Cd40 apc

DCs from patients with T1D (n = 7) were co-cultured with 1 mM PSAB-liposomes (PSAB-DC), 1000 nM Lira (Lira-DC) or combined (PSAB + Lira DC) for 24 h in the presence of 20 μg/ml human insulin (Sigma-Aldrich). DCs were cultured with 20 μg/ml human insulin (Sigma-Aldrich) to obtain immature DCs (iDCs) and adding a cytokine cocktail [1000 IU/ml TNFα and 2000 IU/ml IL-1β (Immunotools) and 1 μM Prostaglandin E₂ (Cayman Chemical, Ann Arbor, USA)] to obtain mature DCs (mDC). To assess DCs phenotype, CD25-PE, CD86-FITC, HLA ABC-FITC, HLA DR-FITC, CD14-PE and CD40-APC (Immunotools), CD36-APCCy7, TIM4-APC, CD54-PECy7, TLR2-FITC, CXCR4-APCCy7, CCR2-APC, PD-L1-PECy7, ILT3-PECy7 (Biolegend, San Diego, USA) and CCR7-PECy7 (BD Biosciences) monoclonal antibodies were used to determine their membrane expression. All DCs conditions from the same patient were analysed at the time. All MFI groups were normalized to the MFI of mDCs.

The phenotype of CD34⁺ HSC exposed to betamethasone or fluticasone after 20 h of culture was determined by flow cytometry. To that end, 20.000 CD34⁺ cells of each group (control, betamethasone, and fluticasone) were stained with anti-human CD34 PE (BD Biosciences), HLA-ABC FITC (Immunotools), CD45 PercP (BD Biosciences), CD40 APC (Immunotools), CD54 PE-Cy7, CXCR4 APC-Cy7 and HLA-DR V500 (BD Biosciences), and median fluorescence intensity values were obtained using FACS Canto II cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

DCs from patients at onset and with established disease were cultured for 24 h with 20 μg/mL human insulin (Sigma-Aldrich, St. Louis, MO, USA) and 1 mmol/L of PSAB-liposomes to determine the effect of insulin chains as autoantigens (tolDCs). As controls, DCs were either cultured with 20 μg/mL human insulin (Sigma-Aldrich) to obtain iDCs or adding a cytokine cocktail —TNF-α (1,000 IU/mL, Immunotools), IL-1β (2,000 IU/mL, Immunotools), and Prostaglandin E₂ (PGE₂, 1 μmol/L, Cayman Chemical, Ann Arbor, MI, USA)— for 24 h to obtain mature DCs (mDCs). Viability and phenotype were analyzed by flow cytometry (FACSCanto II, BD Biosciences). DCs were stained with 7-AAD (BD Biosciences) and antibodies to CD11c-APC, CD86-FITC, HLA-ABC-FITC, HLA-DR-FITC, CD14-PE and CD40-APC (Immunotools), CD36-APCCy7, TIM4-APC, αvβ5 integrin-PE, CD54-PECy7, TLR2-FITC, CXCR4-APCCy7, CCR2-APC (BioLegend, San Diego, CA, USA). Data were analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).