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, 195 (6), 695-704

Generation of Human CD8 T Regulatory Cells by CD40 Ligand-Activated Plasmacytoid Dendritic Cells

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Generation of Human CD8 T Regulatory Cells by CD40 Ligand-Activated Plasmacytoid Dendritic Cells

Michel Gilliet et al. J Exp Med.

Abstract

Although CD8 T cell-mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand-activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand-activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-gamma upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-gamma, and no IL-4, IL-5, nor transforming growth factor (TGF)-beta. The addition of anti-IL-10-neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10-producing anergic CD8 T cells. IL-10-producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-beta. IL-10-producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10-producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10-producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell-mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.

Figures

Figure 1.
Figure 1.
DC1 and DC2 express similar levels of MHC products and costimulatory molecules, but differ in their ability to produce IL-12. (A) Cell surface expression of HLA-A2, HLA-DR, class II–associated peptides (CLIP), CD80, and CD86 on DC1 and DC2. (B) IL-12p70 in the supernatants of DC1 generated in a 5-d culture with GM-CSF and IL-4 (▪) and DC2 generated in a 5-d culture with IL-3 (•) stimulated at 100,000 cells/ml with CD40 ligand–transfected L cells for 6, 12, 24, and 48 h. Mean value ± SD of two independent experiments are represented. (C) DC1 and DC2 induce strong proliferation of naive CD8 T cells. Naive CD8 T cells were stimulated with allogeneic CD40 ligand–activated DC1 (•) and DC2 (○) and proliferative responses were assessed after 4, 5, 6, or 7 d. Results are expressed as mean cpm ± SD of triplicate wells. Background [3H]thymidine incorporation of CD8 T cells cultured in the absence of DC remained <1,000 cpm at all time points.
Figure 1.
Figure 1.
DC1 and DC2 express similar levels of MHC products and costimulatory molecules, but differ in their ability to produce IL-12. (A) Cell surface expression of HLA-A2, HLA-DR, class II–associated peptides (CLIP), CD80, and CD86 on DC1 and DC2. (B) IL-12p70 in the supernatants of DC1 generated in a 5-d culture with GM-CSF and IL-4 (▪) and DC2 generated in a 5-d culture with IL-3 (•) stimulated at 100,000 cells/ml with CD40 ligand–transfected L cells for 6, 12, 24, and 48 h. Mean value ± SD of two independent experiments are represented. (C) DC1 and DC2 induce strong proliferation of naive CD8 T cells. Naive CD8 T cells were stimulated with allogeneic CD40 ligand–activated DC1 (•) and DC2 (○) and proliferative responses were assessed after 4, 5, 6, or 7 d. Results are expressed as mean cpm ± SD of triplicate wells. Background [3H]thymidine incorporation of CD8 T cells cultured in the absence of DC remained <1,000 cpm at all time points.
Figure 1.
Figure 1.
DC1 and DC2 express similar levels of MHC products and costimulatory molecules, but differ in their ability to produce IL-12. (A) Cell surface expression of HLA-A2, HLA-DR, class II–associated peptides (CLIP), CD80, and CD86 on DC1 and DC2. (B) IL-12p70 in the supernatants of DC1 generated in a 5-d culture with GM-CSF and IL-4 (▪) and DC2 generated in a 5-d culture with IL-3 (•) stimulated at 100,000 cells/ml with CD40 ligand–transfected L cells for 6, 12, 24, and 48 h. Mean value ± SD of two independent experiments are represented. (C) DC1 and DC2 induce strong proliferation of naive CD8 T cells. Naive CD8 T cells were stimulated with allogeneic CD40 ligand–activated DC1 (•) and DC2 (○) and proliferative responses were assessed after 4, 5, 6, or 7 d. Results are expressed as mean cpm ± SD of triplicate wells. Background [3H]thymidine incorporation of CD8 T cells cultured in the absence of DC remained <1,000 cpm at all time points.
Figure 2.
Figure 2.
