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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

Schematic illustration of the rapid expansion protocol (REP) and allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP) T-cell expansion protocols and proposed mechanisms of action for the AEP protocol. (a) REP: T cells were expanded for 12 days by stimulation with irradiated feeder cells (peripheral blood mononuclear cells (PBMCs) from three donors), OKT-3 and IL-2. ASAL expansion protocol (AEP): Prior to expansion: Monocytes from one donor were differentiated into immature dendritic cells (DCs) with IL-4 and granulocyte-macrophage colony-stimulating factor for 6 days and then matured for 24 hours with IFN-γ, poly(I:C) and R848. ASALs were generated over 7 days by coculturing PBMCs from one donor with irradiated PBMCs from a second donor (autologous to the DCs) at a ratio of 1:1. The ASALs and mDCs can be frozen and stored or used immediately. Expansion phase: T cells, allogeneic to both the ASAL and mDCs, were expanded for 12 days by coculture with irradiated ASAL, mDCs, OKT-3, and IL-2. When comparing the two protocols, the same amount of T cells from the same donors were expanded and analyzed in parallel. (b) A proposed illustration why the AEP protocol leads to activation of responder T cells, which are insensitive to immunosuppressive and oxidative stress. ASALs provide “help signals”, which lead to upregulation of CD70, CD80, CD64, and CD40 on allogeneic mDCs. The interaction between ASALSs and mDC also leads to secretion of IL-2 and IFN-γ. Furthermore, CD40L expression on helper cells in the ASAL population can interact with CD40 on mDCs and thereby induce IL-12 secretion and Th1 polarization. CD64 expression on the mDCS ensures proper Fc binding of the OKT-3 antibody. The combination of allogeneic anti-CD3-armed Th1-polarizing mDCs and ASALs leads to T cells, which are insensitive to immunosuppressive and oxidative stress.
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fig1: Schematic illustration of the rapid expansion protocol (REP) and allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP) T-cell expansion protocols and proposed mechanisms of action for the AEP protocol. (a) REP: T cells were expanded for 12 days by stimulation with irradiated feeder cells (peripheral blood mononuclear cells (PBMCs) from three donors), OKT-3 and IL-2. ASAL expansion protocol (AEP): Prior to expansion: Monocytes from one donor were differentiated into immature dendritic cells (DCs) with IL-4 and granulocyte-macrophage colony-stimulating factor for 6 days and then matured for 24 hours with IFN-γ, poly(I:C) and R848. ASALs were generated over 7 days by coculturing PBMCs from one donor with irradiated PBMCs from a second donor (autologous to the DCs) at a ratio of 1:1. The ASALs and mDCs can be frozen and stored or used immediately. Expansion phase: T cells, allogeneic to both the ASAL and mDCs, were expanded for 12 days by coculture with irradiated ASAL, mDCs, OKT-3, and IL-2. When comparing the two protocols, the same amount of T cells from the same donors were expanded and analyzed in parallel. (b) A proposed illustration why the AEP protocol leads to activation of responder T cells, which are insensitive to immunosuppressive and oxidative stress. ASALs provide “help signals”, which lead to upregulation of CD70, CD80, CD64, and CD40 on allogeneic mDCs. The interaction between ASALSs and mDC also leads to secretion of IL-2 and IFN-γ. Furthermore, CD40L expression on helper cells in the ASAL population can interact with CD40 on mDCs and thereby induce IL-12 secretion and Th1 polarization. CD64 expression on the mDCS ensures proper Fc binding of the OKT-3 antibody. The combination of allogeneic anti-CD3-armed Th1-polarizing mDCs and ASALs leads to T cells, which are insensitive to immunosuppressive and oxidative stress.

Mentions: The REP and AEP protocols are illustrated in Figure 1a. For the REP protocol, irradiated allogeneic PBMCs from three different donors are used as feeder cells. For the AEP protocol, the ASALs, mDCs, and T cells for expansion are allogeneic with respect to each other. Irradiated PBMCs are used to stimulate allogeneic PBMCs for 7 days to become ASALs. These irradiated PBMCs are from the same donor as the mDCs, meaning that the ASALs will reexperience the allogeneic major histocompatibility complex class I and class II molecules on mDCs when they are mixed for T-cell expansion. ASALs and mDCs can be prepared in advance over 7 days and used either directly or kept frozen until T-cell expansion is initiated. The ASALs are irradiated before they are added to the DCs and T cells.


