse model of autoimmunity, provided that the GrB cleavage site within the autoantigen is conserved between mice and human beings. It is clear that future studies are necessary to elucidate the potential function for GrB cleavage of autoantigens in the generation of autoimmunity and will require studies in the human model.

GrB Cleavage Sites Reside in Unstructured Regions of Antigens and Co-locate with Autoimmune Epitopes

The ability to obtain detailed information about the structure of autoantigens by crystallography as well as by dynamic methods including protein nuclear magnetic resonance (NMR) and deuterium exchange mass spectrometry may provide valuable information regarding the three-dimensional context of granzyme cleavage sites. Although this data is available for very few antigens at this time, some interesting themes are emerging. Analysis of the available crystal structures of autoantigens known to be cleaved by GrB reveals common features of granzyme cleavage sites.60, 61 Partial structures of B23,62 poly(ADP)ribose polymerase 1,63 and HRS/Jo-164 show that GrB cleavage sites are located in unstructured regions or in areas adjacent to highly structured elements such as coiled coils. Similarly, the GrB site (IEAD15) in the scleroderma antigen and DNA replication protein topoisomerase I is located in the highly extended 174 amino-acid N-terminal domain. This region was determined by circular dichroism to be virtually completely unfolded.65 In other cases, known granzyme cleavage sites are located on loosely ordered surface loops or linker regions between structural elements. The structure of scleroderma autoantigen fibrillarin reveals that the GrB cleavage site (VGPD184) is located on a surface loop between an α-helix and β-strand.66

The effect of antigen structure on epitope selection is highlighted by work from Landry67, which showed that disordered regions of antigens lead to increased presentation of epitopes from adjacent, structurally organized sequences. As GrB cleavage sites tend to be located in disordered areas or linker regions connecting structural elements, it is possible that cleavage of autoantigens at the GrB site influences the presentation of downstream or hidden structured epitopes (Figure 3). Again, a complete dataset is not available, but review of cleavage sites as well as known B- and T-cell epitopes of autoantigens reveals that granzymes and the immune system target overlapping regions of these proteins (Figure 4). Epitope mapping of La has revealed overlapping B-68 and T-cell58, 69 epitopes in a region spanning the GrB cleavage site. Similarly, overlapping B-70 and T-cell71 epitopes have been identified in the SLE autoantigen U1-70 kDa in a C-terminal region containing the GrB cleavage site.

Figure 3
figure 3

Cleavage of autoantigens at GrB cleavage sites located in unstructured loops may liberate cryptic epitopes derived from structural elements. (a) Proteolysis by GrB (black triangle) occurring in unstructured loops or linker regions of autoantigens (red star), may enhance presentation of cryptic epitopes derived from adjacent structural elements (blue). (b) GrB cleavage may also induce structural changes leading to increased presentation of cryptic epitopes derived from earlier hidden regions of autoantigens (green)

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Figure 4
figure 4

Autoimmune epitopes are co-located with GrB cleavage sites. B (blue) and T (yellow) cell epitopes of several autoantigens are clustered in regions containing GrB cleavage sites (red). Overlapping B- and T-cell epitopes (green) in La and U1-70 kDa suggest autoimmune ‘hot spots’ in these regions of antigens containing GrB cleavage sites

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Although the discussion of the effect of autoantigen cleavage by granzymes has focused on antigen processing and presentation of peptide epitopes to T cells, it is clear that autoantibody epitopes can also be affected. Granzyme cleavage of autoantigens can lead to the creation of immunogenic protein fragments, which are preferentially recognized by autoantibodies from patients with systemic autoimmune diseases.72, 73 Autoantibodies from some patients with primary SS have been shown to preferentially recognize GrB cleavage fragments of La/SSB. A subset of these patients has autoantibodies that recognize cryptic epitopes generated after GrB cleavage, in that they cannot be blocked by intact La protein.72 Likewise, sera from patients with scleroderma and ischemic digital loss exhibit a striking preference for GrB-generated fragments of antigens, specifically those with autoantibodies to centromere protein CENP-C.73

Although cleavage of autoantigens by granzymes can aid in the generation of antigenic epitopes, granzyme cleavage also has the capacity to destroy immunoreactive epitopes. Circulating autoantibodies74 and T cells75 targeting pyruvate dehydrogenase complex-E2 (PDC-E2) are a frequent finding in patients with primary biliary cirrhosis (PBC). Although cleavage of PDC-E2 by caspases is capable of generating immunogenic fragments, GrB cleavage seems to destroy a dominant autoantibody epitope, abolishing reactivity by PBC patient sera.76 Interestingly, although two CD8+ T-cell epitopes may be destroyed by GrB cleavage,77, 78 another79 seems to be unaffected and perhaps even liberated by proteolysis. A similar pattern is observed for MG autoantigen AchRÃ¥, in which GrB cleavage has the potential to destroy a T-cell epitope while liberating another.80

Conclusions

There is strong evidence implicating the cytotoxic lymphocyte granule pathway in the pathogenesis of systemic autoimmune diseases. Activated cytotoxic lymphocytes are present in target tissues and effector function is positively correlated with disease activity in several diseases (e.g. myositis, SLE, RA). Autoantigens have diverse structures, functions, and subcellular localizations, yet are efficiently cleaved by GrB at a small number of unique sites, distinct from caspase cleavage sites. GrB-sensitive conformations of antigens have been detected in the disease-relevant target tissue in systemic autoimmune diseases. Analysis of available data shows that GrB cleavage sites are often situated in unstructured regions of antigens, adjacent to structured domains. Immune epitopes are also frequently located in close proximity to GrB cleavage sites, suggesting that cleavage by GrB may modulate presentation of epitopes from adjacent structured regions. Although there is clear data that single proteolytic events early during antigen processing can have striking effects on the epitopes presented, such data is not yet directly available for GrB. The observation that human and mouse GrB have distinct cleavage site specificities underscores the challenges to show a direct function for GrB-mediated cleavage of autoantigens in human autoimmunity. Studies in the human system will be needed, and will require an antigen, which is frequently targeted in a phenotypically distinct autoimmune disease, a substrate for human GrB, and associated with known MHC class II alleles. Establishing a mechanistic function for granzymes in the self-amplifying pathway of autoimmunity would have important therapeutic implications.

Conflict of interest

The authors declare no conflict of interest.