**1.2 Lymphocyte diversity in the immune response**

Lymphocytes are present in blood and lymph vessels and include innate and adaptive subtypes. All lymphocytes originate in the bone marrow (organ where all blood cells are created) from a common lymphoid precursor (CLP); however, not all lymphocytes reach mature status while in the bone marrow. For example, to achieve a mature T cell lineage, migration into the thymus is required, so they can complete their maturation through a complex process [16].

Classically, innate lymphocytes are represented by natural killer (NK) cells, whereas their adaptive counterparts include T and B cells. However, other lymphocytic subpopulations also exist in both innate and adaptive subtypes. The main difference between innate and adaptive lymphocytes is that the former express receptors encoded in the germline with limited diversity, such as toll-like receptors (TLRs) and carbohydrate-recognizing receptors (lectins), amongst others [17]. In contrast, adaptive lymphocytes express receptors generated by genetic recombination, which ultimately results in endless diversity. T and B lymphocytes perform gene rearrangements to express surface dimeric (two-chain, membrane-bound) T cell receptor (TCR) and B cell receptor (BCR), respectively. The TCR structure consists of alpha-beta chains, whereas the BCR structure is characterized by heavy and light chains forming a membrane-inserted immunoglobulin [18]. Thus, conventional innate lymphocytes are the NK cells and the conventional adaptive lymphocytes are the T and B cells.

An extended functional and phenotypical characterization of lymphocytes recently uncovered a growing diversity in lymphocytic subpopulations. A group of lymphocytes bearing low diversity TCRs and simultaneously expressing surface markers of NK was identified and named NKT cells [19]. Whereas most T cells express TCRs composed of classical alpha-beta chains, a less-abundant, mucosadwelling subtype of T cells express TCRs composed of gamma-delta chains, which is referred as γδ T cells [20]. Currently, both NKT and γδ T cells are considered unconventional T cells. More recently, other unconventional innate-like lymphocytes have been identified and these include the group 2 and group 3 innate lymphoid cells (ILC2s and ILC3s, respectively) [21] and mucosal-associated invariant T cells (MAITs), whose role has been widely explored in other mucosal surfaces (gut, skin, and lungs) [22].

B cells also have an innate-like counterpart; therefore, B cells are also subdivided into B1 and B2 cells. B1 cells mostly reside in the peritoneal and pleural cavity and produce low-affinity antibodies without stimulation (naturally produced antibodies), whereas B2 cells can produce high-affinity antibodies (affinity maturation process) and highly efficient memory responses [23].

Regarding their role within the immune response, all these lymphocytic cells contribute to a wide variety of both physiological and pathological processes. Lymphocytes residing and circulating during homeostatic conditions participate in immune surveillance; however, lymphocytes can be rapidly activated and collaborate in pathogen clearance. Furthermore, lymphocytes are involved in highly specialized functions such as the generation of memory responses, which allows increased intensity and efficiency in a secondary response. Lymphocytes additionally participate in the amplification of the immune response by rapidly releasing cytokines and chemokines (helper subpopulations) and preventing pathogen and tumor cells dissemination by elimination of infected or transformed cells (cytotoxic and killer types). Conversely, specific lymphocytes are also able to down-modulate

#### *Dry Eye Syndrome - Modern Diagnostic Techniques and Advanced Treatments*

the intensity of the immune response, thus turning these cells into regulatory subtypes, which are central in the resolution phase of the inflammatory process.

As above-mentioned, lymphocytes are active players both as promoters and regulators of inflammatory diseases. In the case of DED, how lymphocytes might be involved in the immunopathology of this disease was only recently described, and the understanding of these lymphocyte-driven pathological pathways may pave the way for new therapeutic opportunities.

## **2. The role of lymphocytes in DED**

#### **2.1 NK cells**

NK cells are an early source of cytokines, such as interferon (IFN)γ and tumor necrosis factor (TNF)α and display cytotoxic features that place them in the firstline defense against intracellular pathogens and tumor cells. NK cells are grouped into the innate arm of the immune response since they lack antigen-specific receptors such as those found on adaptive cells (i.e., TCR and BCR) [24]. To fulfill their primary cytolytic role, NK cells are equipped with killer-activating receptors (KARs) as well as killer-inhibitory receptors (KIRs), whose role is integrating external signals that modulate the release of perforin and granzyme-containing granules that eliminate target cells. NK cells additionally express pattern-recognition receptors (PRRs), cytokine and chemokine receptors, and antibody Fc fragment receptors, all of which also contribute to NK functions [24].

NK cells were reported to be present in human conjunctiva samples obtained by cytology, and DED patients showed similar numbers of NK cells, suggesting that NK cell populations are not increased in DED [25]. Moreover, mouse studies showed that NK cells are found in the healthy conjunctiva, and DED induction caused a rapid infiltration of NK cells into the ocular surface (cornea and conjunctiva) as well as NK cell expansion in the draining cervical lymph node (CLN) [26–28]. A dual role for NK cells in the immunopathology of DED has been described. On one hand, it was reported that NK cells progressively decrease upon DED induction, which was paralleled by lower levels of IL-13 and goblet cell loss [29]. Thus, NK cells were identified as an IL-13 source. In turn, IL-13 prevented goblet cell loss, assigning a protective role to NK cells during DED (**Table 1**). Additionally, cyclosporine A (CsA) administration preserved NK cells and down-regulated pathogenic IFNγ and IL-17 cytokines [29]. On the other hand, a pathogenic role for NK cells in acute mouse DED was suggested since NK depletion with either antibodies (anti NK1.1) [26, 28] or antiasialo rabbit serum [27] ameliorated DED signs as gauged by less corneal damage (**Table 1**). Mechanistically, when IFNγ-producing NK cells were depleted, a reduced expression of costimulatory molecules (CD80 and CD86) in antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), was found [26]. In line with this, immune neutralization of NK cells reduced CXCL9, CXCL10, and CXCL11 chemokines and IFNγ [28]. Furthermore, NK cells were identified as a source of the highly pathogenic cytokine IL-17 [27]. Ablation of NK cells resulted in matrix metalloproteinase (MMP) 3 and MMP9 attenuation in the cornea. Therefore, NK cell depletion strongly impacts pathogenic cytokine and chemokine output as well as APC activation, suggesting that rapid NK cell activation and further cytokine secretion in turn activate APCs to contribute to DED.

