Abstract
Purpose :
Ocular graft versus host disease (oGVHD) affects 40-60% of patients who have undergone allogeneic hematopoietic stem cell transplant resulting in debilitating eye disease characterized by decreased vision, pain, and dry eye disease (DED). The mechanisms underpinning inflammation in oGVHD are not completely understood. Granzyme B (GzmB) is a serine protease associated with granule mediated perforin dependent pathway leading to apoptosis. GzmB is thought to be involved in multiple autoimmune diseases such as Systemic Lupus Erythematous, scleroderma, which share similarities to GvHD. We hypothesize that the production of GzmB increases the severity of oGvHD, playing a critical role in oGVHD pathogenesis via perforin dependent and independent mechanisms.
Methods :
We conducted an allogeneic hematopoietic stem cell transplant using a mouse model. This study included four groups; T and B cell depleted Bone Marrow (TBCD-BM) only, TBCD-BM+Splenocytes (BM+Spl), TBCD-BM+Splenocytes+IL-2c (BM+Spl+IL2), and TBCD-BM+Splenocytes(-CD8depl)+IL-2c (BM+Spl-CD8+IL2). CD8depl is a group in which CD8 T cells were depleted from the spleen prior to transplantation. IL-2c is a complex of murine IL-2 cytokine and IL-2 monoclonal antibody which activate T-cells. GvHD and oGvHD was scored for the duration of the experiment to determine the progression of GvHD and oGvHD. The individual scores for cornea staining, eyelid, eyelid fur, and cornea haze are used to determine the overall oGvHD score by combining the values and then taking the average within the groups at each time point. Grade 1 refers to the least severe and Grade 4 refers to the most severe. In addition, we collected tissue from the cornea, eyelid, conjunctiva, and lacrimal gland from the mice and quantified the gene expressions of GzmB, Arg-1, TNFα, and IFNγR using RT-qPCR.
Results :
BM+Spl-CD8+IL2 group showed a significant increase (75%, p=0.013) in oGvHD severity compared to BM+Spl. In fact, the BM+Spl+IL2 group showed a 7.5% increase in oGvHD compared to BM+Spl though this was not significant. BM+Spl+IL2 showed an increase in GzmB, IFNγR, Arg-1 expression in the conjunctiva (36, 3, and 3-fold), the eyelid (42, 7, and 2-fold), and the cornea (38, 33, 1.2-fold) compared to TBCD-BM only.
Conclusions :
The presence of IL-2c which promotes GzmB production of T cells increases the severity of oGvHD. Further investigation of GzmB in oGvHD is warranted as a potential target for therapy.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.