Ocular graft-versus-host disease (oGVHD): From A to Z

Abstract

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a definitive therapy for a variety of disorders. One of the complications is acute graft-versus-host disease (aGVHD), which has a high mortality rate. Patients can also develop chronic graft-versus-host disease (cGVHD), a more indolent yet afflicting condition that affects up to 70% of patients. Ocular involvement (oGVHD) is one of the most prevalent presentations of cGVHD and can manifest as dry eye disease, meibomian gland dysfunction, keratitis, and conjunctivitis. Early recognition of ocular involvement using regular clinical assessments as well as robust biomarkers can aid in better management and prevention. Currently, the therapeutic strategies for the management of cGVHD, and oGVHD in particular, have mainly focused on the control of symptoms. There is an unmet need for translating the preclinical and molecular understandings of oGVHD into clinical practice. Herein, we have comprehensively reviewed the pathophysiology, pathologic features, and clinical characteristics of oGVHD and summarized the therapeutic landscape available to combat it. We also discuss the direction of future research regarding a more directed delineation of pathophysiologic underpinnings of oGVHD and the development of preventive interventions.

1. Introduction

Since the first successful report in 1968, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become a curative therapy for a variety of malignant and nonmalignant hematologic disorders and certain inherited metabolic diseases.⁴² Acute graft-versus-host disease (aGVHD) is a common lethal complication of allo-HSCT, with an incidence rate of 30–50%.¹⁵⁸ Acute GVHD is an inflammatory condition that classically affects 3 target organs: the integumentary system, the gastrointestinal epithelium, and the liver and bile ducts. Chronic GVHD (cGVHD) is the most common persistent complication in allo-HSCT patients and is the leading cause of nonrelapse deaths. Although the presence of aGVHD is a risk factor for the development of cGVHD, it can also manifest de novo.³⁹ According to the statistical estimates, the incidence of cGVHD among allo-HSCT patients is between 10% to 70%, and, likely due to increased survival rates there has been an increasing trend in its incidence.⁶ The signs and symptoms of cGVHD usually appear at least 100 days after the transplantation, and most patients will be diagnosed within the first year of transplantation.⁵³ The criteria for other forms of GVHD (including the late onset aGVHD and the overlap syndrome, which has presentations of both aGVHD and cGVHD) have been developed in recent years; however, they are not as common as the two mentioned classic forms.⁵³

Unlike aGVHD, cGVHD can affect any organ system, including skin, oral mucosa, gastrointestinal tract and hepatobiliary system, lungs, eyes, genitalia, and joints.⁵³ Less than 10% of aGVHD patients will suffer from ocular involvement that manifests as pseudomembrane conjunctivitis and corneal involvement. Based on early reports, ocular involvement is associated with increased mortality risk.¹¹⁴ On the other hand, it has been reported that ocular involvement can be seen in 30 to 85% of cGVHD cases, making it a relatively common manifestation.⁵⁰ Although ocular involvement usually manifests after the appearance of other signs of cGVHD, it can be the initial sign or symptom of the disease.¹²¹ According to the 2014 National Institutes of Health (NIH) criteria, new-onset signs of keratoconjunctivitis sicca (KCS); cicatricial conjunctivitis; confluent punctate keratopathy, or dry or painful eye--collectively known as dry eye disease (DED)--are cardinal manifestations of ocular GVHD (oGVHD).⁵³ Progression of KCS to corneal infection, ulceration, and perforation may occur in severe cases.¹³⁰ Nevertheless, the presence of DED by itself can significantly dwindle the vision,¹⁰⁷ quality of life,²⁷ and Karnofsky scores in patients.¹⁰⁷

We review the pathophysiology, pathologic features, and clinical characteristics of oGVHD, as well as the therapeutic landscape available to combat it. We also discuss the direction of future research regarding a more directed delineation of pathophysiologic underpinnings of oGVHD and the development of preventive interventions.

2. Pathophysiology of aGVHD and cGVHD

There are fundamental differences between acute and chronic GVHD. The pathophysiology of acute GVHD has been extensively studied, and there is a general agreement that the inciting event is the donor T-cell activation against the recipient antigens.¹⁵⁸ In the scenario of major histocompatibility complex (MHC) mismatch, the donor CD4⁺ T-cells can initiate the development of acute GVHD; however, for subjects with MHC match (almost all the currently encountered cases), the classic ‘three signals’ (e.g. antigen recognition, co-stimulation, and cytokine-mediated differentiation and expansion) are required for the donor T-cell activation.¹¹⁸ At the first step, antigen presenting cells (APC) are activated due to several factors such as underlying disease, prior infection, and tissue damage caused by the conditioning regimen (damage-associated molecular patterns [DAMPs] and pathogen-associated molecular patterns [PAMPs]).¹⁵⁸ The second phase is activation, proliferation, and differentiation of donor T- cells. During this process, co-stimulation between T- cell and APC is established through interaction between CD28 of T-cells and CD80 or CD86 of APCs. After T-cell activation, cytokines such as IL- 2 and IFN- γ are released, IFN- γ, promoting aGVHD. The third phase is the effector phase, leading to cell death. During this phase, tissue damage occurs.¹¹⁸

Currently, the pathophysiological underpinnings of the development of cGVHD are not well-understood.⁴⁰ Various cells such as Tregs and B-cells, effector T-cells, are implicated in the development of cGVHD.^{35, 149} Notably, it seems that patients with cGVHD have fewer Tregs compared to control patients.³⁵ On the other hand, studies have reported that the number of B-cells is increased in cGVHD.^{55, 117} Overall, there is an unmet need to achieve more solid conclusions.

3. Pathologic features of oGVHD

3.1. Lacrimal glands

The most common manifestation of oGVHD is DED. In accordance with the fibrotic changes in different target tissues as the hallmark of cGVHD, lacrimal gland fibrosis is thought to be the main culprit for the development of DED.⁹¹ Fibrotic changes in the conjunctiva, leading to a decrease in tear production by accessory lacrimal glands and tear evaporation due to alterations in the lipid layer of the tear film are also implicated in this condition.⁹¹ Early immunohistochemical investigation of oGVHD-related lacrimal glands showed the presence of activated CD34⁺ fibroblasts.⁹¹ Ogawa and colleagues reported periductal infiltrations with T-cells (mostly CD8⁺ cells),⁹² thickening and multilayering of the vascular and ductal basal lamina, and a positive association between the bulk of fibrosis and the severity of oGVHD-related signs. They hypothesized that residing donor T-cells (mainly CD4⁺ cells) can induce fibroblasts activation,⁹² and more interestingly, such fibroblasts can act as APCs for T-cells, which creates a vicious feedback loop (Figure 1).⁹¹

Figure 1.

