Conjunctival HLA-DR and CD8 expression detected by impression cytology in ocular graft versus host disease

Abstract

Purpose: To assess the expression of human leucocyte antigen (HLA)-DR in epithelial cells and cluster of differentiation (CD8)-positive lymphocytes as possible markers of chronic ocular graft versus host disease (cGvHD) after hematological stem cell transplantation (HSCT).

Methods: Twenty-seven consecutive patients with dry-eye symptoms following HSCT (24 [89%] with peripheral blood stem cell transplantation and 3 [11%] with bone marrow transplants; 17 [63%] familiar allogenic grafts) and 19 age-matched controls were included. Conjunctival impression cytology specimens were stained for HLA-DR, cytokeratin 19, and CD8. Oxford grading scale, blinking frequency, Schirmer test, tear film break-up time (TBUT), and Ocular Surface Disease Index (OSDI) were also recorded. Wilcoxon nonparametric testing was used to compare controls and HSCT recipients and to assess HSCT recipient subgroups with and without clinical cGVHD.

Results: Eighteen patients showed clinical signs of ocular cGVHD. TBUT and Schirmer test scores were significantly lower in patients, while Oxford grades and OSDI were significantly higher than in controls. Epithelial HLA-DR expression was generally higher in HSCT recipients than in controls, but it did not correlate with ocular cGVHD status. CD8-positive lymphocytes were identified in five patients with ocular cGvHD and one control.

Conclusions: A strong HLA-DR expression as detected by impression cytology appears to indicate a general HSCT response and fails to predict ocular cGVHD. However, the detection of CD8-positive lymphocytes using impression cytology was frequently associated with ocular cGvHD. Our data warrant further evaluation of CD8 expression in impression cytology, along with comparison to conjunctival biopsies and brush cytology, as impression cytology may offer a less invasive strategy for assessing cGVHD status.

Figure 1

Immunofluorescence images of cytospins showing cytokeratin (CK)19-positive epithelial cells. A: Shown are examples of human leucocyte antigen (HLA)-DR-positive epithelial cells from different patients with ocular chronic graft versus host disease (cGvHD). B: Image shows single epithelial cells with strong HLA-DR expression. C: Depicted are single epithelial cells with low HLA-DR expression. D: Shown is an epithelial cell sheet with HLA-DR-negative and -positive epithelial cells. E, F: Displayed are two cluster of differentiation (CD)8-positive lymphocytes of patients with ocular cGvHD.

Figure 2

Oxford grades were compared between the control (n=19) and the patient group (n=27). Box plots display the median and quartiles (Q_0,25 and Q_0,75) and the total range. Median grade was 0 in the control group and 3 in the patient group, which was statistically significant (p<0.001, Wilcoxon non-parametric testing), showing that the patient group depicted significantly more ocular surface staining than the control group.

Figure 3

Box plots. Box plots display the median and quartiles (Q_0,25 and Q_0,75) and the total range of the ocular surface disease index score, which was 8.3 in the control group (n=19) and 52.2 in the patient group (n= 27; p<0.001, Wilcoxon non parametric testing), showing that the patient group had significantly more complaints from ocular surface disease than the control group.

Figure 4

Box plots display the median and quartiles (Q_0,25 and Q_0,75) and the total range of human leucocyte antigen (HLA)-DR expression. A median of 5.0% of conjunctival epithelial cells expressed HLA-DR in the control group (n=19), while 29% of conjunctival epithelial cells expressed this marker in the patient group (n=27; p<0.001).