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Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8(+) T lymphocytes.

von Euw EM, Barrio MM, Furman D, Bianchini M, Levy EM, Yee C, Li Y, Wainstok R, Mordoh J - J Transl Med (2007)

Bottom Line: DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta.Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fundación Instituto Leloir, Patricias Argentinas 435 (1405), Buenos Aires, Argentina. evoneuw@leloir.org.ar

ABSTRACT

Background: In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells).

Methods: We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.

Results: Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.

Conclusion: We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.

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CCR7 expression and in vitro DCs migration in response to MIP-1α and MIP-3β. CCR7 expression (left panel) was analyzed by FACS in iDCs (A), LPS-treated DCs (B) and DC/Apo-Nec (C). Grey histograms represent the corresponding isotype matched control Ab. In each histogram the percentage of positive cells and the Mean are indicated. In vitro migration in response to MIP-1α and MIP-3β was evaluated (right panel) for iDC, LPS-treated DCs and DC/Apo-Nec cells as indicated. A control for random migration was done using RPMI medium. The number of migrating cells was calculated as the mean ± SD of cells counts in five medium-power (40×) fields/well performed in triplicate. A representative experiment of three is shown *p < 0.05 (Student's t-Test).
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Figure 5: CCR7 expression and in vitro DCs migration in response to MIP-1α and MIP-3β. CCR7 expression (left panel) was analyzed by FACS in iDCs (A), LPS-treated DCs (B) and DC/Apo-Nec (C). Grey histograms represent the corresponding isotype matched control Ab. In each histogram the percentage of positive cells and the Mean are indicated. In vitro migration in response to MIP-1α and MIP-3β was evaluated (right panel) for iDC, LPS-treated DCs and DC/Apo-Nec cells as indicated. A control for random migration was done using RPMI medium. The number of migrating cells was calculated as the mean ± SD of cells counts in five medium-power (40×) fields/well performed in triplicate. A representative experiment of three is shown *p < 0.05 (Student's t-Test).

Mentions: DCs migration to lymph nodes is crucial to trigger T lymphocyte priming. MIP-3β chemokine drives the homing of mDCs to the lymph node and interacts specifically with its receptor CCR7 expressed on the cell surface. iDCs expressed low levels of CCR7 (7%, Mean: 9) but increased its expression after Apo-Nec phagocytosis (90%; Mean: 18) or LPS maturation (35%, Mean: 13) (Figure 5). We observed that iDC migrated to MIP-1α but did not respond to MIP-3β (Figure 5A); on the contrary DC/Apo-Nec and DC + LPS migrated to MIP-3β but failed to respond to MIP-1α (Figure 5B and 5C). Thus, DC/Apo-Nec cells express MIP-3β receptor CCR7 and are able to migrate in response to MIP-3β, potentially allowing their homing to lymph nodes.


Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8(+) T lymphocytes.

von Euw EM, Barrio MM, Furman D, Bianchini M, Levy EM, Yee C, Li Y, Wainstok R, Mordoh J - J Transl Med (2007)

CCR7 expression and in vitro DCs migration in response to MIP-1α and MIP-3β. CCR7 expression (left panel) was analyzed by FACS in iDCs (A), LPS-treated DCs (B) and DC/Apo-Nec (C). Grey histograms represent the corresponding isotype matched control Ab. In each histogram the percentage of positive cells and the Mean are indicated. In vitro migration in response to MIP-1α and MIP-3β was evaluated (right panel) for iDC, LPS-treated DCs and DC/Apo-Nec cells as indicated. A control for random migration was done using RPMI medium. The number of migrating cells was calculated as the mean ± SD of cells counts in five medium-power (40×) fields/well performed in triplicate. A representative experiment of three is shown *p < 0.05 (Student's t-Test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1863425&req=5

Figure 5: CCR7 expression and in vitro DCs migration in response to MIP-1α and MIP-3β. CCR7 expression (left panel) was analyzed by FACS in iDCs (A), LPS-treated DCs (B) and DC/Apo-Nec (C). Grey histograms represent the corresponding isotype matched control Ab. In each histogram the percentage of positive cells and the Mean are indicated. In vitro migration in response to MIP-1α and MIP-3β was evaluated (right panel) for iDC, LPS-treated DCs and DC/Apo-Nec cells as indicated. A control for random migration was done using RPMI medium. The number of migrating cells was calculated as the mean ± SD of cells counts in five medium-power (40×) fields/well performed in triplicate. A representative experiment of three is shown *p < 0.05 (Student's t-Test).
Mentions: DCs migration to lymph nodes is crucial to trigger T lymphocyte priming. MIP-3β chemokine drives the homing of mDCs to the lymph node and interacts specifically with its receptor CCR7 expressed on the cell surface. iDCs expressed low levels of CCR7 (7%, Mean: 9) but increased its expression after Apo-Nec phagocytosis (90%; Mean: 18) or LPS maturation (35%, Mean: 13) (Figure 5). We observed that iDC migrated to MIP-1α but did not respond to MIP-3β (Figure 5A); on the contrary DC/Apo-Nec and DC + LPS migrated to MIP-3β but failed to respond to MIP-1α (Figure 5B and 5C). Thus, DC/Apo-Nec cells express MIP-3β receptor CCR7 and are able to migrate in response to MIP-3β, potentially allowing their homing to lymph nodes.

Bottom Line: DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta.Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fundación Instituto Leloir, Patricias Argentinas 435 (1405), Buenos Aires, Argentina. evoneuw@leloir.org.ar

ABSTRACT

Background: In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells).

Methods: We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.

Results: Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.

Conclusion: We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.

Show MeSH
Related in: MedlinePlus