Disease severity in CD11c^dnRMog^TCR mice correlates with CNS uncontrolled production of mature DCs lacking TGF-βR signaling.

<p>(A) Flow cytometry of CD11b versus I-A/I-E, CD11c versus CD11b, and CD45.2 versus CD11b in the brain of untreated CD11c^dnR (<i>n</i> = 6), Mog^TCR (<i>n</i> = 3), and CD11c^dnRMog^TCR (<i>n</i> = 6) mice. Plots are from mononuclear cells. Gates delineate two myeloid cell subsets expressing high (red) and low (blue) levels of DC maturation markers, and numbers represent the percentages of cells in red gates. Bar graphs summarize the number of CD45.2^hiCD11b^hiCD11c^hiMHCII^hi DCs (as identified by red gates) recovered from the brain of untreated CD11c^dnR (white), Mog^TCR (gray), and CD11c^dnRMog^TCR (black) mice. (B) Flow cytometry of IL-17 versus IFNγ is shown in total mononuclear cells as well as among gated CD4⁺ T cells in the brain and spleen of untreated CD11c^dnR (<i>n</i> = 3), Mog^TCR (<i>n</i> = 3), and CD11c^dnRMog^TCR (<i>n</i> = 3) mice. Numbers in quadrants indicate the frequency of Th17 cells. (C) Flow cytometry of CD11c versus I-A/I-E in the spleen of untreated CD11c^dnR (<i>n</i> = 6), Mog^TCR (<i>n</i> = 3), and CD11c^dnRMog^TCR (<i>n</i> = 6) mice. Data are representative of three (A, C) and two (B) independent experiments. Mean ± s.e.m. (A). *<i>P</i><0.05 (A).</p

Inflamed CNS of CD11c^dnR mice revealed massive production of highly mature DCs lacking TGF-βR signaling.

<p>(A) Flow cytometry of CD11b versus I-A/I-E, CD11c versus CD11b, and CD45.2 versus CD11b in the brain and spinal cord of CD11c^dnR and wild-type (WT) mice at days 9 (<i>n</i> = 5) and 13 (<i>n</i> = 8) post-immunization. Plots are from mononuclear cells. Gates delineate two myeloid cell subsets expressing high (red) and low (blue) levels of DC maturation markers, and numbers represent the percentages of cells in red gates. (B) Overlay of gated CD11b^lo (black and green) and CD11b^hi (red) cell subsets from the brain (Br; solid) and spinal cord (Sc; dashed) of CD11c^dnR (green and red) and wild-type (WT) (black) mice at peak of EAE (day 13). Histograms display the fluorescence intensity of surface expression of CD11b, CD11c, I-A/I-E, and CD45.2. (C) Numbers of DC subsets (CD11b^lo and CD11b^hi) recovered from the CNS of CD11c^dnR (black) and wild-type (WT) (gray) mice at peak of EAE (day 13). (D) Flow cytometry of CD19 versus CD11b in brain, spinal cord, lymph nodes, and spleen of CD11c^dnR and wild-type (WT) mice at peak of EAE (day 13). (E) Ratio of CD11b^lo (blue) versus CD11b^hi (red) DC subsets in brain, spinal cord, lymph nodes, and spleen of CD11c^dnR and wild-type (WT) mice at peak of EAE (day 13). Data are representative of three independent experiments. Mean ± s.e.m. (C). *<i>P</i><0.05 (C).</p

TGF-β suppresses DC production but has no effect on DC activation.

