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Immature dendritic cells acquire CD8(+) cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli.

Schuurhuis DH, Laban S, Toes RE, Ricciardi-Castagnoli P, Kleijmeer MJ, van der Voort EI, Rea D, Offringa R, Geuze HJ, Melief CJ, Ossendorp F - J. Exp. Med. (2000)

Bottom Line: The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo.Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells.Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

ABSTRACT
The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.

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Induction of primary peptide-specific CTL responses in vivo by D1 cells treated with agonistic CD40 antibody or LPS. Immature D1 cells (A) and FGK45- (B) or LPS-treated (C) D1 cells were used for in vivo CTL induction by loading them with E1ACTL peptide and injecting them into CD4-depleted B6 mice. Three mice were injected with immature D1 cells, and four mice were injected with FGK45- or LPS-treated D1 cells. After 10 d, 5 × 106 spleen cells were restimulated in vitro using 5 × 105 Ad5E1-MECs. After 6 d, cells were used in a cytotoxicity assay using targets loaded with E1ACTL peptide or E7CTL peptide as control. Data shown in the top panels are means of triplicates from one representative experiment out of three experiments performed. The bottom panels show detection of E1ACTL-specific CD8+ cells in bulk cultures. Bulk cultures were analyzed for the presence of CD8+ cells capable of interacting with the H-2Db–E1ACTL tetrameric complexes. Indicated are percentages of the CD8+ cells staining with H-2Db–E1ACTL tetramers.
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Figure 3: Induction of primary peptide-specific CTL responses in vivo by D1 cells treated with agonistic CD40 antibody or LPS. Immature D1 cells (A) and FGK45- (B) or LPS-treated (C) D1 cells were used for in vivo CTL induction by loading them with E1ACTL peptide and injecting them into CD4-depleted B6 mice. Three mice were injected with immature D1 cells, and four mice were injected with FGK45- or LPS-treated D1 cells. After 10 d, 5 × 106 spleen cells were restimulated in vitro using 5 × 105 Ad5E1-MECs. After 6 d, cells were used in a cytotoxicity assay using targets loaded with E1ACTL peptide or E7CTL peptide as control. Data shown in the top panels are means of triplicates from one representative experiment out of three experiments performed. The bottom panels show detection of E1ACTL-specific CD8+ cells in bulk cultures. Bulk cultures were analyzed for the presence of CD8+ cells capable of interacting with the H-2Db–E1ACTL tetrameric complexes. Indicated are percentages of the CD8+ cells staining with H-2Db–E1ACTL tetramers.

Mentions: Next, we investigated whether the phenotypic and functional differences between immature and activated D1 cells as observed in vitro had consequences for their capacity to prime specific CTL immunity in vivo. Immature D1 cells, exogenously loaded with a human adenovirus CTL epitope (E1ACTL), were injected into CD4-depleted mice. 10 d after immunization, spleens were harvested and splenocytes were restimulated in vitro with Ad5E1-MECs. After 6 d, epitope-specific CTL activity was measured in a cytotoxicity assay. E1ACTL peptide–loaded immature D1 cells did not prime peptide-specific CTLs in CD4-depleted mice (Fig. 3 A and 4 C), in agreement with our recent study 15 describing the lack of Ad5E1A-specific CTL priming in the absence of CD4+ T cell help. However, in this study, CTL priming was restored by in vivo CD40 triggering, which is thought to activate bone marrow–derived APCs in vivo. Therefore, we studied whether in vitro–activated D1 cells, using agonistic CD40 antibody or LPS as stimuli 16, were able to induce priming of peptide-specific CTLs in vivo. In contrast to immature D1 cells (Fig. 3 A, top panel), D1 cells activated in vitro by FGK45 (Fig. 3 B, top panel) or LPS (Fig. 3 C, top panel) indeed primed E1ACTL-specific CTLs in vivo in CD4-depleted mice. The induction of E1ACTL-specific cytotoxicity correlated with elevated numbers of CD8+ T cells in the bulk cultures that stained with PE-conjugated H-2Db tetramers containing the E1ACTL peptide (Db/E1A) (Fig. 3, bottom panel).


