Thus, to evaluate in our experimental model if T. cruzi infection upregulates PD-1 and CTLA-4 expression in CD4 T cells, flow cytometry was performed on spleen cells at several time points after infection, and the percentage of CD4+ T cells expressing PD-1 or CTLA-4 on the surface was determined, as shown in Fig 2A and 2C, with representative dot plots displayed in Fig 2B and 2D. Here, CTLA4 is linked to infection.