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, 199 (12), 1607-18

The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells in Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation

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The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells in Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation

Shin-Ichiro Fujii et al. J Exp Med.

Abstract

Dendritic cell (DC) maturation is an innate response that leads to adaptive immunity to coadministered proteins. To begin to identify underlying mechanisms in intact lymphoid tissues, we studied alpha-galactosylceramide. This glycolipid activates innate Valpha14(+) natural killer T cell (NKT) lymphocytes, which drive DC maturation and T cell responses to ovalbumin antigen. Hours after giving glycolipid i.v., tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were released primarily by DCs. These cytokines induced rapid surface remodeling of DCs, including increased CD80/86 costimulatory molecules. Surprisingly, DCs from CD40(-/-) and CD40L(-/-) mice did not elicit CD4(+) and CD8(+) T cell immunity, even though the DCs exhibited presented ovalbumin on major histocompatibility complex class I and II products and expressed high levels of CD80/86. Likewise, an injection of TNF-alpha up-regulated CD80/86 on DCs, but CD40 was required for immunity. CD40 was needed for DC interleukin (IL)-12 production, but IL-12p40(-/-) mice generated normal ovalbumin-specific responses. Therefore, the link between innate and adaptive immunity via splenic DCs and innate NKT cells has several components under distinct controls: antigen presentation in the steady state, increases in costimulatory molecules dependent on inflammatory cytokines, and a distinct CD40/CD40L signal that functions together with antigen presentation ("signal one") and costimulation ("signal two") to generate functioning CD4(+) T helper cell 1 and CD8(+) cytolytic T lymphocytes.

