cobea.org.br/). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institutional Animal Care and Use Committee at the Federal University of Sao Paulo (Id # CEP 0426/09). Female 8-week-old mice (C57BL/6 and A/Sn) were purchased from CEDEME (Federal University of São Paulo). Transgenic mice expressing the diphtheria toxin receptor (DTR) under control of the CD11c promoter (CD11c-DTR) on a C57BL/6 background were derived as described and were maintained in our colony as heterozygotes [30]. Blood-derived trypomastigotes of the Y strain of T. cruzi were obtained from A/Sn mice

infected 7–8 days earlier. Each C57BL/6 or A/Sn mouse was challenged sub-cutaneously (s.c.) at the base of the tail with a final dose containing 104–105 or 150 parasites, respectively, in a final volume of 0.1 mL. Parasite Capmatinib solubility dmso development was monitored by counting the number of blood-derived trypomastigotes in 5 μL of fresh blood collected from the tail vein [10]. Wild type (WT) and CD11c-DTR mice

were treated i.p. with 2 doses of 50 ng diphtheria toxin from Corynebacterium diphteriae (DT, Sigma), 48 h before and on the same day of challenge. In addition, infected WT mice were treated Crizotinib chemical structure every other day, beginning on the same day of infection, with doses of 20 μg FTY720 (Cayman Chemical, Ann Arbor, MI) per mouse (1 mg/kg) in a final volume of 0.2 mL. The control mice were injected with the diluent only. Peptides were purchased from Genscript (Piscataway, NJ). Purity was as follows: VNHRFTLV, 97.2% and TsKb-20 (ANYKFTLV), 99.7%. Plasmid pIgSPCl.9 and the human replication-defective adenovirus type 5 containing the asp-2 gene were described previously [22], [24], [25] and [31]. Heterologous too prime-boost immunization involved priming i.m. with 100 μg of plasmid DNA followed by a dose of viral suspension containing 2 × 108 plaque-forming units (pfu) of adenovirus 21 days later in the same locations. Immunological assays or challenges were performed 14 days after viral inoculation (boost).

The panel of conjugated antibodies used for FACS analyses were CD11c-FITC (clone HL3), CD19-PECy7 (clone 1D3), CD8α-PerCP (clone 53-6.7), CD86-APC (clone GL1), CD80-APC (clone 16-10A1), CD40-APC (clone 3/23) all from BD; PDCA-1-PE (clone JF05-1C2.4.1) from Miltenyi Biotec. Single-cell suspensions from Inguinal lymph nodes or spleen were stained for surface markers on ice for 20 min, and then washed twice in buffer containing PBS, 0.5% BSA, and 2 mM EDTA fixed in 4% PBS-paraformaldehyde solution for 10 min. At least 300,000 events were acquired on a BD FACSCanto II flow cytometer and then analyzed with FlowJo (Tree Star, Ashland, OR). PDCA-1+ cells were isolated from LN collected from C57BL/6 mice infected 5 days earlier s.c. with 104T. cruzi parasites. As controls, we used PDCA-1+ cells isolated from LN of naïve C57BL/6 mice (n = 15).