Abstract
BackgroundDevelopment of chronic Graft Versus Host Disease (cGVHD) represents a major impediment in allogeneic hematopoietic stem cell transplantation (HSCT). This disorder is associated with severe impairment of B-cell homeostasis, regulatory B-cell function and distribution. Conversely, the presence of bone marrow and circulating hematogones is associated with reduced GVHD risks. These findings raised the question whether B-cell progenitors, which provide protection in various autoimmune disease models following activation with the TLR-9 agonist CpG (CpG-proBs), could likewise reduce disease symptoms in a sclerodermatous model of cGVHD. MethodsChronic sclerodermatous GVHD was induced in irradiated Balb/c recipients reconstituted with T- and B-cell-depleted bone marrow cells and splenocytes from C57BL/6J donors. CpG-proB-cell progenitors sorted from in vitro CpG-activated bone marrow cells were then adoptively transferred into GVHD recipients. Their effect on disease symptoms, such as diarrhea, skin fibrosis and survival was evaluated in the therapeutic window defined beforehand. Transferred progenitors were analyzed for migration, differentiation and cytokine expression using flow cytofluorimetric methods, which were also used to establish their impact on T-cell cytokine expression and follicular helper/regulatory T-cell ratios (Tfh/Tfr) in peripheral and mesenteric lymph nodes. Skin fibrosis was assessed by histology, identification of infiltrating cells and gene expression profiles of cytokines and molecules involved in the fibrotic process, using qRT-PCR microarrays in all tissue samples.ResultsWe found that CpG-proBs, adoptively transferred during the initial phase of disease, reduced the diarrhea score and mostly prevented cutaneous fibrosis. Progenitors migrated to the draining lymph nodes and to the skin where they mainly differentiated into follicular B cells. CpG activation and IFN-γ expression were required for the protective effect, which resulted in reduced CD4+ T-cell-derived production of cytokines critically involved in cGVHD, such as TGF-β, IL-13 and IL-21. Adoptive transfer increased the Tfr/Tfh ratio. Moreover, CpG-proBs privileged the accumulation of IL-10-positive CD8+ T cells, B cells and dendritic cells in the skin. ConclusionOur findings support the notion that adoptively transferred CpG-proBs provide an efficient strategy for alleviating sclerodermatous cGVHD either per se or as a beneficial adjunct to the HSC graft.