Demonstration of Donor T cell Recruitment and Correlation with Inflammatory Cytokine Presence in Experimental Ocular Graft-versus-Host Disease

Abstract

Purpose: Graft-versus-host disease is a potentially blinding condition that may affect allogeneic hematopoietic stem cell transplant (HSCT) survivors. Alloreactive donor T cells are essential for the development of GVHD, but their direct role in local ocular GVHD has not been demonstrated. In this study, we examined immunological and ocular changes using a clinically relevant MHC-matched, minor antigen mis-matched HSCT transplantation model of systemic and ocular GVHD.

Methods: After high-dose TBI, C3H.SW (H2b) mice were transplanted with TCD-BM alone or together with T cells from either wild type B6 mice or CXCR6-GFP knockin mice, which express GFP regulated by the CXCR6 promoter (B6-CXCR6-/- GFP). GVHD was monitored weekly by clinical score composite and ocular surface fluorescein staining. In vivo fluorescent microscopy was used to measure the recruitment of B6-CXCR6-/- GFP donor T cells. At 6-7 weeks goblet cells were quantified using PAS staining and T cell phenotype was characterized by flow cytometry analysis of corneal cells suspension. Total RNA was prepared and the production of mediators of inflammation analyzed by qPCR.

Results: Mice that received transplanted BM and T-cells from either wild type B6 or B6-CXCR6-/- GFP developed evidence of systemic GVHD by day 21 as opposed to control animals. Beginning at day 28, the recruitment of donor B6-CXCR6-/- GFP T cells correlated with the development of progressively increasing ocular surface disease in mice with GVHD. Interesting, relative expression of RNA of the T cell chemo-attractant CXCL10 and its receptor CXCR3, also increased in the corneas. Similarly, the relative expression of the inflammatory cytokines, IFN-γ TNF- α and IL-6 were significantly increased, correlating with a significantly decreased number of goblet cells in the conjunctiva of mice with GVHD but not controls.

Conclusions: C3H.SW mice that receive BM with T cells develop ocular GVHD characterized by donor T cell recruitment, increased expression of CXCL10/CXCR3, inflammatory cytokines and the development of corneal staining, ulceration and loss of goblet cells. These findings support a critical role for donor T cells in the immune responses occurring at the ocular surface. Understanding kinetics and pathways will provide targets for the prevention and treatment of ocular GVHD.

Keywords: 555 immunomodulation/immunoregulation • 557 inflammation • 741 transplantation