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

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.

Figure 1. Cbl-b is highly expressed in BMDCs and does not affect DC differentiation.

(A) Cbl-b Protein expression of in vitro generated 85–95% pure immature BMDCs determined by Western blotting. (B) Surface marker expression of wildtype and cblb−/− BMDCs was analyzed by multi-colour flow cytometry. The overlay histograms depict the expression of wildtype versus cblb−/− BMDC surface markers of immature cells after seven days of culture and LPS matured cells after eight days of culture. Representative FACS plots, n = 6 mice per group. The graphs in (C) depict the percentage of CD11c⁺ MHC-II⁺ and surface marker positive BMDCs either immature cells at day seven of culture or LPS matured cells at day eight of culture. Data represent mean value ± SEM; n = 6, **p<0.005.

Figure 2. 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.

Figure 3. 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.

Figure 4. Macropinocytosis is not altered by cblb−/− deficiency.

(A) Macropinocytosis was quantified by incubating immature day seven BMDCs with 2 mg/ml FITC-dextran at 37°C or at 4°C (negative control). After 2 and 4 hours, uptake was stopped and analyzed by FACS. Data represent mean value ± SEM; n = 4. (B) Representative dot plot of 4 independent experiments is shown.

Figure 5. 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.