Fig. 4

MnJ promotes antigen presentation and specific T-cell responses. a, b WT mice were immunized subcutaneously with PBS, phycoerythrin (PE, 10 μg), PE (10 μg) + MnJ (20 μg), or PE (10 μg) + Imject Alum (200 μg). Inguinal lymph nodes were collected 12 or 24 h later (n = 3). The ratio and number of PE⁺ APCs (a) and Mo-DCs (b) among dLN cells were analyzed by FACS. Live cells were identified by DAPI staining. Among live singlet cells, APCs were identified as the cell subset single or double positive for CD11c and F4/80. Among APCs, Mo-DCs were identified as CD8a⁺ CD64⁺ cells. c BMDCs were treated with MnCl₂ (20 μg/ml), MnJ (20 μg/ml), or Imject Alum (20 μg/ml) for 20 h. CD86 expression was analyzed by FACS. d CD45.1⁺ OT-I CD8⁺ or OT-II CD4⁺ T cells were labeled with CFSE and transferred to CD45.2⁺ WT mice. These mice were then immunized with OVA (1 μg), OVA (1 μg) + MnJ (10 μg), or OVA (1 μg) + Imject Alum (1320 μg). After 3 days, T-cell proliferation was analyzed by FACS (n = 3). e WT mice were immunized intramuscularly with OVA (10 μg), OVA (10 μg) + MnJ (10 μg), or OVA (10 μg) + Imject Alum (1320 μg) on days 0, 7, and 14. Splenocytes were collected on day 21 and stimulated with OVA (100 μg/ml). IL-2, IL-4, IFNγ, and IL-10 secretion by T cells was measured by ELISA (n = 3). f OVA-specific cytotoxicity was measured on day 21 in an in vivo killing assay (mice were immunized on days 0, 7, and 14, n = 4). g Numbers of Tfh or GC B cells in dLNs from WT mice were analyzed by FACS. Live cells were identified by DAPI staining. Among live singlet cells, CD4⁺ T cells were identified as the cell subset double positive for CD3 and CD4. Among CD4⁺ T cells, Tfh cells were identified as PD1⁺ CXCR5⁺ cells. B cells were identified as the cell subset double positive for CD45 and B220. Among B cells, GC B cells were identified as Fas⁺ GL7⁺ cells (n = 3). One representative experiment of at least three independent experiments is shown, and each was performed in triplicate. Error bars represent the SEM; data were analyzed by an unpaired t-test. ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001