Pathological alterations in the lacrimal glands of oGVHD cases and its immunological underpinnings. The basement membrane of lacrimal ducts and lacrimal vasculature exhibits multilayering and thickening. The CD8+ and CD4+ T-cells, along with plasma cells, Th17 cells, macrophages, and neutrophils, infiltrate the peri-ductal area and contribute to fibroblast activation, myofibroblast emergence, and eventually, tissue fibrosis. In addition, activated fibroblasts perpetuate immune activation by acting as antigen-presenting cells (by expressing HLA-DR, CD34, CD40, CD54, CD80, and CD86). The involvement of other pro-fibrotic mediators and EMT-inducers, namely TGF-β, neutrophil extracellular traps, renin-angiotensin-aldosterone system, reactive oxygen species, and chemokines is also implicated in the oGVHD-related lacrimal gland destruction.

More recently, the importance of the renin-angiotensin-aldosterone system (RAAS) in the development of cGVHD-related fibrosis has been studied.¹⁵¹ Yaguchi and colleagues have shown that mouse lacrimal glands express mRNAs of angiotensinogen, prorenin, angiotensin-converting enzyme (ACE), and angiotensin II receptors type 1 (AT1R) and type 2 (AT2R), that collectively make the “tissue RAAS”.¹⁵¹ Interestingly, administration of an AT1R antagonist (valsartan) culminated in a lesser degree of fibrosis, decreased densities of CD45⁺ inflammatory cells and fibroblasts, and higher tear production in a cGVHD mouse model. It is presumed that the fibrogenic effects of angiotensin II are mediated via AT1R, which can subsequently trigger the upregulation of the expression of transforming growth factor-beta (TGF-β). TGF- β induces the activation and productivity of fibroblasts.¹⁵² α -SMA is a differentiation marker for activated myofibroblasts (MFBs), and its expression can be induced by the triggering effect of TGF-β.¹²⁰ Shamloo and colleagues have further shown the co-localization of α-SMA and AT1R, which implicates another aspect of the role of RAAS in the induction of ocular fibrosis.¹²⁰ Last but not least, the attachment of angiotensin II to the AT1R can also trigger the activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and causes mitochondrial dysfunction which increases reactive oxygen species (ROS) generation.¹⁴⁰

3.2. Meibomian glands

Meibomian gland dysfunction (MGD) is a common disorder that generally manifests as DED. MGD is the result of the morphological and functional changes in the meibomian glands.¹⁶ Clinically, meibomian gland involvement is present in 47.5 to 80% of patients with oGVHD and may be diagnosed even before the conduction of allo-HSCT.⁹⁰ Studies on a mouse model of cGVHD, however, have shown endothelial damage, fibroblast activation, and immune cell infiltrations in the MGs, and necrosis and apoptosis of MG basal cells.¹⁵⁵ Moreover, the eyelid blood vessels can exhibit several pathologic alterations, including tortuosity, dilation, neovascularization, and peri-vascular inflammatory cell infiltration and fibrosis.¹⁵⁵ Clinically, MGD among oGVHD patients presents with gross abnormalities such as orifice obstruction (by hyperkeratinization), displacement of mucocutaneous (gray) line, vascular tortuosity, altered quality and quantity of sebum, and acinar cell atrophy.¹⁰ Compared to the non-dry eye/non-cGVHD cases, the mean meibomian gland acinar unit density (MGAUD), meibomian gland acinar longest diameter (MGALD), meibomian gland acinar shortest diameter (MGASD) values in confocal microscopy of cGVHD-related dry eye cases are significantly lower, meanwhile the mean fibrosis grade is significantly higher. Also, clinical findings on the lid margin, tear dynamics, and ocular surface findings were significantly worse.¹⁰

In a comparative study, Choi and colleagues aimed to assess and compare the features of meibomian glands of patients with oGVHD, Sjögren’s syndrome (SS), MGD, and healthy controls.¹⁷ They found that oGVHD cases have significantly more severe meibomian gland abnormalities (indicated by higher meibum expressibility score [MES], meibum quality score [MQS], lid margin abnormality score, ocular surface staining, and gland dropout). More importantly, they identified positive correlations between the mentioned scores and the severity of oGVHD.¹⁷

It has been proposed that early, pretransplantation loss of meibomian gland area (MGA) is a predictive indicator for the development of oGVHD.³⁰ Nevertheless, there are controversies regarding this issue. Since the meibum is secreted via the holocrine pathway, meibocytes have a high turnover rate. As a result, chemotherapy and radiotherapy regimens might exert detrimental effects on the meibomian glands’ regeneration.¹⁰⁶ In addition, meibum secretion is stimulated by circulating androgens, and there are reports of hormonal disturbances among allo-HSCT subjects.¹³⁴ Hence, in addition to the importance of inflammation, the role of these hormonal factors should be investigated in future research.

3.3. Cornea

Corneal involvement is common among oGVHD patients (Figure 2). In one report, 66% of cases had superficial punctate keratopathy, and 29% showed evidence of filamentary keratitis.⁹ In another report, the prevalence of superficial punctate keratitis was as high as 91%.²⁴ Persistent epithelial defects (PEDs) are reported to be seen in around 8% of oGVHD cases.¹²⁶ PEDs are associated with limbal stem cell deficiency (LSCD), subconjunctival fibrosis, and filamentary keratitis. The most gruesome corneal complication of OGVHD, corneal perforation, occurs in 3.7% of cases.¹²⁶ Superior limbic keratoconjunctivitis (SLK)-like involvement is not common among oGVHD patients, as its reported prevalence has been only 8.8%.⁶⁶

Figure 2.

Corneal manifestations of oGVHD. a. A case of oGVHD which interestingly presented with corneal perforation. MGD, prominent eyelid margin vascularization, pupil distortion, and iris prolapse in the location of corneal thinning is evident. b. A case of oGVHD with chronic involvement of cornea including neovascularization and opacity.

