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Exogenous addition of arachidonic acid to the culture media enhances the functionality of dendritic cells for their possible use in cancer immunotherapy.

Kumar J, Gurav R, Kale V, Limaye L - PLoS ONE (2014)

Bottom Line: For their successful use in the clinics, the propagation and functionality of DCs is crucial.We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality.Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA(+) DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Lab., National Centre for Cell Science, Ganeshkhind, Pune, India.

ABSTRACT
The development of dendritic cell based vaccines is a promising approach in cancer immunotherapy. For their successful use in the clinics, the propagation and functionality of DCs is crucial. We earlier established a two-step method for the large scale generation of DCs from umbilical cord blood derived MNCs/CD34(+) cells. This work aims at improving their functionality based on the following observations: in vitro generated DCs can be less efficient in migration and other functional activities due to lower eicosanoid levels. The production of eicosanoids from Arachidonic Acid (AA) can be hampered due to suppression of the enzyme phospholipase A2 by IL-4, an essential cytokine required for the differentiation of DCs. We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality. DCs were generated with and without AA. The two DC sets were compared by phenotypic analysis, morphology and functional assays like antigen uptake, MLR, CTL assay and in vitro and in vivo migration. Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA(+) DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2. AA(+) DCs also show a favorable Th1 cytokine profile than AA- DCs. Thus addition of AA to the culture media is skewing the DCs towards the secretion of more IL-12 and less of IL-10 along with the restoration of eicosanoids levels in a COX-2 mediated pathway thereby enhancing the functionality of these cells to be used as a potent cellular vaccine. Taken together, these findings will be helpful in the better contriving of DC based vaccines for cancer immunotherapy.

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Chemotaxis and CCR7 expression: (A) DCs suspended in upper chamber of well before migration.After 3 hours (B) No spontaneous migration was observed in the wells, where CCL-19 was not added. Less no. of AA- DCs (C) migrated towards CCL-19 than AA+ DCs (D). (E) AA+ DCs showed statistically efficient migration towards CCL-19 compared to AA- DCs (n = 3, mean ± SD). (F) FACS histogram profile of a representative experiment showing expression of chemokine receptor CCR-7 along with MFI values in brackets. Filled histograms show the isotype control and open ones show the specific phenotype. (G) Blocking with CCR-7 Ab causes significant reduction in migration of both AA+ DCs and AA- DCs (n = 3, mean ± SD). [P≤0.05(*), P≤0.01(**) & P≤0.001(***)].
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pone-0111759-g002: Chemotaxis and CCR7 expression: (A) DCs suspended in upper chamber of well before migration.After 3 hours (B) No spontaneous migration was observed in the wells, where CCL-19 was not added. Less no. of AA- DCs (C) migrated towards CCL-19 than AA+ DCs (D). (E) AA+ DCs showed statistically efficient migration towards CCL-19 compared to AA- DCs (n = 3, mean ± SD). (F) FACS histogram profile of a representative experiment showing expression of chemokine receptor CCR-7 along with MFI values in brackets. Filled histograms show the isotype control and open ones show the specific phenotype. (G) Blocking with CCR-7 Ab causes significant reduction in migration of both AA+ DCs and AA- DCs (n = 3, mean ± SD). [P≤0.05(*), P≤0.01(**) & P≤0.001(***)].

Mentions: Migration of DCs toward a chemokine gradient is an important functional property because in the immunotherapy protocols they must be capable of migration from the site of injection toward the lymph nodes, where they interact with the T cells and initiate the immune response. Fig. 2A shows, the cells suspended in IMDM added to the upper chamber in well at 0 hrs. No spontaneous migration was observed in the wells after 3 hrs, where CCL-19 was not added (Fig. 2B). More number of AA+ DCs (Fig. 2D) migrated towards CCL-19 as compared to AA- DCs (Fig. 2C) and the difference was statistically significant (*p≤0.05, **p≤0.01; n = 3; Fig. 2E). Migration towards CCL19 is mainly because the DCs express CCR7 receptor. When we stained the DCs of the two sets for the CCR-7 receptor antibody, the AA+ DCs and AA- DCs showed 93.25% and 91.89% positive cells respectively along with MFI values in brackets (Fig. 2F). Mean MFI values of 3 samples for AA- DCs were 821±156 and AA+ DCs were 1089±392. Thus both sets exhibited high and comparable level of CCR7 expression. The specificity of CCR7-CCL19 receptor ligand reaction was further confirmed by blocking the receptor with CCR-7 blocking Ab and then testing the migratory ability. As seen in Fig. 2G there was a significant reduction in migration of cells pretreated with blocking antibody in the two cultures. The migration of the test cells was once again significantly higher than control cells. This data indicated that, the addition of AA during differentiation step of DCs significantly improves their migratory ability.


