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T cells and T cell tumors efficiently generate antigen-specific cytotoxic T cell immunity when modified with an NKT ligand.

Chung Y, Lee YH, Zhang Y, Martin-Orozco N, Yamazaki T, Zhou D, Kang CY, Hwu P, Kwak LW, Dong C - Oncoimmunology (2012)

Bottom Line: While T cells loaded with a class I-restricted peptide induced proliferation but not effector differentiation of antigen-specific CD8 T cells, injection of T cells co-pulsed with αGC strongly induced IFNγ and Granzyme B expression in T cells and complete lysis of target cells in vivo.Of note, the generation of this cytotoxic T cell response was independent of IL-4, IFNγ, IL-12, IL-21 and costimulation.Our data indicate that iNKT cell can license a non-professional APC to directly trigger antigen-specific cytotoxic T cell responses, which provides an alternative cellular vaccine strategy against tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology; Center for Cancer Immunology Research; University of Texas MD Anderson Cancer Center; Houston, TX USA ; Institute of Molecular Medicine; University of Texas Medical School; Houston, TX USA.

ABSTRACT
Various Invariant NKT (iNKT) cell ligands have been shown as potent adjuvants in boosting T cell reactivates to antigens on professional APC. Non-professional APC, such as T cells, also co-expressing MHC class I and CD1d, have been unattractive cell vaccine carriers due to their poor immunogenicity. Here, we report that T cells as well as T cell lymphoma can efficiently generate antigen-specific cytotoxic T lymphocytes (CTL) responses in mice in vivo, when formulated to present iNKT ligand α-galactosylceramide (αGC) on their surface CD1d. Vaccination with αGC-pulsed EG-7 T-cell lymphoma induced tumor-specific CTL response and suppressed the growth of EG-7 in a CD8 T cell-dependent manner. Injection of αGC-loaded CD4 T cells in mice efficiently activated iNKT cells in vivo. While T cells loaded with a class I-restricted peptide induced proliferation but not effector differentiation of antigen-specific CD8 T cells, injection of T cells co-pulsed with αGC strongly induced IFNγ and Granzyme B expression in T cells and complete lysis of target cells in vivo. Presentation of αGC and peptide on the same cells was required for optimal CTL response and vaccinating T cells appeared to directly stimulate both iNKT and cytotoxic CD8 T cells. Of note, the generation of this cytotoxic T cell response was independent of IL-4, IFNγ, IL-12, IL-21 and costimulation. Our data indicate that iNKT cell can license a non-professional APC to directly trigger antigen-specific cytotoxic T cell responses, which provides an alternative cellular vaccine strategy against tumors.

No MeSH data available.