Numerous pathological changes have been documented in the cornea and limbus of patients with cGVHD-related DED. An early study by Wang and colleagues showed that compared to healthy controls, post allo-HSCT patients demonstrated decreased corneal sensitivity (CS).¹⁴⁴ Moreover, the number of inflammatory cells within the conjunctival brush cytology samples was significantly higher for oGVHD-related DED compared to healthy controls and post-HSCT cases without DED. It should be mentioned that, in this study, the decrease in CS was similar for all severity stratifications of DED.¹⁴⁴ Therefore, the authors of this study speculated that the conditioning regimens that are being administered before the conduction of allo-HSCT might be responsible for this finding.¹⁴⁴ In a retrospective study on oGVHD cases,¹²⁵ 14% showed evidences of neurotrophic keratopathy in the course of the disease.

In another attempt, DED cases with or without the underlying oGVHD were recruited and evaluated by in vivo confocal microscopy (IVCM). Both groups had a higher density of conjunctival immune cells and corneal dendritic cells (DCs) but showed decreases in the density of the corneal sub-basal nerves.¹⁰ The pathologic changes of corneal nerves are not limited to their altered densities; the other alterations include increased tortuosity of nerve fibers and DC densities and decreased corneal superficial epithelial cells, nerve fibers’ densities, and nerve reflectivity.¹⁰

Similarly, the corneal densities of DCs and their size and number of dendrites have been higher among patients with DED compared to healthy subjects.⁶⁴ Intriguingly, one study documented significantly higher DC density, size, and related dendrites among those with SS and oGVHD-related DED, compared with non-immune causes.⁶⁴ In addition, a recent study found that the size and dendrites of DCs increase as the DED becomes more severe.²

3.4. Conjunctiva

Conjunctival involvement among aGVHD patients may have different manifestations. A commonly used classification for the conjunctival involvement in aGVHD comprises four stages, i.e., conjunctival hyperemia (stage I), chemosis (stage II), pseudomembrane formation (stage III), and corneal epithelial sloughing (stage IV).⁵¹ Generally, conjunctival involvement is mostly seen among aGVHD cases. In a study on a series of post-HSCT subjects found that the prevalence rates of KCS and pseudomembrane formation are 10% and 9.2%, respectively.⁶³ In another report, 14.2% of aGVHD cases have had acute conjunctivitis.⁹⁰

Involvement in cGVHD can also be graded. In grade 1, conjunctival hyperemia is present. In grade 2, palpebral conjunctival fibrovascular changes occur with or without epithelial sloughing. In grade 3, these changes involve 25–75% of total surface area, and finally, in grade 4, involvement of 75% of total surface area with or without cicatricial entropion can be expected.⁵¹ In a retrospective study on chronic phase of 62 oGVHD cases, conjunctival hyperemia was detectable in 65% of cases, followed by chemosis in 29%, conjunctival ulceration and sloughing in 13%, subconjunctival fibrosis in 6%, and forniceal foreshortening and symblepharon in 5% of cases.⁹ The formation of fibrosis in the conjunctiva can further evolve into cicatricial changes, which eventually lead to eyelid abnormalities, including entropion and lagophthalmos, that will further perpetuate the vicious cycle of DED.⁹ From the molecular point of view, the conjunctiva of oGVHD eyes exhibits fibrosis, loss of secretory function, and metaplastic alterations.⁹³ In one study, Ogawa and colleagues examined the conjunctival biopsies of oGVHD and healthy individuals with electron microscopy and immunohistochemical staining for epithelial and mesenchymal markers.⁹³ Ocular GVHD samples showed fibrosis of subconjunctival stroma containing active fibroblasts, thinning of the conjunctival epithelial and basal lamina, and disruption of the basal epithelial cells.⁹³ Along with these findings, basal epithelial cells exhibited reduced expression of E-cadherin and increased staining for Snail, α-SMA, heat shock protein (HSP) 47, and collagen bundles.⁹³ E-cadherin is responsible for epithelial cell-to-cell adhesions, and its absence in the epithelial layer is seen before the initiation of epithelial to mesenchymal transition (EMT).⁹³ Snail is a potent inhibitor of E-cadherin, and α-SMA, along with HSP47, are markers of mesenchymal cells.⁹³ Moreover, the disruption and segmentation of the basal lamina in the stroma under disrupted segments were seen.⁹³ Based on these observations and the interaction between p63/HSP47 positive cells and lymphocytes, the authors concluded that EMT occurs in the conjunctiva following cytokine release by the immune system and is partially responsible for the fibrotic changes. Furthermore, this study has found similar pathologic alterations in the lacrimal glands, which might be conducive to their fibrosis (Figure 3).⁹³

Figure 3.

Pathological findings of the oGVHD conjunctiva. Thinning of epithelia, loss of goblet cell microvilli and their densities; thickening, multilayering, and disruption of the basal lamina; vacuolization of basal cells; CD8+ T-cell infiltration; and fibroblast activation and myofibroblast generation are seen in the conjunctival samples of oGVHD eyes. The most prominent pathologic feature is the EMT, which is the consequence of immune activation and cytokine release.

The microscopic characteristics of oGVHD-afflicted conjunctiva are not limited to the aforementioned findings. In fact, some studies^{8, 144} have documented squamous cell metaplasia (assessed by impression cytology) and decreased density of goblet cells in the superficial conjunctival epithelium, which has been considerably different from that of normal subjects and SS cases. Furthermore, the epithelial cell microvilli had a shorter height and wider width, as well as decreased densities. The epithelial cells of oGVHD and SS patients had a lower density of secreting vesicles, and more importantly, the oGVHD cases have shown thinner Mucin 1 (MUC1), MUC4, and MUC16, and also MUC5AC layers.¹⁴³ Lubrication, hydration, and protection of the ocular surface against pathogen invasions are the main functions of these mucins.⁴⁴ Goblet cells have also been proposed to have immunomodulatory functions. They produce TGF-β2, which can impede the IL-2-dependent growth of T-cells⁶⁹ and maintain the immature, tolerogenic form of DCs.²⁰ Other studies have shown the presence of intercellular adhesion molecule 1 (ICAM-1) on the oGVHD conjunctiva, which has been correlated with lower Schirmer’s test scores and goblet cell densities.⁷

3.5. Other ocular manifestations

Some studies have reported ocular hypertension and glaucoma as comorbidities of oGVHD. In one report, 33 out of 218 oGVHD patients had ocular hypertension, eight have had possible glaucoma, and one has had definite glaucoma.¹¹² Of note, the authors of this study have mentioned the use of topical and systemic corticosteroids as the principal cause of increased ocular pressure.¹¹² The formation of cataracts (mainly the posterior subcapsular form) generally follows the same pathway as ocular hypertension and glaucoma.¹¹² In fact, it mainly develops as a complication of the administration of corticosteroids and radiation therapy, with a prevalence rate similar to that of glaucoma.¹³⁰ Other conditions, including episcleritis, scleritis, uveitis, central serous chorioretinopathy, serous retinal detachment, microvascular retinopathy, infectious retinitis or endophthalmitis, optic nerve head edema, and intraocular lymphoma are rarely encountered in oGVHD cases.²⁸

4. Microbiome and oGVHD

In recent years, the importance of commensal microflora in the development or prevention of a wide variety of disorders has gained unprecedented interest.¹³⁷ The conjunctival epithelium is home to a variety of microbial agents, and some authors have attempted to investigate the alterations in their populations (concerning both numbers and species) before and after the development of oGVHD.

