The purpose of this study was to evaluate the effect of rapamycin delivery by poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles on the maturation of dendritic cells (DCs). DCs were generated from mouse bone marrow and exposed to particulate and soluble rapamycin without any additional treatment, or with pre- or posttreatment with lipopolysaccharide (LPS). Annexin V-FITC/PI staining was performed on DC cultures to assess the viability of DCs during study. Surface phenotype of DCs was characterized for the expression of maturation markers, that is, MHC class II, CD86, and CD40 by flow cytometry. Cell culture supernatants were analyzed for the production of TGF-beta, IL-12, and IL-10 cytokines using sandwich ELISA method. DCs from Balb/C mice were cocultured with T cells from C57BL/6 mice and allogenic mixed lymphocyte reaction was assessed by [3H]-Thymidine incorporation. Unlike free rapamycin that has shown little if any effect on the expression of maturation markers in immature DCs, PLGA encapsulated rapamycin decreased the expression of all maturation markers under the study, that is, MHC class II, CD86, and CD40, significantly. LPS pre- or posttreated DCs demonstrated decreased expression of MHC class II, CD86, and CD40 in the presence of soluble or encapsulated rapamycin. The cytokine secretion profiles revealed high levels of TGF-beta and very low levels of IL-10 and IL-12 production. Rapamycin in soluble or encapsulated form significantly inhibited mixed lymphocyte reaction in DCs. The inhibitory effect of rapamycin on the maturation of DCs with respect to DC phenotype, cytokine production, and functional effects on the proliferation of T cells was significantly increased by PLGA delivery.
Immune responses of dendritic cells (DCs) can be modulated by delivery of adjuvants to alter their maturation profile. The purpose of this study was to generate DCs from CD34(+) cells of human cord blood and characterize the effects of poly(D,L-lactic-co-glycolic acid) (PLGA)-nanoparticle encapsulated rapamycin in generating an immunosuppressive DC. Expression of ICAM-1 (intercellular adhesion molecule), a key molecule in DC-T cell interaction was increased in mature DCs in response to lipopolysaccharide (LPS). When rapamycin was encapsulated in the nanoparticle to maintain DCs in the immature state, ICAM-1 expression was down regulated. When delivered in the free form, rapamycin did not alter the expression of ICAM-1. Cytokine arrays exhibited an immunosuppressive profile of various cytokines in response to the nanoparticulate delivery of rapamycin. In addition, RT-PCR data demonstrated the presence of toll like receptor (TLR) 9 transcripts, although our DCs are myeloid in nature. In summary, our study demonstrates that DCs may be rendered immunosuppressive upon delivery of rapamycin-containing nanoparticles.
PURPOSE: Tumors can escape immune eradication by harnessing dendritic cell (DC) maturation. However, DC types used as in vitro models to study tumor-mediated immunosuppression possess fundamental variability that could influence research outcomes. Therefore, we assessed the behavior of two distinct murine DC models upon exposure to tumor-conditioned medium of B16.F10 melanoma (B16-CM). METHODS: Using primary bone-marrow derived dendritic cells (BMDCs) or immortalized DC2.4 cell line, we evaluated the level of signal transducer and activator of transcription 3 (STAT3) phosphorylation by Western blot as a molecular parameter. We also examined the surface expression of co-stimulatory molecules on DCs by flow cytometry as a phenotypic parameter. RESULTS: Our results revealed critical discrepancies between the two models in response to tumor-conditioned medium. While conditioned medium was able to induce STAT3 phosphorylation in BMDCs, it did not significantly induce STAT3 phosphorylation in DC2.4 cell line. Moreover, only in BMDCs, the expression of CD86 and CD40 was remarkably downregulated by B16-CM and was not totally recovered after LPS stimulation. In contrast, DC2.4 cells did not show any signs of harnessed maturation upon exposure to B16-CM. CONCLUSIONS: In order to study the effect of tumor-mediated immunosuppression on DC maturation in vitro via tumor-induction of STAT3 activation, primary BMDCs are more reliable as a model than DC2.4.
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