Related in: MedlinePlus

Figure 3. Vaccination with αGC-loaded EG-7 generates OVA-specific cytotoxic T cell response in vivo. (A and B) EG-7 cells were cocultured overnight with 1 μg/ml of αGC (EG-7/αGC) or vehicle (0.5% polysorbate, EG-7/veh) followed by irradiation (50 Gy). C57BL/6 mice (n = 3 per group) were vaccinated with EG-7/veh, EG-7/αGC or left untreated (Nil). (A) One week later, an in vivo CTL assay for SIINFEKL was performed. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. (B) One week after vaccination, CD8+ T cells were isolated and incubated with 1 µg/mL peptide-pulsed splenocytes that had been labeled with DDAO-SE for 2 h. The cells were fixed, permeabilized and stained with anti–cleaved caspase-3 mAb. The levels of cleaved caspase-3 in the DDAO-SE-positive cells were analyzed by flow cytometry. Data shown are representative FACS plots (upper panels) and mean ± SE (lower panel). *p < 0.05, in comparison with EG-7/veh group. (C) One week after the vaccination, splenocytes were isolated and restimulated with SIINFEKL for 5 h in the precence of Golgi-Plug before intracellular staining of granzyme B and IFNγ. (D) Ovalbumin-specific CD8 T cells were isolated, labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice as described in Figure 1. On the following day, mice were i.v. injected with irradiated EG-7 cells manipulated in vitro with indicated conditions. Five days later, lymphoid cells from the spleen of the recipient mice were restimulated with SIINFEKL for 5 h before intracellular staining of granzyme B and IFNγ. Data are representative of at least two separate experiments. *p < 0.05, **p < 0.01, in comparison with non-treated (Nil) group.
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Figure 3: Figure 3. Vaccination with αGC-loaded EG-7 generates OVA-specific cytotoxic T cell response in vivo. (A and B) EG-7 cells were cocultured overnight with 1 μg/ml of αGC (EG-7/αGC) or vehicle (0.5% polysorbate, EG-7/veh) followed by irradiation (50 Gy). C57BL/6 mice (n = 3 per group) were vaccinated with EG-7/veh, EG-7/αGC or left untreated (Nil). (A) One week later, an in vivo CTL assay for SIINFEKL was performed. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. (B) One week after vaccination, CD8+ T cells were isolated and incubated with 1 µg/mL peptide-pulsed splenocytes that had been labeled with DDAO-SE for 2 h. The cells were fixed, permeabilized and stained with anti–cleaved caspase-3 mAb. The levels of cleaved caspase-3 in the DDAO-SE-positive cells were analyzed by flow cytometry. Data shown are representative FACS plots (upper panels) and mean ± SE (lower panel). *p < 0.05, in comparison with EG-7/veh group. (C) One week after the vaccination, splenocytes were isolated and restimulated with SIINFEKL for 5 h in the precence of Golgi-Plug before intracellular staining of granzyme B and IFNγ. (D) Ovalbumin-specific CD8 T cells were isolated, labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice as described in Figure 1. On the following day, mice were i.v. injected with irradiated EG-7 cells manipulated in vitro with indicated conditions. Five days later, lymphoid cells from the spleen of the recipient mice were restimulated with SIINFEKL for 5 h before intracellular staining of granzyme B and IFNγ. Data are representative of at least two separate experiments. *p < 0.05, **p < 0.01, in comparison with non-treated (Nil) group.

Mentions: Our EG-7 tumor models (Fig. 1) and CD8 depletion experiment (Fig. 2) raised a hypothesis that tumor cells of T cell origin, when they present iNKT ligand, are able to induce cytotoxic T cell response specific for endogenous tumor antigens. To test our hypothesis, we co-cultured EG-7 cells with αGC or vehicle before being irradiated (EG-7/αGC and EG-7/veh, respectively). To increase the expression of peptide/MHC-I complex, EG-7 cells were first pre-treated with IFNγ. One week after vaccination, we performed an in vivo CTL assay to determine the antigen-specific cytotoxicity against SIINFEKL-loaded syngenic splenocytes11. When injected into naïve syngenic mice, EG-7/αGC resulted in an evident peptide-loaded target cell lysis whereas EG-7/veh did not (Fig. 3A; -0.37% and 72.8% lysis, respectively). To examine if CD8+ T cells in the vaccinated mice directly kill target cells in an antigen-specific way, we performed an in vitro CTL assay with purified CD8+ T cells from the vaccinated mice by analyzing caspase-3 activation in the target cells. As depicted in Figure 3B, the CD8+ T cells from the EG-7/αGC vaccinated mice induced a significantly higher percentage of caspase-3 cleavage in the target cells than those from the EG-7/veh vaccinated mice. In addition, the effective target lysis in EG-7/αGC-vaccinated mice was associated with increased antigen-specific IFNγ+ granzyme B+ CD8 T cells in the spleen (Fig. 3C). To further characterize the antigen-specific CD8 T cells, we adoptively transferred CFSE-labeled OT-I T cells (CD45.2) into congenic mice (CD45.1). The recipients were vaccinated with either EG-7/veh or EG-7/αGC and CD45.2+ cells were analyzed five days later. OT-I T cells in mice vaccinated with EG-7/veh underwent an extensive proliferation, but expressed little IFNγ (< 10%) or granzyme B (< 3%). By contrast, significantly higher population of OT-I T cells in EG-7/αGC-vaccinated mice expressed IFNγ (> 50%) and granzyme B (> 8%) (Fig. 3D). Taken together, these data demonstrate that αGC-loaded EG-7 thymoma induced tumor-specific CD8+ T cells to differentiate into cytotoxic effector T cells expressing high levels of IFNγ and granzyme B in vivo.