Using the traditional culture-based methods, Shimizua and colleagues¹²³ observed significantly higher rates of positive cultures (75%) and also multiple detections (positive culture for more than 2 species, 45.8%) among the oGVHD cases compared to non-GVHD allo-HSCT cases (36% and 3.6%), and healthy subjects (25% and 0). They found Staphylococcus Epidermidis, α-hemolytic Streptococcus, Corynebacterium species, and Propionibacterium Acnes as the most common species among the oGVHD subjects.¹²³

Using the DNA sequencing methods, another study aimed to evaluate the ocular microbiome of a variety of severe ocular surface disorders.¹⁶⁰ Contrary to the findings of the previous report, this study found that 63% of healthy and 43% of oGVHD eyes have positive results (probably as the consequence of topical antibiotic usage). Furthermore, the authors found that one of the oGVHD eyes has Enterobacteriaceae species, which has been a unique finding in their study.¹⁶⁰ In addition, five of six oGVHD-positive eyes exhibited evidence of Lactobacillus/Streptococcus species, similar to healthy eyes. Last but not least, in one patient with bilateral oGVHD, each eye showed a completely different microbiome.¹⁶⁰ Another study⁷⁴ used shotgun metagenomic sequencing to compare the diversity of the healthy and post-allo-HSCT eyes’ microbiome. This study found a significantly reduced microbiome diversity for post-allo-HSCT eyes. Besides, Gordonia bronchialis and Pseudomonas parafulva were the predominant species, showing a completely different microbiome in oGVHD eyes from normal ones.⁷⁴

Animal studies are available evaluating the effects of the oral administration of antibiotics on the microbiome. It has been proposed that the interactions between the gut microbiome and the immune system might be an effective factor.¹¹⁵ In line with the importance of the microbiome and to evaluate the effects of oral administration of antibiotics, Sato and colleagues used a mouse model and gave them gentamicin, ampicillin, vancomycin, and fradiomycin two weeks before until four weeks after the transplantation and compared their clinicopathologic features with the control group. They observed that the gentamicin-treated group had lower corneal fluorescein staining (CFS) and higher tear film break-up time (TFBT) scores, less fibrosis and fewer inflammatory and α-SMA positive cell infiltration inside the target organs, lower mRNA levels of IL-6 and IL-17; fewer Th17 and higher Treg densities in the lacrimal glands, and lower systemic GVHD scores.¹¹⁵ The molecular and physiologic mechanisms behind such promising effects are yet to be elucidated; however, the investigators have proposed that the interactions between the gut microbiome and the immune system might play a leading role.

5. Tear biomarkers

With respect to the importance of delineating the pathologic processes contributing to the manifestation of oGVHD and diagnosing this condition more accurately and promptly, the cytokine and protein levels of the tear have been of interest to many researchers. Table 1 provides a brief summarization of the status of various cytokines, growth factors, and other immune-related molecules in the tear of oGVHD eyes.

Table 1.

Alteration in the tear levels of various cytokines, growth factors, and other immune-related molecules of oGVHD eyes.

Tear molecule	Status	Reference(s)	Tear molecule	Status	Reference(s)
IL-1β	↑	46	CCL24	↑	18
IL-1Rα	↑	19	CXCL9	↑	146
IL-6	↑	18, 46, 86, 128	CXCL10	↑	105, 146
IL-8	↑	4, 19, 46, 86, 105	CXCR3	↑	146
IL-9	↑	18	ICAM-1	↑	46
IL-10	↑	18, 19, 86	MMP-8	↑	5
IL-12	↑	86	MMP-9	↑	5, 86
IL-17	↑	86	VEGF	↑	86
IFN-γ	↑	18, 86	eDNA	↑	4, 128
CCL2	↑	18	TNFSF14	↑	4
CCL18	↑	18	TNF-α	↑	4, 105, 128
Neutrophil elastase	↑	4, 5, 128	IL-7	↓	46
Myeloperoxidase	↑	4, 5	EGF	↓	19, 46
NGAL	↑	4	EGFR	↓	18

(oGVHD, ocular graft-versus-host disease; IL, interleukin; IFN, interferon; CCL, chemokine (C-C motif) ligand; NGAL, neutrophil gelatinase-associated lipocalin; CXCL, chemokine (C-X-C motif) ligand; CXCR, chemokine (C-X-C motif) receptor; ICAM-1, intercellular adhesion molecule 1; MMP-9, matrix metalloproteinase-9; VEGF, vascular endothelial growth factor; eDNA, extracellular DNA; TNFSF14, tumor necrosis factor superfamily 14; TNF- α, tumor necrosis factor-alpha; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor.)

6. Tear proteomic of oGVHD

The investigators initiated to analyze the tear proteome in the early 2000s and identified more than 1500 proteins in the tears of healthy subjects.^{26, 159} Lactoferrin, lysozyme, lipocalin, and soluble immunoglobulin A (sIgA) are the most abundant proteins in the tears of healthy eyes.¹³⁶ In recent years, researchers have aimed to identify similarities and differences between the tear proteome of oGVHD and healthy eyes, with similar purposes and methods as those of tear cytokine studies.

It is noteworthy that many of the mentioned changes in the tear proteome of oGVHD eyes are similar to those of DED.⁷³ The reduction in the concentration of most of the mentioned proteins is the result of lacrimal gland destruction.²³ Of note, the currently published studies on the tear proteome of oGVHD eyes have not enrolled a sufficient number of patients. Hence, prospective studies should focus on larger cohorts of post-allo-HSCT subjects to illustrate whether the tear proteome alterations occur before the onset of clinical manifestations of oGVHD. The functional roles and status of these proteins during oGVHD are summarized in Table 2.