DC2-primed CD8 T cells exhibit poor cytolytic activity and are hyporesponsive to secondary restimulation. (A) CD8 T cells were primed for 6 d with DC1 (•, ▪) and DC2 (○, □), and collected and tested for their ability to lyse allogeneic (•, ○) or autologous (▪, □) B cells. The results represent six independent experiments. (B) CD8 T cells primed with DC1 and DC2 for 6 d were collected and restimulated with DC1 and DC2 derived from the donor used for priming. CD8 T cell expansion (upper panels), proliferation (middle panels), and increase in cell size (lower panel) were determined after 3 d of restimulation. Histograms represent CD8 T cell size before (filled) and after restimulation (open histograms). The results represent three independent experiments. (C) CD8 T cells from donor A were primed for 6 d with DC1 and DC2 from donor B, and then restimulated with DC1 from donor B or donor C. Proliferation was determined after 3 d of restimulation. (D) IFN-γ and IL-10 in the supernatants of CD8 T cells, restimulated as described in C for 2 d.
Figure 2.
Figure 2.
DC2-primed CD8 T cells exhibit poor cytolytic activity and are hyporesponsive to secondary restimulation. (A) CD8 T cells were primed for 6 d with DC1 (•, ▪) and DC2 (○, □), and collected and tested for their ability to lyse allogeneic (•, ○) or autologous (▪, □) B cells. The results represent six independent experiments. (B) CD8 T cells primed with DC1 and DC2 for 6 d were collected and restimulated with DC1 and DC2 derived from the donor used for priming. CD8 T cell expansion (upper panels), proliferation (middle panels), and increase in cell size (lower panel) were determined after 3 d of restimulation. Histograms represent CD8 T cell size before (filled) and after restimulation (open histograms). The results represent three independent experiments. (C) CD8 T cells from donor A were primed for 6 d with DC1 and DC2 from donor B, and then restimulated with DC1 from donor B or donor C. Proliferation was determined after 3 d of restimulation. (D) IFN-γ and IL-10 in the supernatants of CD8 T cells, restimulated as described in C for 2 d.
Figure 2.
Figure 2.
DC2-primed CD8 T cells exhibit poor cytolytic activity and are hyporesponsive to secondary restimulation. (A) CD8 T cells were primed for 6 d with DC1 (•, ▪) and DC2 (○, □), and collected and tested for their ability to lyse allogeneic (•, ○) or autologous (▪, □) B cells. The results represent six independent experiments. (B) CD8 T cells primed with DC1 and DC2 for 6 d were collected and restimulated with DC1 and DC2 derived from the donor used for priming. CD8 T cell expansion (upper panels), proliferation (middle panels), and increase in cell size (lower panel) were determined after 3 d of restimulation. Histograms represent CD8 T cell size before (filled) and after restimulation (open histograms). The results represent three independent experiments. (C) CD8 T cells from donor A were primed for 6 d with DC1 and DC2 from donor B, and then restimulated with DC1 from donor B or donor C. Proliferation was determined after 3 d of restimulation. (D) IFN-γ and IL-10 in the supernatants of CD8 T cells, restimulated as described in C for 2 d.
Figure 2.
Figure 2.
DC2-primed CD8 T cells exhibit poor cytolytic activity and are hyporesponsive to secondary restimulation. (A) CD8 T cells were primed for 6 d with DC1 (•, ▪) and DC2 (○, □), and collected and tested for their ability to lyse allogeneic (•, ○) or autologous (▪, □) B cells. The results represent six independent experiments. (B) CD8 T cells primed with DC1 and DC2 for 6 d were collected and restimulated with DC1 and DC2 derived from the donor used for priming. CD8 T cell expansion (upper panels), proliferation (middle panels), and increase in cell size (lower panel) were determined after 3 d of restimulation. Histograms represent CD8 T cell size before (filled) and after restimulation (open histograms). The results represent three independent experiments. (C) CD8 T cells from donor A were primed for 6 d with DC1 and DC2 from donor B, and then restimulated with DC1 from donor B or donor C. Proliferation was determined after 3 d of restimulation. (D) IFN-γ and IL-10 in the supernatants of CD8 T cells, restimulated as described in C for 2 d.
Figure 3.
Figure 3.