Figures

Figure 1.
Figure 1.
Cytokine production by splenic DCs in response to i.v. α-GalCer (A) Serum IFN-γ and TNF-α levels 1, 2, and 4 h after mice received 2 μg α-GalCer i.v.; no cytokines were detected in mice given vehicle only. (B) Production of cytokines by the indicated subsets (x axis) of splenocytes isolated 2 h after vehicle or α-GalCer i.v., which is before the time that NK cells are activated. (C) FACS® for intracellular cytokines produced by splenic CD11c+ DCs taken from mice 2 h after injection of vehicle or α-GalCer and cultured for 4 h in brefeldin A; numbers are the percentage of CD8+ and CD8 CD11c+ cells producing cytokines. (D and E) Maturation of splenic DCs, as assessed by CD86 expression, after blockade of both TNF-α and IFN-γ. Vehicle or α-GalCer was given to wild-type or TNF-α−/− mice treated with α-IFN-γ antibody or control Ig (300 μg/mouse) 3 h before injection of α-GalCer. Mean values for CD86 expression are shown for three experiments in four groups of mice. All results represent the mean of three or more independent experiments (A, B, and D) or are representative (C and E).
Figure 2.
Figure 2.
Function of DCs in response to IFN-γ and TNF-α. (A) Up-regulation of CD86 expression on splenic DC subsets 4 h after administration of a single low (20 ng IFN-γ and 2 ng TNF-α/mouse), medium (200 ng IFN-γ and 20 ng TNF-α), or high (2 μg IFN-γ and 200 ng TNF-α) dose of TNF-α (left) or a combination of high dose TNF-α and IFN-γ (right). (B) Induction of some CD8+ T cell immunity when OVA-loaded TAP−/− splenocytes were given to mice followed 2 h later by TNF-α (inset). Immunity was monitored at 7 d by formation of IFN-γ–secreting, OVA-specific CD8+ T cells.
Figure 3.
Figure 3.
CD40 is required for the immunogenicity of DCs maturing in response to α-GalCer. (A) CD40−/− or wild-type B6 mice were primed with 2 × 107 OVA-loaded TAP−/− spleen cells plus i.v. α-GalCer 7 d later. Spleen cells were evaluated for the production of IFN-γ in response to restimulation by MHC class I (CD8+ T)– or MHC class II (CD4+ T)–binding OVA peptides. Insets indicate newly formed CD8+ and CD4+ T cells that produced IFN-γ upon OVA peptide reexposure. (B) As in A, except that the mean values for three experiments are shown. (C) Immunization of naive mice with 106 CD11c+ DCs taken from wild-type or CD40−/− mice 4 h after administration of α-GalCer together with TAP−/− OVA-loaded splenocytes. Immunity was monitored 7 d later by enumerating OVA-responsive CD4+ and CD8+ T cells as in A.
Figure 4.
Figure 4.
Several forms of CD4+ and CD8+ T cell priming are blocked in mice lacking CD40, but occur substantially during blockade of TNF-α and IFN-γ (mouse groups defined in the top row of labels). (A) As in Fig. 3 A, mice were immunized with 2 × 107 OVA-loaded TAP−/− spleen cells and i.v. α-GalCer, and 7 d later, antigen (OVA peptide)-responsive, cytokine (IL-2 and IFN-γ)-producing T cells were monitored in spleen and expressed as a percentage of CD4+ (top) or CD8+ (bottom) T cells (insets). (B) As in A, but recall proliferative responses (*) were used to monitor T cell priming. CFSE-labeled spleen cells from the different groups of mice were challenged during 3 d of culture with 500 μg/ml OVA protein. (C) As in A, but the development of cytolytic T cells was monitored 7 d after immunization. The groups of mice were given a mixture of CFSE spleen cells, 107 each loaded with OVA257-264 peptide (high CFSE) or not (low CFSE). 12 h later, killing of antigen-loaded spleen cells was detected (arrow) in wild-type mice and in TNF−/− mice treated with anti–IFN-γ antibody, but not in CD40−/− mice. Data are representative of three individual mice.
Figure 5.
Figure 5.
CD40 ablation does not interfere with efficient DC expression of signal one (antigen presentation) and signal two (CD80/86 costimulation). (A) Presentation of cell-associated OVA to OT-I, CD8+, and OT-II, CD4+ TCR transgenic T cells in wild-type C57BL/6 or CD40−/− mice in the absence or presence of α-GalCer. Total numbers of transgenic T cells (the mean of two experiments) are shown in each panel. (B) As in Fig. 1 E, 8 h after i.v. α-GalCer to C57BL/6 or CD40−/− mice, the maturation of spleen CD11c+ DC subsets was assessed at the level of three surface markers.
Figure 6.
Figure 6.
Requirement for CD80/86 for the adjuvant action of α-GalCer. (A) As in Fig. 1 E, 8 h after i.v. administration of α-GalCer to C57BL/6, CD40−/−, or CD80/86−/− mice, the maturation of spleen CD11c+ DC subsets was assessed at the level of surface markers using α-CD205 and α-CD119 antibodies. (B) As in Fig. 3, CD80/86−/− or wild-type B6 mice were primed with a combination of 2 × 107 OVA-loaded TAP−/− spleen cells and i.v. α-GalCer. 7 d later, spleen cells were evaluated for the production of IFN-γ in response to restimulation by MHC class I (CD8+ T cells)– or MHC class II (CD4+ T cells)–binding OVA peptides. The results (insets) expressed as the percentage of CD4+ or CD8+ T cells producing IFN-γ, are representative of four individual mice.
Figure 7.
Figure 7.
Cytokine and CD40 requirements for increased MLR stimulatory activity. (A) 8 h after i.v. administration of α-GalCer, CD11c+ DCs were used to stimulate T cell proliferation (y axis) in the MLR. Graded numbers of spleen DCs from wild-type B6, TNF-α−/−, or IFN-γ−/− mice were irradiated (30 Gy) and added for 3 d in flat bottomed 96-well plates to 2 × 105 allogeneic BALB/c T cells (Allo MLR, left) and C57BL/6 T cells (Syn MLR, middle). To block DC maturation in the T cell coculture, the DCs were fixed with paraformaldehyde for 30 min and irradiated followed by the addition to allogeneic T cells (right). T cell proliferation was measured by [3H]thymidine incorporation; representative results of three independent experiments are shown. (B) As in A, DCs from mice given TNF-α and IFN-γ i.v. were fixed with paraformaldehyde for 30 min and irradiated followed by the addition of allogeneic T cells, and [3H]thymidine uptake was measured at 88–96 h. (C) As in A, DCs from α-GalCer–treated wild-type and CD40−/− mice were fixed, irradiated, and used to stimulate the MLR.

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