Table 2.

Commonly reported tear proteins that exhibit alterations in their concentration after the development of oGVHD.

Protein name	Functions	Status	Reference(s)
Histone H2B and H4	Nucleosome formation; histone and its proteins get released from the dying cells77 and constitute a part of the NETs,4 which have substantial roles in the induction of ocular surface inflammation.	↑	23, 38
Periplakin	An intracellular scaffold protein that is involved in the cornification of epithelial cells.110	↑	38
Ribosome binding protein 1	Similar to histone proteins.	↑	38
Annexin A1 and A2	Membrane trafficking, mediation of IFN-γ induced inflammation,141 and induction of epithelial cell migration during corneal wound healing.78	↑	23, 89
Actin, cytoplasmic 1	One of the cytoskeletal proteins. Its extracellular form is a sign of cell death and is an inducer of inflammation.71	↑	23, 89
Prelamin-A/C	A nuclear membrane stabilizer.43 Its overexpression is related to the shortening telomerase, hence, linked to the senescence processes,47 which are implicated as one of the underpinnings of the oGVHD.154	↑	38
S100 proteins A8 and A9	S100 family contains calcium-binding proteins which are involved in the induction of inflammation, ROS production, and cornification of keratinocytes.29	↑	23, 38
Ig gamma-1 and −3 chain C region	Complement activation, immune-cell chemoattractant, and antigen-binding capacities.38	↑	23, 38
Lactotransferrin	Antimicrobial and anti-inflammatory functions via binding to and reducing the levels of free iron.33	↓	23, 38
Proline-rich protein 1	Unknown.38 Decreased in many studies on DED subjects.104	↓	23, 38
Lipocalin-1	An integral part of tear antimicrobial components and is also a stabilizer of the lipid layer of tear22 and, due to its endonuclease activities,157 an eliminator of extracellular DNAs.	↓	23, 38
Cystatin-S	Cysteine protease inhibitors	↓	23, 38
Lysozyme C	Antimicrobial activities via the hydrolysis of the bacterial cell wall.80	↓	23, 38
Polymeric immunoglobulin receptor	Transportation of dimeric IgA through the apical surface of epithelial cells, also known as transmembrane secretory component.58	↓	23, 89
Prolactin-inducible protein	Might have roles in maintaining tear quality and modifying tear evaporation.60	↓	23, 89
Ig heavy constant α1 and α2	-	↓	23, 89
Ig J chain	Formation of the binding site for polymeric Ig receptor within the polymeric IgA, which is essential for its secretion.57	↓	23, 89

(oGVHD, ocular graft-versus-host disease; NET, neutrophil extracellular trap; IFN-γ, interferon-gamma; ROS, reactive oxygen species; Ig, immunoglobulin; DNA, deoxyribonucleic acid)

7. The importance of senescence in the pathogenesis of oGVHD

Cellular senescence is defined as the arrest of the cell cycle, with the resultant loss of proliferative and regenerative capacities of cells.⁶² The DNA damage that is caused by conditioning chemo-radiotherapeutic regimens, as well as the inflammatory milieu, will induce the activation of senescence-associated molecular pathways.⁶²

Stress-induced senescence can also instigate a “senescence-associated secretory phenotype” (SASP) in the affected cells, which is characterized by the secretion of several cytokines, including IL-1β, IL-6, IL-8, CXCL-1, and CXCL-9.¹⁰⁹ A preclinical study on an oGVHD mouse model¹⁵⁴ has confirmed the higher concentrations of the mentioned cytokines in the lacrimal gland of affected mice compared to healthy controls. In addition, immune cells, fibroblasts, and other cellular infiltration of the oGVHD-affected lacrimal glands have shown significantly higher levels of senescence-associated molecules (p16, p21, and osteopontin), markers of DNA damage response (53BP1), and also the proliferation biomarker Ki-67.¹⁵⁴ Subsequently, the administration of the senolytic ABT-263 (navitoclax), an inhibitor of the Bcl-2 anti-apoptotic family and inducer of pro-apoptotic molecules Bax and Bak, has resulted in the alleviation of lacrimal gland fibrosis and immune-cells infiltration, decreased cytokine production, and enhanced tear secretion.¹⁵⁴

8. Clinical risk factors for oGVHD

Apart from different underlying pathologic mechanisms, various cross-sectional and cohort studies have documented clinical parameters that are associated with an increased risk of developing oGVHD. In fact, there is considerable heterogeneity in the reports of studies regarding risk factors and epidemiologic characteristics of oGVHD. However, most of it stems from the lack of widely accepted criteria for the diagnosis of this disorder, as discussed earlier. Factors positively associated with the risk of developing oGVHD are listed in Table 3.

Table 3.

Signs, symptoms, and co-morbidities that are positively associated with the increased risk of oGVHD.

Risk factor	Reference(s)	Risk factor	References(s)
Female-to-male allo-HSCT	11, 52, 54, 61	Chronic or acute myeloid leukemia (compared to aplastic anemia)	72
The female sex of recipients	102	Presence of malignant disease	54
Matched related donors	11	Higher age of recipients	54, 102
Matched unrelated donors	54	Age over 27 years of recipients	72
Diabetes mellitus	85	PBSCT	54, 102
Repeated allo-HSCT	85	PBSCT compared to BMT	72
History of aGVHD	102	PBSCT, compared to both BMT and CBT (only for severe oGVHD)	138
History of acute skin GVHD	52	Schirmer’s test value of <10 mm	72
Number of affected organs by stage I cGVHD	85	Schirmer’s test values of ≤10 mm/5 minute before transplantation	54
Grade III-IV aGVHD	54	Presence of dry mouth	72
Grade II/III overall cGVHD	142	Epstein-Barr virus-positive donor	142
Grade I-III oral and skin cGVHD	142	Conjunctival fibrosis and hyperemia	11
Presence of systemic cGVHD	72

(oGVHD, ocular graft-versus-host disease; allo-HSCT, allogeneic hematopoietic stem cell transplantation; aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; PBSCT, peripheral blood stem cell transplantation; BMT, bone marrow stem cell transplantation; CBT, cord blood stem cell transplantation; mm, millimeters)

9. Diagnosis

Complete ophthalmological assessment should be performed including visual acuity testing, slit-lamp examination with vital dye staining (e.g., fluorescein dye, lissamine green, or Rose bengal), evaluation of tear breakup time (TBUT), Schirmer testing, and corneal sensitivity and also funduscopy.

The two widely acknowledged diagnostic criteria for oGVHD are as follows: NIH CC 2014 criteria: The diagnostic criteria were based on the Schirmer test and slit- lamp examination.⁵³ The International Chronic oGVHD (ICCGVHD) consensus group diagnostic criteria are based on scores derived from the Ocular Surface Disease Index (OSDI), Schirmer’s test without anesthesia, CFS, conjunctival injection, and presence of systemic GVHD. The diagnostic categories included no oGVHD, probable oGVHD, and definite oGVHD^{87, 95} (Table 4).

Table 4:

Ocular GVHD diagnostic criteria and grading scale according to the NIH criteria (2014) and Ocular GVHD diagnostic criteria and grading scale according to the International Consensus Criteria on chronic ocular graft- versus- host disease (ICCGVHD)

A. Ocular GVHD diagnostic criteria and grading scale according to the NIH criteria (2014)
Severity Grade	Score 0	Score 1	Score 2	Score 3
Symptoms	KCS confirmed by an ophthalmologist in the absence of symptoms, the requirement of eye drops or ADL	Mild	Moderate	Severe
The requirement of lubricant eye drops		≤3 times/day	>3 times/day or punctal plug	Special eyewear required to relieve pain
ADL impairment		Not affected	Partially affected without new vision impairment due to KCS	Significantly affected or unable to work or loss of vision due to KCS
B. Ocular GVHD diagnostic criteria and grading scale according to the International Consensus Criteria on chronic ocular graft-versus-host disease (ICCGVHD)
Diagnosis	None (points)	Probable ocular GVHD (points)	Definite ocular GVHD (points)
Systemic GVHD (−)	0–5	6–7	≥8
Systemic GVHD (+)	0–3	4–5	≥6
Severity scale	Schirmer test (mm)	CFS (points)	OSDI (points)	Conjunctival injection
0	>15	0 (No staining)	<13	None
1	11–15	<2 (Minimal Staining)	13–22	Mild/Moderate
2	6–10	2–3 (Mild-moderate staining)	23–32	Severe
3	≤5	>4 (Severe staining)	≥33	-
oGVHD disease severity	None	Mild-Moderate	Severe
The total score is obtained by adding the severity score for Schirmer test + CFS + OSDI + conjunctival injection	0–4	5–8	9–11

(KCS, keratoconjunctivitis sicca; ADL, activities of daily living; CFS, corneal fluorescein staining; OSDI, ocular surface disease index)

A moderate agreement between the two mentioned diagnostic criteria has been found.¹⁰¹ However, it seems that ICCGVHD is superior in differentiation between oGVHD patients and non- oGVHD DED.¹⁰¹ Regarding the agreement between ICCGVHD and Best Clinical Practices (BCPs), which is defined assessment of oGVHD cases by a highly trained single expert with extensive [>20 years] clinical experience on oGVHD, it has been reported that mild cases of oGVHD were over-diagnosed by BCPs while a stronger agreement was found in more severe cases.¹⁰¹ Besides, the Tear Film and Ocular Surface Society Dry Eye Workshop II (TFOS DEWS II) criteria and the Japanese Dry Eye Society criteria are the other diagnostic criteria, which have been used in studies on GVHD.^{41, 122, 133, 148} However, these criteria were originally meant for conventional DED.

Besides clinical examination, several diagnostic tools such as meibography, tear interferometry, confocal scanning, and tear film osmolarity are available to help ophthalmologists in the evaluation of oGVHD patients. Meibography provides the chance of in vivo evaluation of the structure of meibomian glands and ducts. A cutoff value of 40% of MG area has been adopted for diagnosing MGD in oGVHD patients.¹⁰⁶ Also, the function of meibomian glands in oGVHD cases can be assessed by tear interferometry.¹⁰ As discussed earlier, structural alteration in the ocular surface of oGVHD patients can be studied by confocal microscopy at a cellular level.¹⁰ Regarding tear film osmolarity, cutoff value of >310 mOsm/L has sensitivity of 98.4% and specificity of 60.7% in diagnosing oGVHD.⁸⁴

10. Prevention

GVHD prevention strategies are based on proper HLA-matching. Number, activity, and proliferation of donor T-cells can be targeted via prophylactic agents. For this purpose, several immunosuppressives can be applied, including calcineurin inhibitors (e.g., cyclosporine and tacrolimus), methotrexate, mycophenolate mofetil, Anti-thymocyte globulin (ATG), monoclonal antibody alemtuzumab (anti-CD52), and anti-TNF drugs. Currently, tacrolimus plus methotrexate, with or without ATG, is the standard prophylactic regimen.¹³⁵ Also, it has been shown that targeting Aurora kinase A and JAK2 can prevent GVHD.¹²

11. Therapeutic strategies to combat oGVHD

Like most other immune system-related disorders, corticosteroids have been an integral part of the therapeutic approach for both aGVHD and cGVHD.¹⁰³ The administration of systemic steroids is generally initiated for moderate and severe cGVHD,^{53, 147} and depending on the site of involvement, mild cGVHD can be managed by the application of topical immunosuppressants.¹⁴⁷ Nevertheless, systemic steroids are not curative. In a cohort of 283 cGVHD patients, the 6-month overall response after the administration of systemic therapies (prednisone and/or a calcineurin inhibitor) was 32%. The response rate was even worse for ophthalmic involvement, as only 23% of patients showed a response.⁴⁹ As a consequence, around 50% to 60% of cGVHD cases will eventually require second-line systemic therapies due to either steroid-refractory disease¹¹⁶ or the development of steroid-related debilitating complications.³⁴ Until now, there was no accepted consensus on the agent(s) of choice for the second-line therapies. Tyrosine kinase inhibitors (TKIs, e.g., ibrutinib and nintedanib), Janus-associated kinase (JAK1/2) inhibitors (e.g., itacitinib and ruxolitinib), mammalian target of rapamycin (mTOR) inhibitors (e.g., sirolimus), Rho-associated coiled-coil-containing protein kinase-2 (ROCK2) inhibitor (Belumosudil), proteasome inhibitors (ixazomib), inhibitors of TGF-β and fibroblast proliferation (pirfenidone) and extracorporeal photopheresis (ECP) have been used, among other immunosuppressive agents.⁷⁹ Early reports on such therapies revealed more favorable response rates, but at the expense of severe toxicities. The combination of methylprednisolone with tacrolimus and sirolimus led to an overall response rate of 63% (22/35) among cGVHD cases. Also, a response rate of 64% (7/11) was reported for oGVHD patients.²¹ The outcome of therapy with ruxolitinib was 26% (6/23) in one study⁸¹ although higher response rates are also reported.³¹

Due to the pathophysiologic underpinnings of oGVHD and the toxicity of systemic therapies, as well as the acceptable effectiveness of topical therapies and interventions, such formulas comprise the mainstay of the therapeutic approach for oGVHD.¹⁰³ Topical therapies can be broadly categorized into those that prevent tear evaporation, reduce ocular surface inflammation, and increase surface lubrication.

