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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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Cblb−/− BMDCs show increased allogeneic T cell proliferation but are not more effective in the induction of antigen-specific T cell responses and cross-presentation.(A) Increased allogeneic T cell proliferation induced by cblb−/− BMDCs. LPS matured wildtype or cblb−/− BMDCs were added in increasing numbers to 2×105 allogeneic T cells for two days. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 4), representative of 4 independent experiments is shown, *p<0.05 wildtype BMDC versus cblb−/− BMDCs (Student t-test). (B/C) No difference in antigen specific T cell proliferation between wildtype and cblb−/− BMDCs. Wildtype and cblb−/− BMDCs were matured with 100 ng/ml LPS and primed with (B) 10 µM SIINFEKL-peptide (OT-I) or (C) 10 µM ISQAVHAAHAEINEAGR–peptide (OT-II) on day seven of culture overnight. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific CD8+ (OT-I) or CD4+ (OT-II) transgenic T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 3), representative of at least 3 independent experiments is shown. (D) Unaltered antigen specific T cell priming of cblb−/− BMDCs in vivo. C57BL/6 recipient mice were injected intravenously with 5×106 CFSE-labeled transgenic CD8+ T cells. 24 hours thereafter 5×106 wildtype versus cblb−/− BMDCs matured with 100 ng/ml LPS and pulsed in vitro with 10 µM SIINFKEL peptide or solvent as control were injected subcutanously in the flank of the mouse. Representative FACS histogram of CFSE dilution in OT-I transgenic T cells in the draining lymph node after injection of SIINFEKL peptide loaded wildtype versus cblb−/− BMDCs in wildtype recipients is shown. Data represent % proliferated OT-I+ T cells (mean value ± SEM, one representative experiment is shown with n = 3 mice per group), G = Generation. (E/F) No difference between wildtype and cblb−/− BMDCs in cross-presentation of protein antigen. Wildtype and cblb−/− BMDCs were cultured from day seven to day eight with 1 mg/ml OVA-protein and 100 ng/mL LPS. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific (E) CD8+ (OT-I) or (F) CD4+ (OT-II) T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16 to 18 hours of culture was measured. Data are counts per minute (mean value ± SD, n = 2), representative of 2 independent experiments is shown.
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pone-0065178-g003: Cblb−/− BMDCs show increased allogeneic T cell proliferation but are not more effective in the induction of antigen-specific T cell responses and cross-presentation.(A) Increased allogeneic T cell proliferation induced by cblb−/− BMDCs. LPS matured wildtype or cblb−/− BMDCs were added in increasing numbers to 2×105 allogeneic T cells for two days. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 4), representative of 4 independent experiments is shown, *p<0.05 wildtype BMDC versus cblb−/− BMDCs (Student t-test). (B/C) No difference in antigen specific T cell proliferation between wildtype and cblb−/− BMDCs. Wildtype and cblb−/− BMDCs were matured with 100 ng/ml LPS and primed with (B) 10 µM SIINFEKL-peptide (OT-I) or (C) 10 µM ISQAVHAAHAEINEAGR–peptide (OT-II) on day seven of culture overnight. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific CD8+ (OT-I) or CD4+ (OT-II) transgenic T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 3), representative of at least 3 independent experiments is shown. (D) Unaltered antigen specific T cell priming of cblb−/− BMDCs in vivo. C57BL/6 recipient mice were injected intravenously with 5×106 CFSE-labeled transgenic CD8+ T cells. 24 hours thereafter 5×106 wildtype versus cblb−/− BMDCs matured with 100 ng/ml LPS and pulsed in vitro with 10 µM SIINFKEL peptide or solvent as control were injected subcutanously in the flank of the mouse. Representative FACS histogram of CFSE dilution in OT-I transgenic T cells in the draining lymph node after injection of SIINFEKL peptide loaded wildtype versus cblb−/− BMDCs in wildtype recipients is shown. Data represent % proliferated OT-I+ T cells (mean value ± SEM, one representative experiment is shown with n = 3 mice per group), G = Generation. (E/F) No difference between wildtype and cblb−/− BMDCs in cross-presentation of protein antigen. Wildtype and cblb−/− BMDCs were cultured from day seven to day eight with 1 mg/ml OVA-protein and 100 ng/mL LPS. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific (E) CD8+ (OT-I) or (F) CD4+ (OT-II) T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16 to 18 hours of culture was measured. Data are counts per minute (mean value ± SD, n = 2), representative of 2 independent experiments is shown.

Mentions: Due to the increased production of cytokines, it is tempting to speculate that the T cell stimulatory capacity of cblb−/− BMDCs is modulated. Indeed,cblb−/− BMDCs were able to induce stronger allogeneic T cell priming capacity compared to wildtype BMDCs. To demonstrate this, T cells were prepared from spleens of BALB/c mice and co-cultured with LPS-matured wildtype or cblb−/− BMDCs. Increasing numbers of wildtype and cblb−/− BMDCs were incubated with a fixed number of allogeneic T cells for 48 hours. Our results in Figure 3A depict that BMDCs of cblb−/− mice are significantly more active MLR stimulators compared to wildtype BMDCs. Figure 3A shows that approximately 300 cblb−/− BMDCs could already trigger substantial T cell responses. The difference in induced T cell proliferation between wildtype and cblb−/− BMDCs increased with graded doses of BMDCs (Figure 3A). The proliferation responses by T cells alone or BMDCs alone served as control and were always low and not distinguishable between the genotypes.


