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Tumor rejection induced by CD70-mediated quantitative and qualitative effects on effector CD8+ T cell formation.

Arens R, Schepers K, Nolte MA, van Oosterwijk MF, van Lier RA, Schumacher TN, van Oers MH - J. Exp. Med. (2004)

Bottom Line: However, it is unclear to what extent CD8+ T cell instruction in vivo is modulated by costimulatory signals.Concomitantly, the quality of antigen-specific T cells improved as evidenced by increased interferon (IFN)-gamma production and a greater cytotoxic potential on a per cell basis.Thus, CD70 costimulation enhances both the expansion and per cell activity of antigen-specific CD8+ T cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Immunology, Academic Medical Center, University of Amsterdam, Netherlands.

ABSTRACT
In vivo priming of antigen-specific CD8+ T cells results in their expansion and differentiation into effector T cells followed by contraction into a memory T cell population that can be maintained for life. Recent evidence suggests that after initial antigenic stimulation, the magnitude and kinetics of the CD8+ T cell response are programmed. However, it is unclear to what extent CD8+ T cell instruction in vivo is modulated by costimulatory signals. Here, we demonstrate that constitutive ligation of the tumor necrosis factor receptor family member CD27 by its ligand CD70 quantitatively augments CD8+ T cell responses to influenza virus infection and EL-4 tumor challenge in vivo by incrementing initial expansion and maintaining higher numbers of antigen-specific T cells in the memory phase. Concomitantly, the quality of antigen-specific T cells improved as evidenced by increased interferon (IFN)-gamma production and a greater cytotoxic potential on a per cell basis. As an apparent consequence, the superior effector T cell formation induced by CD70 protected against a lethal dose of poorly immunogenic EL4 tumor cells in a CD8+ T cell- and IFN-gamma-dependent manner. Thus, CD70 costimulation enhances both the expansion and per cell activity of antigen-specific CD8+ T cells.

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Related in: MedlinePlus

Increased IFN-γ production and cytotoxic activity in CD70 Tg mice. Phenotypic analysis of splenic CD8+ T cells of wild-type and CD70 Tg mice collected 12 d after challenge with 106 EL4-NP tumor cells. (A) Intracellular IFN-γ and TNF-α staining of NP366-374-specific CD8+ T cells. Intracellular IFN-γ and TNF-α levels were measured in spleen cells after 5 h of incubation in the presence or absence of NP366-374 peptide. The percentage and MFI of IFN-γ+ and TNF+ cells within the CD8+ gate are indicated. Background (no peptide) was <0.2%. (B) Cytotoxic activity of NP366-374-specific CD8+ T cells. Target cells (EL4 cells) were fluorescently labeled, pulsed with NP366-374 peptide or unpulsed, and subsequently cocultured with effector cells (NP366-374-specific CD8+ T cells) at different effector to target cell ratios in which effector populations from wild-type and CD70 Tg mice were equalized based on the percentage of NP-specific cells. Killing of target cells was assessed by a flow cytometric CTL assay detecting the induction of caspase activity in target cells. Histograms are gated on EL4 target cells, and the numbers indicate the percentage caspase positive cells. The solid and dotted lines represent peptide-pulsed or unpulsed target cells, respectively. (C) Measurement of cytotoxicity as described in B with different effector to target cell ratios. Data are displayed as mean and standard error (n = 5). (D) Ex vivo intracellular granzyme B staining. CD8+ T cells and NP366-374-specific CD8+ T cells were stained for CD43 and intracellular granzyme B. Gated CD8+ and NP366-374-specific CD8+ T cells are shown. The numbers indicate the percentages of cells within the designated quadrant and are representative of five mice.
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fig5: Increased IFN-γ production and cytotoxic activity in CD70 Tg mice. Phenotypic analysis of splenic CD8+ T cells of wild-type and CD70 Tg mice collected 12 d after challenge with 106 EL4-NP tumor cells. (A) Intracellular IFN-γ and TNF-α staining of NP366-374-specific CD8+ T cells. Intracellular IFN-γ and TNF-α levels were measured in spleen cells after 5 h of incubation in the presence or absence of NP366-374 peptide. The percentage and MFI of IFN-γ+ and TNF+ cells within the CD8+ gate are indicated. Background (no peptide) was <0.2%. (B) Cytotoxic activity of NP366-374-specific CD8+ T cells. Target cells (EL4 cells) were fluorescently labeled, pulsed with NP366-374 peptide or unpulsed, and subsequently cocultured with effector cells (NP366-374-specific CD8+ T cells) at different effector to target cell ratios in which effector populations from wild-type and CD70 Tg mice were equalized based on the percentage of NP-specific cells. Killing of target cells was assessed by a flow cytometric CTL assay detecting the induction of caspase activity in target cells. Histograms are gated on EL4 target cells, and the numbers indicate the percentage caspase positive cells. The solid and dotted lines represent peptide-pulsed or unpulsed target cells, respectively. (C) Measurement of cytotoxicity as described in B with different effector to target cell ratios. Data are displayed as mean and standard error (n = 5). (D) Ex vivo intracellular granzyme B staining. CD8+ T cells and NP366-374-specific CD8+ T cells were stained for CD43 and intracellular granzyme B. Gated CD8+ and NP366-374-specific CD8+ T cells are shown. The numbers indicate the percentages of cells within the designated quadrant and are representative of five mice.