11.1. Medical management

11.1.1. Lubrication

Preservative-free artificial tears and viscous eye drops reduce surface friction, dilute the concentration of inflammatory cytokines, and enhance the visual quality.¹⁴ The prescription of artificial tears might serve as the sole therapy for those with mild DED.¹⁴ The addition of acetylcysteine drops can be beneficial by enhancing TBUT and Schirmer test values among those with moderate/severe DED, probably by strengthening the adherence of the tear layer to the ocular surface.⁸⁸ The administration of oral muscarinic agonists (namely pilocarpine and cevimeline) is effective in improving tear production among patients with SS,¹⁰⁰ but their side effects and contraindications have prevented their routine prescription by physicians.

Diquafosol is another option for oGVHD DED. As a purinergic agonist of the P2Y2 receptor, diquafosol is dispersed on the ocular surface and, after stimulation, can induce mucin and aqueous tear secretion by the influx of calcium ions into the goblet cells.¹⁵³ Treatment of mild-to-moderate oGVHD with 3.0% diquafosol drops has resulted in improved TBUT and CFS and reduced pain sensation, but without noticeable effects on the Schirmer’s test values.¹⁵³ Rebamipide is another mucin secretagogue¹³² with anti-inflammatory properties as it inhibits IL-6, IL-8, and CCL-11 production and also attenuates the degradation of NF-κB inhibitor IκBα.³⁶ Furthermore, it can stimulate the proliferation of conjunctival goblet cells.¹³⁹ Rebamipide drop has shown favorable outcomes in studies and randomized trials enrolling patients with DED.⁶⁷ In the case of oGVHD, rebamipide eye drop has been able to partially maintain tear film layer volume and decrease the corneal keratopathy score in a mouse model of cGVHD.¹¹⁹ Prospective trials are required to assess its efficacy for oGVHD patients. Finally, some studies have evidenced the efficacy of topical progesterone on enhanced tear production in mice.⁷⁵ Based on such descriptions and the antinociceptive activities of progesterone, a randomized phase II trial aimed to evaluate the effects of the forehead application of progesterone 1% topical gel on oGVHD cases with moderate to severe ocular discomfort. This therapy resulted in significantly reduced severity and frequency of symptoms and central-inferior CFS.⁷⁵

11.1.2. Control of inflammation

Owing to the inflammatory nature of oGVHD, topical anti-inflammatory agents should be administered for those who do not respond to the initial treatment with artificial tears, as well as those with moderate to severe disease.¹⁴ Topical corticosteroids might be used for cases with cicatricial¹⁰⁸ and pseudomembranous conjunctivitis,⁶⁶ acute exacerbations of both acute and chronic oGVHD,²⁷ and moderate to severe disease.¹⁴ However, one study has documented a minimal response to low-dose topical steroids in moderate to severe oGVHD.¹⁵⁶ Higher doses of these agents are also contraindicated in the presence of CEDs, ocular infections, and stromal thinning. Moreover, concerns about their debilitating side effects and the frequent use of systemic steroids in the situation of exacerbations has limited the use of topical steroids.¹⁴

Topical cyclosporine 0.05% is a well-known agent of this group, and various trials have supported its promising efficacy in enhancing tear secretion and TBUT and Schirmer test values, increasing the densities of goblet cells and MUC5AC mRNA expression, and diminishing the apoptosis and the number of inflammatory cells.¹⁴³ Of note, the ocular response to calcineurin inhibitors might take weeks.²⁷

Tacrolimus, a more potent calcineurin inhibitor than cyclosporine, might serve as a suitable substitute for steroids. The findings of a retrospective case series showed that the administration of topical tacrolimus 0.02% can significantly reduce ocular surface inflammation and steroid usage.⁵⁹ In a prospective study on seven oGVHD cases with anterior segment inflammation refractory to topical steroids, the application of tacrolimus 0.03% ointment, along with infrequent steroid use, was able to significantly alleviate corneal and conjunctival inflammation.¹¹¹ In one trial on patients with refractory oGVHD, a 10-week application of topical tacrolimus 0.05% resulted in a significant reduction in CFS, OSDI, and ICAM-1 and HLA-DR scores, while methylprednisolone 0.5% showed inferior efficacy in reducing HLA-DR score, CFS, and OSDI.¹ However, these improvements have come at the cost of a severe burning sensation in 75% of the users, which has led to its withdrawal in three out of 24 cases.¹

There are reports on the efficacy of several novel topical anti-inflammatory solutions for oGVHD. One study showed the potency of anakinra, an antagonist of IL-1Rα, in alleviating corneal epitheliopathy among patients with refractory DED and concomitant MGD.³ Lifitegrast is an antagonist of lymphocyte function antigen-1 (LFA-1), an integrin that is essential for the migration of lymphocytes.¹²⁷ Several randomized trials have demonstrated its efficacy in reducing the eye dryness score (EDS), and to a lesser extent, the ocular discomfort score (ODS) of DED patients.⁴⁵ Based on these results, the United States Food and Drug Administration (FDA) has approved lifitegrast for the treatment of DED.^{15, 45} Regarding oGVHD, a study on 18 cases showed the ability of lifitegrast to reduce the NIH severity score in 8 (44%) cases. However, it should be noted that 72% of the included cases have been receiving concurrent systemic immunosuppressives.¹⁵

