IL-1R signaling is required in vivo for full Bhlhe40 expression by Th cells, and systemic PTX induces IL-1β production by lymph node cells. (A) GFP (Bhlhe40) expression by CD4⁺ T cells from the DLNs of Bhlhe40^GFP or Il1r1^−/− Bhlhe40^GFP mice on day 7 after immunization with MOG/CFA + PTX. One representative experiment out of three is shown (n = 9–10 mice/group). (B) Quantitation of the percentage (left) of GFP^pos cells (of CD4⁺ T cells) and the (right) number of GFP^pos CD4⁺ T cells in the DLNs from A. Data are combined from three experiments (n = 9–10/group). (C) GFP (Bhlhe40) expression by CD4⁺ T cells from the DLNs of Bhlhe40^GFP mice treated with control Ab or IL-1 blockade in vivo on days −1, 1, and 4. Mice were left unimmunized or were immunized with MOG/CFA ± PTX treatment (days 0 and 2). One experiment out of two is shown (n = 1–3 mice/group). (D) Quantitation of the percentage (left) of GFP^pos cells (of CD4⁺ T cells) and the (right) number of GFP^pos CD4⁺ T cells in the DLNs from (C). One experiment out of two is shown (n = 1–3 mice/group). (E) Mean EAE scores of WT, Bhlhe40^−/−, and Il1r1^−/− mice immunized with MOG/CFA + PTX. Data are combined from two experiments (n = 6–12). Incidence of clinical disease is indicated. (F) CD4⁺ T cells from the DLN of MOG/CFA + PTX-immunized Bhlhe40^GFP (CD45.1/CD45.2) mice and Il1r1^−/−Bhlhe40^GFP (CD45.2) mice were purified on day 7 and co-transferred to WT (CD45.1) recipients 1 d before immunization with MOG/CFA + PTX. 7 d after this immunization, transferred Bhlhe40^GFP or Il1r1^−/−Bhlhe40^GFP GFP^pos cells were identified. One experiment out of two is shown (n = 6). (G) Quantitation of the percentage of transferred Bhlhe40^GFP or Il1r1^−/−Bhlhe40^GFP GFP^pos cells (of GFP^pos cells) in the DLNs from F. One experiment out of two is shown (n = 6). (H) Groups of WT mice were either unimmunized or immunized with MOG/CFA. PTX or mPTX was administered i.p. on days 0 and 2. DLN cells, collected on day 7, were cultured with or without 50 µg/ml heat-killed Mtb for 2 d and supernatants were analyzed for IL-1β. Data are combined from three experiments (n = 3–7 mice/group). (I) Quantitation of the percentage of pro–IL-1β⁺ cells (of B220⁻ DLN cells) from WT mice treated as in H. Data are combined from four experiments (n = 3–12/group). (J) ICS for pro–IL-1β expression by B220⁻ DLN cells from representative mice shown in I. (K; left) Ly6G and Ly6C expression on B220⁻ DLN cells from the mice shown in (J). Numbers show the percentage of Ly6G⁺Ly6C⁺ neutrophils. (right) MHC class II and Ly6C expression by Ly6G⁻B220⁻ cells. Numbers show the percentage of monocytes/moDCs. One experiment out of four is shown (n = 3–12/group). (L) Quantitation of the number of pro–IL-1β⁺ neutrophils, MHC II⁺Ly6C⁺ monocytes/moDCs, migratory DCs, and resident DCs in the DLNs from (I-K). Data are combined from two experiments (n = 2–7/group). Data are mean ± SEM. Dots represent individual mice except in E. Unpaired (B, D, H, I, and L) or paired (G) two-tailed Student's t tests were performed to determine significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.