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Priming of Salmonella enterica serovar typhi-specific CD8(+) T cells by suicide dendritic cell cross-presentation in humans.

Salerno-Goncalves R, Sztein MB - PLoS ONE (2009)

Bottom Line: Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.This study is the first to demonstrate the effect of S.Typhi.

View Article: PubMed Central - PubMed

Affiliation: Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, USA. rmezghan@medicine.umaryland.edu

ABSTRACT

Background: The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8(+) T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-gamma secretion. However, how S. Typhi regulates the development of specific CD8(+) responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8(+) T cells.

Methodology/principal findings: We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-alpha, but low levels of IL-12 p70 and IFN-gamma. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.

Conclusions/significance: This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8(+) cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.

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DC priming of S. Typhi-specific T cell responses.PBMC from volunteer CVD4000#63 were co-cultured with DC alone (media), or pre-mixed with live or heat-killed S. Typhi at a MOI of 10∶1, or uninfected or S. Typhi-infected blasts at a 1∶1 blast∶DC ratio. In some cases, DC were pre-treated with ZVA-D or CCD before exposure to S. Typhi or S. Typhi-infected blasts respectively. In other cases, blasts were treated with ZVA-D before co-culture with DC. After 20 hours of incubation, cells were surface stained with a combination of mAb to CD3, CD4, CD8, CD14 and CD19 as well as ViViD. After fixation and permeabilization, cells were intracellularly stained for IL-2, IFN-γ, TNF-α and CD69 and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their light scatter characteristics. Single lymphocytes were gated based on forward scatter height vs. forward scatter area. A “dump” channel was used to eliminate dead cells (ViViD+) as well as CD14+ and CD19+ cells from analysis. This was followed by additional gating on CD3, CD4 and CD8, to identify cytokine-producing CD8+ T cells. Each cytokine was gated individually. Numbers correspond to the percentage of positive cells in the indicated regions in each histogram. These results are representative of 1 of 4 volunteers with similar results. Additional data is provided in Fig. 7.
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pone-0005879-g006: DC priming of S. Typhi-specific T cell responses.PBMC from volunteer CVD4000#63 were co-cultured with DC alone (media), or pre-mixed with live or heat-killed S. Typhi at a MOI of 10∶1, or uninfected or S. Typhi-infected blasts at a 1∶1 blast∶DC ratio. In some cases, DC were pre-treated with ZVA-D or CCD before exposure to S. Typhi or S. Typhi-infected blasts respectively. In other cases, blasts were treated with ZVA-D before co-culture with DC. After 20 hours of incubation, cells were surface stained with a combination of mAb to CD3, CD4, CD8, CD14 and CD19 as well as ViViD. After fixation and permeabilization, cells were intracellularly stained for IL-2, IFN-γ, TNF-α and CD69 and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their light scatter characteristics. Single lymphocytes were gated based on forward scatter height vs. forward scatter area. A “dump” channel was used to eliminate dead cells (ViViD+) as well as CD14+ and CD19+ cells from analysis. This was followed by additional gating on CD3, CD4 and CD8, to identify cytokine-producing CD8+ T cells. Each cytokine was gated individually. Numbers correspond to the percentage of positive cells in the indicated regions in each histogram. These results are representative of 1 of 4 volunteers with similar results. Additional data is provided in Fig. 7.

Mentions: DC from volunteer CVD4000#64 treated or not with ZVA-D or CCD agents were incubated with uninfected or S. Typhi-infected CD45-labeled blasts at a DC∶blast ratio of 1∶5 at 37°C. After 2 hours of incubation, DC were stained with ViViD, followed by surface staining with mAbs to HLA-DR, DC-Sign, Salmonella common structural antigens (CSA) and caspase-3 and analysed by flow cytometry. DC were gated based on their scatter characteristics. Single DC were selected by gating on forward scatter height vs. forward scatter area and then on HLA-DR and DC-Sign. (A) endocytosis and apoptosis on DC was analyzed by studying expression of CD45 and caspase-3, respectively. (B) The presence of S. Typhi antigens on caspase-3+ cells were analyzed by determining their expression of CSA. (C) Viability of DC under different culture conditions was evaluated by ViViD as a dead cell exclusion marker. Dotted lines represent the cut-offs between positive and negative cells. Numbers correspond to the percentage of positive cells in the indicated quadrants or regions in each histogram. These results are representative of 1 of 3 volunteers with similar results. Additional data is provided in Fig. 5.


