Novel Scoring Criteria for the Evaluation of Ocular Graft-versus-Host Disease in a Preclinical Allogeneic Hematopoietic Stem Cell Transplantation Animal Model

Abstract

Ocular complications occur after transplantation in 60% to 90% of chronic graft-versus-host disease (GVHD) patients and significantly impair vision-related quality of life. Ocular surface inflammation and dry eye disease are the most common manifestations of ocular GVHD. Ocular GVHD can be viewed as an excellent preclinical model that can be studied to understand the immune pathogenesis of this common and debilitating disease. A limitation of this is that only a few experimental models mimic the ocular complications after hematopoietic stem cell transplantation (HSCT) and have focused on the acute GVHD process. To address this issue, we used a preclinical animal model developed by our group where ocular involvement was preceded by systemic GVHD to gain insight regarding the contributing immune mechanisms. Employing this "matched unrelated donor" model enabled the development of clinical scoring criteria, which readily identified different degrees of ocular pathology at both the ocular surface and adnexa, dependent on the level of conditioning before HSCT. As far as we are aware, we report for the first time that these clinical and immune responses occur not only on the ocular surface, but they also heavily involve the lid margin region. In total, the present study reports a preclinical scoring model that can be applied to animal models as investigators look to further explore GVHD's immunologic effects at the level of the ocular surface and eyelid adnexa compartments. We speculate that future studies will use this clinical scoring index in combination with what is recognized histologically and correlated with serum biomarkers identified in chronic/ocular GVHD.

Keywords: Allogeneic hematopoietic stem cell transplantation; Graft-versus-host disease (GVHD); Lacrimal gland; Ocular GVHT; Ocular adenexa; Ocular scoring.

Fig 1. Clinical and immunological criteria for pre-clinical ocular GVHD scoring

Lid margin and cornea involvement are the predominant clinical manifestations occurring in the ophthalmic compartment in the ocular adnexa following allogeneic HSCT. These clinical changes correlate with the fluorescence intensity of infiltrating cells labeled with eGFP. A) Individual mice received a score of 0 to 4 for grade of both clinical and eGFP degree of lid margin involvement. Clinical score was based on the severity of lid edema and to the degree of lid involvement causing local skin swelling and lid closure. EGFP parameters were based on the presence and intensity of eGFP cells in the upper or lower lid, and the degree of extension in the surrounding skin. B) Clinical and eGFP scoring criteria used to identify the degree of corneal involvement throughout GVHD progression. The clinical spectrum of corneal involvement ranged from clear to varying degrees of keratopathy to corneal ulceration. EGFP parameters were based on the percentage of cornea/limbus surface area infiltrated by eGFP cells.

Fig 2. Tempo of clinical and immune infiltrative changes in pre-clinical model of GHVD

Animals were conditioned with a 10.5 Gy and transplanted with B6-eGFP bone marrow and CD90.1 T-cells. Clinical photographs and eGFP expression was captured by light and in vivo stereo fluorescent microscopy at different time-points post-allogeneic HSCT. A) external photographs demonstrating disease progression at the lid margin. At weeks 4–5, lid edema was present and by week 6, partial lid closure was evident. Severe lid closure occurred by week 7. Clinical examination revealed mild corneal pathology between weeks 6–7 post transplant. B) Fluorescent stereomicroscopy photographs demonstrating increasing eGFP cell infiltrate, with prominent lid margin involvement by week 6. Note the presence of eGFP infiltrate at week 3 correlating with worsening clinical lid edema and closure, with increased infiltrate by weeks 6 and 7. eGFP cell infiltrate localization to the ocular surface precedes development of mild clinical corneal pathology.

Fig 3. Utilization of scoring system to quantitate disease progression and discriminate between severity of eyelid disease induced by level of total body irradiation (TBI) conditioning

Animals were conditioned with low (7.5Gy) or high (10.5 Gy) TBI and transplanted as in Fig. 2. The scoring system (Table 1) was applied to monitor and score the development of oGVHD. A) Clinical lid margin score with 10.5Gy BM+T cells demonstrated significant eyelid involvement relative to control, with peak score at week 7. In contrast, mice with low conditioning exhibited marginal changes vs. control, B) eGFP lid margin score in mice receiving high conditioning exhibited significantly more eGFP cell infiltrate compared to control. 105Gy+ T cells reached near peak infiltrate by week 4. Similarly to clinical scoring, mice receiving low conditioning exhibited marginal changes. C) Quantification of eGFP by MGI also demonstrated significant changes in high but not low conditioned animals. Mean values are mean score +/− SEM of data from all mice in the injected T cell or control groups. *p < .05. **p < .0.01, paired t-test

Fig 4. Corneal scoring of disease and infiltration using scoring system

Animals from the same experiment described in Figure 3 scored (Table 1) for corneal changes and disease. A) Clinical cornea score did not detect any significant differences between all groups, B) eGFP cornea score in mice receiving high conditioning demonstrated significantly more eGFP labelled inflammatory cell infiltrate compared to controls. Similar to lid margin involvement, this reached near peak score by week 4. Marginal changes were observed in mice receiving low conditioning before transplant. C) Quantification of eGFP by MGI illustrated more intense eGFP than controls. Mean values are mean score +/− SEM of data from all mice in the injected T cell or control groups. *p < .05. **p < .0.01, paired t-test.

Fig 5. Total ocular GVHD score identifies differences between animals expressing comparable systemic GVHD changes

Animals as described in Figs. 3 and 4 were assessed for both overall systemic GVHD clinical changes and total ocular GVHD scoring. A) Total ocular GVHD score, combining clinical and eGFP scores for both anatomic domains, reflect overall ocular GVHD progression and illustrate significant differences between low and high conditioned groups. B) Total systemic GVHD score for all groups of conditioned mice did not identify differences between low and high conditioned animals. Mean values are mean score +/− SEM of data from all mice in the injected T cell or control groups. *p < .05. **p < .0.01, paired t-test