MOESM1 of Glycogen synthase kinase 3 promotes multicellular development over unicellular encystation in encysting Dictyostelia

Additional file 1. Additional figures 1–3 and additional table 1

Additional file 2: of Subcellular dissemination of prothymosin alpha at normal physiology: immunohistochemical vis-a-vis western blotting perspective

Densitometric quantification of the western blot bands of PTMA expression across peripheral tissues. Each value is the Mean ± SD of at least three independent experiments. Arbitrary units were obtained by normalizing the signal to the internal control (values shown are PTMA signal/GAPDH signal). (DOCX 60 kb

Interaction between γδ T cells and CD56^brightCD11c⁺cells.

<p>(A) Cell aggregates observed in culture of γδ T cells mixed with CD56^brightCD11c⁺ cells without pulse (left), pulsed with ZOL (center), and further incubated with ZOL (right) (upper panels) and confocal microscopic observation of the culture of γδ T cells (green) and CD56^brightCD11c⁺ cells (red) in the presence of ZOL (lower panels). Data shown are representative of three independent experiments. (B) Flow cytometric analysis of γδ T cells incubated with CD56^brightCD11c+ cells (upper panels) and the numbers of γδ T cells after expansion for 7 days (lower panel). Dot plots shown are representative of three independent experiments, and data of cell counts show mean ± SD (n = 5), *p<0.05, **p<0.01. (C) There was no sustained proliferation of freshly isolated γδ T cells in the absence of accessory cells even after stimulation with IL-2, IL-2/ZOL, IL-2/IL-18/ZOL, or IL-2/2M3B1PP for 7 days. Data show the mean ± SD (n = 5). (D) Expression of co-stimulatory molecules and chemokine receptors on γδ T cells after culture with ZOL, IL-2, or ZOL/IL-2 for day 7. The result of flow cytometric analysis is representative of three independent experiments. (E) Induction of CCL21 by IL-18 in CD56^brightCD11c⁺cells. CCL21 concentration in the culture supernatant at 7 days was measured by ELISA. Data show mean ± SD (n = 5), *p<0.05.</p

Development, change of phenotype and function of CD56^brightCD11c⁺ cells induced by IL-18.

<p>(A) Time course of the development of CD56^brightCD11c⁺ cells. PBMCs (1×10⁵ cells/0.5 ml/well) were stimulated with ZOL/IL-2/IL-18. The absolute numbers of CD56⁺ or CD56^brightCD11c⁺ cells (white bar), γδ T cells (black bar) and αβ T cells (gray bar) were determined by flow cytometry and trypan blue dye exclusion test at day 0, day 3 (left), and day 7 (right). Data show mean ± SD (n = 10), **p<0.01. (B) Proliferation of CD56⁺ or CD56^brightCD11c⁺ cells (red line), γδ T cells (black line), and αβ T cells (gray line). Data show mean ± SD (n = 10), **p<0.01. (C) Requirement of CD56^brightCD11c⁺ cells for maximal sustained proliferation of γδ T cells. PBMCs were pre-stimulated with ZOL/IL-2/IL-18, and harvested on day 7. The proliferating cells were divided into 2 groups: one group was incubated with anti-CD56 antibody-conjugated beads and CD56⁺ cells were selectively removed. The other group was incubated with mouse IgG1-conjugated beads and used as a control. Both groups were re-incubated with ZOL/IL-2/IL-18 for another 14 days. Data show mean ± SD (n = 5), **p<0.01. (D) Development of CD56^brightCD11c⁺cells and gradual reduced expression of CD11c. The number and CD11c expression of proliferated cells were analyzed by flow cytometry during the culture of CD3⁺ T cell-depleted PBMCs. (Grey shadow: isotype control; blue: CD56^intCD11c⁺cells on day 0; red: CD56^brightCD11c⁺cells on day 7; thin black line: day 14; and bold black line: day 21). Data show mean ± SD (n = 5). A histogram shown is a representative of five independent experiments. (E) Comparison among CD56^brightCD11c⁺ cells, monocytes, and several subsets of DCs in helper activity for γδ T cells proliferation. Freshly isolated γδ T cells(5×104/well) were labeled with CFSE and co-cultured for 7 days with fresh CD14⁺ monocytes, IFN-α-DCs, IL-4-DCs, CD56^brightCD11c⁺, or pDCs, at a ratio of 1∶1 in the presence of ZOL/IL-2/IL-18. Then, proliferative responses of γδ T cells were analyzed based on flow cytometry and trypan blue dye exclusion test. R1: γδ T cells labeled with CFSE undergoing cell division; R2: Unlabeled CD56^brightCD11c⁺ cells. Data show mean ± SD (n = 5), **p<0.01. The result is representative of five independent experiments. (F) Differential cytotoxicity of CD56^brightCD11c⁺ cells against normal osteoblast cells (NHOst) and tumor cells (MG-63). The cytotoxicity was assessed using standard propidium iodide staining. Dot plots shown are representative of three independent experiments.</p

Putative model for the regulation of the development and expansion of CD56^brightCD11c⁺ cells and γδ T cells.

