**2. Disease Development**

Like other autoimmune diseases, SS is considered a multifactorial disease where a susceptible genetic background requires an environmental factor trigger, such as viral infection [13], to initiate the development of disease. Genome wide association studies (GWAS) have identified several genetic risk factors for SS. Two GWAS in SS have been performed, one with patients of European descent, and another with Han Chinese patients [14,15]. Both studies identified alleles within human leukocyte antigen (HLA) Class II to be the most associated with SS, particularly alleles of the HLA-DR and HLA-DQ isotypes. While major histocompatibility complex (MHC) Class II alleles show the greatest association with SS, several non-MHC genes also possess a significant association. Many of these susceptibility genes, including *IRF5*, *STAT4*, and *IL12A* are involved in the regulation of the interferon (IFN) system [14,15]. The upregulation of IFN pathways and its stimulated genes are associated with the clinical symptoms of SS [16,17]. Over half of all pSS patients exhibit an IFN signature, and these patients typically present higher titers of anti–Sjögren's-syndrome-related antigen A (SSA/Ro) and anti–Sjögren's-syndrome-related antigen B (SSB/La) autoantibodies and higher disease activity as measured by the European League Against Rheumatism Disease Activity Index (ESSDAI) [18]. Additionally, an increased IFN gene signature in the salivary glands (SG) has been linked to poorer patient response to Rituximab, a chimeric mouse/human monoclonal antibody (mAb) therapy with binding specificity to CD20 [19]. Both Type I and type II IFN signatures have been detected for SS patients and genetic ablation of interferon α receptor 1 (IFNAR1), IFN-γ, or its receptor IFNγR prevent the onset of disease in the spontaneous SS models: the non-obese diabetic (NOD) mouse and it's derivative C57BL/6.NOD-*Aec1Aec2* [20–23]. The initial events activating the IFN system remain unclear, as does how the precise nature of how the IFN signature of SS mediates disease. Type I IFNs (T1-IFN) are driven by toll-like receptor (TLR) stimulation and while capable of being produced by all nucleated cell types, they are strongly associated with cells of the innate immune system, whereas Type II IFNs are largely produced by T cells, NK cells and macrophages [24,25]. The apparent necessity of IFNs in the SS disease process together with IFN regulatory risk genes in humans, indicates a role for cells of the innate immune system as well as the adaptive in the development of disease. Indeed, therapies targeting T1-IFN and the IFN pathway continue to be investigated in SS [26].

The insidious onset of SS, coupled with generalized symptoms, overlap with other autoimmune diseases, and the complex classification/diagnostic parameters contributes partially to a frequent delay in diagnosis [2]. Due to the challenges of identifying "pre-SS patients", understanding of the disease in humans has been limited to studying patients with advanced symptomatic disease. This deficit has contributed to a lack of understanding of pathological events preceding observable symptoms. Therefore, in an attempt to elucidate the early patho-immunological processes, many induced and spontaneous mouse models for SS have been developed and used to study disease progression [27]. These mouse models can differ greatly in their SS disease manifestations. For example, NOD mice develop well characterized salivary gland pathology with less lacrimal gland (LG) involvement, whereas thrombospondin-1 (TSP-1) deficient mice experience more severe LG disease [28]. These discrepancies between models mirror the heterogenous presentation of SS in human patients. Critically, the disease profile of individual mouse models can mimic that of subgroups of patients (IFN+, IFN−, etc.) thereby facilitating understanding of disease in these subgroups. Much of the work on spontaneous models has been done using the NOD mouse and its derivatives [29]. In studying SS progression in C57BL/6.NOD-*Aec1Aec2* mice, we were able to identify 3 distinct but overlapping phases of disease [27]. C57BL/6.NOD-*Aec1Aec2* mice develop SS symptoms temporally and phenotypically similar to NOD mice but without the presence of diabetes, making them an ideal candidate to study spontaneous pSS [30–32]. Phase 1 (0–8 weeks) is characterized by acinar epithelial cell death and delayed salivary gland (SG) morphogenesis. Phase 2 (8–16 weeks), where IFN

stimulated genes become activated, coincides with migration of macrophages and dendritic cells (DCs), followed by CD4+T and B220+B lymphocytes, and the emergence of autoantibodies. Finally, in Phase 3 (16 weeks onward) there is overt clinical disease where a progressive measurable loss of exocrine gland function occurs. Disease development in C57BL/6.NOD-*Aec1Aec2* mice shares some similarities to other SS mouse models, even if different glands are targeted. For example, increased apoptosis was observed at 8 weeks in the LGs of TSP-1-deficient mice, which can also be seen in the SG of C57BL/6.NOD-*Aec1Aec2* mice. Additionally, TSP-1 mice displayed ocular surface damage at 12 weeks with an increase in SSA/Ro and SSB/La antibodies detected at 12–16 weeks. Finally, infiltrates primarily composed of CD4<sup>+</sup> T cells were discovered in the LGs of TSP-1-deficient mice with increased expression of Th1 and Th17 and related transcription factors. Similar observation was seen in the SG of C57BL/6.NOD-*Aec1Aec2* mice at similar age [28]. Together these findings offer insights as to how the aberrant activity of both innate and adaptive immune cells mediate the pathogenesis of SS.
