FUNCTIONAL CAPACITY OF TUMOR LYSATE-PULSED MONOCYTE-DERIVED DENDRITIC CELLS INDUCING IN VITRO IMMUNE RESPONSES
(Abstract release date: 05/19/16)
EHA Library. Pinho C. 06/09/16; 134744; PB1844
Mr. Catarina Pinho
Contributions
Contributions
Abstract
Abstract: PB1844
Type: Publication Only
Background
Dendritic cells (DC) have been shown to be a promising adjuvant to initiate antitumor immune responses. Over the last years, several methods have been developed to isolate DC from cancer patients, ex vivo expand and pulse them, aiming to generate highly immunogenic clinical grade infusion products.
Aims
In order to validate our previously established in vitro methodology for DC generation, the present study aims to assess the functional capacity of the DC final product inducing immune responses. usion products.
Methods
As part of an authorized pre-clinical study with solid tumors, four experiments were performed using tumor lysates (2 glioblastomas, 1 sarcoma and 1 breast cancer) for DC stimulation. DC were differentiated from monocytes obtained from peripheral blood (PB) and subsequently matured and pulsed with previously prepared tumor lysate, during 8 days of culture in cytokines-supplemented medium. Loaded mature DC (mDC) were evaluated for cell counting, viability, morphology and immunophenotype. DC functionality was evaluated via one-way MLR using peripheral blood mononuclear cells (PBMC) which were isolated from PB by density gradient centrifugation and pre-labeled with the ‘green’ fluorescent dye CFSE. The CFSE+ PBMC (responder cells) were co-cultured with mDC (stimulating cells) in a 96-well plate. PBMC without DC stimulation was used as negative control. In both MLR and negative control, the same tumor lysate used in DC stimulation was added or not. After 7 days of incubation at 37°C, 5% CO2, proliferation of responder cells was measured by flow cytometry.
Results
The single-cell suspension obtained at the end of culture showed: numerous cells presenting extended and multiple dendrites; up-regulation of the characteristic maturation markers CD83/CD86; and down-regulation of CD14, marker of the precursor cells. Taken together these results prove the achievement of the DC maturation state. These final loaded mDC were assessed for the antigen presentation skills by their in vitro allostimulatory capacity of PBMC. The average percentage of proliferating PBMC obtained when co-cultured with mDC was greater than that verified without DC stimulation (47.7±3.0% vs. 19.3±8.4% and 47.0±5.0% vs. 8.8±3.5%, with and without tumor lysate, respectively). Despite the small sample size, DC stimulation consistently induced more than double the PBMC proliferation.
Conclusion
This study demonstrated the functionality of the mDC final product to induce in vitro immune responses. With our mDC manufacturing protocol validated, we are motivated to obtain the legal authorization for the implementation of this Advanced-Therapy Medicinal Product in clinical grade. It may be beneficial for the patients to have cell collection, production and administration available in the same hospital setting.
Session topic: E-poster
Keyword(s): Dendritic cell, Dendritic cell vaccine
Type: Publication Only
Background
Dendritic cells (DC) have been shown to be a promising adjuvant to initiate antitumor immune responses. Over the last years, several methods have been developed to isolate DC from cancer patients, ex vivo expand and pulse them, aiming to generate highly immunogenic clinical grade infusion products.
Aims
In order to validate our previously established in vitro methodology for DC generation, the present study aims to assess the functional capacity of the DC final product inducing immune responses. usion products.
Methods
As part of an authorized pre-clinical study with solid tumors, four experiments were performed using tumor lysates (2 glioblastomas, 1 sarcoma and 1 breast cancer) for DC stimulation. DC were differentiated from monocytes obtained from peripheral blood (PB) and subsequently matured and pulsed with previously prepared tumor lysate, during 8 days of culture in cytokines-supplemented medium. Loaded mature DC (mDC) were evaluated for cell counting, viability, morphology and immunophenotype. DC functionality was evaluated via one-way MLR using peripheral blood mononuclear cells (PBMC) which were isolated from PB by density gradient centrifugation and pre-labeled with the ‘green’ fluorescent dye CFSE. The CFSE+ PBMC (responder cells) were co-cultured with mDC (stimulating cells) in a 96-well plate. PBMC without DC stimulation was used as negative control. In both MLR and negative control, the same tumor lysate used in DC stimulation was added or not. After 7 days of incubation at 37°C, 5% CO2, proliferation of responder cells was measured by flow cytometry.
Results
The single-cell suspension obtained at the end of culture showed: numerous cells presenting extended and multiple dendrites; up-regulation of the characteristic maturation markers CD83/CD86; and down-regulation of CD14, marker of the precursor cells. Taken together these results prove the achievement of the DC maturation state. These final loaded mDC were assessed for the antigen presentation skills by their in vitro allostimulatory capacity of PBMC. The average percentage of proliferating PBMC obtained when co-cultured with mDC was greater than that verified without DC stimulation (47.7±3.0% vs. 19.3±8.4% and 47.0±5.0% vs. 8.8±3.5%, with and without tumor lysate, respectively). Despite the small sample size, DC stimulation consistently induced more than double the PBMC proliferation.
Conclusion
This study demonstrated the functionality of the mDC final product to induce in vitro immune responses. With our mDC manufacturing protocol validated, we are motivated to obtain the legal authorization for the implementation of this Advanced-Therapy Medicinal Product in clinical grade. It may be beneficial for the patients to have cell collection, production and administration available in the same hospital setting.