<p>(A) Flow cytometry of DCs derived from bone-marrow precursors for 6 days in the absence or presence of 5 ng/ml TGF-β. Plots show the distribution of CD11c versus I-A/I-E, and numbers in quadrants indicate the percentage of CD11c⁺MHCII⁺ DCs. Bar graphs indicate the number of CD11c⁺MHCII⁺ DCs derived from CD11c^dnR (<i>n</i> = 6) and wild-type (WT) (<i>n</i> = 6) cultures in the absence (black) versus presence (gray) of TGF-β. (B) Flow cytometry of DCs derived from wild-type bone-marrow precursors (<i>n</i> = 4) cultured for 6 days in the presence of different doses of TGF-β (0, 1, 5, 10, and 25 ng/ml). Plots show the distribution of CD11b versus I-A/I-E, and numbers in quadrants indicate the percentage of CD11b⁺MHCII⁺ DCs. (C) Flow cytometry of DCs from wild-type bone-marrow precursors cultured in the presence of TGF-β at different times and for different durations as indicated by red arrows (each red arrow indicates the addition of TGF-β). Plots show the distribution of CD11c versus I-A/I-E and CD86 versus I-A/I-E in each combination. (D) Flow cytometry of bone marrow derived DCs stimulated with LPS (black arrow) for 16 hrs in the absence or presence of TGF-β (red arrow). Plots show the distribution of CD11c versus I-A/I-E and CD11c versus CD86 from wild-type cultures (<i>n</i> = 4). Bar graphs summarize the average frequency of DCs in response to two doses (1 and 100 ng) and two sources (<i>E. coli</i> and <i>S. enterica</i>) of LPS stimulation in the absence (black) or presence (gray) of 5 ng/ml TGF-β. (E-F) Cytokine production measured by ELISA in the supernatants collected from bone-marrow-derived DCs stimulated with soluble LPS (E–F) or LPS-A20 (E) for 16 hrs in the absence or presence of TGF-β at 5 ng/ml (E) or TGF-β at 1, 5, 10, 25, and 100 ng/ml (F). Bar graphs summarize the average production of IL-12, IL-6, and IL-10 from CD11c^dnR (<i>n</i> = 4) and wild-type (WT) (<i>n</i> = 4) cultures. Data are representative of four (A), two (B, C), and three (E, F) independent experiments. Mean ± s.e.m. (A and D–F). *<i>P</i><0.05 (A).</p

Location of TGF-β activity during EAE.

<p>(A) Bioluminescence imaging of SBE-Luc mice immunized subcutaneously with MOG peptide emulsified in CFA. Bioluminescence indicating pSMAD signaling was recorded on both the dorsal (upper panel for signal detection in the brain and spinal cord) and ventral (lower panel for signal detection in spleen and draining lymph nodes) sides of live animals at different times after EAE induction, including steady state (day 0), priming (days 3–5), pre-clinical (day 9), disease peak (day 13), and disease remission (days 17–21) phases. One representative mouse (<i>n</i> = 5) is shown. (B) SYBR Green quantitative PCR of TGF-β1 expression in the CNS and periphery of CD11c^dnR (<i>n</i> = 6) and wild-type (<i>n</i> = 6) mice on days 0 and 13 post-immunization. Data were analyzed using the 2^−ΔΔCt (cycle threshold) method, and results are expressed as the fold of change in CD11c^dnR versus wild-type organs. Data are representative of one (A) and two (B) independent experiments. Mean ± s.e.m. (B).</p

Potent Th17 differentiation uncovered in the inflamed CNS of CD11c^dnR mice.

<p>(A–D) Flow cytometry of CD4 versus IL-17 among total cells (A) and IL-17 versus IFNγ among gated CD4⁺ T cells (B) in brain, spinal cord, lymph nodes, and spleen of CD11c^dnR (<i>n</i> = 8) and wild-type (WT) (<i>n</i> = 8) mice at days 9 and 13 post-immunization. (C) Numbers of Th17 (CD4⁺IL-17⁺) cells in brain, spinal cord, lymph nodes, and spleen of CD11c^dnR (black) versus wild-type (WT) (gray) mice at the peak of EAE (day 13). (D) Percentages of Th17 (IL-17⁺IFNγ⁻) (gray), Th1 (IL-17⁻IFNγ⁺) (white), and Th1/Th17 (IL-17⁺IFNγ⁺) (black) cells in brain, spinal cord, lymph nodes, and spleen of CD11c^dnR versus wild-type (WT) mice at the peak of EAE (day 13). (E) Spinal cord and draining lymph nodes were isolated from CD11c^dnR (<i>n</i> = 4) and wild-type (WT) (<i>n</i> = 4) mice on day 13 post-immunization and total mononuclear cells were cultured in the presence of 50 µg/ml MOG peptide for 24 hours. Plots show the distribution of IL-17 versus IFNγ among gated CD4⁺ T cells in response to MOG re-stimulation from spinal cord versus draining lymph nodes. (F) SYBR Green quantitative PCR of the indicated genes in the CNS (black) and periphery (gray) of CD11c^dnR (<i>n</i> = 6) and wild-type (<i>n</i> = 6) mice at the peak of EAE (day 13). Data were analyzed using the 2^−ΔΔCt (cycle threshold) method, and results are expressed as the fold of change in CD11c^dnR versus wild-type organs. Data are representative of three (A–D) and two (E–F) independent experiments. Mean ± s.e.m. (C–D). *<i>P</i><0.05 (C).</p