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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: 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.

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

Stimulatory molecule expression and cytokine release are increased on mature dendritic cells (DCs) after coculture with allogeneic allosensitized allogeneic lymphocytes (ASALs). (a) Mature DCs (CD83+) either cultured alone or cultured with ASALs for 48 hours were analyzed for CD80, CD86, CD70, and CD64 expression by flow cytometry. (b) Enzyme-linked immunosorbent assay (ELISA) was used to analyze IFN-γ, IL-2, and IL-12p70 secretion from mDCs cultured alone, ASALs cultured alone, and a coculture of mDCs and ASALs after 48 hours. (a, b) The experiments were performed at least three times with three new and different donors each time. Error bars represent SD, and statistical significance was depicted by symbols where there was a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001).
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fig3: Stimulatory molecule expression and cytokine release are increased on mature dendritic cells (DCs) after coculture with allogeneic allosensitized allogeneic lymphocytes (ASALs). (a) Mature DCs (CD83+) either cultured alone or cultured with ASALs for 48 hours were analyzed for CD80, CD86, CD70, and CD64 expression by flow cytometry. (b) Enzyme-linked immunosorbent assay (ELISA) was used to analyze IFN-γ, IL-2, and IL-12p70 secretion from mDCs cultured alone, ASALs cultured alone, and a coculture of mDCs and ASALs after 48 hours. (a, b) The experiments were performed at least three times with three new and different donors each time. Error bars represent SD, and statistical significance was depicted by symbols where there was a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001).

Mentions: Licensing of DCs by helper cells is supposed to enhance the expression of costimulatory molecules on DCs and to induce secretion of bioactive IL-12.16 Moreover, helper cells may secrete factors like IL-2 and IFN-γ that directly affect CD8+ T-cell survival, cell cycle progression, and differentiation to either effector or memory T cells.17,18 The frequency of mature DCs expressing CD80 and CD70 significantly increased (P = 0.04 and P = 0.006, respectively) after coculture with ASALs, while the already high frequency of CD86 on mature DCs did not further increase (Figure 3a). There was also a tendency that the frequency of mature DCs expressing FcγRI (CD64) and CD40 were increased (Figure 3a). Representative flow cytometry histograms for CD70 and CD40 expression on DCs are shown as Supplementary Figure S2a,b. Coculture of mDCs and ASALs for 24 hours induced a substantial release of IFN-γ, IL-2, and IL-12p70 (Figure 3b). Depletion experiments of CD4+, CD8+, and CD56+ cells from the ASAL population showed that both T cells and NK cells are needed as helper cells in this process. Depletion of either CD4+ or CD56+ cells led to a significant drop in CD70 expression on the mature DCs (Supplementary Figure S2c). For T-cell expansion, it was observed that depletion of CD4+, CD8+, or CD56+ cells from the ASAL population translated to a reduction in T-cell expansion (Supplementary Figure S2d), a shift in phenotype with relatively less expanded CD8+ T cells (Supplementary Figure S2e) and reduced T-cell viability after exposure to oxidative stress in the form of H2O2 (Supplementary Figure S2f).


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)

Stimulatory molecule expression and cytokine release are increased on mature dendritic cells (DCs) after coculture with allogeneic allosensitized allogeneic lymphocytes (ASALs). (a) Mature DCs (CD83+) either cultured alone or cultured with ASALs for 48 hours were analyzed for CD80, CD86, CD70, and CD64 expression by flow cytometry. (b) Enzyme-linked immunosorbent assay (ELISA) was used to analyze IFN-γ, IL-2, and IL-12p70 secretion from mDCs cultured alone, ASALs cultured alone, and a coculture of mDCs and ASALs after 48 hours. (a, b) The experiments were performed at least three times with three new and different donors each time. Error bars represent SD, and statistical significance was depicted by symbols where there was a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Stimulatory molecule expression and cytokine release are increased on mature dendritic cells (DCs) after coculture with allogeneic allosensitized allogeneic lymphocytes (ASALs). (a) Mature DCs (CD83+) either cultured alone or cultured with ASALs for 48 hours were analyzed for CD80, CD86, CD70, and CD64 expression by flow cytometry. (b) Enzyme-linked immunosorbent assay (ELISA) was used to analyze IFN-γ, IL-2, and IL-12p70 secretion from mDCs cultured alone, ASALs cultured alone, and a coculture of mDCs and ASALs after 48 hours. (a, b) The experiments were performed at least three times with three new and different donors each time. Error bars represent SD, and statistical significance was depicted by symbols where there was a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001).
Mentions: Licensing of DCs by helper cells is supposed to enhance the expression of costimulatory molecules on DCs and to induce secretion of bioactive IL-12.16 Moreover, helper cells may secrete factors like IL-2 and IFN-γ that directly affect CD8+ T-cell survival, cell cycle progression, and differentiation to either effector or memory T cells.17,18 The frequency of mature DCs expressing CD80 and CD70 significantly increased (P = 0.04 and P = 0.006, respectively) after coculture with ASALs, while the already high frequency of CD86 on mature DCs did not further increase (Figure 3a). There was also a tendency that the frequency of mature DCs expressing FcγRI (CD64) and CD40 were increased (Figure 3a). Representative flow cytometry histograms for CD70 and CD40 expression on DCs are shown as Supplementary Figure S2a,b. Coculture of mDCs and ASALs for 24 hours induced a substantial release of IFN-γ, IL-2, and IL-12p70 (Figure 3b). Depletion experiments of CD4+, CD8+, and CD56+ cells from the ASAL population showed that both T cells and NK cells are needed as helper cells in this process. Depletion of either CD4+ or CD56+ cells led to a significant drop in CD70 expression on the mature DCs (Supplementary Figure S2c). For T-cell expansion, it was observed that depletion of CD4+, CD8+, or CD56+ cells from the ASAL population translated to a reduction in T-cell expansion (Supplementary Figure S2d), a shift in phenotype with relatively less expanded CD8+ T cells (Supplementary Figure S2e) and reduced T-cell viability after exposure to oxidative stress in the form of H2O2 (Supplementary Figure S2f).

Bottom Line: 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.

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