Figure 3: Pam3 and L-pampo promote the expansion of follicular helper T cells in the periphery, but only L-pampo adjuvanticity prominently maintains OVA-specific CD4⁺ T cells during the memory phase.

Naïve Ly5.2⁺ B6/J mice were injected with Ly5.1⁺ OT-II cells (5 × 10⁵ cells each) before the immunization and immunized with 100 μg of OVA alone or in combination with alum, polyI:C, Pam3, or L-pampo as adjuvants. (a) Mouse PBMCs were isolated from the blood, and the frequency of the transferred OT-II cells (CD4⁺ CD44⁺ Ly5.1⁺) among CD4⁺ T cells was analyzed kinetically by flow cytometry at the indicated time points after immunization. (b–d) Eight days after the immunization, splenocytes were isolated and analyzed by flow cytometry. (b) Representative FACS plots and the frequencies and numbers of the transferred OT-II cells among CD4⁺ T cells (gated as CD4⁺ CD44⁺ Ly5.1⁺). (c) Representative FACS plots and the frequencies of PD-1⁺ CXCR5⁺ follicular helper T cells (Tfh) or GL-7⁺ CXCR5⁺ germinal center Tfh (GC-Tfh). (d) Concatenated FACS plots of cytokine-producing OT-II cells and the frequencies and numbers of IFN-γ⁺ cells among the OT-II cells. (e) Forty-eight days after immunization, splenocytes were isolated and analyzed by flow cytometry. Representative FACS plots and the frequencies and numbers of the transferred OT-II (CD4⁺ CD44⁺ Ly5.1⁺) cells among CD4⁺ T cells. (f,g) Mouse serum was isolated at the indicated time points, and OVA-specific IgG (f) and the indicated isotypes at day 29 (g) were determined by an ELISA. The data are represented as the mean ± SEM with each dot indicating one mouse (n = 3–4). The data are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.