ELISA measurement of IL-15Rα (A) and IL-15 (B) proteins and IL-15–IL-15Rα complex (D) expression in lysates of poly I:C–stimulated BMDCs at the indicated time points

(C) ELISA determination of IL-15 protein levels after dissociation of IL-15–IL-15Rα protein complexes with 0.01% SDS and boiling. Lines represent data from WT (▪), (▴), and (▾) BMDCs. All data are representative of at least three separate experiments.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

ELISA determination of sIL-15Rα, IL-15, and IL-15–sIL-15Rα proteins in sera from intact (A–C, on left) or chimeric (D–F, on right) mice stimulated with 25 μg/g poly I:C or PBS

Sera were collected 24 h after stimulation and IL-15 (B and E), sIL-15Rα (A and D), and IL-15–sIL-15Rα complex (C and F) protein levels were measured by ELISA. Each symbol reflects values obtained from individual mice.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

Activation of IFN-γ secretion by NK cells co-cultured with various combinations of poly I:C–stimulated BMDCs and supernatants from these BMDCs

BMDCs from various genotypes (indicated along the x axis of graph) were activated with poly I:C and then supplemented with supernatants exchanged from similarly activated BMDCs. Genotypes of DCs from which supernatants were derived are indicated by individual columns (gray, WT; white, 15KO; black, RαKO; checkered, mixture of 15KO and RαKO). ELISA quantitation of NK cell IFN-γ secretion is indicated on the y axis. Note that WT DCs activate NK cells regardless of the type of supernatant added. Note also that WT DC–derived supernatants containing IL-15–sIL-15Rα complexes fail to support or augment NK cell activation.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

(A) ELISA measurement of IL-12 secretion by poly I:C–stimulated BMDCs of the indicated genotypes

(B) Flow cytometric analysis of DC activation after poly I:C stimulation. Histograms of poly I:C–stimulated DCs (gray-filled histograms) of the indicated genotypes indicating surface expression levels of the surface activation markers CD40 and CD86. (C) ELISA determination of IFN-γ secretion by NK cells co-cultured with WT, 15KO, RαKO, or mixed 15KO:RαKO BMDCs. BMDCs were treated with PBS or poly I:C for 18 h, after which NK cells were co-cultured with activated DCs for an additional 6 h. Poly I:C–stimulated cultures are indicated by black bars, and PBS-stimulated control cultures are indicated by white bars. Note that significant poly I:C–induced NK cell activation occurs only in the presence of WT DCs. (D and E) Flow cytometric measurement of NK cell (NK1.1) activation by poly I:C (p(I:C)) –stimulated DC–NK cell co-cultures. Elevated CD69 surface staining reflects initial (TLR-dependent, IL-15–independent) activation of NK cells. IFN-γ and granzyme B expression by NK cells were performed by intracellular staining 6 and 12 h after stimulation, respectively. Numbers indicate the percentage of cells in the indicated gate. Note that NK cells express significant levels of both IFN-γ and granzyme B only when activated by WT DCs. Plots are representative of three separate experiments.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

Up-regulation of E-cadherin expression by rhIL-15 on RPTEC is dependent of the βc-dependent signaling pathway.

<p>The γc neutralization, as well as JAK3 or STAT5 inhibition, hamper the maintenance of E-cadherin surface expression induced by rhIL-15 on RPTEC without interfering on the E-cadherin down-regulation on rhIL-15-treated RCC7. Cells were pretreated with 1 µg/ml of neutralizing anti-IL2Rγ antibody mAb2842, 0.25 µM of JAK3 inhibitor (CP-690, 550, Calbiochem) or 100 µM of STAT5 inhibitor (STAT5 Inh., 573108, Calbiochem) for 1 h before adding the recombinant cytokine (10 pg/mL) for 5 days. Treatment with rhIL-15 and STAT5 inhibitor was renewed at day 3. White histograms refer to isotype-matched control. Mean fluorescence intensity values for each marker are shown in each histogram. The data are representative of 3 separate experiments performed using different RCC (RCC5, RCC8) and RPTEC batches.</p

Immunohistochemical staining for IL-2Rβ, IL-2Rγ and JAK3 in normal and neoplastic kidney specimens.

