Figure 5.

Exosomal miR-21 affects the CD4⁺ T cell proliferation. (A) Exosomes from the culture supernatants of murine DCs infected with LdWT or LdCen^−/− parasites were purified and the expression of miR-21 was measured in qRT-PCR. Exosomes from uninfected DCs were used as control. Fold increase in miR-21 expression was based on the uninfected control DCs. (B) Schematic of the experiment to determine the impact of exosomal miR-21 on the CD4⁺ T cell proliferation. Murine BMDCs were infected with live or dead LdWT or LdCen^−/− parasites. DCs were treated with anti-miR-21 LNAs to block the miR-21 as indicated. Exosomes were purified from the infected and or treated DC culture supernatants. The exosomes were transferred to independent BMDC cultures infected with LdCen^−/− parasites. These cultures were treated with ova peptide and co-cultured in presence of CFSE stained T cells from transgenic OT-II mice for 5 days. (C) Gating scheme to identify proliferation of CFSE stained CD4⁺T cells in presence of exosomes is shown. (D) Proliferation of CFSE stained CD4⁺ T cells in presence of exosomes isolated from uninfected DCs, (E) LdWT infection derived exosomes or (F) LdCen^−/− infection derived exosomes is shown. Proliferation in presence of exosomes derived from anti-miR-21 LNA treated cultures is also shown in these groups. (G) CD4⁺ OT-II cell proliferation in presence of exosomes that contain miR-21 and in presence of anti-miR-21 oligonucleotides is shown. Statistical significance between CD4⁺ T cell proliferation in presence of LdWT and LdCen^−/− infection derived exosomes 5 days post co-culture infection is shown (t-test; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).