A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines

Abstract

BackgroundGeneration of tolerogenic dendritic cells (TolDCs) for therapy is challenging due to its implications for the design of protocols suitable for clinical applications, which means not only using safe products, but also working at defining specific biomarkers for TolDCs identification, developing shorter DCs differentiation methods and obtaining TolDCs with a stable phenotype. We describe here, a short-term protocol for TolDCs generation, which are characterized in terms of phenotypic markers, cytokines secretion profile, CD4+ T cell-stimulatory ability and migratory capacity.MethodsTolDCs from healthy donors were generated by modulation with dexamethasone plus monophosphoryl lipid A (MPLA-tDCs). We performed an analysis of MPLA-tDCs in terms of yield, viability, morphology, phenotypic markers, cytokines secretion profile, stability, allogeneic and antigen-specific CD4+ T-cell stimulatory ability and migration capacity.ResultsAfter a 5-day culture, MPLA-tDCs displayed reduced expression of costimulatory and maturation molecules together to an anti-inflammatory cytokines secretion profile, being able to maintain these tolerogenic features even after the engagement of CD40 by its cognate ligand. In addition, MPLA-tDCs exhibited reduced capabilities to stimulate allogeneic and antigen-specific CD4+ T cell proliferation, and induced an anti-inflammatory cytokine secretion pattern. Among potential tolerogenic markers studied, only TLR-2 was highly expressed in MPLA-tDCs when compared to mature and immature DCs. Remarkable, like mature DCs, MPLA-tDCs displayed a high CCR7 and CXCR4 expression, both chemokine receptors involved in migration to secondary lymphoid organs, and even more, in an in vitro assay they exhibited a high migration response towards CCL19 and CXCL12.ConclusionWe describe a short-term protocol for TolDC generation, which confers them a stable phenotype and migratory capacity to lymphoid chemokines, essential features for TolDCs to be used as therapeutics for autoimmunity and prevention of graft rejection.

Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

“…moDCs were generated as previously described (20) in AIM-V medium (Gibco BLR, Grand Island, NE, USA), supplemented with 500 U/ml of recombinant human GM-CSF and IL-4 (eBioscience, San Diego, CA, USA) within 5 days. At days 3 and 4, cells were modulated with 1 μM dexamethasone (tDCs) (Sigma-Aldrich, St. Louis, MO, USA) and activated with 1 μg/ml cGMP-grade MPLA (MPLA-tDCs) (Avanti, Alabaster, AL, USA).…”

“…Different strategies have been described for the in vitro generation of TolDCs derived from peripheral blood monocytes, including modulation by IL-10 or TGF-β (8, 13), dexamethasone (14), Bay11-7082 (15), rapamycin (16), aspirin (17) or vitamin D3 (18), short stimulation with lipopolysaccharide (LPS) (19), or its non-toxic analog monophosphoryl lipid A (MPLA) (20). Although TolDC properties may vary according to the applied protocol, TolDC features include reduced expression of co-stimulatory and antigen presentation molecules, low IL-12 production, and suppression of effector T cell responses in vitro (21, 22).…”

“…Recently, we described a shortened protocol for the differentiation of monocytes from healthy donors into TolDCs, using dexamethasone for immunomodulation, and current good manufacture practice (cGMP)-grade MPLA to trigger toll-like receptor (TLR)-mediated activation, including the upregulation of chemokine receptors that mediate the migration to secondary lymphoid organs (20). These MPLA-tDCs expressed low levels of co-stimulatory molecules and maturation markers, and secreted high levels of IL-10 and low levels of IL-12.…”

Co-delivery of autoantigen and dexamethasone in incomplete Freund's adjuvant ameliorates experimental autoimmune encephalomyelitis

“…CD11c+ DCs have been shown to act as tolerogenic DCs in the spleen [50]. Also, the treatment of DCs with DEX has previously been demonstrated to produce higher levels of tolerogenic DCs [49, 52-54]. Additionally, although not statistically significant, a slight trend in the decrease of CD3+ T-cells with the addition of DEX was also noted (Figure 6A).…”

EFFECT OF DEXAMETHASONE ON INTERFERON-α-INDUCED DIFFERENTIATION OF MONOCYTES TO DENDRITIC CELLS

“…Так, например, возрастание экспрессии PD-L1 было продемонстрировано в культурах незрелых IL4-ДК при добавлении дексаметазона с начала культивирования клеток [32]. В то же время, при анализе LPS-активированных IL4-ДК и более позднем внесении дексаметазона, стимулирую-щий эффект дексаметазона на экспрессию PD-L1 не выявлялся [8,29].…”