Fig 2: TNF signaling promotes Tfh-B cell interactions(A and B) PBMC from young adults were freshly isolated and sorted for coculture of T cell subsets (naive, gated as CD4+CD45RA+CD27+; CXCR5− memory, non-naive CD4+CXCR5−; cTfh, non-naive CD4+CXCR5+PD-1+) with autologous naive B cells (CD3−CD19+CD27loIgD+). Supernatant IgM (A) and IgG1 (B) were measured after 7 days, as shown for the following conditions: unstimulated (gray), SEB alone (0.5 μg/mL, green), SEB with recombinant human TNF (125 ng/mL, tan), or SEB with α-TNF antibodies (2 μg/mL, sienna) (1-way repeated-measures ANOVA with Holm-Sidak’s test; n = 12 per group).(C and D) Correlation shown for the TNFRSF1A in ICOS+CD38+ cTfh at day 7 compared with the H1N1-specific (C) hemagglutinin inhibitory (HAI) titer (n = 14) or H3N2-specific (D) HAI titer (n = 14) in young (orange) and elderly (purple) adults.(E) Correlation of apoptosis and NF-κB gene set GSVA scores for ICOS+CD38+ cTfh at day 7 for young (orange, n = 6) and elderly (purple, n = 8) adults.(F) Pearson coefficients for BCL family member genes compared to GSVA scores for TNF-NF-κB signaling in ICOS+CD38+ cTfh at day 7 for young (x axis) and elderly (y axis) adults. Genes classified as either pro-survival (dark blue) or apoptotic (sea green).

Fig 3: Neutralizing maternal TNFα is protective against embryonic malformation induced by the combination of high maternal iron and Western diet.a Clustering analysis of 32-plex cytokine panel in maternal serum from iron-adequate (gray circles), dietary iron-loaded (Fe diet, light blue circles), and genetically iron-loaded hepcidin KO (HKO, dark blue circles) dams fed standard or Western diet (WD). Color key indicates Z-score for each cytokine. b–e E18.5 TNFα measurements in b maternal serum, c embryo serum pooled from each litter, d pooled amniotic fluid, and e placental Tnf gene expression. Placentas were randomly selected for analysis. f Hepcidin KO females were fed Western diet (100 ppm iron) starting at 3 weeks of age and were mated after 9 weeks. Pregnant dams received intravenous injections of neutralizing TNFα antibody (white circles) or isotype IgG (dark blue circles) targeting trinitrophenol as a control (250 µg/injection) on E5.5, E8.5, E12.5, and E15.5, and embryo outcome was evaluated at E18.5. g Embryo gross morphology. h–j Incidence of eye malformations. b–e, h, j Error bars represent mean ± s.e.m. Statistical differences were determined by one-way ANOVA on ranks followed by Dunn’s method for multiple comparisons (denoted by #) or two-tailed Fisher’s exact test (indicated by ‡). P-values are indicated in each figure panel. Source data are provided as a Source data file.

Fig 4: L-WRN CM activity is maintained over several weeks of storage following thaw.(A-E) L-WRN CM was stored at 4C for 0WK, 1WK, 2WK, 3WK or subjected to a second freeze-thaw cycle (2XFT). An aliquot of the 2XFT sample was removed prior to the second freeze-thaw cycle to serve as a direct control (2XFT Cont). Spheroids cultured in differentiation medium with EP4 inhibitor (DM + EP4i), treated with cycloheximide and tumor necrosis factor (CHX + TNF), or treated with butyrate served as negative controls. (A) Schematic of experimental time line for assays in (B) and (C). (B) Graph of CellTiter-Glo data presented as fold change (mean ± s.e.m.) relative to DM + EP4i; n = 3 independent experiments. ****P < 0.0001 by 1-way ANOVA and Dunnett’s post test relative to 0WK. (C) Graphs of mRNA gene expression for indicated genes as determined by qPCR. Data are presented as fold change (mean ± s.e.m.) relative to 0WK; n = 3 independent experiments. (D) Schematic of experimental time line for (E). (E) Graph of Cdc25A-CBRluc data normalized to the average 0 h value of all samples; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA (B, C) or by 2-way repeated measures ANOVA (E) using Dunnett’s post test relative to 0WK (B, C, E).

Fig 5: The S513A mutation destabilizes Regnase-1 protein but does not affect target mRNA abundance.(A–C) Immunoblot analysis of Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) (A), BMDMs stimulated with LPS (100 ng/ml) (B), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) (C) for indicated time. PECs were pretreated with MG-132 (5 μM) 2 hr before the stimulation. (D)-(F) mRNA expression of Zc3h12a and Il6 in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) for 4 hr (D), BMDMs stimulated with LPS (100 ng/ml) for 4 hr (E), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) for indicated time (F). (G)-(I) IL-6 secretion in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml), IL-17A (50 ng/ml), or TNF (10 ng/ml) for 24 hr (G), BMDMs stimulated with Pam3CSK4 (1 or 10 ng/ml), poly I:C (10 or 100 μg/ml), LPS (10 or 100 ng/ml), R848 (10 or 100 nM), or CpG DNA (0.1 or 1 μM) for 24 hr (H), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml), R848 (100 nM), or IL-1β (10 ng/ml) for 24 hr (I). (J) Schematic representation of Model 1 in which 14-3-3-bound Regnase-1 does not have the function of degrading its target mRNAs. This model could explain the experimental observations. (K) Schematic representation of Model 2 in which 14-3-3-bound Regnase-1 maintains some ability to degrade its target mRNAs. This model is not consistent with the experimental observations. In (D)-(I), bars represent mean values of biological replicates (n = 3), and error bars represent standard deviation. Data is representative of two independent experiments, each with three biological replicates.