Mouse studies suggested that NK cells reside in the ocular surface and, upon DED induction, a highly dynamic cytokine response in these cells is initiated. However, depending on the cytokine profile released by NK cells, a differential impact on the DED outcome is observed. Altogether this mouse evidence suggested

#### *Lymphocytes in Dry Eye Disease DOI: http://dx.doi.org/10.5772/intechopen.98969*



#### **Table 1.**

*The diverse roles of lymphocytes in rodent DED.*

a dual role of NK cells in DED; a switch from normal protective IL-13-mediated to a pathogenic IFNγ- and IL-17-mediated role has been proposed for the eye-resident NK population [27]. What triggers either a protective or pathogenic program in NK cells and the role of NK cells in human DED remains to be determined.

#### **2.2 NKT cells**

NKT cells are a subgroup of innate-like lymphocytes that recognize lipid antigens presented by MHC class-I-like molecule (CD1d) and are identified by the coexpression of TCR and the NK-related NK1.1 marker. NKT cells are further divided into type I NKT or invariant NKT cells (iNKT) and type II NKT cells. Type I NKT cells express a semi-invariant TCR, whereas type II NKT cells possess a more diverse TCR repertoire. Upon NKT activation by either lipid antigens or bacterial products sensed by TLRs, these cells rapidly secrete large amounts of cytokines, such as IFNγ, TNF-α, IL-2, IL-4, IL-5, and IL-13, which modulate the function of neighbouring innate and adaptive cells. In organs such as the liver, NKT cells are abundant and the main players of the local response; however, in the eye, NKT cells are shown to be a relevant cell type [45].

Pioneer studies identified NKT cells as the resident population in the mouse ocular surface. Cells isolated through immunobeads from the mouse ocular surface under homeostatic conditions were identified using reliable NKT markers (TCR and NK1.1). Moreover, these cells were found to be an important source of IL-13; through the secretion of this molecule, NKT cells help to preserve the goblet cells, which promote ocular surface stability by producing mucins (**Table 1**) [29]. The same group of researchers subsequently confirmed their findings by identifying cells positively stained for CD3 and NK1.1 markers, a phenotype compatible with NKT cells in conjunctiva samples from healthy mice [30]. Interestingly, these authors reported that the actual number of NKT cells was higher than the number of conventional T cells (CD4 and CD8 lymphocytes), suggesting that, like in other epithelial tissues, NKT cells are abundant in the ocular surface [30].

Therefore, the evidence, although minimal but convincing, shows that NKT cells are fundamental for maintenance of the ocular surface through communication with goblet cells. Impaired crosstalk between these cells adds to the development of DED, so a protective role can therefore be inferred for NKT cells in DED.

#### **2.3** γδ **T cells**

T lymphocytes bearing a TCR composed of gamma-delta chains (γδ T cells) are less abundant than αβ T cells; however, γδ T cells represent a major T cell population in the epithelial tissues such as the skin, and gastrointestinal and reproductive

#### *Lymphocytes in Dry Eye Disease DOI: http://dx.doi.org/10.5772/intechopen.98969*

tracts. As part of the intraepithelial lymphocytes (IELs), γδ T cells are central players in the protection and homeostasis of surfaces in constant contact with the external environment. Specifically in the eye, γδ T cells collaborate in maintaining ocular immune privilege [46].

Early evidence arose from studies on the non-diabetic obese (NOD) mouse strain, which, upon ageing, develop a Sjögren's-syndrome-like disease. When DED was induced in NOD mice via scopolamine delivery, symptoms such as a decreased tear volume and goblet cell density as well as increased corneal permeability were observed (**Table 1**). The authors noted a significant decline in the numbers of γδ T cells in the conjunctival epithelium during the acute phase of DED [31]. Intriguingly, using the same DED model (scopolamine administration) in a different mouse strain (C57BL/6), decreased numbers of γδ T cells present in the conjunctival epithelium were observed when DED was induced, but increased γδ T cells were visible in flow cytometry samples [30]. Moreover, a strain-dependent effect of γδ T cells on tear volume was found where C57Bl/10 J (B10) mice lacking γδ T cells presented higher tear volume compared with C57BL/6 J (B6) similarly lacking γδ T cells [47].

Regarding human DED, the role of γδ T cells has not yet been explored; however, Sjögren's syndrome patients are reported to present altered numbers of γδ T cells. One may speculate that as DED is frequently observed in Sjögren's syndrome patients, in human DED secondary to autoimmune disease, a modified γδ T cell response is expected.

Salient evidence suggests that the depletion of γδ T cells is a hallmark of experimental DED, supporting an immunoregulatory role of the γδ T cells despite them being a well-known source of pathogenic IL-17 (**Table 1**). This regulatory role is further supported by mice lacking γδ T cells not developing anterior-chamber-associated immune deviation (ACAID), and corneal grafts are more tolerated when γδ T cells are present [48]. An anti-inflammatory role for γδ T cells in DED is currently accepted.