DC2-primed CD8 T cells produce IL-10 and low levels of IFN-γ. (A) Secretion of IFN-γ, TNF-α, IL-4, IL-5, and IL-10 by CD8 T cells primed for 6 d with DC1, DC2, or anti-CD3 plus anti-CD28, and restimulated with anti-CD3 plus anti-CD28 for 24 h. Each symbol represent an independent experiment. (B) Two-color staining of intracellular IFN-γ and IL-10, or IL-4 and IL-10, produced by DC1- and DC2-primed CD8 T cells after a 5-h restimulation with anti-CD3 plus anti-CD28 (left), or PMA plus ionomycin (middle and right). 104 cells were analyzed and the percentages of each population are indicated in the plots.
Figure 3.
Figure 3.
DC2-primed CD8 T cells produce IL-10 and low levels of IFN-γ. (A) Secretion of IFN-γ, TNF-α, IL-4, IL-5, and IL-10 by CD8 T cells primed for 6 d with DC1, DC2, or anti-CD3 plus anti-CD28, and restimulated with anti-CD3 plus anti-CD28 for 24 h. Each symbol represent an independent experiment. (B) Two-color staining of intracellular IFN-γ and IL-10, or IL-4 and IL-10, produced by DC1- and DC2-primed CD8 T cells after a 5-h restimulation with anti-CD3 plus anti-CD28 (left), or PMA plus ionomycin (middle and right). 104 cells were analyzed and the percentages of each population are indicated in the plots.
Figure 4.
Figure 4.
The generation of IL-10–producing CD8 T cells is not due to the absence of IL-12 nor to DC exhaustion caused by prolonged CD40 ligand activation. (A) Cytokine secretion by CD8 T cells primed for 6 d with DC1 plus neutralizing anti–IL-12, DC2, or DC2 plus IL-12. (B) Cytokine production of CD8 T cells primed for 6 d with DC1 and DC2 activated with CD40 ligand for either 1 d (5 d of GM-CSF/IL-4 or IL-3, respectively, plus an additional day of CD40 ligand), or 6 d (6 d of GM-CSF/IL-4 or IL-3, respectively, in the presence of CD40 ligand).
Figure 4.
Figure 4.
The generation of IL-10–producing CD8 T cells is not due to the absence of IL-12 nor to DC exhaustion caused by prolonged CD40 ligand activation. (A) Cytokine secretion by CD8 T cells primed for 6 d with DC1 plus neutralizing anti–IL-12, DC2, or DC2 plus IL-12. (B) Cytokine production of CD8 T cells primed for 6 d with DC1 and DC2 activated with CD40 ligand for either 1 d (5 d of GM-CSF/IL-4 or IL-3, respectively, plus an additional day of CD40 ligand), or 6 d (6 d of GM-CSF/IL-4 or IL-3, respectively, in the presence of CD40 ligand).
Figure 5.
Figure 5.
IL-10 is required for the generation of IL-10–producing CD8 T cells and mediates the poor cytolytic activity and secondary hyporesponsiveness of DC2-primed CD8 T cells. (A) Cytokine profile of CD8 T cells primed for 6 d with DC1, DC2, or DC2 plus neutralizing anti–IL-10 (10 μg/ml). (B) CD8 T cells primed with DC1 (•), DC2 (○), or DC2 plus anti–IL-10 (□) were tested for the ability to lyse allogeneic B cells in a 5-h Cr-release assay. Specific lysis of autologous B cells remained below 15% in all conditions (unpublished data). (C) Secondary proliferation of CD8 T cells primed with DC1, DC2, or DC2 plus anti–IL-10 after a 3-d restimulation with DC2. Equivalent results were obtained by restimulation with DC1 (unpublished data). Results are representative of three independent experiments.
Figure 5.
Figure 5.
IL-10 is required for the generation of IL-10–producing CD8 T cells and mediates the poor cytolytic activity and secondary hyporesponsiveness of DC2-primed CD8 T cells. (A) Cytokine profile of CD8 T cells primed for 6 d with DC1, DC2, or DC2 plus neutralizing anti–IL-10 (10 μg/ml). (B) CD8 T cells primed with DC1 (•), DC2 (○), or DC2 plus anti–IL-10 (□) were tested for the ability to lyse allogeneic B cells in a 5-h Cr-release assay. Specific lysis of autologous B cells remained below 15% in all conditions (unpublished data). (C) Secondary proliferation of CD8 T cells primed with DC1, DC2, or DC2 plus anti–IL-10 after a 3-d restimulation with DC2. Equivalent results were obtained by restimulation with DC1 (unpublished data). Results are representative of three independent experiments.
