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. 2015 Jun 3:13:175.
doi: 10.1186/s12967-015-0528-7.

Different maturation cocktails provide dendritic cells with different chemoattractive properties

Chiara Massa  1 , Carolin Thomas  2 , Ena Wang  3   4 , Francesco Marincola  5   6 , Barbara Seliger  7
Affiliations

VSports手机版 - Different maturation cocktails provide dendritic cells with different chemoattractive properties

Chiara Massa et al. J Transl Med. .

Abstract

Background: Dendritic cells (DC) are currently implemented as immunotherapeutic strategy for the treatment of tumor patients based on their central role in the immune system. Despite good results were obtained in vitro and in animal models, their clinical use has provided limited success suggesting the requirement to optimise the protocol for their production VSports手机版. .

Methods: A cDNA array was performed on FastDC obtained from the differentiation of human peripheral blood monocytes stimulated with the clinical gold standard or with two alternative maturation cocktails combining interferon (IFN)γ and ligands for different toll like receptors (TLR). V体育安卓版.

Results: A stronger modulation of the DC transcriptome with respect to immature DC was found in alternatively stimulated DC when compared to DC stimulated with the clinical gold standard. A major class of molecules differentially expressed using distinct DC stimulation protocols were chemokines V体育ios版. Validation of their differential expression pattern at the mRNA and protein level confirmed the secretion of inflammatory chemokines by the alternative DC. Functional analyses of the chemotactic properties of DC "wash out" supernatants highlighted the ability of alternative, but not of gold standard DC to efficiently recruit immune cells with a prevalence of monocytes. Effector cells belonging to the innate as well as adaptive immunity were also attracted and the interaction with alternative DC resulted in enhanced secretion of IFNγ and induction of cytotoxic activity. Using leukocytes from cancer patients, it was demonstrated that the monocyte-attracting activity targeted cells with an inflammatory phenotype characterised by high levels of HLA-DR expression. .

Conclusions: Despite other classes of immune modulatory genes differently expressed in the alternative DC require to be investigated and characterised regarding their functional consequences, the reduced maturation state and chemoattractive properties of the gold standard versus alternative DC clearly promote the necessity to change the clinically used maturation cocktail of DC in order to improve the outcome of patients treated with DC-based vaccines. VSports最新版本.