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)

Cblb−/− BMDCs show increased allogeneic T cell proliferation but are not more effective in the induction of antigen-specific T cell responses and cross-presentation.(A) Increased allogeneic T cell proliferation induced by cblb−/− BMDCs. LPS matured wildtype or cblb−/− BMDCs were added in increasing numbers to 2×105 allogeneic T cells for two days. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 4), representative of 4 independent experiments is shown, *p<0.05 wildtype BMDC versus cblb−/− BMDCs (Student t-test). (B/C) No difference in antigen specific T cell proliferation between wildtype and cblb−/− BMDCs. Wildtype and cblb−/− BMDCs were matured with 100 ng/ml LPS and primed with (B) 10 µM SIINFEKL-peptide (OT-I) or (C) 10 µM ISQAVHAAHAEINEAGR–peptide (OT-II) on day seven of culture overnight. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific CD8+ (OT-I) or CD4+ (OT-II) transgenic T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 3), representative of at least 3 independent experiments is shown. (D) Unaltered antigen specific T cell priming of cblb−/− BMDCs in vivo. C57BL/6 recipient mice were injected intravenously with 5×106 CFSE-labeled transgenic CD8+ T cells. 24 hours thereafter 5×106 wildtype versus cblb−/− BMDCs matured with 100 ng/ml LPS and pulsed in vitro with 10 µM SIINFKEL peptide or solvent as control were injected subcutanously in the flank of the mouse. Representative FACS histogram of CFSE dilution in OT-I transgenic T cells in the draining lymph node after injection of SIINFEKL peptide loaded wildtype versus cblb−/− BMDCs in wildtype recipients is shown. Data represent % proliferated OT-I+ T cells (mean value ± SEM, one representative experiment is shown with n = 3 mice per group), G = Generation. (E/F) No difference between wildtype and cblb−/− BMDCs in cross-presentation of protein antigen. Wildtype and cblb−/− BMDCs were cultured from day seven to day eight with 1 mg/ml OVA-protein and 100 ng/mL LPS. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific (E) CD8+ (OT-I) or (F) CD4+ (OT-II) T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16 to 18 hours of culture was measured. Data are counts per minute (mean value ± SD, n = 2), representative of 2 independent experiments is shown.
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getmorefigures.php?uid=PMC3675148&req=5

pone-0065178-g003: Cblb−/− BMDCs show increased allogeneic T cell proliferation but are not more effective in the induction of antigen-specific T cell responses and cross-presentation.(A) Increased allogeneic T cell proliferation induced by cblb−/− BMDCs. LPS matured wildtype or cblb−/− BMDCs were added in increasing numbers to 2×105 allogeneic T cells for two days. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 4), representative of 4 independent experiments is shown, *p<0.05 wildtype BMDC versus cblb−/− BMDCs (Student t-test). (B/C) No difference in antigen specific T cell proliferation between wildtype and cblb−/− BMDCs. Wildtype and cblb−/− BMDCs were matured with 100 ng/ml LPS and primed with (B) 10 µM SIINFEKL-peptide (OT-I) or (C) 10 µM ISQAVHAAHAEINEAGR–peptide (OT-II) on day seven of culture overnight. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific CD8+ (OT-I) or CD4+ (OT-II) transgenic T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16–18 hours of culture was measured. Data represent counts per minute (mean value ± SD, n = 3), representative of at least 3 independent experiments is shown. (D) Unaltered antigen specific T cell priming of cblb−/− BMDCs in vivo. C57BL/6 recipient mice were injected intravenously with 5×106 CFSE-labeled transgenic CD8+ T cells. 24 hours thereafter 5×106 wildtype versus cblb−/− BMDCs matured with 100 ng/ml LPS and pulsed in vitro with 10 µM SIINFKEL peptide or solvent as control were injected subcutanously in the flank of the mouse. Representative FACS histogram of CFSE dilution in OT-I transgenic T cells in the draining lymph node after injection of SIINFEKL peptide loaded wildtype versus cblb−/− BMDCs in wildtype recipients is shown. Data represent % proliferated OT-I+ T cells (mean value ± SEM, one representative experiment is shown with n = 3 mice per group), G = Generation. (E/F) No difference between wildtype and cblb−/− BMDCs in cross-presentation of protein antigen. Wildtype and cblb−/− BMDCs were cultured from day seven to day eight with 1 mg/ml OVA-protein and 100 ng/mL LPS. Graded doses of BMDCs were then co-cultured with 2×105 antigen-specific (E) CD8+ (OT-I) or (F) CD4+ (OT-II) T cells for 48 hours. Incorporation of 3H-Thymidine radioactivity during the last 16 to 18 hours of culture was measured. Data are counts per minute (mean value ± SD, n = 2), representative of 2 independent experiments is shown.
Mentions: Due to the increased production of cytokines, it is tempting to speculate that the T cell stimulatory capacity of cblb−/− BMDCs is modulated. Indeed,cblb−/− BMDCs were able to induce stronger allogeneic T cell priming capacity compared to wildtype BMDCs. To demonstrate this, T cells were prepared from spleens of BALB/c mice and co-cultured with LPS-matured wildtype or cblb−/− BMDCs. Increasing numbers of wildtype and cblb−/− BMDCs were incubated with a fixed number of allogeneic T cells for 48 hours. Our results in Figure 3A depict that BMDCs of cblb−/− mice are significantly more active MLR stimulators compared to wildtype BMDCs. Figure 3A shows that approximately 300 cblb−/− BMDCs could already trigger substantial T cell responses. The difference in induced T cell proliferation between wildtype and cblb−/− BMDCs increased with graded doses of BMDCs (Figure 3A). The proliferation responses by T cells alone or BMDCs alone served as control and were always low and not distinguishable between the genotypes.

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