Vitro Characterization of IRIV

To characterize IRIV-elicited immune responses in vitro, we addressed cell proliferation and cytokine expression in peripheral blood mononuclear cell (PBMC) cultures, as well as IRIV effects on dendritic cells (DC). In all experiments, PBMC were obtained from healthy donors and, if needed, further separated into different cell subsets. Finally, cells were cultured in the presence or absence of IRIV as indicated. 

Cell proliferation was addressed by conventional H-thymidine incorporation assays. Briefly, PBMCs were cultured in the presence of IRIV and liposomes, and in absence of any stimuli. On day 5 of culture, cells were pulsed with H-thymidine for 18 hours, then harvested, and cell proliferation was determined by tracer incorporation measurement. 

Proliferation assays performed with PBMC cultures from a number of healthy donors demonstrated that IRIV indeed elicited cell proliferation in all tested cultures to an extent variable from donor to donor (Fig. 1 A). Further proliferation assays with CD4+ T-cells or CD8-I- T-cells in coculture with CD 14+ cells demonstrated that IRIV induce CD4+ T-cell proliferation, whereas no proliferation of CD8+ T-cells could be observed (Fig. 1C). Dissection of the CD4+ T-cell population into CD4+CD45RO+ and CD4+CD45RA+ cell subsets pointed to CD4+CD45RO+ cells as proliferative responders (Fig. ID). Importantly, culture of cord blood mononuclear cells in presence of IRIV did not result in major cell proliferation (Fig. IB), underlining antigen experience as prerequisite for IRIV-induced cell proliferation. 

IRIV Induce a Cytokine Expression Profile Consistent with a T Helper 1 Response 

In addition to above-mentioned cytokines, IRIV were also shown to induce expression and secretion of various chemokines, such as IP-10, MIG, and Rantes (Schumacher R, unpublished). Secretion of chemokines is important for the recruitment of immune cells however, the relevance of this in vitro finding has not been addressed by migration assays or by in vivo studies so far. 

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