Figure 3.

Virus replicon particle (VRP) immunization induces specific immune cell proliferation in the draining lymph nodes. (A) The gating strategy used to quantify B and T cell proliferation, based on the expression of CD3, CD4, CD8, CD21, IgM, and CellTrace™ markers, is shown. (B) On the left side is shown a representative histogram, where FCS^low and FSC^high gates can be distinguished. Then, CD4⁺ T cells (CD3⁺CD21^-IgM^-CD4⁺CD8^-), CD4⁺CD8⁺ T cells (CD3⁺CD21^-IgM^-CD4⁺CD8⁺), CD8⁺ T cells (CD3⁺CD21^-IgM^-CD4^-CD8⁺), and B cells (CD3^-CD21⁺IgM⁺CD4^-CD8^-) where analyzed in parallel from those two independent FSC^low and FSC^high gates. The representative examples on the right side clearly show that proliferative cells are CellTrace^low FSC^high. (C, D) At day 56 post-first immunization, freshly isolated PBMCs and LN cells where exposed against recombinant CSFV E2 and influenza virus HA and NP, or were let unstimulated (“-”). For all samples, Con A was used as a positive control. (C) Induction of FSC^high cell population following Ag restimulation. Cells from the individual animals are represented by separate symbols. The percentage of FSC^high cells was determined by flow cytometry with FlowJo. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). (D) After 3 or 5 days of cell culture, the numbers of division cycles using CellTrace™ of proliferating cells from FSC^high subset from restimulated LN cells were determined by flow cytometry with FlowJo. Representative animals are shown for the three animal groups “RepRNA + c-di-AMP”, “[RepRNA/PEI] + c-di-AMP” and “VRP”.