Clinical Study on Cytokines Panel in the Diagnosis of Ocular Chronic Graft-Versus-Host Disease
Authors: Cheng Xianjing , Ji Rui , Huang Ruihao , Huang Shiqin , Fan Wei , Zhao Yuancheng , Yuan Rongdi , Wang Xiaoqi , Zhang Xi 
Published: December 2023

Background
Chronic graft-versus-host disease (cGVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), affecting 30-70% of patients after allo-HSCT. Sixty to ninety percent of cGVHD patients suffer from ocular chronic GVHD (ocGVHD), which is characterized by new-onset dry, “gritty”, or painful eyes, cicatricial conjunctivitis, keratoconjunctivitis sicca, and confluent areas of punctate keratopathy, significantly affecting patients' quality of life. Nevertheless, the clinical diagnosis of ocGVHD relies heavily on observing symptoms and clinical signs. Treatment is typically initiated during the symptomatic stage, by which time the damage to the ocular tissues may be irreversible. Consequently, it is crucial to investigate methods of early diagnosis for early intervention.
Methods
The cGVHD mouse model was established to explore the correlation between cGVHD and serum cytokines. Based on the findings from animal experiments and literature review, a panel of 16 cytokine combinations was identified. Enzyme-linked immunosorbent assay (ELISA) was employed to compare the levels of cytokines in serum and tear samples obtained from patients who underwent allogeneic hematopoietic stem cell transplantation. The inclusion criteria were set as post-allo-HSCT patients between the ages of 18 and 60 who were willing to participate in the study. The visual acuity, ocular pressure, Ocular Surface Disease Index (OSDI) score, Schirmer's test without anesthesia, corneal fluorescein staining (CFS), and fluorescein tear breakup time (FTBUT) were evaluated. The tear sample was collected via Schirmer's strips. Qualitative data were compared with the chi-square test. The Spearman test was used to analyze correlations (ChiCTR2200062516).
Results
In comparison to the control group, cGVHD mice exhibited elevated concentrations of serum IL-1β, IL-6, IL-8, IL-17, IFN-γ, CX3CL1, CXCL11, CXCL13, CCL11, and CCL19 (P&lt;0.05). A total of 26 post-allo-HSCT ocGVHD patients (38±11y; range 19-57y) and 27 patients without ocGVHD (34±9y; range 20-52y) were recruited. Between patients with and without ocGVHD, there were significant differences in ocular surface parameters (p&lt;0.0001). Analysis of serum and tear cytokine profiles in patients revealed that, when compared to patients without ocular cGVHD, those with ocular cGVHD exhibited increased serum IL-8 levels (P=0.0319, AUC value=0.678), decreased serum IL-10 levels (P=0.0305, AUC value=0.701), elevated tear fluid levels of IL-8, IFN-γ, CXCL9, and CCL17, and lower tear fluid levels of IL-10 and CCL19 (P&lt;0.05, AUC values were &gt;0.7). Moreover, the correlation analysis between tear cytokine levels and ophthalmic indices showed that 3 cytokines were correlated with ophthalmic indices: IL-10, IFN-γ and CXCL9, indicating the superior diagnostic capabilities and correlation with ocGVHD severity. (Figure 1)
Conclusions
Tear fluid demonstrates greater specificity and sensitivity as biomarkers for diagnosing ocular cGVHD compared to serum biomarkers. Among the identified tear cytokines, IL-8, IL-10, IFN-γ, CXCL9, CCL17, and CCL19 can serve as diagnostic biomarkers for ocular cGVHD post-transplantation offering practical reference value for diagnosis. IL-10, IFN-γ and CXCL9 levels could evaluate the severity of ocGVHD. In addition, large-scale prospective studies are warranted to confirm our findings.