Tranilast, an inhibitor of TGF-β and NF-κB, showed promising results in a few mild, early oGVHD patients⁹⁴ and mice models of systemic cGVHD.⁸³ In a pilot study⁷⁰ enrolling DED patients with evidence of anti-citrullinated protein autoantibodies on the ocular surface, the administration of pooled human immune globulin-eye drops resulted in diminished OSDI, CFS, and SLK. Such outcomes are attributed to the ability of immune globulins to decrease cytokine release.⁷⁰ Heavy chain-Hyaluronan/Pentraxin 3 is an amniotic membrane-derived compound with anti-inflammatory functions.⁹⁶ In a mouse model of oGVHD, its subconjunctival and subcutaneous injection resulted in the reduced infiltration of T-cells and HSP47⁺ fibroblasts into the lacrimal glands, increased conjunctival goblet cell density, and preservation of tear layer volume.⁹⁶ In another attempt to combat the pro-fibrogenic effects of HSP47, a vitamin A-coupled liposome that contains anti-HSP47 small interfering RNAs was administered to a mouse model of oGVHD.⁹⁷ This compound could both prevent and reduce the collagen deposits in the lacrimal glands and increase the volume of the tear layer.⁹⁷

Another study showed that the natural homeostatic glycoprotein clusterin (CLU) can inhibit MMPs and bind to and heal the damaged ocular surface.³² In another study, administration of 4-phenyl butyric acid (PBA) in a cGVHD mouse model showed reduced levels of inflammatory cytokines (namely, IL-6), HSP47, lacrimal gland fibrosis, and MGD.⁸² The role of nutritional supplement linoleic acid and omega-3 and omega-6 fatty acids in reducing the DED-related ocular surface inflammation are also implicated by some studies.²⁵

Autologous serum eye drop (ASED) is another option for oGVHD cases, as it has both lubricating and anti-inflammatory advantages.⁶⁵ The prominent feature of ASED is the containment of various nutrients, growth factors, inhibitors of MMPs, and electrolytes that make it similar to the natural tear.⁶⁵ It should be mentioned that ASED has higher concentrations of fibronectin, vitamin A, lysozyme, and TGF-X, and lower concentrations of IgA, EGF, and vitamin C compared to natural tears.⁹⁹ Despite anti-inflammatory features, a study demonstrated increased densities of HLA-DR positive conjunctival epithelial and Langerhans cells after the administration of ASED.⁵⁶. In addition, the ocular surface HLA-DR density of SS cases has decreased after the administration of ASED.⁵⁶ Finally, therapeutic levels of some systemically administered immunosuppressants can be found in ASED,¹³¹ but their potential impacts on the course of the disease must be determined by future studies. Regarding another blood-derived product, a phase I/II randomized trial aimed to assess the safety and effectiveness of fibrinogen-depleted human platelet lysate (FD hPL) eye drops for oGVHD cases. However, despite significant improvements in various ocular symptoms, TBUT and CFS showed no significant changes.¹²⁹

Based on our experience, early start of anti-inflammatory agents such as tacrolimus and cyclosporine is so critical and can prevent progression of the disease and further damage. The practice of using anti-inflammatory agents is supported by the preponderance of literature on the topic.

11.1.3. Control of evaporation

Meibomian gland dysfunction and blepharitis are associated with the loss of the tear layer as a result of increased evaporation. Along with the beneficial effects of topical anti-inflammatory drugs and regular application of warm compresses,⁹⁸ the usage of topical antibiotic ointments (e.g., tetracycline or doxycycline), drops (e.g., azithromycin), and also systemic antibiotics (e.g., tetracycline or doxycycline) can combat the superimposed infections.^{27, 65} Of note, the latter has the advantage of alleviating MGD. Using swim goggles to reduce tear evaporation can aid in diminishing the experienced discomfort by DED patients.⁶⁸ Placement of fluid-ventilated, gas-permeable scleral lenses can diminish tear evaporation and is another therapeutic option for severe DED, including oGVHD cases.⁷⁶ Such devices are able to attenuate pain and photophobia, enhance OSDI, and improve visual acuity in oGVHD eyes refractory to conventional therapies.⁷⁶

11.2. Surgical management

Obstruction of canalicular puncta with silicone or collagen plugs may be conducive to retaining the tear layer, and their potential side effect of increasing the concentrations of inflammatory cytokines does not seem to be a major drawback.^{113, 124} For oGVHD-related KCS, punctal plugs have been able to improve subjective symptoms, CFS, and TBUT;^{113, 124} however, their extrusion is common,¹²⁴ and the next steps to reduce the lacrimal duct drainage are surgical ligation or cauterization of puncta.³⁷ Punctal thermal cauterization has been effective in improving subjective symptoms, CFS, and TBUT,¹⁵⁰ and stepping down the severity of DED, but recanalization can occur in as many as 21% of cases.¹⁴⁵

Tarsorrhaphy is both a prophylactic and therapeutic approach to various ocular surface disorders, including CED and DED. Partial tarsorrhaphy is proposed as an option for severe oGVHD cases.^{14, 27}

Amniotic membrane transplantation serves as an appropriate intervention for those with refractory disease resulting in persistent CED, SLK, corneal perforation, and symblepharon.⁴⁸ The amniotic membrane is rich in growth factors and cytokines and will provide a physical barrier to the ocular surface and diminish the eyelid friction forces, which, along with the lack of HLA antigens, makes it a promising option for such severe cases. Nevertheless, keratoplasty might be needed as the final resort for the advanced form of the disease.⁴⁸ Conjunctival-limbal stem cell transplantation is another option for severe cases, although its efficacy is only supported by case reports.¹³

12. Conclusion

Ocular GVHD is a common disorder in those who are affected by cGVHD. Its detrimental effects on the quality of life have been well established, and serious complications including corneal perforation and loss of vision can occur. Although the pathological mechanisms that lead to oGVHD are not fully understood, there is now solid evidence regarding the pathologic alterations that occur after the initiation of the disease. Such data can provide insight into the development of novel agents that can inhibit crucial signaling cascades during the development of oGVHD. Future research should focus on translating preclinical data into large clinical studies. In addition, it is prudent to amass and summarize the currently available diagnostic criteria and reach well-accepted robust criteria.

13. Methods of literature search

A PubMed search was performed to identify relevant articles (with no time restrictions) using the search terms: “graft-versus host disease” or “GVHD” in combination with “dry eye” or “DED” or “ophthalmology” or “lacrimal” or “keratoconjunctivitis sicca” or “tear” or “eye” or “ocular”. Searches were performed on August 1, 2022. Articles are excluded if they were not referenced in English.