Generation of Tolerogenic Dendritic Cells from Systemic Lupus Erythematosus Patients
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1
Pontificia Universidad Catolica de Chile, Departamento de Inmunología Clínica y Reumatología, Chile
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2
Pontificia Universidad Catolica de Chile, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas., Chile
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3
Pontificia Universidad Catolica de Chile, Departamento de Salud Pública, Escuela de Medicina, Chile
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4
Pontificia Universidad Catolica de Chile, Departamento de Enfermedades Hematológicas Escuela de Medicina, Chile
Introduction: Tolerogenic dendritic cells (TolDCs) are a promising strategy for several autoimmune diseases based on their specific suppressive effect on autoreactive T cells that target self-tissue damage. Specific and more effective therapies are needed for systemic lupus erythematosus (SLE) due to side-effects associated to available therapeutic strategies. Unfortunately, a predominant auto-antigen that drives the autoimmune response has not been identified yet, which has hampered the development of specific therapies. However, large evidence from in vitro and in vivo studies have linked SLE to apoptotic cells, suggesting that autoantibodies are directed to nuclear components contained in these cells. Therefore, in this study we propose to use apoptotic cells as a source of autoantigens, to specifically target TolDCs generated from SLE using NF-κB inhibitors to autoreactive lymphocytes.
Patients and Methods: 15 SLE patients that fulfilled the American College of Rheumatology criteria 1997 criteria for SLE and signed an informed consent form in accordance with the regulations of the Ethics Committee were included. Exclusion criteria were pregnancy, patients undergoing dialysis or who were severely ill.
Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll density gradient centrifugation and seeded at a density of 1x107 cells per well on culture plates for 2 hours at 37°C to allow adherence. Cells were washed 3 times with pre-warmed PBS to remove lymphocytes then serum-free AIM-V medium was added. Whole lymphocyte fraction was collected maintained in complete RPMI 1640 medium prior to use for apoptotic T cells generation. Monocytes were differentiated into DCs by addition of 1000 UI/mL IL-4 and 1000 UI/mL GM-CSF at days 0, 2 and 4 to medium. At the end of day 5, 10 µM rosiglitazone (RGZ) and 1 µM dexamethasone (DEXA) were added to medium for 24 hours. Then DCs were pulsed with apoptotic T cells (UV induced) for 24 hours and challenged with lipopolysaccharide (LPS) for additional 48 hours.
Results: TolDCs from peripheral monocytes of SLE patients (12F:3M; 34 ± 11,6 years; SLEDAI 4 ± 3,7) were generated using RGZ and DEXA and pulsed with apoptotic cells. TolDCs were next challenged with LPS to evaluate the effect of NF-κB inhibitors in maintaining a tolerogenic phenotype after a pro-inflammatory stimulus. Interestingly, the expression of surface maturation markers CD80, CD83 and CD86 was decreased in TolDCs (P<0.05, Friedman Test) and the expression of HLA-DR was unaltered when compare to mature DCs treated only with LPS. In addition, TolDCs secrete lower levels of proinflammatory cytokines IL-6 and IL-12p70. To better characterize the tolerogenic potential of cytokines secreted by TolDCs, the effect of the supernatants on T lymphocyte activation was evaluated. CD4+ T cells activated with anti-CD3 and anti-CD28 showed lower expression of the activation markers CD25, CD69 and CD71 in the presence of tolDCs supernatants compared to T-cells stimulated with anti-CD3 and anti-CD28 in fresh medium. Preliminary data on mixed lymphocyte reaction with allogeneic T cells suggest suppressor properties of these TolDCs, impairing their proliferation and expression of activation markers CD25 and CD71.
Conclusions: We were able to successfully generate DC with a tolerogenic phenotype from peripheral blood of SLE patients. The use of apoptotic cells as a source of autoantigens, makes them suitable for the generation of a specific immunotherapy for SLE.
Keywords:
Inmunotherapy,
Tolerogenic dendritic cells,
systemic lupus erythematosus,
Autoimmunity,
Immune Tolerance,
cell therapy
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Immunotherapy
Citation:
Torres Baeza
A,
Vega Tapia
F,
Mackern-Oberti
J,
Viviani Garcia
P,
Pereira Garcés
J,
Kalergis
AM and
Llanos Muñoz
C
(2015). Generation of Tolerogenic Dendritic Cells from Systemic Lupus Erythematosus Patients.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00095
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Received:
14 Apr 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Dr. Carolina Llanos Muñoz, Pontificia Universidad Catolica de Chile, Departamento de Inmunología Clínica y Reumatología, Santiago, Santiago, Chile, cllanos@med.puc.cl