<p>In response to ZOL, CD14⁺ monocytes stimulate γδ T cells in a TCR-dependent manner. Concomitantly, CD14⁺ monocytes induce the IL-2/IL-18-mediated generation of CD56^brightCD11c⁺ cells from their putative precursor CD56^intCD11c⁺ cells. IFN-α inhibits this process possibly through the production of IFN-α-DCs. The resulting CD56^brightCD11c⁺ cells initiate and promote the expansion of γδ T cells for several days and gradually lose their helper function as they lose CD11c expression.</p

Negative regulation of CD56^brightCD11c⁺ cell development by IFN-α.

<p>(A) Inhibition of γδ T cell proliferation by IFN-α in PBMC cultures. PBMCs were stimulated with ZOL/IL-2 for 10 days in presence of various doses of IFN-α. Data show mean ± SD (n = 5), **p<0.01. (B) Attenuation by IFN-α of IL-18-mediated γδ T cell expansion in PBMC cultures with ZOL/IL-2. Data show mean ± SD (n = 4), **p<0.01. (C) Abrogation by IFN-α of the development and proliferation of CD56^brightCD11c⁺ cells in cultures of CD3⁺ T cells-depleted PBMCs. Data show mean ± SD (n = 4), **p<0.01. (D) Inhibition of cell aggregation by IFN-α in cultures of CD56^intCD11c⁺ cells and CD14⁺ monocytes, in the absence and presence of freshly isolated γδ T cells. CD14⁺ monocytes were pretreated with IL-2/IL-18 for 3 days, with or without IFN-α then CD56^intCD11c⁺ cells were added into the culture (upper panels). Next, freshly isolated γδ T cells were added and cellular clusters were observed by microscope (lower panels). The cell aggregates image is representative of three independent experiments. (E) Proliferation of γδ T cells in cultures containing mature CD56^brightCD11c⁺ cells even in the presence of IFN-α. Freshly isolated γδ T cells and mature CD56^brightCD11c⁺ cells were stimulated with ZOL/IL-2, with or without further addition of IFN-α since day 7 onwards and were continuously incubated. The number of proliferating cells was assayed after another 7 days' culture. Data show mean ± SD (n = 5).</p

Role of CD14⁺ monocytes in the development of CD56^brightCD11c⁺ cells.

<p>(A) CD14⁺, CD56⁺, and CD11c⁺ cells were required for the development of CD56^brightCD11c⁺ cells. CD14⁺, CD56⁺, or CD11c⁺ cells were depleted from T cell-depleted PBMCs (2×10⁴ cells/0.5 ml) and stimulated with IL-2/ IL-18 for 7 days. The number of CD56^brightCD11c⁺ cells was counted based on trypan blue dye exclusion. Data show mean ± SD (n = 5), **p<0.01. (B) Effect of CD14⁺ monocytes on the division of CD56⁺ cells. CFSE-labeled CD56⁺ cells were cultured with purified CD14⁺ monocytes at a ratio of 1:1 at a cell density of 2×105/well. After co-culture for 4 days, proliferating cells were analyzed by CFSE, CD56 and CD11c expression. A representative result of three independent experiments is shown. Division of CFSE-labeled CD14⁺ cells is also shown (black line). (C) Formation of large aggregates of CD56^brightCD11c⁺ cells in the co-culture of CD56^intCD11c⁺ cells and CD14⁺ monocytes in the presence of IL-2/IL-18 for 7 days, not in cultures of individual cell types. Data show mean ± SD (n = 4), **p<0.01, and the morphological data are representative of three independent experiments. (D) IL-2/IL-18-dependent cell aggregation in culture of CD14⁺ monocytes and CD56^intCD11c⁺ cells. IL-4-DCs, IL-18-induced CD14⁺ monocytes and IFN-α-DCs were co-cultured with freshly isolated CD56^intCD11c⁺ cells (1×105 cells/well) at a ratio of 1:1, respectively. After 12 h incubation, cell aggregates were observed under a microscope. Flow cytometric analyses are carried out after 3 days of co-culture. A representative result of three independent experiments is shown.</p

Phenotypic and functional analyses of CD14⁺ monocytes involved in the generation of CD56^brightCD11c⁺ cells.

<p>(A) Effect of IL-2/IL-18 on CD14⁺ monocytes proliferation. Data show mean ± SD (n = 4). (B) Analysis of IL-18 receptors and LFA-1 expressed by fresh CD14⁺ monocytes by flow cytometry. A representative result of three independent experiments is shown. (C) Flow cytometric analysis of CD14⁺ monocytes stimulated with IL-2/IL-18 after 24h culture. A representative result of three independent experiments is shown. (D) Analysis of cellular interactions between enriched CD56⁺ cells and purified CD14⁺ monocytes. Purified CD14⁺ monocytes were placed in the lower chambers and CD56⁺ cells containing CD56^intCD11c⁺ cells in the upper chambers. After incubation for 5 days in the presence of IL-2/IL-18, the number of cells in lower chambers was counted and the frequency of cells in the lower chambers was analyzed by flow cytometry. Data show mean ± SD (n = 4), **p<0.01.</p