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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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Migration capacity and therapeutic potential as tumor vaccine of cblb−/− BMDCs.(A) 0.125 mg of TB in FIA was injected in a total volume of 50 µl per hock into wildtype recipients. On day two CFSE labeled wildtype and TAMRA labeled cblb−/− BMDCs were injected in the hock in wildtype control recipients and TB injected recipients. 24 hours thereafter migration of the BMDCs in the draining lymph node and non-draining lymph node (not shown) was measured by flow cytometry. Data represent mean value ± SEM, n = 4 mice per group, two independent experiments. (B) 1×105 B16-OVA cells were injected subcutaneously into the left flank of wildtype recipients. Tumor-bearing mice were subcutaneously vaccinated into the opposite right flank on day five with 2×105 10 µM SIINFEKL primed semi-mature wildtype versus cblb−/− BMDCs. Tumor growth was monitored thereafter every two/three days. Control animals received PBS. All data points represent tumor volume (mean value ± SEM, n = 4 mice), representative of two independent experiments is shown. (C) Survival of the same animals described in (B) was monitored.
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pone-0065178-g005: Migration capacity and therapeutic potential as tumor vaccine of cblb−/− BMDCs.(A) 0.125 mg of TB in FIA was injected in a total volume of 50 µl per hock into wildtype recipients. On day two CFSE labeled wildtype and TAMRA labeled cblb−/− BMDCs were injected in the hock in wildtype control recipients and TB injected recipients. 24 hours thereafter migration of the BMDCs in the draining lymph node and non-draining lymph node (not shown) was measured by flow cytometry. Data represent mean value ± SEM, n = 4 mice per group, two independent experiments. (B) 1×105 B16-OVA cells were injected subcutaneously into the left flank of wildtype recipients. Tumor-bearing mice were subcutaneously vaccinated into the opposite right flank on day five with 2×105 10 µM SIINFEKL primed semi-mature wildtype versus cblb−/− BMDCs. Tumor growth was monitored thereafter every two/three days. Control animals received PBS. All data points represent tumor volume (mean value ± SEM, n = 4 mice), representative of two independent experiments is shown. (C) Survival of the same animals described in (B) was monitored.

Mentions: Using an in vivo migration assay we investigated whether the absence of Cbl-b could influence the migration potential of BMDCs. Therefore, we induced peripheral inflammation by the injection of TB-FIA into the hock of mice and subsequently measured migration of differentially labeled wildtype and cblb−/− BMDCs to the draining lymph node. CFSE labeled wildtype and TAMRA labeled cblb−/− BMDCs were injected into the hock two days after induction of inflammation. 24 hours thereafter, migration of the dye-labeled BMDCs in the draining lymph node (Figure 5A) and non-draining lymph node as control was measured by flow cytometry. In all experiments, dye-labeled BMDCs were clearly detectable. BMDCs migrate best into the local draining (popliteal) lymph node and were not detectable in the inguinal lymph node. In solvent injected control mice the transferred BMDCs failed to migrate into the draining lymph node regardless of genotype. This migration assay revealed that both, cblb−/− and wildtype BMDCs were able to migrate properly to local draining lymph nodes (Figure 5A).


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)

Migration capacity and therapeutic potential as tumor vaccine of cblb−/− BMDCs.(A) 0.125 mg of TB in FIA was injected in a total volume of 50 µl per hock into wildtype recipients. On day two CFSE labeled wildtype and TAMRA labeled cblb−/− BMDCs were injected in the hock in wildtype control recipients and TB injected recipients. 24 hours thereafter migration of the BMDCs in the draining lymph node and non-draining lymph node (not shown) was measured by flow cytometry. Data represent mean value ± SEM, n = 4 mice per group, two independent experiments. (B) 1×105 B16-OVA cells were injected subcutaneously into the left flank of wildtype recipients. Tumor-bearing mice were subcutaneously vaccinated into the opposite right flank on day five with 2×105 10 µM SIINFEKL primed semi-mature wildtype versus cblb−/− BMDCs. Tumor growth was monitored thereafter every two/three days. Control animals received PBS. All data points represent tumor volume (mean value ± SEM, n = 4 mice), representative of two independent experiments is shown. (C) Survival of the same animals described in (B) was monitored.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065178-g005: Migration capacity and therapeutic potential as tumor vaccine of cblb−/− BMDCs.(A) 0.125 mg of TB in FIA was injected in a total volume of 50 µl per hock into wildtype recipients. On day two CFSE labeled wildtype and TAMRA labeled cblb−/− BMDCs were injected in the hock in wildtype control recipients and TB injected recipients. 24 hours thereafter migration of the BMDCs in the draining lymph node and non-draining lymph node (not shown) was measured by flow cytometry. Data represent mean value ± SEM, n = 4 mice per group, two independent experiments. (B) 1×105 B16-OVA cells were injected subcutaneously into the left flank of wildtype recipients. Tumor-bearing mice were subcutaneously vaccinated into the opposite right flank on day five with 2×105 10 µM SIINFEKL primed semi-mature wildtype versus cblb−/− BMDCs. Tumor growth was monitored thereafter every two/three days. Control animals received PBS. All data points represent tumor volume (mean value ± SEM, n = 4 mice), representative of two independent experiments is shown. (C) Survival of the same animals described in (B) was monitored.
Mentions: Using an in vivo migration assay we investigated whether the absence of Cbl-b could influence the migration potential of BMDCs. Therefore, we induced peripheral inflammation by the injection of TB-FIA into the hock of mice and subsequently measured migration of differentially labeled wildtype and cblb−/− BMDCs to the draining lymph node. CFSE labeled wildtype and TAMRA labeled cblb−/− BMDCs were injected into the hock two days after induction of inflammation. 24 hours thereafter, migration of the dye-labeled BMDCs in the draining lymph node (Figure 5A) and non-draining lymph node as control was measured by flow cytometry. In all experiments, dye-labeled BMDCs were clearly detectable. BMDCs migrate best into the local draining (popliteal) lymph node and were not detectable in the inguinal lymph node. In solvent injected control mice the transferred BMDCs failed to migrate into the draining lymph node regardless of genotype. This migration assay revealed that both, cblb−/− and wildtype BMDCs were able to migrate properly to local draining lymph nodes (Figure 5A).

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