Disturbed B cell and DC-Homeostasis in Pediatric cGVHD Patients-Cocultivation Experiments and Review of the Literature

B cells and DCs are suspected to play an important role in the pathogenesis of cGvHD, which is a serious complication of HSCT with high morbidity. It is characterized by immune responses of donor immune cells against recipient-derived antigens. Pathogenesis is not yet fully understood, however reconstitution of B cells after HSCT has similarities to physiologic ontogeny. Immunophenotyping and co-cultivation-experiments of B cells and DCs from pediatric patients with cGvHD as well as healthy donors were conducted. Significant differences between patients and healthy donors were observed with increased memory, transitional, CD69+ and CD86+ phenotype and lower levels of naïve B cells due to apoptosis. Co-cultivation revealed this to be primarily B cell-dependent without major effects of and with DCs. There was a decreased CD11cphenotype in patients and less apoptosis of DCs. Our data suggest a disturbed homeostasis in B cells with increased memory phenotype in patients, whereas DCs could not influence these differences, therefore DCs are not imposing as promising targets. B cell-dependent approaches should be further investigated. Julian Zipfel*, Matthias Eyrich, Paul-Gerhardt Schlegel and Verena Wiegering Department of Pediatric Hematology/Oncology and Stem Cell Transplantation, University Hospital Würzburg, Germany Julian Zipfel, et al. Clinics in Oncology General Oncology Remedy Publications LLC. , | http://clinicsinoncology.com/ 2016 | Volume 1 | Article 1097 2 [9]. The possible essential steps of its pathogenesis could be triggered by damage to the thymus following conditioning prior to HSCT or, more significantly, aGvHD. Thus, a decrease in negative selection of CD4+ T cells may occur, leading to a deviated immune response with increased production of IL-4, IL-5 and IL-11, being part of the Th2 cytokine pattern. Tissue fibroblasts can be stimulated both directly by Th2-released IL-2, IL-10 and TGFβ1, and indirectly via activation of macrophages, which produce PDGF and TGFβ1. This process can be enhanced by low Treg levels resulting in further deviation of the immune system towards Th2 and Th17 response [10]. Th17 cells, a CD4+ T cell subset expressing the IL-23-receptor [11], can act as B cell helpers and have been shown to increase the formation of germinal centres as well as promoting proliferation and activation of B cells with their signature cytokines IL-17, IL-21 and IL-22 [12]. This dysregulation of B cell homeostasis can, as well as high levels of BAFF, result in B cell autoreactivity [10]. In summation, all these steps contribute to systemic effects similar to autoimmune diseases affecting multiple organs as skin, nails, mouth, eyes, muscles, gastrointestinal tract, liver, lungs, kidneys, heart and bone marrow [13-16]. However, an improvement of therapy opportunities is desperately needed, as standard therapy consisting of glucocorticoids with or without tacrolimus or ciclosporin [1], has many side effectsespecially in pediatric patients [6] and we are still lacking specific therapies. Several biomarkers have been introduced and proposed for the identification of patients at risk of cGvHD, including Tregs, B cells, BAFF, IL-10 [17], as well as development of HY antibodies [18]. While various ways of interaction between DCs and T cells not only via antigen presentation are known [19], their influence on B cells has just lately become the focus of investigation. Indeed, many of the aforementioned mechanisms directly involved in the pathogenesis of autoimmunity are complemented or redundantly synergize with effects of DCs and B cells on each other. In response to TLR stimulation or BCR ligation, pDCs can enhance auto reactive B cell proliferation and production of anti-snRNP auto antibodies as well as B cell survival [20]. Furthermore, dendritic cells have been shown to be the most important APCs. In mouse models anti-CD40 activated B cells were unable to fully prime CD4+ T cells in vivo without DCs expressing the correct restriction element MHC II I-E [21]. This would rather implicate an auxiliary role of B cells in optimizing CD4+ T cell priming. Therefore only concerted presentation of antigens by both dendritic and B cells leads to an optimal and effective CD4+ T cellimmunity in-vivo [21]. Additionally, pDCs can induce differentiation of CD40-activated B cells into antibody-producing plasma cells via sequential secretion of IL-6 and type I IFN [22,23]. This explains, how these cytokines, together with BAFF may partially contribute to the effects of pDCs on B cell activation in addition to cell-to-cell contact [20,24]. Besides these means of communication, a direct involvement of the ICAM-1-LFA-1 pathway in the signaling between pDCs and auto reactive B cells has been described [20]. As for type I IFN, it is able to upregulated CD38 on B cells and enhance secretion of BAFF and APRIL by B cells, monocytes and mDCs, confirming its importance in the context of cGvHD [25,26]. Follicular DCs are involved in the development of somatically mutated and switched memory cells [27]. Following activation via TLR7/8-L and, less pronounced, via TLR9, pDCs have been shown to be able to induce upregulation of BLIMP1 and X-box binding protein 1, to increase B cellproliferation as well as the differentiation into CD27high plasmablasts [28] which indicates their involvement in B cellhomeostasis. The aims of our study were to compare patterns of B cells and DCs from patients with cGvHD and healthy donors. Furthermore, apoptosis was measured and co-cultivation was used to observe possible interactions and evaluate the effects of B cell activation on immune cells. Materials and Methods We performed immunophenotyping of B cells and DCs from 7 pediatric patients (Table 1) with cGvHD and 13 healthy donors. Mononuclear cells were isolated from peripheral blood via Ficoll, followed by positive selection of CD19+ and CD14+ cells via MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells were cultivated with IL-10 (Sigma, Taufkirchen, Germany), CD14+ cells matured and differentiated to fast DCs within 48 hours with GMCSF, IL-4, IL-1b, IL-6, TNFα (CellGenix, Freiburg, Germany) and PGE2 (Pharmacia Limited, Kent, UK) [29].