Figure 5: L-pampo expands multi-cytokine-producing antigen-specific CD4⁺ T cells more synergistically as the number of boosts increases.

Naïve Ly5.2⁺ B6/J mice were injected with Ly5.1⁺ OT-II T cells (5 × 10⁵ each) before the immunization, then immunized with 100 μg of OVA alone or in combination with alum, polyI:C, Pam3, or L-pampo as adjuvants, followed by the same immunization at day 30 (a–d) or 3 or 6 weeks after the first immunization (e–h). (A) Mouse PBMCs were isolated from the blood, and the frequencies of the transferred OT-II cells (CD4⁺ CD44⁺ Ly5.1⁺) among CD4⁺ T cells were analyzed kinetically by flow cytometry at the indicated time points. (b–h) Six days after the secondary immunization (b–d) or 7 days after the tertiary immunization (e–h), splenocytes were isolated and analyzed by flow cytometry. (b,e) Representative FACS plots and the frequencies and numbers of the transferred OT-II cells (CD4⁺ CD44⁺ Ly5.1⁺) among CD4⁺ T cells are shown. (c,f) Representative (c) or concatenated (f) FACS plots and the frequencies and numbers of IFN-γ⁺-producing CD4⁺ T cells among total CD4⁺ T cells are shown. (d,g) Representative (d) or concatenated (g) FACS plots of cytokine-producing OT-II cells and the frequencies of IFN-γ⁺ cells among the OT-II cells and of IFN-γ⁺ TNF-α⁺ cells among IFN-γ⁺ OT-II cells and the IFN-γ expression levels of IFN-γ⁺ OT-II cells were determined using the mean fluorescence intensity (MFI) of IFN-γ. (h) Splenocytes were stimulated ex vivo with OVA_323–339 peptides. The levels of IFN-γ, IL-6, and TNF in the supernatants of the re-stimulated splenocytes were measured by a cytokine ELISA (left column). Splenocytes were analyzed for IFN-γ or IL-4 secretion by an ELISpot assay (right column). The data are represented as the mean ± SEM with each dot indicating one mouse (n = 4). *p < 0.05; **p < 0.01; ***p < 0.001.