Two major unanswered questions related to the pathogenesis of type 1 diabetes (T1D) prevent progress in our ability to intervene in disease development: What breaks immune tolerance to the pancreatic β-cells? And what kills the β-cells?
By the surprising demonstration that knockout of the tryptase granzyme A (GrzmA), conventionally considered to be a key proapoptotic mediator of immune killer cell cytotoxicity, does not protect but markedly accelerates and increases incidence of T1D in the nonobese diabetic (NOD) mouse, Mollah et al. (1) add to answering both of these questions. On one hand, they show that GrzmA does not contribute to β-cell killing. On the other hand, their striking finding that diabetes resistance in the proinsulin II transgenic mouse is broken by GrzmA knockout provides evidence of an important role of GrzmA for maintenance of peripheral tolerance.
By answering “No” to the question as to whether GrzmA is important in β-cell killing, this study adds to reports showing that granzyme B (GrzmB) (2), Fas (3,4), and perforin (5,6) are dispensable in β-cell killing in the NOD mouse. These observations challenge the view that β-cell destruction in T1D is caused by classic effector T-cell mechanisms and support the notion that these cells do ensure antigen-specific homing and reactivity but then interact with innate immune cells to orchestrate an intraislet inflammatory reaction leading to β-cell killing, the selectivity of which is in part determined by differentiation-dependent sensitivity of β-cells to cytotoxic inflammatory mediators (7).
The “Yes” to the question as to whether GrzmA contributes to maintain peripheral tolerance is perhaps even more important because it identifies new potential therapeutic targets. T-cell GrzmA expression and production is reduced in human T1D by yet unknown mechanisms (8). Understanding of these mechanisms and restoration of GrzmA levels may have therapeutic perspectives. Key in this respect would be clarification of the regulation and mechanism of action of GrzmA.
GrzmA, discovered by Tschopp and colleagues (9) in 1986, belongs to the granzyme family of serine proteases encompassing 10 members in the mouse and 5 members in the human (10,11). They have trypsin-, chymotrypsin-, and elastase-like proteolytic activities, but in contrast to these broad serine proteases, granzymes have higher substrate specificity conferred by a substrate binding pocket. Granzymes are expressed mainly in cytotoxic T cells and natural killer (NK) cells in response to interleukin (IL)-2 in synergy with IL-12 (12,13), with GrzmA being the most abundantly expressed but also having lower cytotoxic activity than GrzmB. GrzmA and GrzmB are also expressed in γδT cells, a subset of the intraepithelial lymphocytes abundant in the intestinal mucosa, and in thymocytes, suggesting roles in both central tolerance and gut immunity. GrzmB, but as yet not GrzmA, is also found to be expressed in plasmacytoid dendritic cells (pDCs) (14), the innate master producer of the antiviral type I interferon (IFN) IFNα. Apart from mediating T-cell and NK-cell granule-dependent cytotoxicity, granzymes regulate B lymphocyte proliferation, inhibit viral replication, and kill bacteria.
In granule-dependent cytotoxicity granzymes are delivered to early endosomes by clathrin- and dynamin-dependent endocytosis after entering the target cell via perforin, a membrane pore-forming molecule cosecreted with granzymes (Fig. 1). Granzymes enter the cytosol from the endosomal compartment and are then activated by the near-neutral pH of the cytosol, where they in turn trigger the caspase-dependent intrinsic death pathway (11). Until recently it was believed that GrzmA was a mere “backup” for GrzmB in perforin-mediated apoptosis and activated identical death pathways. It is now clear that not only do GrzmA- and GrzmB-deficient mice have differential susceptibility to different infections, but GrzmA also triggers noncaspase-dependent cell death by activating the DNA-degrading SET complex (Fig. 1) (10,11). This activity relates to a unique ability of GrzmA to homodimerize, thereby exposing an extended exosite that determines its substrate specificity.
Having shown that enhanced diabetes development in GrzmA−/− NOD mice depended on type I IFN signaling and that innate immune cell accumulation of ssDNA was associated with a type I IFN–related islet gene expression signature, Mollah et al. (1) make the case that GrzmA deficiency permits liberation of ssDNA fragments into the cytosol in pDCs due to lack of GrzmA-mediated activation of the SET complex nuclease activity and that the ssDNA fragments subsequently promote type I IFN production (likely via Toll-like receptors) that in turn breaks tolerance (Fig. 1). However, it remains to be shown that pDCs express GrzmA, and GrzmA deficiency did not increase IFNα in pDC culture medium or in circulation. ssDNA was increased in GrzmA-expressing NK cells correlating with increased NK numbers, but NK cells are IFNγ—not type I IFN—producers. Although ssDNA did not accumulate in GrzmA-expressing T cells, β-cell antigen–specific CD8+ T cells were increased in islets, lymph nodes, and spleen. Regulatory T-cell number and function were unaffected. Taken together, these observations suggest that GrzmA may directly regulate the autoreactive T-cell pool. Thus GrzmB has been shown to mediate activation-induced cell death in T cells (15), and pDC-derived GrzmB suppresses effector T-cell proliferation by degrading the T-cell receptor ζ-chain (16). It would be relevant to determine whether GrzmA has similar actions.
What is the human relevance of this study? T1D risk is increased in clinical trials of type I IFN (17), underpinning that aberrant production of type I IFN may break tolerance. Yet targeting type I IFN alone does not prevent diabetes development in the NOD mouse (18). There are no human studies of anti-IFNα in T1D. The authors raise the caveat that NOD mice carry endogenous nonpathogenic retroviruses that may be the source of accumulating cytosolic ssDNA. However, common human diseases are caused by DNA viruses (adenovirus, herpes simplex virus, poxviruses, and hepatitis B virus), prompting human studies. Also since IL-2 deficiency is a central defect in T1D pathogenesis (19) and since GrzmA expression is driven by IL-2, the observation that T-cell GrzmA is reduced in children with T1D (8) further emphasizes the translational implications of the study by Mollah et al. (1). Larger clinical trials of IL-2 in T1D, now shown in small studies to be safe and yielding increases in regulatory T cells (20), are under way.
See accompanying article, p. 3041.
Duality of Interest. No potential conflicts of interest relevant to this article were reported.