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Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors.

Jin C, Yu D, Hillerdal V, Wallgren A, Karlsson-Parra A, Essand M - Mol Ther Methods Clin Dev (2014)

Bottom Line: The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation.When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol.Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University , Uppsala, Sweden.

ABSTRACT
Adoptive T-cell therapy of cancer is a treatment strategy where T cells are isolated, activated, in some cases engineered, and expanded ex vivo before being reinfused to the patient. The most commonly used T-cell expansion methods are either anti-CD3/CD28 antibody beads or the "rapid expansion protocol" (REP), which utilizes OKT-3, interleukin (IL)-2, and irradiated allogeneic feeder cells. However, REP-expanded or bead-expanded T cells are sensitive to the harsh tumor microenvironment and often short-lived after reinfusion. Here, we demonstrate that when irradiated and preactivated allosensitized allogeneic lymphocytes (ASALs) are used as helper cells to license OKT3-armed allogeneic mature dendritic cells (DCs), together they expand target T cells of high quality. The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation. When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol. Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

No MeSH data available.


Related in: MedlinePlus

GD2-CAR-engineered T cells, expanded by the allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP), are more resistant to immunosuppressive cytokines and oxidative and apoptotic stress than GD2-CAR T cells expanded by the rapid expansion protocol (REP). (a) An illustration of the GD2-CAR-encoding lentiviral vector used to transduce T cells. (b) CD8+ T cells were transduced, isolated by magnetic beads, and expanded by the REP or AEP protocols. The starting number of GD2-CAR T cells was 1 × 105 for both protocols and a ratio of 1:1:4 (T-cell:mDC:ASAL) was used in the AEP protocol. Fold expansion of GD2-CAR T cells is presented. (c) The cytotoxic ability of GD2-CAR T cells was analyzed by coculture with luciferase-expressing GD2+ (IMR-32) or GD2− (SK-N-FI) neuroblastoma target cells at different E:T ratios. Viability of target cells were measured by luminescence and related to signals from target cells alone. (d) GD2-CAR T cells expanded by the REP and AEP protocols were exposed to either 25 μmol/l H2O2 or 0.1 μmol/l doxorubicin for 24 hours. Viability was analyzed by flow cytometry using Annexin-V and PI staining and relative viability was normalized against nontreated GD2-CAR T cells. (e–f) The REP- and AEP-expanded GD2-CAR T cells were treated with IL-10/TGF-β for 4 hours or with H2O2 for 24 hours, followed by 4 hours treatment with IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 24 hours. (e) Flow cytometry was used to analyze CD107a expression on GD2-CAR T cells (CD3+) after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (f) ELISA was used to analyze IFN-γ production from GD2-CAR T cells after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (g) GD2-CAR T cells were treated with H2O2 for 24 hours, followed by 4 hours labeling with CFSE in the presence of IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 4 days before proliferation analysis by flow cytometry. (a–g) The experiments were performed three times with three new and different donors each time. Error bars represent SD, and statistical significance is depicted by symbols (*P < 0.05, **P < 0.01, ***P < 0.001).
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fig5: GD2-CAR-engineered T cells, expanded by the allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP), are more resistant to immunosuppressive cytokines and oxidative and apoptotic stress than GD2-CAR T cells expanded by the rapid expansion protocol (REP). (a) An illustration of the GD2-CAR-encoding lentiviral vector used to transduce T cells. (b) CD8+ T cells were transduced, isolated by magnetic beads, and expanded by the REP or AEP protocols. The starting number of GD2-CAR T cells was 1 × 105 for both protocols and a ratio of 1:1:4 (T-cell:mDC:ASAL) was used in the AEP protocol. Fold expansion of GD2-CAR T cells is presented. (c) The cytotoxic ability of GD2-CAR T cells was analyzed by coculture with luciferase-expressing GD2+ (IMR-32) or GD2− (SK-N-FI) neuroblastoma target cells at different E:T ratios. Viability of target cells were measured by luminescence and related to signals from target cells alone. (d) GD2-CAR T cells expanded by the REP and AEP protocols were exposed to either 25 μmol/l H2O2 or 0.1 μmol/l doxorubicin for 24 hours. Viability was analyzed by flow cytometry using Annexin-V and PI staining and relative viability was normalized against nontreated GD2-CAR T cells. (e–f) The REP- and AEP-expanded GD2-CAR T cells were treated with IL-10/TGF-β for 4 hours or with H2O2 for 24 hours, followed by 4 hours treatment with IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 24 hours. (e) Flow cytometry was used to analyze CD107a expression on GD2-CAR T cells (CD3+) after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (f) ELISA was used to analyze IFN-γ production from GD2-CAR T cells after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (g) GD2-CAR T cells were treated with H2O2 for 24 hours, followed by 4 hours labeling with CFSE in the presence of IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 4 days before proliferation analysis by flow cytometry. (a–g) The experiments were performed three times with three new and different donors each time. Error bars represent SD, and statistical significance is depicted by symbols (*P < 0.05, **P < 0.01, ***P < 0.001).