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)

Schematic illustration of the rapid expansion protocol (REP) and allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP) T-cell expansion protocols and proposed mechanisms of action for the AEP protocol. (a) REP: T cells were expanded for 12 days by stimulation with irradiated feeder cells (peripheral blood mononuclear cells (PBMCs) from three donors), OKT-3 and IL-2. ASAL expansion protocol (AEP): Prior to expansion: Monocytes from one donor were differentiated into immature dendritic cells (DCs) with IL-4 and granulocyte-macrophage colony-stimulating factor for 6 days and then matured for 24 hours with IFN-γ, poly(I:C) and R848. ASALs were generated over 7 days by coculturing PBMCs from one donor with irradiated PBMCs from a second donor (autologous to the DCs) at a ratio of 1:1. The ASALs and mDCs can be frozen and stored or used immediately. Expansion phase: T cells, allogeneic to both the ASAL and mDCs, were expanded for 12 days by coculture with irradiated ASAL, mDCs, OKT-3, and IL-2. When comparing the two protocols, the same amount of T cells from the same donors were expanded and analyzed in parallel. (b) A proposed illustration why the AEP protocol leads to activation of responder T cells, which are insensitive to immunosuppressive and oxidative stress. ASALs provide “help signals”, which lead to upregulation of CD70, CD80, CD64, and CD40 on allogeneic mDCs. The interaction between ASALSs and mDC also leads to secretion of IL-2 and IFN-γ. Furthermore, CD40L expression on helper cells in the ASAL population can interact with CD40 on mDCs and thereby induce IL-12 secretion and Th1 polarization. CD64 expression on the mDCS ensures proper Fc binding of the OKT-3 antibody. The combination of allogeneic anti-CD3-armed Th1-polarizing mDCs and ASALs leads to T cells, which are insensitive to immunosuppressive and oxidative stress.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362340&req=5

fig1: Schematic illustration of the rapid expansion protocol (REP) and allosensitized allogeneic lymphocytes (ASAL) expansion protocol (AEP) T-cell expansion protocols and proposed mechanisms of action for the AEP protocol. (a) REP: T cells were expanded for 12 days by stimulation with irradiated feeder cells (peripheral blood mononuclear cells (PBMCs) from three donors), OKT-3 and IL-2. ASAL expansion protocol (AEP): Prior to expansion: Monocytes from one donor were differentiated into immature dendritic cells (DCs) with IL-4 and granulocyte-macrophage colony-stimulating factor for 6 days and then matured for 24 hours with IFN-γ, poly(I:C) and R848. ASALs were generated over 7 days by coculturing PBMCs from one donor with irradiated PBMCs from a second donor (autologous to the DCs) at a ratio of 1:1. The ASALs and mDCs can be frozen and stored or used immediately. Expansion phase: T cells, allogeneic to both the ASAL and mDCs, were expanded for 12 days by coculture with irradiated ASAL, mDCs, OKT-3, and IL-2. When comparing the two protocols, the same amount of T cells from the same donors were expanded and analyzed in parallel. (b) A proposed illustration why the AEP protocol leads to activation of responder T cells, which are insensitive to immunosuppressive and oxidative stress. ASALs provide “help signals”, which lead to upregulation of CD70, CD80, CD64, and CD40 on allogeneic mDCs. The interaction between ASALSs and mDC also leads to secretion of IL-2 and IFN-γ. Furthermore, CD40L expression on helper cells in the ASAL population can interact with CD40 on mDCs and thereby induce IL-12 secretion and Th1 polarization. CD64 expression on the mDCS ensures proper Fc binding of the OKT-3 antibody. The combination of allogeneic anti-CD3-armed Th1-polarizing mDCs and ASALs leads to T cells, which are insensitive to immunosuppressive and oxidative stress.
Mentions: The REP and AEP protocols are illustrated in Figure 1a. For the REP protocol, irradiated allogeneic PBMCs from three different donors are used as feeder cells. For the AEP protocol, the ASALs, mDCs, and T cells for expansion are allogeneic with respect to each other. Irradiated PBMCs are used to stimulate allogeneic PBMCs for 7 days to become ASALs. These irradiated PBMCs are from the same donor as the mDCs, meaning that the ASALs will reexperience the allogeneic major histocompatibility complex class I and class II molecules on mDCs when they are mixed for T-cell expansion. ASALs and mDCs can be prepared in advance over 7 days and used either directly or kept frozen until T-cell expansion is initiated. The ASALs are irradiated before they are added to the DCs and T cells.

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