Figure 5.
Figure 5.
IL-10 is required for the generation of IL-10–producing CD8 T cells and mediates the poor cytolytic activity and secondary hyporesponsiveness of DC2-primed CD8 T cells. (A) Cytokine profile of CD8 T cells primed for 6 d with DC1, DC2, or DC2 plus neutralizing anti–IL-10 (10 μg/ml). (B) CD8 T cells primed with DC1 (•), DC2 (○), or DC2 plus anti–IL-10 (□) were tested for the ability to lyse allogeneic B cells in a 5-h Cr-release assay. Specific lysis of autologous B cells remained below 15% in all conditions (unpublished data). (C) Secondary proliferation of CD8 T cells primed with DC1, DC2, or DC2 plus anti–IL-10 after a 3-d restimulation with DC2. Equivalent results were obtained by restimulation with DC1 (unpublished data). Results are representative of three independent experiments.
Figure 6.
Figure 6.
DC2-primed CD8 T cells inhibit primary CD8 T cell proliferation induced by various types of APCs, in a dose-dependent manner. (A) Primary CD8 T cell proliferation in response to allogeneic monocytes in the presence of increasing numbers of syngeneic DC2-primed CD8 Tr cells (▪). Primary proliferation of naive CD8 T cells without CD8 Tr (▴) and proliferation of CD8 T regulatory cells (•) are also represented. (B) Proliferation of CD8 T cells stimulated with allogeneic monocytes, (C) immature DC1, or (D) mature DC1 in the presence of equal numbers of CD8 Tr (1:1 ratio). Results are representative of two independent experiments.
Figure 6.
Figure 6.
DC2-primed CD8 T cells inhibit primary CD8 T cell proliferation induced by various types of APCs, in a dose-dependent manner. (A) Primary CD8 T cell proliferation in response to allogeneic monocytes in the presence of increasing numbers of syngeneic DC2-primed CD8 Tr cells (▪). Primary proliferation of naive CD8 T cells without CD8 Tr (▴) and proliferation of CD8 T regulatory cells (•) are also represented. (B) Proliferation of CD8 T cells stimulated with allogeneic monocytes, (C) immature DC1, or (D) mature DC1 in the presence of equal numbers of CD8 Tr (1:1 ratio). Results are representative of two independent experiments.
Figure 6.
Figure 6.
DC2-primed CD8 T cells inhibit primary CD8 T cell proliferation induced by various types of APCs, in a dose-dependent manner. (A) Primary CD8 T cell proliferation in response to allogeneic monocytes in the presence of increasing numbers of syngeneic DC2-primed CD8 Tr cells (▪). Primary proliferation of naive CD8 T cells without CD8 Tr (▴) and proliferation of CD8 T regulatory cells (•) are also represented. (B) Proliferation of CD8 T cells stimulated with allogeneic monocytes, (C) immature DC1, or (D) mature DC1 in the presence of equal numbers of CD8 Tr (1:1 ratio). Results are representative of two independent experiments.
Figure 6.
Figure 6.
DC2-primed CD8 T cells inhibit primary CD8 T cell proliferation induced by various types of APCs, in a dose-dependent manner. (A) Primary CD8 T cell proliferation in response to allogeneic monocytes in the presence of increasing numbers of syngeneic DC2-primed CD8 Tr cells (▪). Primary proliferation of naive CD8 T cells without CD8 Tr (▴) and proliferation of CD8 T regulatory cells (•) are also represented. (B) Proliferation of CD8 T cells stimulated with allogeneic monocytes, (C) immature DC1, or (D) mature DC1 in the presence of equal numbers of CD8 Tr (1:1 ratio). Results are representative of two independent experiments.
Figure 7.
Figure 7.
