Quantification of CD4+ T Cell Response

Cell suspension from lymph nodes and spleens were incubated for 1.5 hours at 37 °C with the MHC class II-restricted peptides mixture, which is recognized by CD4+ T cells. Cells were stained with CellTrace^TM CFSE dye (ThermoFisher Scientific). MHC class II-restricted peptides recognized by CD4+ T cells were described previously^{33 (link), 49 (link)} and purchased from Peptides&elephants GmbH (Potsdam, Germany). Unloaded cells had been stained with CellTrace^TM Violet (ThermoFisher Scientific). Peptide loaded and unloaded cells were suspended in PBS in a 1:1 ratio. Cell suspension containing 3.0 × 10⁶ cells of each population was injected intravenously into each mouse. 20 hours later, lymph nodes and spleens have been harvested. The ratio of live CFSE+ target cells loaded with peptides recognized by CD4+ versus Violet+ unloaded target cells have been calculated normalized to the ratio in naive animals.