Conjunctival polyploid cells and donor-derived myofibroblasts in ocular GvHD

Abstract

After allogeneic hematopoietic stem cell transplantation (allo-SCT), ocular GvHD is a common complication, typical symptoms being dry eye syndrome with features of fibrosis. In this study, we have identified and quantified two cell types—myofibroblasts (MFB) and polyploid (PP) cells—in the conjunctival surface of allo-SCT patients (pts) and have explored their kinetics and association with local and systemic GvHD. Results are compared with control groups of (a) pretransplant samples from allo-SCT patients, (b) recipients of autologous transplantation (auto-SCT) and (c) healthy controls. Imprint cytologies were obtained by pressing the conjunctival surface with a sterile, non-abrasive cellulose acetate filter (Millipore). After retraction, typically a monolayer of the outermost cells of the epithelium were retrieved. MFB were identified by immunofluorescent (IF) staining for alpha-smooth muscle protein. PP cells were detected by aberrant chromosome content analyzed via X/Y-FISH (X/Y fluorescence in situ hybridization). In female pts with a male donor (M→F group), donor genotype were identified by sex chromosome detection using FISH methodology. IF and FISH methods were applied in situ on the same filter, and amounts of MFB and PP cells are expressed as the percentage of all cells on the filter. In all, 70 samples from 46 pts were obtained 1–122 months after allo-SCT. The total MFB density (MFB^TOT) was higher in allo-SCT pts compared with healthy individuals and auto-SCT pts and increased by time after transplantation (P<0.001). In M→F recipients, this increase proved to be due to a significant (P<0.001) and gradual elevation of donor-derived MFB (MFB^XY), whereas recipient-derived MFB (MFB^XX) did not vary over time. Clinical ocular GvHD correlated with MFB^XY/MFB^TOT ratio (P=0.034), whereas no association between MFB^TOT or MFB^XY systemic GvHD was observed. In the M→F group (n=25), both MFB^XY and MFB^XX were detected on 28 of the 37 imprints (76%). In pts >36 months post transplant, on 11/12 imprints, a median of 9.4% (1.4–39%) MFB^XY and 3.6% (0–11%) MFB^XX was found. In one patient, 1.6% MFB^XY were detected at 3 weeks post transplant. PP cells (6–24n), exclusively of recipient origin, were found to a median of 0.6% (0–37%). The PP cell density differed significantly (P<0.001) between time intervals, with a maximum 8.9% (0–35%) of all cells at 3–12 months. No correlation between PP cells and GvHD (ocular or systemic) was observed. The MFB has been indicated as a culprit in chronic inflammation and fibrosis. The observation that MFB^XY/MFB^TOT ratio correlated with ocular GvHD suggests a role of donor MFB in GvHD pathogenesis. The constant finding of recipient-derived MFB^XX cells many years after transplant in pts with 100% donor hematopoiesis indicates that there is a non-hematopoietic differentiation route to MFB. The origin and role of PP cells after allo-SCT remains obscure.