Exogenous addition of arachidonic acid to the culture media enhances the functionality of dendritic cells for their possible use in cancer immunotherapy.

Kumar J, Gurav R, Kale V, Limaye L - PLoS ONE (2014)

Chemotaxis and CCR7 expression: (A) DCs suspended in upper chamber of well before migration.After 3 hours (B) No spontaneous migration was observed in the wells, where CCL-19 was not added. Less no. of AA- DCs (C) migrated towards CCL-19 than AA+ DCs (D). (E) AA+ DCs showed statistically efficient migration towards CCL-19 compared to AA- DCs (n = 3, mean ± SD). (F) FACS histogram profile of a representative experiment showing expression of chemokine receptor CCR-7 along with MFI values in brackets. Filled histograms show the isotype control and open ones show the specific phenotype. (G) Blocking with CCR-7 Ab causes significant reduction in migration of both AA+ DCs and AA- DCs (n = 3, mean ± SD). [P≤0.05(*), P≤0.01(**) & P≤0.001(***)].
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219773&req=5

pone-0111759-g002: Chemotaxis and CCR7 expression: (A) DCs suspended in upper chamber of well before migration.After 3 hours (B) No spontaneous migration was observed in the wells, where CCL-19 was not added. Less no. of AA- DCs (C) migrated towards CCL-19 than AA+ DCs (D). (E) AA+ DCs showed statistically efficient migration towards CCL-19 compared to AA- DCs (n = 3, mean ± SD). (F) FACS histogram profile of a representative experiment showing expression of chemokine receptor CCR-7 along with MFI values in brackets. Filled histograms show the isotype control and open ones show the specific phenotype. (G) Blocking with CCR-7 Ab causes significant reduction in migration of both AA+ DCs and AA- DCs (n = 3, mean ± SD). [P≤0.05(*), P≤0.01(**) & P≤0.001(***)].
Mentions: Migration of DCs toward a chemokine gradient is an important functional property because in the immunotherapy protocols they must be capable of migration from the site of injection toward the lymph nodes, where they interact with the T cells and initiate the immune response. Fig. 2A shows, the cells suspended in IMDM added to the upper chamber in well at 0 hrs. No spontaneous migration was observed in the wells after 3 hrs, where CCL-19 was not added (Fig. 2B). More number of AA+ DCs (Fig. 2D) migrated towards CCL-19 as compared to AA- DCs (Fig. 2C) and the difference was statistically significant (*p≤0.05, **p≤0.01; n = 3; Fig. 2E). Migration towards CCL19 is mainly because the DCs express CCR7 receptor. When we stained the DCs of the two sets for the CCR-7 receptor antibody, the AA+ DCs and AA- DCs showed 93.25% and 91.89% positive cells respectively along with MFI values in brackets (Fig. 2F). Mean MFI values of 3 samples for AA- DCs were 821±156 and AA+ DCs were 1089±392. Thus both sets exhibited high and comparable level of CCR7 expression. The specificity of CCR7-CCL19 receptor ligand reaction was further confirmed by blocking the receptor with CCR-7 blocking Ab and then testing the migratory ability. As seen in Fig. 2G there was a significant reduction in migration of cells pretreated with blocking antibody in the two cultures. The migration of the test cells was once again significantly higher than control cells. This data indicated that, the addition of AA during differentiation step of DCs significantly improves their migratory ability.

Bottom Line: For their successful use in the clinics, the propagation and functionality of DCs is crucial.We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality.Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA(+) DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Lab., National Centre for Cell Science, Ganeshkhind, Pune, India.

ABSTRACT
The development of dendritic cell based vaccines is a promising approach in cancer immunotherapy. For their successful use in the clinics, the propagation and functionality of DCs is crucial. We earlier established a two-step method for the large scale generation of DCs from umbilical cord blood derived MNCs/CD34(+) cells. This work aims at improving their functionality based on the following observations: in vitro generated DCs can be less efficient in migration and other functional activities due to lower eicosanoid levels. The production of eicosanoids from Arachidonic Acid (AA) can be hampered due to suppression of the enzyme phospholipase A2 by IL-4, an essential cytokine required for the differentiation of DCs. We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality. DCs were generated with and without AA. The two DC sets were compared by phenotypic analysis, morphology and functional assays like antigen uptake, MLR, CTL assay and in vitro and in vivo migration. Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA(+) DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2. AA(+) DCs also show a favorable Th1 cytokine profile than AA- DCs. Thus addition of AA to the culture media is skewing the DCs towards the secretion of more IL-12 and less of IL-10 along with the restoration of eicosanoids levels in a COX-2 mediated pathway thereby enhancing the functionality of these cells to be used as a potent cellular vaccine. Taken together, these findings will be helpful in the better contriving of DC based vaccines for cancer immunotherapy.

Show MeSH
Related in: MedlinePlus