Mentions: It is unconfirmed whether signals transmitted via TNF receptor family members improve effector T cell responses in a qualitative fashion (36). To assess whether CD27 stimulation contributes to the competence of antigen-specific T cells, we analyzed the acquisition of effector cell properties of NP366-374-specific T cells directly ex vivo. At the peak of the CD8+ T cell response to EL4-NP tumor cells, splenic NP366-374-specific CD8+ T cells were analyzed for their capacity to produce IFN-γ and TNF-α after in vitro stimulation with NP366-374 peptide. The percentage of splenic CD8+ T cells producing IFN-γ after NP366-374 peptide stimulation was significantly increased in CD70 Tg mice (4.1 ± 1.0% in wild-type vs. 7.2 ± 1.2% in CD70 Tg mice; n = 5, P < 0.05). This may in large part be explained by the increased size of the antigen-specific CD8+ T cell compartment. Remarkably, the IFN-γ production on a per cell basis was reproducibly higher (Fig. 5 A, 316 ± 68 mean fluorescence intensity (MFI) wild-type vs. 582 ± 98 MFI CD70 Tg mice; n = 5, P < 0.05). Constitutive CD27 ligation had no effect on TNF-α production in CD8+ T cells (Fig. 5 A). The contribution of CD27–CD70 interaction to the development of cytotoxic activity to NP366-374 peptide–loaded EL4 cells was examined using a recently described flow cytometric CTL assay (34). On a per cell basis, the ex vivo cytotoxicity of splenic NP-specific CD8+ T cells was enhanced in CD70 Tg mice as compared with wild-type mice (Fig. 5, B and C). Furthermore, the increase in cytotoxic activity in T cells of CD70 Tg mice was associated with augmented granzyme B expression in both CD8+ T cells and NP-specific CD8+ T cells in terms of intensity and percentages (Fig. 5 D). As predicted, granzyme B expression was primarily found in the CD43+ effector T cell subset. Also, after influenza infection, splenic antigen-specific CD8+ T cells in CD70 Tg mice had increased IFN-γ expression and cytotoxic activity (unpublished data). Thus, apart from increasing the size of the effector T cell pool, CD27 stimulation enhances the capacity of CD8+ effector T cells to produce IFN-γ and to execute cytolysis of antigen-bearing target cells.


Tumor rejection induced by CD70-mediated quantitative and qualitative effects on effector CD8+ T cell formation.

Arens R, Schepers K, Nolte MA, van Oosterwijk MF, van Lier RA, Schumacher TN, van Oers MH - J. Exp. Med. (2004)

Increased IFN-γ production and cytotoxic activity in CD70 Tg mice. Phenotypic analysis of splenic CD8+ T cells of wild-type and CD70 Tg mice collected 12 d after challenge with 106 EL4-NP tumor cells. (A) Intracellular IFN-γ and TNF-α staining of NP366-374-specific CD8+ T cells. Intracellular IFN-γ and TNF-α levels were measured in spleen cells after 5 h of incubation in the presence or absence of NP366-374 peptide. The percentage and MFI of IFN-γ+ and TNF+ cells within the CD8+ gate are indicated. Background (no peptide) was <0.2%. (B) Cytotoxic activity of NP366-374-specific CD8+ T cells. Target cells (EL4 cells) were fluorescently labeled, pulsed with NP366-374 peptide or unpulsed, and subsequently cocultured with effector cells (NP366-374-specific CD8+ T cells) at different effector to target cell ratios in which effector populations from wild-type and CD70 Tg mice were equalized based on the percentage of NP-specific cells. Killing of target cells was assessed by a flow cytometric CTL assay detecting the induction of caspase activity in target cells. Histograms are gated on EL4 target cells, and the numbers indicate the percentage caspase positive cells. The solid and dotted lines represent peptide-pulsed or unpulsed target cells, respectively. (C) Measurement of cytotoxicity as described in B with different effector to target cell ratios. Data are displayed as mean and standard error (n = 5). (D) Ex vivo intracellular granzyme B staining. CD8+ T cells and NP366-374-specific CD8+ T cells were stained for CD43 and intracellular granzyme B. Gated CD8+ and NP366-374-specific CD8+ T cells are shown. The numbers indicate the percentages of cells within the designated quadrant and are representative of five mice.
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Related In: Results  -  Collection

