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

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

Treatment of hairy-cell leukemia with recombinant alpha2-interferon

Eleven patients with hairy-cell leukemia (eight with progressive and three with non-progressive disease) were treated with low dose recombinant human alpha2-interferon. After a 3-month treatment period, nine patients showed an improvement and one patient a partial remission. By then, transfusions were not required any more and serious infections were no longer encountered. Four patients were further treated: three for a total period of 9 months and one for 6 months; all of them reached a partial or complete remission. The treatment was equally effective in patients with both progressive and non-progressive disease. Previous absence of response to splenectomy did not preclude a positive effect of IFN therapy. In two patients, IFN dose reduction was necessary due to unremitting flu-like symptoms. © 1986.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Macropinocytosis is not altered by <i>cblb</i>−/− deficiency.

<p>(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.</p

Migration capacity and therapeutic potential as tumor vaccine of <i>cblb</i>−/− BMDCs.

<p>(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 <i>cblb</i>−/− 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 <i>versus cblb</i>−/− 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.</p

Cytokine and chemokine production by wildtype <i>versus cblb</i>−/− BMDs after stimulation with different TLR agonists.

<p>Wildtype and <i>cblb</i>−/− 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 <i>versus cblb</i>−/− BMDCs.</p

GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal–related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib