Tear cytokine profiling for ocular graft versus host disease utilizing a new multiplex assay

Abstract Many patients who have undergone allogeneic hematopoietic stem cell transplantation (aHSCT) as part of cancer therapy achieve remission. However, nearly 50% of these patients develop Graft versus Host Disease (GVHD), a chronic autoimmune disease that can damage eyes. Quantifying specific cytokine changes in tears may reveal biomarkers and future treatment targets for ocular GVHD (oGVHD). Compared to other assays, the IsoLight Codeplex Secretome multiplex assay (Isoplexis) has advantages of high-throughput analyses and small sample requirements, but has yet to be validated for tears. This study evaluates the tears of a patient with oGVHD utilizing Isoplexis and correlates the cytokine profile with clinical disease severity. As a pilot study, we collected normal and oGVHD patient tears which we analyzed on two backgrounds– standard Bovine Serum Albumin (BSA) or artificial tears (ATs). The controls were ATs (negative) and a concentrated cytokine solution (positive). The cytokine levels of artificial tears alone were below the limit of detection (LOD) as expected. Out of a 22 cytokine panel, oGVHD patient tears had elevated TNF-α, TNF-β, perforin, MIP-1α, MIP-1β, MCP-1, IL-2, IL-4, IL-5, IL-7A, IL-9, IL-13, IL-15, IFN-γ, granzyme B, and GM-CSF with ATS background, but no cytokines above the LOD in the BSA background plate. The control tears had elevated IP-10. Elevated cytokines for the oGVHD patient corresponded to clinical symptom findings. The results suggest that using ATs as the background improves sensitivity to detect tear cytokines on Isoplexis. Elevated IL-7A and GM-CSF in oGVHD tears parallel literature findings. Further sample evaluation will continue to validate Isoplexis for tear cytokine analyses. Supported by Program for Research Initiated by Students and Mentors (PRISM), University of Maryland School of Medicine Office of Student Research and Eversight, Cigarette Restitution Fund, University of Maryland Greenebaum Comprehensive Cancer Center