<p>Hematoxylin staining of biopsies from 2 normal samples reveals the presence of the normal kidney structures (glomerulus (Gl), distal (Dt) and proximal (Pt) tubules), while analysis of two different cancer specimens shows that the normal tissue architecture is totally lost and are replaced by tumor cells with clear cell morphology, characterized by optically clear cytoplasm and sharply outlined cell membrane. Whereas no difference on IL-2Rβ is observed between normal and tumoral tissue samples, the IL-2Rγ staining, localized to both proximal and distal tubuli in normal tissue samples, is not found in the tumor samples. A strong JAK3 staining is localized to both proximal and distal tubular cells of normal tissues, while a very faint JAK3 protein expression is detected in tumor samples. Two representative samples of a total of ten are shown for each staining. Negative control was subjected to all treatments omitting primary antibody. Scale bars, 50 µm. Staining was quantified using a morphometric TRIBVN software (Montrouge, France) and results are presented as histograms.</p

Normal and tumoral renal epithelial cells express different IL-15R subtypes.

<p>Analysis of IL-15R and JAK3 expression was performed by RT-PCR (<b>A</b>) and immunoblotting (<b>B</b>) on primary normal (RPTEC) and tumoral (RCC5, RCC7, RCC8) epithelial cells and the ACHN cell line. Data show that RPTEC express the three chains of the IL-15R (αβγ) and JAK3 whereas γc and JAK3 proteins were not detected in RCC. Specific primers or Abs against IL-15Rα (AF247), IL-2Rβ (sc-1046), IL-2Rγ (sc-670) and JAK3 (sc-513) were used. PBL, TF1β, MCF7 and IFNγ-activated U937 cells were used as controls. Housekeeping β-actin was used as loading control.</p

Soluble IL-15, at physiologic concentration, differently controls E-cadherin expression in RCC and RPTEC.

<p>Immunofluorescence analysis (<b>A</b>) and immunoblot (<b>B</b>) show that 5 days rhIL-15 treatment (10 pg/mL) preserves membrane E-cadherin expression on primary normal epithelial cells RPTEC, whereas it induces its down-regulation on RCC7. The medium culture of RPTEC was not changed in order to induce the decrease of E-cadherin expression. Treatment with rhIL-15 was renewed at day 3. Histograms represent densitometry comparison of E-cadherin immunoblots normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) cells and 3 RPTEC batches. * P<0.05.</p

Reestablishment of IL-2Rγ chain-dependent signal transduction pathway in RCC interferes with the rhIL-15-induced E-cadherin down-regulation.

<p>RCC7 were transiently transfected for 48 hours with vectors containing IL-2Rγ and/or JAK3 Human cDNA. <b>A</b>) Transient expression of IL-2Rγ and JAK3 was analyzed by immunoblotting in each transfected RCC. Immunoblotting for β-actin was used as a control for equal protein loading and transfer. <b>B</b>) Flow cytometry shows that 40 min rhIL-15 treatment did not induce STAT5 phosphorylation in IL-2Rγ- or JAK3-transfected RCC while rhIL-15 treatment induced STAT5 phosphorylation in co-transfected cells. <b>C</b>) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating E-cadherin expression by flow cytometry. The introduction of either IL-2Rγ chain, JAK3 or both molecules do not modify E-cadherin expression on untreated rhIL-15 cells, while the E-cadherin down-regulation observed after 48 hours of rhIL-15 treatment was counterbalanced only in co-transfected cells. Mean fluorescence intensity values for each marker are shown in each histogram. One experiment representative of a total of three is shown.</p

E-cadherin modulation by soluble IL-15 controls epithelial-mesenchymal transition on renal epithelial cells.

<p><b>A</b>) Immunofluorescence of cell–cell adhesion molecules show that IL-15 favors epithelial-mesenchymal transition (EMT) on RCC7, whereas it preserves the EMT commitment of RPTEC. The medium culture of RPTEC was not changed in order to induce the EMT process. Cells stimulated or not with 10 pg/ml of rhIL-15 for 5 days, were fixed and stained using standard immunofluorescence procedures with Abs against epithelial (cytokeratins and ZO-1) and mesenchymal markers (F-actin, ASO2 and vimentin). Similar results were obtained using different RCC (RCC5, RCC8) and RPTEC batches. <b>B</b>) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating the epithelial (cytokeratins) and mesenchymal (vimentin) markers expression by flow cytometry. RhIL-15 induced EMT was counterbalanced only in IL-2Rγ/JAK3 co-transfected RCC. Mean fluorescence intensity values for each marker are shown in each histogram. Results are representative of three experiments.</p