Suppression is mediated by soluble IL-10 and requires Ag-specific stimulation of IL-10+CD8 Tr cells. (A) 5-d primary CD8+T cell proliferation induced by allogeneic monocytes plus equal numbers of CD8 Tr cells in the presence or absence of neutralizing anti–IL-10 or anti–TGF-β. To determine if IL-10 could display a similar inhibitory effect of CD8+ Tr cells, primary CD8+T cell proliferation induced by allogeneic monocytes was performed in the presence of IL-10, or IL-10 plus anti–IL-10. The stimulation index of naive CD8 T cells was calculated by the following formula: cpm [(naive T + T reg + mono) − (T reg + mono)]/cpm (naive T). Results are representative of three independent experiments. (B) Naive CD8 T cells (donor A) were stimulated with allogeneic monocytes from donor B or C in the presence of CD8 Tr cells primed to donor B or C. Results are representative of two independent experiments. (C) CD8 Tr cells stimulated separately with monocytes were added to the primary cultures of naive CD8 T cells plus monocytes in Transwell chambers. The stimulation index of naive CD8 T cells was calculated by the following formula: cpm (naive T + mono)/cpm (naive T).
Figure 7.
Figure 7.
Suppression is mediated by soluble IL-10 and requires Ag-specific stimulation of IL-10+CD8 Tr cells. (A) 5-d primary CD8+T cell proliferation induced by allogeneic monocytes plus equal numbers of CD8 Tr cells in the presence or absence of neutralizing anti–IL-10 or anti–TGF-β. To determine if IL-10 could display a similar inhibitory effect of CD8+ Tr cells, primary CD8+T cell proliferation induced by allogeneic monocytes was performed in the presence of IL-10, or IL-10 plus anti–IL-10. The stimulation index of naive CD8 T cells was calculated by the following formula: cpm [(naive T + T reg + mono) − (T reg + mono)]/cpm (naive T). Results are representative of three independent experiments. (B) Naive CD8 T cells (donor A) were stimulated with allogeneic monocytes from donor B or C in the presence of CD8 Tr cells primed to donor B or C. Results are representative of two independent experiments. (C) CD8 Tr cells stimulated separately with monocytes were added to the primary cultures of naive CD8 T cells plus monocytes in Transwell chambers. The stimulation index of naive CD8 T cells was calculated by the following formula: cpm (naive T + mono)/cpm (naive T).
Figure 7.
Figure 7.
Suppression is mediated by soluble IL-10 and requires Ag-specific stimulation of IL-10+CD8 Tr cells. (A) 5-d primary CD8+T cell proliferation induced by allogeneic monocytes plus equal numbers of CD8 Tr cells in the presence or absence of neutralizing anti–IL-10 or anti–TGF-β. To determine if IL-10 could display a similar inhibitory effect of CD8+ Tr cells, primary CD8+T cell proliferation induced by allogeneic monocytes was performed in the presence of IL-10, or IL-10 plus anti–IL-10. The stimulation index of naive CD8 T cells was calculated by the following formula: cpm [(naive T + T reg + mono) − (T reg + mono)]/cpm (naive T). Results are representative of three independent experiments. (B) Naive CD8 T cells (donor A) were stimulated with allogeneic monocytes from donor B or C in the presence of CD8 Tr cells primed to donor B or C. Results are representative of two independent experiments. (C) CD8 Tr cells stimulated separately with monocytes were added to the primary cultures of naive CD8 T cells plus monocytes in Transwell chambers. The stimulation index of naive CD8 T cells was calculated by the following formula: cpm (naive T + mono)/cpm (naive T).
Figure 8.
Figure 8.
CD8 Tr cells do not suppress secondary responses. (A) 3-d secondary proliferation and (B) IFN-γ production by DC1-primed CD8 T cells induced by allogeneic monocytes plus CD8 Tr cells (1:1, 1:2, and 1:4 ratio). The stimulation index of naive CD8 T cells was calculated by the following formula: cpm [(DC1 − T + T reg + mono) − (T reg + mono)]/cpm (DC1 − T).
Figure 8.
Figure 8.
CD8 Tr cells do not suppress secondary responses. (A) 3-d secondary proliferation and (B) IFN-γ production by DC1-primed CD8 T cells induced by allogeneic monocytes plus CD8 Tr cells (1:1, 1:2, and 1:4 ratio). The stimulation index of naive CD8 T cells was calculated by the following formula: cpm [(DC1 − T + T reg + mono) − (T reg + mono)]/cpm (DC1 − T).

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