<i>In vivo</i> immune response induced by de-iniDCs and BM-DCs.

<p>(A) 48 hours after intratracheal application of cells, BAL fluid was collected and cells were counted in a haemocytometer. (B) The percentage of the CD66a⁺ neutrophils in the BAL fluid was analyzed by flow cytometry. (C) Percentage of CD3⁺ T cells in the BAL fluid of provoked mice were analyzed by flow cytometry. (D) Numbers of F4/80⁺ macrophages in the BAL fluid were analyzed by flow cytometry. (E) T cell cytokine secretion was measured in the BAL fluid by CBA. (F) May-Grünwald-Giemsa stained cytospin preparations demonstrate recruited eosinophils. (G) Paraffin-embedded lung sections were stained with Hematoxylin and Eosin. Results are expressed as mean ± SEM from 5 mice per group. Statistical significance is indicated, *(P<0.05) and **(P<0.01).</p

CD11c expression and IL-12 production in single cell clones.

<p>De-iniDC single cell clones were stimulated with LPS or left untreated for 24 hours in the presence of the protein transport inhibitor Monensin. Afterwards, cells were stained for the surface marker CD11c, permeabilized and stained for intracellular IL-12. (A) CD11c expression (black) of LPS stimulated cells is displayed. (B) Intracellular IL-12 expression level of CD11c⁺ LPS stimulated (black) and non-stimulated cells (grey) are shown. Isotype control is displayed as grey, dotted curve (A, B).</p

Lentiviral vector mediated transgene expression in iniDCs.

<p>(A) RFP expression level was measured in untransduced (grey dotted) and lentiviral vector particle-transduced iniDCs before (grey) and after (black) puromycin selection. (B) Expression level of maturation markers MHCII, CD40 and CD86 were determined in transduced iniDCs and after their deinduction (de-iniDCs) using flow cytometry. Transduced iniDCs and de-iniDCs (grey) and LPS-stimulated transduced iniDCs and de-iniDCs (black) are shown. Isotype controls are displayed as grey dotted lines. One representative experiment out of 3 is shown.</p

Antigen presentation of de-iniDC clone #1 and BM-DCs to T cells.

<p>De-iniDC clone #1 or BM-DCs were incubated with OVA (13.5 µg/mL) for 24 hours prior to co-culture with OTII/CD45.1 CD4⁺ T cells or OTI CD8⁺ T cells. (A) Proliferation of CD4⁺ T cells was measured using CFSE staining and analyzed by flow cytometry. (B) Secretion of IL-2 was measured with CBA. (C) CD4⁺ T cell secreted cytokines IFNγ, IL-13 and IL-17 were measured in the cell culture supernatant using CBA after 48 hours. (D) Proliferation of CD8⁺ T cells was measured using CFSE staining and flow cytometry. (E) CD8⁺ T cell secreted cytokines IL-2 and IFNγ were measured in the supernatant using CBA after 48 hours. Results of three to four independent experiments are given as mean ± SEM, (n.d.) not detectable. Statistical significance is indicated, *(P<0.05), **(P<0.01) and ***(P<0.001).</p

Dendritic cell surface marker expression.

<p>(A) BM-DCs, iniDCs and 3-days cultured de-iniDCs were stained with antibodies against the dendritic cell subset markers CD11c, CD8α, CD11b, B220 and Ly6C. CD11c⁺ cells (black curve) were further gated for CD8α and CD11b, Ly6C and B220 (contour blots). Gates for CD8α and CD11b, Ly6C and B220 were set on the respective unstained control (red). (B) Immature and mature BM-DCs, iniDCs and de-iniDCs were stained for MHCII, CD40, and CD86. Dead cells (DAPI staining) and cell doublets were excluded. Histograms show the isotype control (grey, dotted), immature cells (grey) and LPS-matured cells (black). The result of one representative experiment is given.</p

Morphology, cell cycle and proliferation.

<p>(A) Microscopic images of BM-DCs, iniDCs and de-iniDCs 3-days after de-induction at 10× magnification. Both, BM-DCs and de-iniDCs show an adherent phenotype with the typical formation of dendrites. (B) Proliferation of iniDCs and de-iniDCs was analyzed by counting the cells in a haemocytometer over a time period of 6 days. (C) Percentage of dead cells counted over a time period of 6 days. (D) Apoptosis and necrosis of iniDCs, 3- and 5-days cultured de-iniDCs were analyzed using anti-AnnexinV-PE antibody and DAPI. Dot blots display AnnexinV and DAPI stained cells. (E) For cell cycle analysis, iniDCs, 3- and 5-days cultured de-iniDCs were stained with PI and analyzed by flow cytometry. Cell cycle stages G1 (left peak), S (middle) and G2 (right peak) were calculated with the Dean-Jett-Fox model using FlowJo software. Proliferation, apoptosis and cell cycle analyses were performed in three independent experiments. For apoptosis and cell cycle analysis the result of a representative experiment is given.</p

Induction of T cell response.

<p>(A) <i>In vivo</i> immunization model with OVA-V antigen. (B, C) Proliferation of CD4+ T cells (B) and effector memory T cells (CD4+ TEM; C) activated with OVA-V-pulsed Dox-pDC. (D, E) Proliferation of CD4+CD8lo T cells (D) and effector memory T cells (CD4+CD8lo TEM; E) activated with OVA-V-pulsed Dox-pDC. (F-H) Frequency of Th1 (IFNγ+CD4+; F), Th17 (RORγt+CD4+; G) and CD8+ T cells (IFNγ+CD8+; H) polarized with OVA-LE-pulsed and TLR9-activated Dox-pDC or BM-pDC. Results are expressed as means ± SD from 3–9 mice per group. Statistical significance is indicated, ***(P<0.005) and ****(P<0.001).</p

Phenotype of immature Dox-pDC.

<p>(A) Scheme of generating Dox-pDC line. (B) Expression of pDC marker by the final 10 single cell clones. (C) IFNα secretion of the final 10 single cell clones. (D) Flow cytometric analysis of BM-pDC and Dox-pDC for different cell-specific markers. (E) Proliferation of Dox-pDC in the presence or absence of Dox and Flt3l and TLR9 activation detected with the viability proliferation dye 450 at day 0, 3, 5, 7 and analysed by flow cytometry. (F) Apoptosis staining of Dox-pDC in the presence or absence of Dox and Flt3l analysed by flow cytometry. (G) Doubling time of Dox-pDC calculated with an exponential growth equation. The figure shows representative results out of 2–4 experiments.</p