In-vivo cytotoxicity assay
RAW 264.7 macrophages were used as target cells for in-vivocytotoxicity assay. RAW 264.7 cell line was acquired from American Type Culture Collection (ATCC TIB-71). Cells were cultured in supplemented DMEM in an atmosphere of 5% CO2 and 95% humidity at 37 ºC. RAW 264.7 Macrophages were stimulated with 5 μg/mL of soluble antigen of T. cruzi (H1 strain) for 2 h, consecutively were stained with CFSE 2 μM (CFSE High) and were mixed in equal proportion with non-stimulated macrophages previously stained with CFSE 0.2 μM (CFSE Low). At day 49 p.i., a total of 4x106 mixed macrophages (CFSE High and Low) were transferred intravenously to non-infected, infected, TSA-1-C4+E6020-SE, Tc24-C4+E6020-SE and TSA-1-C4+Tc24-C4+E6020-SE treated mice. Approximately 20 h later, when mice were euthanized, a total of 10x106 spleen mononuclear cells were recovered and resuspended in FACS buffer. Samples were acquired using BD FACSVerse flow cytometer, and at least 1x106 events in macrophage cell gate were obtained using FACSuiteTM software version 1.0.5. Data were analyzed in FlowJo software version 10.0.7r2. Flow cytometry gating strategies for cytotoxicity assay is presented in Supplementary Fig. 2 . The percentage of cytotoxicity was determined using the following formula40:
\begin{equation} \left[\mathbf{1-}\frac{\mathbf{(\%CFSE\ }\text{High}\mathbf{\ INF\ /\ \%CFSE\ }\text{Low}\mathbf{\ INF)\ }}{\mathbf{(\%CFSE\ }\text{High}\mathbf{\ NI\ /\ \%CFSE\ }\text{Low}\mathbf{\ NI)}}\right]\mathbf{X\ 100\%}\nonumber \\ \end{equation}
Percentage of cytotoxicity was calculated by the difference between CFSE High versus CFSE Low in infected mice (INF) vs the differences of CFSE High and Low in non-infected mice (NI).