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fig5: Increased IFN-γ production and cytotoxic activity in CD70 Tg mice. Phenotypic analysis of splenic CD8+ T cells of wild-type and CD70 Tg mice collected 12 d after challenge with 106 EL4-NP tumor cells. (A) Intracellular IFN-γ and TNF-α staining of NP366-374-specific CD8+ T cells. Intracellular IFN-γ and TNF-α levels were measured in spleen cells after 5 h of incubation in the presence or absence of NP366-374 peptide. The percentage and MFI of IFN-γ+ and TNF+ cells within the CD8+ gate are indicated. Background (no peptide) was <0.2%. (B) Cytotoxic activity of NP366-374-specific CD8+ T cells. Target cells (EL4 cells) were fluorescently labeled, pulsed with NP366-374 peptide or unpulsed, and subsequently cocultured with effector cells (NP366-374-specific CD8+ T cells) at different effector to target cell ratios in which effector populations from wild-type and CD70 Tg mice were equalized based on the percentage of NP-specific cells. Killing of target cells was assessed by a flow cytometric CTL assay detecting the induction of caspase activity in target cells. Histograms are gated on EL4 target cells, and the numbers indicate the percentage caspase positive cells. The solid and dotted lines represent peptide-pulsed or unpulsed target cells, respectively. (C) Measurement of cytotoxicity as described in B with different effector to target cell ratios. Data are displayed as mean and standard error (n = 5). (D) Ex vivo intracellular granzyme B staining. CD8+ T cells and NP366-374-specific CD8+ T cells were stained for CD43 and intracellular granzyme B. Gated CD8+ and NP366-374-specific CD8+ T cells are shown. The numbers indicate the percentages of cells within the designated quadrant and are representative of five mice.
Mentions: It is unconfirmed whether signals transmitted via TNF receptor family members improve effector T cell responses in a qualitative fashion (36). To assess whether CD27 stimulation contributes to the competence of antigen-specific T cells, we analyzed the acquisition of effector cell properties of NP366-374-specific T cells directly ex vivo. At the peak of the CD8+ T cell response to EL4-NP tumor cells, splenic NP366-374-specific CD8+ T cells were analyzed for their capacity to produce IFN-γ and TNF-α after in vitro stimulation with NP366-374 peptide. The percentage of splenic CD8+ T cells producing IFN-γ after NP366-374 peptide stimulation was significantly increased in CD70 Tg mice (4.1 ± 1.0% in wild-type vs. 7.2 ± 1.2% in CD70 Tg mice; n = 5, P < 0.05). This may in large part be explained by the increased size of the antigen-specific CD8+ T cell compartment. Remarkably, the IFN-γ production on a per cell basis was reproducibly higher (Fig. 5 A, 316 ± 68 mean fluorescence intensity (MFI) wild-type vs. 582 ± 98 MFI CD70 Tg mice; n = 5, P < 0.05). Constitutive CD27 ligation had no effect on TNF-α production in CD8+ T cells (Fig. 5 A). The contribution of CD27–CD70 interaction to the development of cytotoxic activity to NP366-374 peptide–loaded EL4 cells was examined using a recently described flow cytometric CTL assay (34). On a per cell basis, the ex vivo cytotoxicity of splenic NP-specific CD8+ T cells was enhanced in CD70 Tg mice as compared with wild-type mice (Fig. 5, B and C). Furthermore, the increase in cytotoxic activity in T cells of CD70 Tg mice was associated with augmented granzyme B expression in both CD8+ T cells and NP-specific CD8+ T cells in terms of intensity and percentages (Fig. 5 D). As predicted, granzyme B expression was primarily found in the CD43+ effector T cell subset. Also, after influenza infection, splenic antigen-specific CD8+ T cells in CD70 Tg mice had increased IFN-γ expression and cytotoxic activity (unpublished data). Thus, apart from increasing the size of the effector T cell pool, CD27 stimulation enhances the capacity of CD8+ effector T cells to produce IFN-γ and to execute cytolysis of antigen-bearing target cells.

Bottom Line: However, it is unclear to what extent CD8+ T cell instruction in vivo is modulated by costimulatory signals.Concomitantly, the quality of antigen-specific T cells improved as evidenced by increased interferon (IFN)-gamma production and a greater cytotoxic potential on a per cell basis.Thus, CD70 costimulation enhances both the expansion and per cell activity of antigen-specific CD8+ T cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Immunology, Academic Medical Center, University of Amsterdam, Netherlands.

ABSTRACT
In vivo priming of antigen-specific CD8+ T cells results in their expansion and differentiation into effector T cells followed by contraction into a memory T cell population that can be maintained for life. Recent evidence suggests that after initial antigenic stimulation, the magnitude and kinetics of the CD8+ T cell response are programmed. However, it is unclear to what extent CD8+ T cell instruction in vivo is modulated by costimulatory signals. Here, we demonstrate that constitutive ligation of the tumor necrosis factor receptor family member CD27 by its ligand CD70 quantitatively augments CD8+ T cell responses to influenza virus infection and EL-4 tumor challenge in vivo by incrementing initial expansion and maintaining higher numbers of antigen-specific T cells in the memory phase. Concomitantly, the quality of antigen-specific T cells improved as evidenced by increased interferon (IFN)-gamma production and a greater cytotoxic potential on a per cell basis. As an apparent consequence, the superior effector T cell formation induced by CD70 protected against a lethal dose of poorly immunogenic EL4 tumor cells in a CD8+ T cell- and IFN-gamma-dependent manner. Thus, CD70 costimulation enhances both the expansion and per cell activity of antigen-specific CD8+ T cells.

Show MeSH
Related in: MedlinePlus