Mentions: Given the recent impressive responses in cancer patients for adoptive T-cell therapy using genetically engineered T cells,25,26 it was of interest to investigate the performance of such T cells using the AEP protocol. We therefore engineered T cells to express a chimeric antigen receptor (CAR) against the disialoganglioside GD2, which is overexpressed on neuroblastoma cells and a relevant target for T-cell therapy of neuroblastoma.27 CD8+ T cells isolated from peripheral blood were transduced with a lentiviral vector encoding the GD2-CAR (Figure 5a), in order to generate GD2-specific T cells. The engineered T cells were then used for comparison of the AEP and REP protocols. Fold expansion was similar between the two protocols (Figure 5b). Specific cytotoxicity against GD2-expressing neuroblastoma target cells (IMR-32) was also similar, but with a tendency that AEP-expanded T cells performed somewhat better (Figure 5c). Importantly, the ability to resist oxidative (H2O2) and apoptotic (doxorubicin) stress was significantly better for AEP-expanded T cells (Figure 5d).


Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors.

Jin C, Yu D, Hillerdal V, Wallgren A, Karlsson-Parra A, Essand M - Mol Ther Methods Clin Dev (2014)

GD2-CAR-engineered T cells, expanded by the allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP), are more resistant to immunosuppressive cytokines and oxidative and apoptotic stress than GD2-CAR T cells expanded by the rapid expansion protocol (REP). (a) An illustration of the GD2-CAR-encoding lentiviral vector used to transduce T cells. (b) CD8+ T cells were transduced, isolated by magnetic beads, and expanded by the REP or AEP protocols. The starting number of GD2-CAR T cells was 1 × 105 for both protocols and a ratio of 1:1:4 (T-cell:mDC:ASAL) was used in the AEP protocol. Fold expansion of GD2-CAR T cells is presented. (c) The cytotoxic ability of GD2-CAR T cells was analyzed by coculture with luciferase-expressing GD2+ (IMR-32) or GD2− (SK-N-FI) neuroblastoma target cells at different E:T ratios. Viability of target cells were measured by luminescence and related to signals from target cells alone. (d) GD2-CAR T cells expanded by the REP and AEP protocols were exposed to either 25 μmol/l H2O2 or 0.1 μmol/l doxorubicin for 24 hours. Viability was analyzed by flow cytometry using Annexin-V and PI staining and relative viability was normalized against nontreated GD2-CAR T cells. (e–f) The REP- and AEP-expanded GD2-CAR T cells were treated with IL-10/TGF-β for 4 hours or with H2O2 for 24 hours, followed by 4 hours treatment with IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 24 hours. (e) Flow cytometry was used to analyze CD107a expression on GD2-CAR T cells (CD3+) after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (f) ELISA was used to analyze IFN-γ production from GD2-CAR T cells after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (g) GD2-CAR T cells were treated with H2O2 for 24 hours, followed by 4 hours labeling with CFSE in the presence of IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 4 days before proliferation analysis by flow cytometry. (a–g) The experiments were performed three times with three new and different donors each time. Error bars represent SD, and statistical significance is depicted by symbols (*P < 0.05, **P < 0.01, ***P < 0.001).
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Related In: Results  -  Collection