Priming of Salmonella enterica serovar typhi-specific CD8(+) T cells by suicide dendritic cell cross-presentation in humans.

Salerno-Goncalves R, Sztein MB - PLoS ONE (2009)

DC priming of S. Typhi-specific T cell responses.PBMC from volunteer CVD4000#63 were co-cultured with DC alone (media), or pre-mixed with live or heat-killed S. Typhi at a MOI of 10∶1, or uninfected or S. Typhi-infected blasts at a 1∶1 blast∶DC ratio. In some cases, DC were pre-treated with ZVA-D or CCD before exposure to S. Typhi or S. Typhi-infected blasts respectively. In other cases, blasts were treated with ZVA-D before co-culture with DC. After 20 hours of incubation, cells were surface stained with a combination of mAb to CD3, CD4, CD8, CD14 and CD19 as well as ViViD. After fixation and permeabilization, cells were intracellularly stained for IL-2, IFN-γ, TNF-α and CD69 and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their light scatter characteristics. Single lymphocytes were gated based on forward scatter height vs. forward scatter area. A “dump” channel was used to eliminate dead cells (ViViD+) as well as CD14+ and CD19+ cells from analysis. This was followed by additional gating on CD3, CD4 and CD8, to identify cytokine-producing CD8+ T cells. Each cytokine was gated individually. Numbers correspond to the percentage of positive cells in the indicated regions in each histogram. These results are representative of 1 of 4 volunteers with similar results. Additional data is provided in Fig. 7.
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Related In: Results  -  Collection

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pone-0005879-g006: DC priming of S. Typhi-specific T cell responses.PBMC from volunteer CVD4000#63 were co-cultured with DC alone (media), or pre-mixed with live or heat-killed S. Typhi at a MOI of 10∶1, or uninfected or S. Typhi-infected blasts at a 1∶1 blast∶DC ratio. In some cases, DC were pre-treated with ZVA-D or CCD before exposure to S. Typhi or S. Typhi-infected blasts respectively. In other cases, blasts were treated with ZVA-D before co-culture with DC. After 20 hours of incubation, cells were surface stained with a combination of mAb to CD3, CD4, CD8, CD14 and CD19 as well as ViViD. After fixation and permeabilization, cells were intracellularly stained for IL-2, IFN-γ, TNF-α and CD69 and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their light scatter characteristics. Single lymphocytes were gated based on forward scatter height vs. forward scatter area. A “dump” channel was used to eliminate dead cells (ViViD+) as well as CD14+ and CD19+ cells from analysis. This was followed by additional gating on CD3, CD4 and CD8, to identify cytokine-producing CD8+ T cells. Each cytokine was gated individually. Numbers correspond to the percentage of positive cells in the indicated regions in each histogram. These results are representative of 1 of 4 volunteers with similar results. Additional data is provided in Fig. 7.
Mentions: DC from volunteer CVD4000#64 treated or not with ZVA-D or CCD agents were incubated with uninfected or S. Typhi-infected CD45-labeled blasts at a DC∶blast ratio of 1∶5 at 37°C. After 2 hours of incubation, DC were stained with ViViD, followed by surface staining with mAbs to HLA-DR, DC-Sign, Salmonella common structural antigens (CSA) and caspase-3 and analysed by flow cytometry. DC were gated based on their scatter characteristics. Single DC were selected by gating on forward scatter height vs. forward scatter area and then on HLA-DR and DC-Sign. (A) endocytosis and apoptosis on DC was analyzed by studying expression of CD45 and caspase-3, respectively. (B) The presence of S. Typhi antigens on caspase-3+ cells were analyzed by determining their expression of CSA. (C) Viability of DC under different culture conditions was evaluated by ViViD as a dead cell exclusion marker. Dotted lines represent the cut-offs between positive and negative cells. Numbers correspond to the percentage of positive cells in the indicated quadrants or regions in each histogram. These results are representative of 1 of 3 volunteers with similar results. Additional data is provided in Fig. 5.

Bottom Line: Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.This study is the first to demonstrate the effect of S.Typhi.

View Article: PubMed Central - PubMed

Affiliation: Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, USA. rmezghan@medicine.umaryland.edu

ABSTRACT

Background: The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8(+) T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-gamma secretion. However, how S. Typhi regulates the development of specific CD8(+) responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8(+) T cells.

Methodology/principal findings: We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-alpha, but low levels of IL-12 p70 and IFN-gamma. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells.

Conclusions/significance: This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8(+) cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.

Show MeSH
Related in: MedlinePlus