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Figures

Figure 1
Figure 1
Enhanced migration of healthy donor PBMC toward conditioned supernatants from alternative DC. “Wash out” supernatants collected 24 h after seeding mature DC in the absence of exogenous stimuli were used as a source of chemoattractants for immune cells. Total PBMC from healthy donors were loaded in a trans-well insert (3 μm pores) and allowed to migrate toward medium or DC “wash out” supernatants for 3 h. Migrated cells were analysed by flow cytometry to determine their total number (a) as well as their distribution into the different immune cell subpopulations (b) by using the indicated gating strategy (c). Data from seven different experiments are reported as mean ± SE after normalization to supernatants from gold standard DC. *p < 0.05 in the Tukey post hoc test.
Figure 2
Figure 2
Effects of DC conditioned supernatants on PBMC from cancer patients. Total PBMC from melanoma or RCC patients were added into trans-well insert (3 μm pores) and allowed to migrate toward DC “wash out” supernatants for 5 h. Migrated cells were evaluated by flow cytometry to determine absolute number and immune composition. a Mean ± SE of the x-fold increase in the migration of the different immune populations over gold standard DC-derived supernatants from 4 to 6 different experiments is shown. b x-fold increase in the HLA-DR MFI of migrated CD14+ monocytes versus the original population. c Gating strategy (insert) and percentage of CD25high CD127low Treg of the original PBMC and among migrated cells shown as percentage of CD4+ T cells or of total leukocytes. *p < 0.05 in the Tukey post hoc test.
Figure 3
Figure 3
Effect of different maturation cocktails on DC transcriptome. Immature DC were compared to cells stimulated with gold standard, alt-1 and alt-2 maturation cocktails using cDNA array. Three different donors were used for the evaluation. The heat map of genes differentially regulated among the four DC types (a) or just the two alternative DC (c) are shown. b Distribution of the differentially expressed genes in the canonical pathways is shown for each mature DC (gold standard DC, top; alt-1 DC, middle; alt-2 DC, bottom) versus the immature DC. The numbers above the bars indicate the number of genes belonging to the different pathways, whereas the yellow line indicates the -Log10 of the p value of the modulation for each family.
Figure 4
Figure 4
qPCR evaluation of chemokine gene expression in differently matured DC. The expression levels of chemokine genes in immature DC as well as in DC stimulated with gold standard, alt-1, alt-2 and alt-3 maturation cocktails were evaluated by qPCR. Results were first normalised to the mean expression of two housekeeping genes (GAPDH and HPRT1) and then toward the gold standard DC. The results are represented as the mean ± SE of 9–12 different donors. *p < 0.05, ns p > 0.05 in the Tukey post hoc test.
Figure 5
Figure 5
Evaluation of chemokine production at the protein level in differently matured DC. a “Wash out” supernatants collected 24 h after seeding mature DC in the absence of exogenous stimuli were used to evaluate chemokine secretion by sandwich ELISA or flow cytometry based plexing. The results are represented as the mean ± SE of 4–7 different donors. b The presence of transmembrane (TM)-CXCL16 on mature DC was evaluated by flow cytometry. The results are shown as mean ± SE of the x-fold increase in MFI over immature DC determined from ten different donors. *p < 0.05 in the Tukey post hoc test.
Figure 6
Figure 6
qPCR evaluation of chemokine gene expression in 24 h “wash out” DC. Mature DC were seeded for 24 h in the absence of exogenous stimuli and then collected for mRNA extraction and qPCR evaluation. Results were first normalised to the mean expression of two housekeeping genes (GAPDH and HPRT1) and then toward the gold standard DC. The results are represented as the mean ± SE of three different donors. *p < 0.05 in the Tukey post hoc test.
Figure 7
Figure 7
Expression and functionality of CCR7 by differently matured DC. a Mature DC were evaluated by flow cytometry for the expression of CCR7. The x-fold increase in MFI over immature DC is show as mean ± SE of 13 different donors. b The ability of mature DC to migrate toward the CCR7 ligands CCL21 was evaluated in a trans-well migration assay. After 3 h incubation at 37°C, DC migrated in the lower chamber were counted by flow cytometry. The results of three experiments are represented as mean ± SE upon normalization to the gold standard DC. *p < 0.05 in the Tukey post hoc test.
Figure 8
Figure 8
Determination of chemokines involved in monocyte chemoattraction. a The expression of different members of the monocyte chemoattractant protein family was evaluated by qPCR. Results are first normalised to the mean expression of two housekeeping genes (GAPDH and HPRT1) and then toward gold standard DC. The relative expression of nine different donor is represented as mean ± SE. *p < 0.05, ns p > 0.05 in the Tukey post hoc test. b The CCL2 and CCL8 concentration of “wash out” supernatants from mature DC seeded in the absence of exogenous stimuli for 24 h were determined by sandwich ELISA. The results are represented as mean ± SE of 8–12 different donors. *p < 0.05, ns p > 0.05 in the Tukey post hoc test.
Figure 9
Figure 9
Functional activation of lymphocytes migrated to alternatively matured DC. a, b 5 × 105 mature DC were seeded in 12 well plate for 4 h, after which 3 × 106 autologous PBMC were added above a 3 μm insert. After 18 h incubation the cells that had migrated into the bottom chamber were taken and either immediately evaluated for IFNγ secretion (a) or incubated for 4 h in the presence of Daudi cells and evaluated for CD107a degranulation (b). c 5 × 105 mature DC were pulsed with SEB for 30 min at 37°C, washed and then seeded in 12 well plate for 4 h, after which 3 × 106 autologous PBMC were added above a 3 μm trans-well insert. After 4 h incubation the cells in the bottom chamber were taken and evaluated for IFNγ secretion. Shown is one representative experiment out of two (c) or three (a, b) with similar results.
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