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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 4. Injection of conventional CD4 T cells coated with SIINFEKL and αGC induces a functional cytotoxic T cell response. (A) Lymphoid cells from spleen and lymph nodes of C57BL/6 mice were stained with FITC-conjugated anti-CD19, anti-NK1.1, anti-Gr-1, anti-CD11b, anti-CD11c, anti-CD8a, anti-I-Ab antibodies together with APC-conjugated CD4 Ab. Lineage negative and CD4+ cells were sorted by FACSAria. The sorted cells were stained with PE-conjugated anti-CD3 or anti-CD1d Ab. Filled histogram is isotype control. (B) Sorted CD4+ T cells were co-cultured with αGC (1 μg/ml). Cells were washed and intravenously injected into syngenic mice. Six hours later, lymphoid cells from spleen were stained with CD1d-tetramer and CD19 before intracellular IFNγ staining. CD1d-tetramer+CD19- cells were gated and analyzed. (C) The sorted CD4+ T cells were co-cultured with αGC (1 μg/ml) or vehicle (0.5% polysorbate) for 16 h including 1 h pulse with SIINFEKL. Cells were washed and intravenously injected into syngenic mice. One week later, syngenic lymphocytes were either loaded with 1 µmol/L peptides or left untouched before being labeled with CFSE at different concentrations (10 and 1 µmol/L, respectively). Equal numbers of the two populations were mixed and injected i.v. into mice. Eighteen to 24 h later, lymphoid cells from spleen and lymph nodes were analyzed to assess peptide-specific killing. (D) OT-I T cells were isolated using anti-CD8 microbeads and AutoMacs. These cells were labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice. On the following day, mice were i.v. injected with T cells manipulated in vitro with indicated conditions. Forty-eight hours later, lymphoid cells from the spleen of the recipient mice were stained with phycoerythrin (PE)-conjugated anti-Vα2 antibody and then analyzed by flow cytometry. For intracellular Granzyme B and IFNγ staining, cells were restimulated with SIINFEKL for 5 h before intracellular staining of these molecules according to manufacturer’s instruction. Data are representative of at least two independent experiments.
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Figure 4: Figure 4. Injection of conventional CD4 T cells coated with SIINFEKL and αGC induces a functional cytotoxic T cell response. (A) Lymphoid cells from spleen and lymph nodes of C57BL/6 mice were stained with FITC-conjugated anti-CD19, anti-NK1.1, anti-Gr-1, anti-CD11b, anti-CD11c, anti-CD8a, anti-I-Ab antibodies together with APC-conjugated CD4 Ab. Lineage negative and CD4+ cells were sorted by FACSAria. The sorted cells were stained with PE-conjugated anti-CD3 or anti-CD1d Ab. Filled histogram is isotype control. (B) Sorted CD4+ T cells were co-cultured with αGC (1 μg/ml). Cells were washed and intravenously injected into syngenic mice. Six hours later, lymphoid cells from spleen were stained with CD1d-tetramer and CD19 before intracellular IFNγ staining. CD1d-tetramer+CD19- cells were gated and analyzed. (C) The sorted CD4+ T cells were co-cultured with αGC (1 μg/ml) or vehicle (0.5% polysorbate) for 16 h including 1 h pulse with SIINFEKL. Cells were washed and intravenously injected into syngenic mice. One week later, syngenic lymphocytes were either loaded with 1 µmol/L peptides or left untouched before being labeled with CFSE at different concentrations (10 and 1 µmol/L, respectively). Equal numbers of the two populations were mixed and injected i.v. into mice. Eighteen to 24 h later, lymphoid cells from spleen and lymph nodes were analyzed to assess peptide-specific killing. (D) OT-I T cells were isolated using anti-CD8 microbeads and AutoMacs. These cells were labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice. On the following day, mice were i.v. injected with T cells manipulated in vitro with indicated conditions. Forty-eight hours later, lymphoid cells from the spleen of the recipient mice were stained with phycoerythrin (PE)-conjugated anti-Vα2 antibody and then analyzed by flow cytometry. For intracellular Granzyme B and IFNγ staining, cells were restimulated with SIINFEKL for 5 h before intracellular staining of these molecules according to manufacturer’s instruction. Data are representative of at least two independent experiments.

Mentions: Our study thus far showed that, with help of αGC, tumors of T cell origin can trigger tumor-specific cytotoxic T cell responses. Therefore we next asked if conventional T cells can also act as antigen presenting cells and trigger cytotoxic T cell responses in vivo, when formulated to present αGC. A previous study by Shimizu et al. showed that T cells, purified by negative selection using magnetic beads and when loaded with αGC, stimulate iNKT cells in vivo.13 To further assess iNKT cell activation by T cells, we first sorted NK1.1−CD8−CD19−CD11b−CD11c−Gr-1−I-Ab−CD4+ cells from the lymphoid cells of C57BL/6 mice. These cells were virtually all CD3+CD1d+, indicating no contamination of professional APC (Fig. 4A). The sorted cells were loaded with αGC (T/αGC) or with vehicle (T/veh) and were i.v. injected into syngenic naïve mice. When we analyzed splenocytes six hours after the injection, we observed that CD1d-tetramer+ cells in mice receiving T/αGC produced IFNγ+ whereas the same population in mice receiving T/veh did not produce IFNγ (Fig. 4B). Therefore CD4+ T cells efficiently triggered iNKT cell activation in vivo when they were manipulated to present αGC.