Immature dendritic cells acquire CD8(+) cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli.

Schuurhuis DH, Laban S, Toes RE, Ricciardi-Castagnoli P, Kleijmeer MJ, van der Voort EI, Rea D, Offringa R, Geuze HJ, Melief CJ, Ossendorp F - J. Exp. Med. (2000)

Induction of primary peptide-specific CTL responses in vivo by D1 cells treated with agonistic CD40 antibody or LPS. Immature D1 cells (A) and FGK45- (B) or LPS-treated (C) D1 cells were used for in vivo CTL induction by loading them with E1ACTL peptide and injecting them into CD4-depleted B6 mice. Three mice were injected with immature D1 cells, and four mice were injected with FGK45- or LPS-treated D1 cells. After 10 d, 5 × 106 spleen cells were restimulated in vitro using 5 × 105 Ad5E1-MECs. After 6 d, cells were used in a cytotoxicity assay using targets loaded with E1ACTL peptide or E7CTL peptide as control. Data shown in the top panels are means of triplicates from one representative experiment out of three experiments performed. The bottom panels show detection of E1ACTL-specific CD8+ cells in bulk cultures. Bulk cultures were analyzed for the presence of CD8+ cells capable of interacting with the H-2Db–E1ACTL tetrameric complexes. Indicated are percentages of the CD8+ cells staining with H-2Db–E1ACTL tetramers.
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Related In: Results  -  Collection

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Figure 3: Induction of primary peptide-specific CTL responses in vivo by D1 cells treated with agonistic CD40 antibody or LPS. Immature D1 cells (A) and FGK45- (B) or LPS-treated (C) D1 cells were used for in vivo CTL induction by loading them with E1ACTL peptide and injecting them into CD4-depleted B6 mice. Three mice were injected with immature D1 cells, and four mice were injected with FGK45- or LPS-treated D1 cells. After 10 d, 5 × 106 spleen cells were restimulated in vitro using 5 × 105 Ad5E1-MECs. After 6 d, cells were used in a cytotoxicity assay using targets loaded with E1ACTL peptide or E7CTL peptide as control. Data shown in the top panels are means of triplicates from one representative experiment out of three experiments performed. The bottom panels show detection of E1ACTL-specific CD8+ cells in bulk cultures. Bulk cultures were analyzed for the presence of CD8+ cells capable of interacting with the H-2Db–E1ACTL tetrameric complexes. Indicated are percentages of the CD8+ cells staining with H-2Db–E1ACTL tetramers.
Mentions: Next, we investigated whether the phenotypic and functional differences between immature and activated D1 cells as observed in vitro had consequences for their capacity to prime specific CTL immunity in vivo. Immature D1 cells, exogenously loaded with a human adenovirus CTL epitope (E1ACTL), were injected into CD4-depleted mice. 10 d after immunization, spleens were harvested and splenocytes were restimulated in vitro with Ad5E1-MECs. After 6 d, epitope-specific CTL activity was measured in a cytotoxicity assay. E1ACTL peptide–loaded immature D1 cells did not prime peptide-specific CTLs in CD4-depleted mice (Fig. 3 A and 4 C), in agreement with our recent study 15 describing the lack of Ad5E1A-specific CTL priming in the absence of CD4+ T cell help. However, in this study, CTL priming was restored by in vivo CD40 triggering, which is thought to activate bone marrow–derived APCs in vivo. Therefore, we studied whether in vitro–activated D1 cells, using agonistic CD40 antibody or LPS as stimuli 16, were able to induce priming of peptide-specific CTLs in vivo. In contrast to immature D1 cells (Fig. 3 A, top panel), D1 cells activated in vitro by FGK45 (Fig. 3 B, top panel) or LPS (Fig. 3 C, top panel) indeed primed E1ACTL-specific CTLs in vivo in CD4-depleted mice. The induction of E1ACTL-specific cytotoxicity correlated with elevated numbers of CD8+ T cells in the bulk cultures that stained with PE-conjugated H-2Db tetramers containing the E1ACTL peptide (Db/E1A) (Fig. 3, bottom panel).

Bottom Line: The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo.Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells.Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

ABSTRACT
The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.

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