Systemic lupus erythematosus (SLE) is an autoimmune disease with unrestrained T-cell and B-cell activity towards self-antigens. Evidence shows that apoptotic cells (ApoCells) trigger an autoreactive response against nuclear antigens in susceptible individuals. In this study, we focus on generating and characterizing tolerogenic dendritic cells (tolDCs) to restore tolerance to ApoCells. Monocyte-derived dendritic cells (DCs) from healthy controls and patients with SLE were treated with dexamethasone and rosiglitazone to induce tolDCs. Autologous apoptotic lymphocytes generated by UV irradiation were given to tolDCs as a source of self-antigens. Lipopolysaccharide (LPS) was used as a maturation stimulus to induce the expression of co-stimulatory molecules and secretion of cytokines. TolDCs generated from patients with SLE showed a reduced expression of co-stimulatory molecules after LPS stimulation compared with mature DCs. The same phenomenon was observed in tolDCs treated with ApoCells and LPS. In addition, ApoCell-loaded tolDCs stimulated with LPS secreted lower levels of interleukin-6 (IL-6) and IL-12p70 than mature DCs without differences in IL-10 secretion. The functionality of tolDCs was assessed by their capacity to prime allogeneic T cells. TolDCs displayed suppressor properties as demonstrated by a significantly reduced capacity to induce allogeneic T-cell proliferation and activation. ApoCell-loaded tolDCs generated from SLE monocytes have a stable immature/tolerogenic phenotype that can modulate CD4 T-cell activation. These properties make them suitable for an antigen-specific immunotherapy for SLE.

Background Tolerogenic dendritic cells (tolDCs) can be considered as an attractive strategy to treat autoimmune diseases. The tolerogenic potential of autologous tolDCs has not yet been evaluated to treat systemic lupus erythematosus (SLE). Although the specific autoantigens involved in the pathogenesis of SLE are still unknown, a failure in the clearance of apoptotic debris seems to be associated with the onset of disease. Objectives To evaluate in vitro the regulatory function and phenotype stability of tolDCs generated from peripheral blood monocytes of SLE patients and to assess whether apoptotic cells affect the tolerogenic function of tolDCs. Methods Peripheral blood mononuclear cells were collected from SLE patients and healthy controls and seeded on cell culture plates, non-adherent cells were thoroughly washed out. Monocytes were differentiated into dendritic cells (DCs) by addition of GM-CSF and IL-4 every other day in AIM-V medium for 5 days. tolDCs were generated by addition of rosiglitazone (RGZ) and/or dexametasone (DEXA) for 24 hours. Tolerogenic phenotype stability was evaluated by challenging tolDCs with lipopolysaccharide (LPS) for 48 hours. Autologous apoptotic T cells were generated by exposure to UV-B radiation (1–1,5 mW/cm2 for 90 minutes) then stained with CFSE for tracking and feeded to tolDCs for 24 hours. Capture of apoptotic cells by tolDCs was assessed by flow cytometry and confocal microscopy. Costimulatory molecules of DCs were evaluated by flow cytometry. Results At the end of the differentiation process, DCs from SLE patients and healthy controls showed a CD11c+ HLA-DR+ CD14– DC phenotype. Induction of maturation leads to increase of maturation markers such as CD83 and PD-L1. tolDCs generated from SLE patients with RGZ and/or DEXA showed a reduced expression of maturation markers after stimulation with LPS for 48 hours in comparison to mature DCs. tolDCs pulsed with CFSE-stained autologous apoptotic T cells displayed positive staining for CFSE by flow cytometry, suggesting internalization of apoptotic cells by tolDCs. This result was confirmed by confocal microscopy. The phenotype of RGZ-tolDCs from SLE patients showed no difference when treated with autologous apoptotic cells. Conclusions We were able to generate DCs from SLE monocytes with a stable immature/tolerogenic phenotype. These cells are capable of capturing apoptotic cells without changing their phenotype, which may provide a suitable source for the autoantigens involved in the SLE onset. References Kuhn A, Wenzel J, Weyd H. Photosensitivity, Apoptosis, and Cytokines in the Pathogenesis of Lupus Erythematosus: a Critical Review. Clinical reviews in allergy & immunology. Jan 14 2014. Llanos C, Mackern-Oberti JP, Vega F, Jacobelli SH, Kalergis AM. Tolerogenic dendritic cells as a therapy for treating lupus.ClinImmunol. Aug 2013;148(2):237-245. Kalergis AM, Iruretagoyena MI, Barrientos MJ, et al. Modulation of nuclear factor-kappaB activity can influence the susceptibility to systemic lupus erythematosus. Immu...

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