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 4. Injection of conventional CD4 T cells coated with SIINFEKL and αGC induces a functional cytotoxic T cell response. (A) Lymphoid cells from spleen and lymph nodes of C57BL/6 mice were stained with FITC-conjugated anti-CD19, anti-NK1.1, anti-Gr-1, anti-CD11b, anti-CD11c, anti-CD8a, anti-I-Ab antibodies together with APC-conjugated CD4 Ab. Lineage negative and CD4+ cells were sorted by FACSAria. The sorted cells were stained with PE-conjugated anti-CD3 or anti-CD1d Ab. Filled histogram is isotype control. (B) Sorted CD4+ T cells were co-cultured with αGC (1 μg/ml). Cells were washed and intravenously injected into syngenic mice. Six hours later, lymphoid cells from spleen were stained with CD1d-tetramer and CD19 before intracellular IFNγ staining. CD1d-tetramer+CD19- cells were gated and analyzed. (C) The sorted CD4+ T cells were co-cultured with αGC (1 μg/ml) or vehicle (0.5% polysorbate) for 16 h including 1 h pulse with SIINFEKL. Cells were washed and intravenously injected into syngenic mice. One week later, syngenic lymphocytes were either loaded with 1 µmol/L peptides or left untouched before being labeled with CFSE at different concentrations (10 and 1 µmol/L, respectively). Equal numbers of the two populations were mixed and injected i.v. into mice. Eighteen to 24 h later, lymphoid cells from spleen and lymph nodes were analyzed to assess peptide-specific killing. (D) OT-I T cells were isolated using anti-CD8 microbeads and AutoMacs. These cells were labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice. On the following day, mice were i.v. injected with T cells manipulated in vitro with indicated conditions. Forty-eight hours later, lymphoid cells from the spleen of the recipient mice were stained with phycoerythrin (PE)-conjugated anti-Vα2 antibody and then analyzed by flow cytometry. For intracellular Granzyme B and IFNγ staining, cells were restimulated with SIINFEKL for 5 h before intracellular staining of these molecules according to manufacturer’s instruction. Data are representative of at least two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376985&req=5

Figure 4: Figure 4. Injection of conventional CD4 T cells coated with SIINFEKL and αGC induces a functional cytotoxic T cell response. (A) Lymphoid cells from spleen and lymph nodes of C57BL/6 mice were stained with FITC-conjugated anti-CD19, anti-NK1.1, anti-Gr-1, anti-CD11b, anti-CD11c, anti-CD8a, anti-I-Ab antibodies together with APC-conjugated CD4 Ab. Lineage negative and CD4+ cells were sorted by FACSAria. The sorted cells were stained with PE-conjugated anti-CD3 or anti-CD1d Ab. Filled histogram is isotype control. (B) Sorted CD4+ T cells were co-cultured with αGC (1 μg/ml). Cells were washed and intravenously injected into syngenic mice. Six hours later, lymphoid cells from spleen were stained with CD1d-tetramer and CD19 before intracellular IFNγ staining. CD1d-tetramer+CD19- cells were gated and analyzed. (C) The sorted CD4+ T cells were co-cultured with αGC (1 μg/ml) or vehicle (0.5% polysorbate) for 16 h including 1 h pulse with SIINFEKL. Cells were washed and intravenously injected into syngenic mice. One week later, syngenic lymphocytes were either loaded with 1 µmol/L peptides or left untouched before being labeled with CFSE at different concentrations (10 and 1 µmol/L, respectively). Equal numbers of the two populations were mixed and injected i.v. into mice. Eighteen to 24 h later, lymphoid cells from spleen and lymph nodes were analyzed to assess peptide-specific killing. (D) OT-I T cells were isolated using anti-CD8 microbeads and AutoMacs. These cells were labeled with 10 µmol/L CFSE and i.v. transferred into their syngenic mice. On the following day, mice were i.v. injected with T cells manipulated in vitro with indicated conditions. Forty-eight hours later, lymphoid cells from the spleen of the recipient mice were stained with phycoerythrin (PE)-conjugated anti-Vα2 antibody and then analyzed by flow cytometry. For intracellular Granzyme B and IFNγ staining, cells were restimulated with SIINFEKL for 5 h before intracellular staining of these molecules according to manufacturer’s instruction. Data are representative of at least two independent experiments.
Mentions: Our study thus far showed that, with help of αGC, tumors of T cell origin can trigger tumor-specific cytotoxic T cell responses. Therefore we next asked if conventional T cells can also act as antigen presenting cells and trigger cytotoxic T cell responses in vivo, when formulated to present αGC. A previous study by Shimizu et al. showed that T cells, purified by negative selection using magnetic beads and when loaded with αGC, stimulate iNKT cells in vivo.13 To further assess iNKT cell activation by T cells, we first sorted NK1.1−CD8−CD19−CD11b−CD11c−Gr-1−I-Ab−CD4+ cells from the lymphoid cells of C57BL/6 mice. These cells were virtually all CD3+CD1d+, indicating no contamination of professional APC (Fig. 4A). The sorted cells were loaded with αGC (T/αGC) or with vehicle (T/veh) and were i.v. injected into syngenic naïve mice. When we analyzed splenocytes six hours after the injection, we observed that CD1d-tetramer+ cells in mice receiving T/αGC produced IFNγ+ whereas the same population in mice receiving T/veh did not produce IFNγ (Fig. 4B). Therefore CD4+ T cells efficiently triggered iNKT cell activation in vivo when they were manipulated to present αGC.

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