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. 2021 Feb 26;11:4735. doi: 10.1038/s41598-021-84023-0

Figure 3.

Figure 3

Fate and functional properties of CpG-pre-pDCs in EAE mice. CD45.1 (a) or GFP+ (b) CpG-pre-pDCs were injected at d-12 into MOG35–55 immunized CD45.2 mice. (a) Absolute numbers of the CD45.1 cells were analyzed in the spleen, draining lymph nodes and spinal cord of the animals at d-18 (mean ± s.e.m.), n = 17 mice with CpG-pre-pDCs, n = 5 mice with PBS-pre-pDCs, from two cumulated experiments, *p = 0.02, using unpaired Students’ t-test. (b) Phenotype of the spinal cord GFP+ cells, assessed at d-18 using flow cytometry analysis with FMO controls. One representative experiment out of 4. (c–e) FACS analysis of intranuclear IRF7 expression (c) in bone marrow CpG-pre-pDCs and spleen derived pDCs, (d) in CD45.1 CpG-pre-pDCs recovered from the spinal cord of EAE recipients, compared to host CD45.2 PDCA-1+ cells, either from CpG-pre-pDC recipients or from control mice with EAE injected with PBS medium. (e) MFI values of IRF7 were plotted for CD45.1 PDCA-1+ and host CD45.2 PDCA-1+ cells from 4 CpG-pre-pDC recipient mice with EAE. Mean ± s.e.m. Statistical analysis was performed using unpaired t-test. *** p < 0.001. (f) Cytokine production of the CD45.1 cells recovered from the spinal cord analyzed at d-18 by flow cytometry with isotype controls after 4 h activation with PMA/ionomycin in presence of brefeldin. Representative flow cytometry analysis out of 5. (g) Histograms summarizing percentages of CD45.1 cells expressing a given cytokine. n = 10 mice for CpG-pre-pDC recipients, n = 3 mice for PBS-pre-pDC recipients, mean ± s.e.m., data are from two cumulated experiments. *, p = 0.0111 for GM-CSF, *, p = 0.0176, for IL-27, by unpaired Students’t-test (h) CpG-pre-pDCs were isolated from the BM culture of either WT or IL-27−/− mice in presence of 1 µg/ml CpG-B and 80,000 of them injected i.v into EAE immunized mice at d-12. Clinical score (mean ± s.e.m.) was assessed until d-45. n = 7 for the IL-27−/− CpG-pre-pDCs treated group, n = 8 for the WT CpG-pre-pDCs treated group, n = 14 for the control group, injected with PBS. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test. Controls vs + IL-27−/− CpG-pre-pDCs, d12–21, **p = 0.0024, NS afterwards, Controls vs + WT CpG-pre-pDCs, d15–d35, **, p = 0.002, d40–d44, p = 0.0012.