T cells and T cell tumors efficiently generate antigen-specific cytotoxic T cell immunity when modified with an NKT ligand.

Chung Y, Lee YH, Zhang Y, Martin-Orozco N, Yamazaki T, Zhou D, Kang CY, Hwu P, Kwak LW, Dong C - Oncoimmunology (2012)

Figure 3. Vaccination with αGC-loaded EG-7 generates OVA-specific cytotoxic T cell response in vivo. (A and B) EG-7 cells were cocultured overnight with 1 μg/ml of αGC (EG-7/αGC) or vehicle (0.5% polysorbate, EG-7/veh) followed by irradiation (50 Gy). C57BL/6 mice (n = 3 per group) were vaccinated with EG-7/veh, EG-7/αGC or left untreated (Nil). (A) One week later, an in vivo CTL assay for SIINFEKL was performed. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. (B) One week after vaccination, CD8+ T cells were isolated and incubated with 1 µg/mL peptide-pulsed splenocytes that had been labeled with DDAO-SE for 2 h. The cells were fixed, permeabilized and stained with anti–cleaved caspase-3 mAb. The levels of cleaved caspase-3 in the DDAO-SE-positive cells were analyzed by flow cytometry. Data shown are representative FACS plots (upper panels) and mean ± SE (lower panel). *p < 0.05, in comparison with EG-7/veh group. (C) One week after the vaccination, splenocytes were isolated and restimulated with SIINFEKL for 5 h in the precence of Golgi-Plug before intracellular staining of granzyme B and IFNγ. (D) Ovalbumin-specific CD8 T cells were isolated, labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice as described in Figure 1. On the following day, mice were i.v. injected with irradiated EG-7 cells manipulated in vitro with indicated conditions. Five days later, lymphoid cells from the spleen of the recipient mice were restimulated with SIINFEKL for 5 h before intracellular staining of granzyme B and IFNγ. Data are representative of at least two separate experiments. *p < 0.05, **p < 0.01, in comparison with non-treated (Nil) group.
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Figure 3: Figure 3. Vaccination with αGC-loaded EG-7 generates OVA-specific cytotoxic T cell response in vivo. (A and B) EG-7 cells were cocultured overnight with 1 μg/ml of αGC (EG-7/αGC) or vehicle (0.5% polysorbate, EG-7/veh) followed by irradiation (50 Gy). C57BL/6 mice (n = 3 per group) were vaccinated with EG-7/veh, EG-7/αGC or left untreated (Nil). (A) One week later, an in vivo CTL assay for SIINFEKL was performed. CFSEhigh, peptide-pulsed target; CFSElow, peptide-unpulsed control. (B) One week after vaccination, CD8+ T cells were isolated and incubated with 1 µg/mL peptide-pulsed splenocytes that had been labeled with DDAO-SE for 2 h. The cells were fixed, permeabilized and stained with anti–cleaved caspase-3 mAb. The levels of cleaved caspase-3 in the DDAO-SE-positive cells were analyzed by flow cytometry. Data shown are representative FACS plots (upper panels) and mean ± SE (lower panel). *p < 0.05, in comparison with EG-7/veh group. (C) One week after the vaccination, splenocytes were isolated and restimulated with SIINFEKL for 5 h in the precence of Golgi-Plug before intracellular staining of granzyme B and IFNγ. (D) Ovalbumin-specific CD8 T cells were isolated, labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice as described in Figure 1. On the following day, mice were i.v. injected with irradiated EG-7 cells manipulated in vitro with indicated conditions. Five days later, lymphoid cells from the spleen of the recipient mice were restimulated with SIINFEKL for 5 h before intracellular staining of granzyme B and IFNγ. Data are representative of at least two separate experiments. *p < 0.05, **p < 0.01, in comparison with non-treated (Nil) group.
Mentions: Our EG-7 tumor models (Fig. 1) and CD8 depletion experiment (Fig. 2) raised a hypothesis that tumor cells of T cell origin, when they present iNKT ligand, are able to induce cytotoxic T cell response specific for endogenous tumor antigens. To test our hypothesis, we co-cultured EG-7 cells with αGC or vehicle before being irradiated (EG-7/αGC and EG-7/veh, respectively). To increase the expression of peptide/MHC-I complex, EG-7 cells were first pre-treated with IFNγ. One week after vaccination, we performed an in vivo CTL assay to determine the antigen-specific cytotoxicity against SIINFEKL-loaded syngenic splenocytes11. When injected into naïve syngenic mice, EG-7/αGC resulted in an evident peptide-loaded target cell lysis whereas EG-7/veh did not (Fig. 3A; -0.37% and 72.8% lysis, respectively). To examine if CD8+ T cells in the vaccinated mice directly kill target cells in an antigen-specific way, we performed an in vitro CTL assay with purified CD8+ T cells from the vaccinated mice by analyzing caspase-3 activation in the target cells. As depicted in Figure 3B, the CD8+ T cells from the EG-7/αGC vaccinated mice induced a significantly higher percentage of caspase-3 cleavage in the target cells than those from the EG-7/veh vaccinated mice. In addition, the effective target lysis in EG-7/αGC-vaccinated mice was associated with increased antigen-specific IFNγ+ granzyme B+ CD8 T cells in the spleen (Fig. 3C). To further characterize the antigen-specific CD8 T cells, we adoptively transferred CFSE-labeled OT-I T cells (CD45.2) into congenic mice (CD45.1). The recipients were vaccinated with either EG-7/veh or EG-7/αGC and CD45.2+ cells were analyzed five days later. OT-I T cells in mice vaccinated with EG-7/veh underwent an extensive proliferation, but expressed little IFNγ (< 10%) or granzyme B (< 3%). By contrast, significantly higher population of OT-I T cells in EG-7/αGC-vaccinated mice expressed IFNγ (> 50%) and granzyme B (> 8%) (Fig. 3D). Taken together, these data demonstrate that αGC-loaded EG-7 thymoma induced tumor-specific CD8+ T cells to differentiate into cytotoxic effector T cells expressing high levels of IFNγ and granzyme B in vivo.