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fig5: GD2-CAR-engineered T cells, expanded by the allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP), are more resistant to immunosuppressive cytokines and oxidative and apoptotic stress than GD2-CAR T cells expanded by the rapid expansion protocol (REP). (a) An illustration of the GD2-CAR-encoding lentiviral vector used to transduce T cells. (b) CD8+ T cells were transduced, isolated by magnetic beads, and expanded by the REP or AEP protocols. The starting number of GD2-CAR T cells was 1 × 105 for both protocols and a ratio of 1:1:4 (T-cell:mDC:ASAL) was used in the AEP protocol. Fold expansion of GD2-CAR T cells is presented. (c) The cytotoxic ability of GD2-CAR T cells was analyzed by coculture with luciferase-expressing GD2+ (IMR-32) or GD2− (SK-N-FI) neuroblastoma target cells at different E:T ratios. Viability of target cells were measured by luminescence and related to signals from target cells alone. (d) GD2-CAR T cells expanded by the REP and AEP protocols were exposed to either 25 μmol/l H2O2 or 0.1 μmol/l doxorubicin for 24 hours. Viability was analyzed by flow cytometry using Annexin-V and PI staining and relative viability was normalized against nontreated GD2-CAR T cells. (e–f) The REP- and AEP-expanded GD2-CAR T cells were treated with IL-10/TGF-β for 4 hours or with H2O2 for 24 hours, followed by 4 hours treatment with IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 24 hours. (e) Flow cytometry was used to analyze CD107a expression on GD2-CAR T cells (CD3+) after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (f) ELISA was used to analyze IFN-γ production from GD2-CAR T cells after exposure to IL-10/TGF-β or H2O2/IL-10/TGF-β and normalized against nontreated GD2-CAR T cells. (g) GD2-CAR T cells were treated with H2O2 for 24 hours, followed by 4 hours labeling with CFSE in the presence of IL-10/TGF-β and then cocultured with GD2+ target cells (IMR-32) in the presence of IL-10/TGF-β for 4 days before proliferation analysis by flow cytometry. (a–g) The experiments were performed three times with three new and different donors each time. Error bars represent SD, and statistical significance is depicted by symbols (*P < 0.05, **P < 0.01, ***P < 0.001).
Mentions: Given the recent impressive responses in cancer patients for adoptive T-cell therapy using genetically engineered T cells,25,26 it was of interest to investigate the performance of such T cells using the AEP protocol. We therefore engineered T cells to express a chimeric antigen receptor (CAR) against the disialoganglioside GD2, which is overexpressed on neuroblastoma cells and a relevant target for T-cell therapy of neuroblastoma.27 CD8+ T cells isolated from peripheral blood were transduced with a lentiviral vector encoding the GD2-CAR (Figure 5a), in order to generate GD2-specific T cells. The engineered T cells were then used for comparison of the AEP and REP protocols. Fold expansion was similar between the two protocols (Figure 5b). Specific cytotoxicity against GD2-expressing neuroblastoma target cells (IMR-32) was also similar, but with a tendency that AEP-expanded T cells performed somewhat better (Figure 5c). Importantly, the ability to resist oxidative (H2O2) and apoptotic (doxorubicin) stress was significantly better for AEP-expanded T cells (Figure 5d).

Bottom Line: The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation.When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol.Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University , Uppsala, Sweden.

ABSTRACT
Adoptive T-cell therapy of cancer is a treatment strategy where T cells are isolated, activated, in some cases engineered, and expanded ex vivo before being reinfused to the patient. The most commonly used T-cell expansion methods are either anti-CD3/CD28 antibody beads or the "rapid expansion protocol" (REP), which utilizes OKT-3, interleukin (IL)-2, and irradiated allogeneic feeder cells. However, REP-expanded or bead-expanded T cells are sensitive to the harsh tumor microenvironment and often short-lived after reinfusion. Here, we demonstrate that when irradiated and preactivated allosensitized allogeneic lymphocytes (ASALs) are used as helper cells to license OKT3-armed allogeneic mature dendritic cells (DCs), together they expand target T cells of high quality. The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation. When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol. Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

No MeSH data available.


Related in: MedlinePlus