Increased Type I Interferon Signaling and Inflammatory Factors Associated with T-Bet+ Cytotoxic Effectors in Chronic Graft Versus Host Disease of Oral Mucosa.

Although chronic Graft versus Host Disease (cGVHD) is a major long-term complication of allogeneic hematopoietic stem cell transplantation, little is known of its pathogenesis. CGVHD of the oral mucosa is a significant contributor to overall morbidity that occurs at a frequency second only to that of cutaneous cGVHD. We examined oral mucosa in patients undergoing a multidisciplinary clinical evaluation as part of a cross-sectional natural history protocol of cGVHD (NCI protocol NCT00331968). Standardized buccal biopsies were obtained at the time of clinical evaluation from 12 patients with severe ulcerative oral cGVHD, 16 patients with lichen-planus-like oral cGVHD and 9 with no evidence of oral involvement. Paraffin embedded tissues were analyzed using multiparameter immunofluorescent staining and confocal microscopy and the results were correlated with clinical severity scoring of the disease. We have reported that the clinical severity of oral cGVHD was correlated with the frequency of apoptotic epithelial cells, often found adjacent to infiltrating T cells expressing both the T-bet transcription factor, characteristic of type I cytokine polarization, and markers of cytotoxic effectors (TIA+, granzyme B+). Although T-bet expression was found on both CD4 and CD8 T cells, T-bet+ CD8 T cells predominated in the patients with severe oral cGVHD (ASH 2007). We have extended these studies to investigate the mechanisms supporting the infiltrate. We determined that the selective accumulation of T cell effectors was associated with evidence of both proliferative expansion and directed migration; an elevated proportion of T cells were in cycle (Ki-67+) and most T cells expressed the T-bet induced chemokine receptor CXCR3. CXCL9 (MIG), a ligand for CXCR3, was produced in both the epithelial keratinocytes and the CD68+ cells of the submucosal inflammatory infiltrate; we reported this same distribution for IL-15, a cytokine known to stimulate CD8 memory/effector proliferation, survival and cytotoxic differentiation. Both of these factors are inducible by interferons (IFN). Involvement of IFN was demonstrated by the presence of the phosphorylated form of STAT1 translocated into keratinocyte nuclei, an indicator of Type I or Type II IFN signaling. Finally, we determined that CD2AP+ CD68+ plasmacytoid dendritic cells (pDC) were present in the submucosal infiltrate. Because pDC are recognized to be the most active producers of Type I IFN, we assessed indicators of local cytokine production, MxA, a definitive indicator of Type I as opposed to Type II IFN, was strongly expressed in both oral epithelial keratinocytes and CD68+ cells in the infiltrate in severely affected tissues. T cell infiltrates were much less frequent and production of MIG, IL-15 and MxA were absent in oral mucosa of cGVHD patients lacking oral symptoms. Infiltration of epithelial layers by CD8 positive cells expressing T-Bet and cytotoxic markers suggests direct involvement of these cells in disease pathogenesis. Our findings support the hypothesis that Type I IFN induced inflammatory factors drive the migration, expansion and cytotoxic differentiation of these cells.