Potential Involvement of Chemokine Fractalkine/CX3CL1 and CX3CR1high Monocytes in Chronic Graft Versus Host Disease.
Authors: Namba Noriko , Katayama Yoshio , Shinagawa Katsuji , Tanimoto Mitsune 
Published: April 2020

Abstract
Chronic graft versus host disease (cGVHD) is the most common late complications of allogeneic stem cell transplantation (alloSCT). Pathogenesis of cGVHD is largely unknown. Immunosuppressive agents are widely used in management of cGVHD, but frequently with unfavorable control. For the establishment of better therapeutic strategy, it is important to determine the cell types that are responsible for cGVHD and the factors that play critical roles to recruit these cells into inflammatory sites. Fractalkine/CX3CL1 (FKN) is an unique chemokine that exists not only as a soluble chemoattractant but also as a membrane-anchored form to mediate firm adhesion of leukocytes, mainly monocytes/macrophages, via its cognitive receptor CX3CR1. Here, we report the potential involvement of FKN-CX3CR1 axis in cGVHD. First, we assessed CX3CR1 expression on peripheral blood CD14+ monocytes by flow cytometry. Peripheral blood samples were obtained from patients who had undergone alloSCT (8 men, 9 women; age: range 20–64, median 45; days after transplantation: range 97–2786, median 681). To assess the clinical severity of cGVHD, we generated a scoring system with a modification of “GVHD Morbidity Assessment” established for Long-term Follow-up Program in Fred Hutchinson Cancer Research Center. We noticed that CD14+ monocytes in patients without cGVHD displayed certain expression level of CX3CR1 (single peak in a histogram) similar to that in healthy controls, and much lower expression in patients with cGVHD. Statistically, the intensity of CX3CR1 expression on CD14+ monocytes showed strong inverse correlation with cGVHD score (n=17, p&lt;0.01, Spearman’s rank correlation coefficient). This was unlikely due to the internalization of this receptor by the binding of soluble FKN in the blood or the effect of immunosuppressive agents for the patients with severe clinical symptoms since the histogram of CX3CR1 in lymphocytes characterized by low forward and side scatter showed two distinct peaks (positive and negative fractions) and the intensity of positive fraction was similar in all patients and healthy controls with no significant correlation between cGVHD score and percentage of positive fraction. Next, we examined the expression of FKN in two cGVHD regions, 1) lungs obtained from patients who had undergone lung transplantation due to respiratory failure caused by bronchiolitis obliterans, and 2) skin, by immunohistochemical staining. FKN was strongly expressed on epithelium of bronchioles in cGVHD as well as in normal lungs. Obliterated bronchioles lost the FKN-positive epithelium and were replaced with fibrous tissues, sometimes extensively surrounded by CD14+ monocytes/macrophages. In normal skin, FKN was faintly expressed in the basal layer of epidermis. In contrast, in cGVHD patients, it was strongly expressed in epidermis where we observed a hyperproliferation of keratinocytes, known as a main FKN producer in the skin. Interestingly, dramatic infiltration of CD14+ monocytes/macrophages was observed in dermis in most patients, and sometimes even in epidermis, suggesting that CX3CR1high monocytes might be recruited from the circulation into dermis by the chemoattraction of FKN supplied by epidermis. Together, our study suggests that monocytes/macrophages, which can function as antigen presenting cells, may play a role in pathogenesis of cGVHD via the Fractalkine-CX3CR1 pathway, and highlights these cells and this chemokine-receptor axis as additional targets for cGVHD therapy.