Session topic: E-poster
Keyword(s): Dendritic cell, Dendritic cell vaccine
Abstract: PB1844
Type: Publication Only
Background
Dendritic cells (DC) have been shown to be a promising adjuvant to initiate antitumor immune responses. Over the last years, several methods have been developed to isolate DC from cancer patients, ex vivo expand and pulse them, aiming to generate highly immunogenic clinical grade infusion products.
Aims
In order to validate our previously established in vitro methodology for DC generation, the present study aims to assess the functional capacity of the DC final product inducing immune responses. usion products.
Methods
As part of an authorized pre-clinical study with solid tumors, four experiments were performed using tumor lysates (2 glioblastomas, 1 sarcoma and 1 breast cancer) for DC stimulation. DC were differentiated from monocytes obtained from peripheral blood (PB) and subsequently matured and pulsed with previously prepared tumor lysate, during 8 days of culture in cytokines-supplemented medium. Loaded mature DC (mDC) were evaluated for cell counting, viability, morphology and immunophenotype. DC functionality was evaluated via one-way MLR using peripheral blood mononuclear cells (PBMC) which were isolated from PB by density gradient centrifugation and pre-labeled with the ‘green’ fluorescent dye CFSE. The CFSE+ PBMC (responder cells) were co-cultured with mDC (stimulating cells) in a 96-well plate. PBMC without DC stimulation was used as negative control. In both MLR and negative control, the same tumor lysate used in DC stimulation was added or not. After 7 days of incubation at 37°C, 5% CO2, proliferation of responder cells was measured by flow cytometry.
Results
The single-cell suspension obtained at the end of culture showed: numerous cells presenting extended and multiple dendrites; up-regulation of the characteristic maturation markers CD83/CD86; and down-regulation of CD14, marker of the precursor cells. Taken together these results prove the achievement of the DC maturation state. These final loaded mDC were assessed for the antigen presentation skills by their in vitro allostimulatory capacity of PBMC. The average percentage of proliferating PBMC obtained when co-cultured with mDC was greater than that verified without DC stimulation (47.7±3.0% vs. 19.3±8.4% and 47.0±5.0% vs. 8.8±3.5%, with and without tumor lysate, respectively). Despite the small sample size, DC stimulation consistently induced more than double the PBMC proliferation.
Conclusion
This study demonstrated the functionality of the mDC final product to induce in vitro immune responses. With our mDC manufacturing protocol validated, we are motivated to obtain the legal authorization for the implementation of this Advanced-Therapy Medicinal Product in clinical grade. It may be beneficial for the patients to have cell collection, production and administration available in the same hospital setting.
Session topic: E-poster
Keyword(s): Dendritic cell, Dendritic cell vaccine
Type: Publication Only
Background
Dendritic cells (DC) have been shown to be a promising adjuvant to initiate antitumor immune responses. Over the last years, several methods have been developed to isolate DC from cancer patients, ex vivo expand and pulse them, aiming to generate highly immunogenic clinical grade infusion products.
Aims
In order to validate our previously established in vitro methodology for DC generation, the present study aims to assess the functional capacity of the DC final product inducing immune responses. usion products.
Methods
As part of an authorized pre-clinical study with solid tumors, four experiments were performed using tumor lysates (2 glioblastomas, 1 sarcoma and 1 breast cancer) for DC stimulation. DC were differentiated from monocytes obtained from peripheral blood (PB) and subsequently matured and pulsed with previously prepared tumor lysate, during 8 days of culture in cytokines-supplemented medium. Loaded mature DC (mDC) were evaluated for cell counting, viability, morphology and immunophenotype. DC functionality was evaluated via one-way MLR using peripheral blood mononuclear cells (PBMC) which were isolated from PB by density gradient centrifugation and pre-labeled with the ‘green’ fluorescent dye CFSE. The CFSE+ PBMC (responder cells) were co-cultured with mDC (stimulating cells) in a 96-well plate. PBMC without DC stimulation was used as negative control. In both MLR and negative control, the same tumor lysate used in DC stimulation was added or not. After 7 days of incubation at 37°C, 5% CO2, proliferation of responder cells was measured by flow cytometry.
Results
The single-cell suspension obtained at the end of culture showed: numerous cells presenting extended and multiple dendrites; up-regulation of the characteristic maturation markers CD83/CD86; and down-regulation of CD14, marker of the precursor cells. Taken together these results prove the achievement of the DC maturation state. These final loaded mDC were assessed for the antigen presentation skills by their in vitro allostimulatory capacity of PBMC. The average percentage of proliferating PBMC obtained when co-cultured with mDC was greater than that verified without DC stimulation (47.7±3.0% vs. 19.3±8.4% and 47.0±5.0% vs. 8.8±3.5%, with and without tumor lysate, respectively). Despite the small sample size, DC stimulation consistently induced more than double the PBMC proliferation.
Conclusion
This study demonstrated the functionality of the mDC final product to induce in vitro immune responses. With our mDC manufacturing protocol validated, we are motivated to obtain the legal authorization for the implementation of this Advanced-Therapy Medicinal Product in clinical grade. It may be beneficial for the patients to have cell collection, production and administration available in the same hospital setting.
Session topic: E-poster
Keyword(s): Dendritic cell, Dendritic cell vaccine
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