The Balance of Effector and Regulatory Cell Populations in Oral Chronic GVHD: Potential Role of IL-15.
Authors: Imanguli Matin , Hakim Frances , Swaim William , Guadagnini Jean-Pierre , League Stacy , Gress Ronald , Pavletic Steven 
Published: October 2019

Abstract
Although chronic Graft versus Host Disease (cGVHD) is the most important long-term complication of allogeneic hematopoietic stem cell transplantation, little is known of its pathogenesis. Most of the work on cGVHD in humans has examined peripheral blood populations which may not accurately represent events in the tissues. Oral cGVHD is a significant contributor to overall morbidity and occurs at a frequency second only to that of cGVHD of skin. We examined oral mucosa in 29 patients who underwent multi-specialty clinical evaluation in a cross-sectional natural history of cGVHD protocol (NCI protocol #04-C-0281). Standardized buccal biopsies were obtained at the time of the clinical evaluation and oral cGVHD grading. We analyzed and quantified the pathologic changes in oral cGVHD using multiparameter immunofluorescent staining and confocal microscopy. We then evaluated cytokine production and transcription factors by quantitative real-time PCR. The results were correlated with clinical aspects of the disease, such as severity scoring, established in the comprehensive cGVHD assessment (Filipovich AH et al, BBMT 2005). We observed that the numbers of cleaved caspase-3 positive apoptotic cells within the epithelial layer correlated with clinical severity assessed using the oral cGVHD grading scale (r=0.60). In severely affected tissues the predominant CD3+ T-cells were CD8+ cells expressing markers of cytotoxic effectors, such as TIA-1. Both CD8 and CD4 T-cells expressed the T-bet transcription factor characteristic of Type I cytokine producing cells. Consistent with this finding, IFN-gamma levels as measured by quantitative PCR positively correlated with the apoptotic index within the affected oral epithelium (r=0.40). We further examined tissues for the balance of effector and regulatory T-cells by immunofluorescent staining for FoxP3 and measuring FoxP3 mRNA levels. FoxP3 expressing cells were quantitatively increased in oral tissues affected by cGVHD, but remained in constant proportion to the total number of infiltrating T-cells. FoxP3 mRNA levels did not correlate with either clinical or histological severity of oral cGVHD. Finally we examined the expression of IL-15, a key cytokine in the generation, proliferation and maintenance of effector-memory CD8 cells that can directly suppress regulatory T cell function (Ruprecht CR et al, JEM 2005). We observed IL-15 expression in both the epithelial keratinocytes and in the sub-epithelial inflammatory infiltrates in GVHD affected oral mucosa. We thereby propose that increased expression of IL-15 in the tissues of oral cGVHD may play a role in supporting the effector cell populations while simultaneously interfering with the regulatory function of the FoxP3+ cells. This is the first study examining the balance of effector and regulatory populations in the oral tissues affected by cGVHD. This study demonstrates that epithelial cell apoptosis is an easily quantifiable histological marker of the oral cGVHD activity. Infiltration of epithelial layers by CD8 cells expressing T-Bet and cytotoxic markers suggests direct involvement of these cells in disease pathogenesis. Our findings provide the initial evidence and potential basis for the targeting of effector-memory CD8 cells and/or IL-15 in oral and possibly other manifestations of cGVHD.