Fig 2: MiR-34a drives DC activation.(a) miR-34a-/- DCs show reduced production of pro-inflammatory cytokines. Bone marrow DCs from WT and miR-34a-/- mice (n=6 pooled) were stimulated with LPS or CL097 for 24 h. (b,c) miR-34a-/- DCs show reduced expression of MHC class II. WT and miR-34a-/- DCs were stimulated as in a for 24 h and the expression of co-stimulatory molecules was evaluated as median fluorescent index (MFI) by flow cytometry. Representative MFI expression of MHC class II (b), and quantitative expression of MHC class II, co-stimulatory (CD40 and CD86), and inhibitory (PD-L1) molecules is shown (c). *P<0.05 Mann–Whitney u-test between genotypes. Data are presented as mean±s.e.m. of 4–6 replicates from two independent experiments. (d,e) miR-34a-/- DCs show a reduced interaction with antigen-specific T cells. CMTPX-labelled WT and miR-34a-/- DCs were co-cultured with CFSE-labelled OT-II CD4+ T cells at 1:1 or 1:3 ratios in the presence of OVA peptide or OVA plus LPS for 24. The contact area was analysed with In Cell Analyzer 2000. Representative pictures of 1:1 cultures (d) and quantitative evaluation (e) presented as mean±s.e.m. of % of overlapping areas between DC and T cells compared to the total cell surface area (six technical replicates in total) are shown. *P<0.05 between conditions, &P<0.05 between genotypes, two-way ANOVA followed by Tukey’s multiple comparison test or unpaired t-test. (f,g) miR-34a regulates pro-inflammatory cytokine production by human DCs. (f–h) Healthy donor monocyte-derived inflammatory DCs (n=4–5) were transfected with miR-34a mimic (34am) or miR-34a inhibitor (34i) or appropriate controls (Cm=mimic or Ci=inhibitor); and (g) DCs (conventional CD1c+ DCs; n=7) were sorted from RA patients’ blood and transfected with miR-34a inhibitor (34i) or control inhibitor (Ci). The DCs were then stimulated with LPS or CL097 for 24 h. (f) Enforced expression of miR-34a increased TNF production; *P<0.05, Kruskal–Wallis followed with Dunn’s multiple comparison tests or paired t-test. (g,h) miR-34a inhibitor reduced the production of cytokines by healthy donor monocyte-derived DCs (g) and sorted PB RA CD1c+ DCs (h). *P<0.05 paired t-test. (f,g) Data are presented as mean±s.e.m of 4–7 biological replicates obtained in separate experiments. ANOVA, analysis of variance.

Fig 3: Ppef2 is predominantly expressed by CD8+ cDC1. (A) Gene expression profiling of spleen and blood cells after sorting. Shown is the quantitative real-time PCR result for Ppef2 of three independent sort experiments (n = 3 pooled mice per sort) ± SEM analyzed by the ΔCt method with HPRT as housekeeping gene. Cells were identified and sorted as ESAMhi (CD11c+MHCII+CD11b+ESAMhi); ESAMlo (CD11c+MHCII+CD11b+ESAMlo); CD8+DC (CD11c+MHCII+CD11b−CD8+); pDC (CD11b−SiglecH+); CD4+ T cells (CD3+CD4+); CD8+ T cells (CD3+CD8+); B cells (CD19+B220+); blood monocytes NK1.1−B200−CD115+CD11b+Ly6G− cells with differential Ly6C and MHCII expression as indicated. Gating strategies are shown in Supplemental Figure 1B. (B) Ppef2 expression in GM-CSF cultured BMDCs of C57BL/6 mice after cell sorting on day 7 of culture based on the expression of CD11c, MHCII, and CD86. Error bars represent SEM of 3 independent experiments. (C) Ppef2 expression 16h after in vitro stimulation with the indicated TLR-ligands of GM-CSF- or Flt3L-cultured BMDCs of C57BL/6 mice, as well as spleen CD8+ DCs 16h after intravenous injection of 10 μg LPS as determined by qPCR. Bar graphs with SEM represent pooled data from independently performed cell cultures [GM-CSF BMDCs: unstim., LPS (n = 5); Flagellin, Poly(I:C), Pam3CSK4, CLO97, (n = 4); anti-CD40, LPS+anti-CD40 (n = 3); Flt3L BMDCs (n = 3); sorted CD8+ DCs (n = 4)]. Statistical analysis was performed using Student's t-test, with *p < 0.05; **p < 0.01; ***p < 0.001.