**2.5 T cytotoxic cells**

Although T cytotoxic cells (CD8<sup>+</sup> T cells) also contribute by producing cytokines such as IFNγ, they are best known for their cytolytic functions. CD8+ T cells contain granules with perforin and granzyme, which are delivered to the target cells with the goal of inducing cell death via membrane damage and cellular content release. A few studies have addressed the role of CD8<sup>+</sup> T cells in DED, and the evidence is controversial. It was suggested that CD8+ T cells might play a regulatory role, since a significant loss of CD8+ T cells in the conjunctiva was found to accompany the development of DED [30] and DED patients showed increased CD4/CD8 compared with healthy donors (**Table 2**) [25]. Furthermore, thrombospondin 1 (TSP1)-deficient mice exhibited DED symptoms accompanied by dramatic lacrimal gland cell infiltration, where CD4+ T cells were significantly increased; CD8<sup>+</sup> T cells were not increased compared with those in mice expressing TSP1 [65]. Conversely, CD8+ T cells were the dominant cell type in severely damaged ducts within lacrimal glands of rats induced with an autoimmune DED model [40]. In line with this,


#### **Table 2.**

*The role of lymphocytes in human DED.*

using a mouse autoimmune model of Sjögren's syndrome, lacrimal glands presented a massive infiltration of CD8+ T cells [53], and aged mice displaying ocular surface pathology (corneal irregularity and conjunctival goblet cell loss) presented increased numbers of CD8+ T cells [42].

Therefore, additional studies are needed to better understand the role of CD8<sup>+</sup> T cells during DED development. In the current literature, most of the mouse studies investigate immunological changes during the acute stage of disease; however, it is well-known that DED is a chronic disease. Thus, the role of resident cells might contribute to the infiltration of additional pathogenic populations responsible for perpetuating the inflammatory process. A differential role of CD8+ T cells occurring during early versus late stages of disease cannot be ruled out.

#### **2.6 B cells**

B lymphocytes complement T cells in adaptive immunity and in generating immune memory responses. B cells perform a variety of functions in homeostatic conditions and following the initiation of an adaptive immune response. These functions include cytokine release, antigen processing and presentation, and their signature role as antibody-producing cells. In mucosal surface immunosurveillance, the presence of IgA is pivotal in limiting pathogen invasion. In the EALT, B cells are present in both diffuse and organized (follicles) forms to support their function. B cells are essential in eye-associated immune responses ranging from surveillance to autoimmune-mediated diseases and allergies.

As mentioned above, human DED can arise from autoimmune diseases (Sjögren's syndrome and rheumatic) and DED was proposed to be a mucosal autoimmune disease [66]. Despite being proposed as an autoimmune disease, the role of B cells in both human and mouse DED has not been completely addressed.

In terms of studies in patients with Sjögren's syndrome, autoimmune-responsetargeting exocrine glands (salivary and lacrimal) are the driving force of the disease; however, human studies mostly focused on the salivary glands rather than lacrimal glands and the ocular surface. Information learned from patients with Sjögren's syndrome linking B cell subpopulations and eye manifestations is still lacking. More thorough research on B cells and their participation during mouse DED must be conducted.

Thus far, the role of B cells in mouse models of DED may be model-dependent. When DED was induced via pharmacological inhibition of the lacrimal gland function (desiccant stress), no significant changes were observed regarding the percentage of B cells present in the tissue (**Table 1**) [30]. Likewise, the NOD autoimmune model of DED was attenuated by blocking high-mobility group box 1 (HMGB1) with neutralizing antibodies; however, no substantial changes in either the percent of B cells or in IL-10-producing B cells were found [67]. Conversely, when DED symptoms were evaluated in aged mice, without any additional chemical agent, B cell numbers were found to be increased accompanying DED development [42]. A pathogenic role of B cells in DED is supported by the findings showing that a DED-like disease can be generated by transferring antibody-containing serum (purified IgG isotype) obtained from mice previously induced with DED for three weeks [43]. The transfer of antibodies required the presence of complement proteins to cause ocular surface damage [43], suggesting that in eye tissue exposed to desiccant stress, antibodies targeting lacrimal gland components like kallikerin 13 are induced (**Table 1**). Additionally, IL-17 collaborates in B cell proliferation and plasma cells generation [44].

Recent findings show that B cells are instrumental in DED, either human or mouse; however, the only mechanism through which these B cells induce eye damage is proposed to be by secreting antibodies targeting lacrimal gland antigens. To prove that additional pathways are also regulated by B cells resulting in Sjögrenassociated and non-Sjögren-related DED, future studies are required.
