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The role of the e3 ligase cbl-B in murine dendritic cells.

Wallner S, Lutz-Nicoladoni C, Tripp CH, Gastl G, Baier G, Penninger JM, Stoitzner P, Wolf D - PLoS ONE (2013)

Bottom Line: However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo.In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs.We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumor Immunology, Tyrolean Cancer Research Institute, Innsbruck, Austria.

ABSTRACT
Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/- BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8(+) OT-I or CD4(+) OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

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Cytokine and chemokine production by wildtype versus cblb−/− BMDs after stimulation with different TLR agonists.Wildtype and cblb−/− BMDCs were stimulated on day seven of culture with 1 µg/mL LPS or 100 nM CpG overnight for, IL-12p70, IL-10, IL-1α, IL-6, TNF-α, IFN-γ, KC, MIP-1α and MCP-1 measurement from cell culture supernatants were performed using Bioplex-Technology. Data represent mean value ± SEM of at least 4 independent experiments, *p<0.05 wildtype BMDCs versus cblb−/− BMDCs.
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pone-0065178-g002: Cytokine and chemokine production by wildtype versus cblb−/− BMDs after stimulation with different TLR agonists.Wildtype and cblb−/− BMDCs were stimulated on day seven of culture with 1 µg/mL LPS or 100 nM CpG overnight for, IL-12p70, IL-10, IL-1α, IL-6, TNF-α, IFN-γ, KC, MIP-1α and MCP-1 measurement from cell culture supernatants were performed using Bioplex-Technology. Data represent mean value ± SEM of at least 4 independent experiments, *p<0.05 wildtype BMDCs versus cblb−/− BMDCs.

Mentions: Production of cytokines and chemokines upon activation is a key feature of DC function [37], [38]. Therefore, we analyzed proinflammatory cytokine (IL-1α, IL-6, TNF-α, IL-10, IL-12p70, IFN-γ) and chemokine (KC, MCP-1, MIP-1α) secretion of cblb−/− BMDCs activated by different TLR agonists. Upon stimulation of wildtype and cblb−/− BMDCs by the TLR4 agonist LPS, significantly higher amounts of TNF-α, IL-6 and the chemokine MIP-1α could be detected in the supernatant of stimulated cblb−/− BMDCs (Figure 2). Only a slight and statistically not significant increase in IL-1α could be detected in LPS-stimulated cblb−/− BMDCs, whereas secretion of IFN-γ, IL-10, MCP-1 and CXCL1 (KC) was not affected by cblb-deficiency. Upon stimulation with the TLR-9 agonist CpG high amounts of the proinflammatory mediators IL-1α, TNF-α and the chemokine MCP-1 were detectable in cblb−/− versus wildtype BMDCs (Figure 2). In contrast, treating cells with the TLR3 agonist Poly(I:C) did not significantly alter their cytokine or chemokine secretion pattern (data not shown). Immature, non-stimulated BMDCs served as control and did not produce detectable amounts of cytokines (data not shown).


The role of the e3 ligase cbl-B in murine dendritic cells.

Wallner S, Lutz-Nicoladoni C, Tripp CH, Gastl G, Baier G, Penninger JM, Stoitzner P, Wolf D - PLoS ONE (2013)

Cytokine and chemokine production by wildtype versus cblb−/− BMDs after stimulation with different TLR agonists.Wildtype and cblb−/− BMDCs were stimulated on day seven of culture with 1 µg/mL LPS or 100 nM CpG overnight for, IL-12p70, IL-10, IL-1α, IL-6, TNF-α, IFN-γ, KC, MIP-1α and MCP-1 measurement from cell culture supernatants were performed using Bioplex-Technology. Data represent mean value ± SEM of at least 4 independent experiments, *p<0.05 wildtype BMDCs versus cblb−/− BMDCs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675148&req=5

pone-0065178-g002: Cytokine and chemokine production by wildtype versus cblb−/− BMDs after stimulation with different TLR agonists.Wildtype and cblb−/− BMDCs were stimulated on day seven of culture with 1 µg/mL LPS or 100 nM CpG overnight for, IL-12p70, IL-10, IL-1α, IL-6, TNF-α, IFN-γ, KC, MIP-1α and MCP-1 measurement from cell culture supernatants were performed using Bioplex-Technology. Data represent mean value ± SEM of at least 4 independent experiments, *p<0.05 wildtype BMDCs versus cblb−/− BMDCs.
Mentions: Production of cytokines and chemokines upon activation is a key feature of DC function [37], [38]. Therefore, we analyzed proinflammatory cytokine (IL-1α, IL-6, TNF-α, IL-10, IL-12p70, IFN-γ) and chemokine (KC, MCP-1, MIP-1α) secretion of cblb−/− BMDCs activated by different TLR agonists. Upon stimulation of wildtype and cblb−/− BMDCs by the TLR4 agonist LPS, significantly higher amounts of TNF-α, IL-6 and the chemokine MIP-1α could be detected in the supernatant of stimulated cblb−/− BMDCs (Figure 2). Only a slight and statistically not significant increase in IL-1α could be detected in LPS-stimulated cblb−/− BMDCs, whereas secretion of IFN-γ, IL-10, MCP-1 and CXCL1 (KC) was not affected by cblb-deficiency. Upon stimulation with the TLR-9 agonist CpG high amounts of the proinflammatory mediators IL-1α, TNF-α and the chemokine MCP-1 were detectable in cblb−/− versus wildtype BMDCs (Figure 2). In contrast, treating cells with the TLR3 agonist Poly(I:C) did not significantly alter their cytokine or chemokine secretion pattern (data not shown). Immature, non-stimulated BMDCs served as control and did not produce detectable amounts of cytokines (data not shown).

Bottom Line: However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo.In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs.We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumor Immunology, Tyrolean Cancer Research Institute, Innsbruck, Austria.

ABSTRACT
Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/- BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8(+) OT-I or CD4(+) OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

Show MeSH
Related in: MedlinePlus