Bottom Line: While T cells loaded with a class I-restricted peptide induced proliferation but not effector differentiation of antigen-specific CD8 T cells, injection of T cells co-pulsed with αGC strongly induced IFNγ and Granzyme B expression in T cells and complete lysis of target cells in vivo.Of note, the generation of this cytotoxic T cell response was independent of IL-4, IFNγ, IL-12, IL-21 and costimulation.Our data indicate that iNKT cell can license a non-professional APC to directly trigger antigen-specific cytotoxic T cell responses, which provides an alternative cellular vaccine strategy against tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology; Center for Cancer Immunology Research; University of Texas MD Anderson Cancer Center; Houston, TX USA ; Institute of Molecular Medicine; University of Texas Medical School; Houston, TX USA.

ABSTRACT
Various Invariant NKT (iNKT) cell ligands have been shown as potent adjuvants in boosting T cell reactivates to antigens on professional APC. Non-professional APC, such as T cells, also co-expressing MHC class I and CD1d, have been unattractive cell vaccine carriers due to their poor immunogenicity. Here, we report that T cells as well as T cell lymphoma can efficiently generate antigen-specific cytotoxic T lymphocytes (CTL) responses in mice in vivo, when formulated to present iNKT ligand α-galactosylceramide (αGC) on their surface CD1d. Vaccination with αGC-pulsed EG-7 T-cell lymphoma induced tumor-specific CTL response and suppressed the growth of EG-7 in a CD8 T cell-dependent manner. Injection of αGC-loaded CD4 T cells in mice efficiently activated iNKT cells in vivo. While T cells loaded with a class I-restricted peptide induced proliferation but not effector differentiation of antigen-specific CD8 T cells, injection of T cells co-pulsed with αGC strongly induced IFNγ and Granzyme B expression in T cells and complete lysis of target cells in vivo. Presentation of αGC and peptide on the same cells was required for optimal CTL response and vaccinating T cells appeared to directly stimulate both iNKT and cytotoxic CD8 T cells. Of note, the generation of this cytotoxic T cell response was independent of IL-4, IFNγ, IL-12, IL-21 and costimulation. Our data indicate that iNKT cell can license a non-professional APC to directly trigger antigen-specific cytotoxic T cell responses, which provides an alternative cellular vaccine strategy against tumors.

No MeSH data available.


Related in: MedlinePlus