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 o0.001). In M → F recipients, this increase proved to be due to a significant (P o 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 436 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 o0.001) 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.

A Systematic Review of the Evidence of Hematopoietic Stem Cell Differentiation to Fibroblasts

“…Three studies were found describing BMDCs in the conjunctiva of humans with ocular grafts versus host disease [ 101 , 102 , 103 ]. All three employed sex-mismatch lineage tracing and found that BMDCs contributed to (myo)fibroblasts in the conjunctiva.…”

“…Donor-derived endothelial cells and myofibroblasts were found to constitute around 5.4% of all cells with no clear relation with time since transplantation. Hallberg et al performed two studies for which the first [ 103 ] 17 samples from nine patients were collected, whereas the second [ 102 ] included 70 samples from 46 patients. In both studies, approximately 9% of myofibroblasts were determined to be of donor origin, which increased gradually over time.…”

Recent advances in ocular graft-versus-host disease

“…Conjunctival subepithelial fibrosis also referred to as subtarsal fibrosis, is present in 50% of patients with ocular GVHD but is absent in all non-GVHD moderate or severe DED patients. Animal studies have also shown that GVHD-associated conjunctival fibrosis is accompanied by donor-derived myofibroblast formation and Ras system activation (72)(73)(74). Additionally, there was marked infiltration of neutrophils at the upper palpebral conjunctiva in patients with ocular GVHD, which might be associated with the clinical manifestations and inflammatory status of the ocular surface.…”

How does ocular graft‐versus‐host disease fit under the dry eye umbrella? A review

“…One Swedish study examined conjunctival specimens of 25 females with male donor recipients and found the ratio of donor myofibroblasts to total myofibroblasts correlated with coGVHD severity. Additionally, the ratio of donor myofibroblasts (XY) to recipient myofibroblasts increased with time post‐transplant 49 . Donor fibroblasts and subsequent fibrosis also contribute to signs of tear dysfunction and abnormal anatomy in coGVHD.…”

“…Additionally, the ratio of donor myofibroblasts (XY) to recipient myofibroblasts increased with time posttransplant. 49 Donor fibroblasts and subsequent fibrosis also contribute to signs of tear dysfunction and abnormal anatomy in coGVHD.…”