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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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Related in: MedlinePlus

DC priming of S. Typhi-specific T cell responses.PBMC from volunteer CVD4000#65 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 and TNF-α and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their 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. During sample acquisition, routinely 300,000-500,000 events were collected in the forward and side scatter (FS/SS) lymphocyte gate. This large number of gated lymphocyte events was necessary to ensure that a sufficient number of positive cells for a defined subset would be collected for each tube analyzed. 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.
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pone-0005879-g007: DC priming of S. Typhi-specific T cell responses.PBMC from volunteer CVD4000#65 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 and TNF-α and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their 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. During sample acquisition, routinely 300,000-500,000 events were collected in the forward and side scatter (FS/SS) lymphocyte gate. This large number of gated lymphocyte events was necessary to ensure that a sufficient number of positive cells for a defined subset would be collected for each tube analyzed. 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.

Mentions: The effect of Salmonella on DC maturation and cytokine production described above, as well as their capacity to induce apoptosis in the infected cells, prompted us to investigate the ability of Salmonella antigen-loaded DC to prime naïve human T cells. Previous studies have shown that by exposing DC to antigen and then to naïve T cells in vitro, it is possible to mimic the process of antigen presentation in vivo [37], [38]. We used this approach to evaluate the ability of DC to either directly (e.g., priming upon uptake and processing of Salmonella) or indirectly (e.g., by cross-priming through a bystander mechanism) present Salmonella antigens to naïve T cells. This was studied by co-culturing ex vivo PBMC from individuals non-exposed to S. Typhi (naïve subjects) with DC that had been pulsed with live or heat-killed S. Typhi at 10∶1 MOI (priming), or pulsed with uninfected or S. Typhi-infected blasts at a 1∶1 blast to DC ratio (cross-priming). After 30 hours of incubation, the S. Typhi specificity of primed PBMC was evaluated by their ability to secrete IL-2, IFN-γ and TNF-α detected as measured by intracellular staining. To avoid infection of DC by live bacteria from dying infected-blasts, blasts were harvested, washed 3 times with RMPI containing gentamicin (100 µg/ml) and incubated for 1 hour at 37°C to kill and remove extracellular bacteria before being added to PBMC. Moreover, to assess the pre-existing background from cross-reactive effector T cells, a control consisting of PBMC co-cultured with the S. Typhi-infected blasts in the absence of DC was also included. Although PBMC in the presence of S. Typhi-infected blasts alone (in the absence of DC) produced moderate levels of cytokines, these levels were considerably lower than those observed in PBMC co-cultured with DC pre-mixed with S. Typhi-infected blasts (Figs. 6 and 7). Thus, these results clearly indicate that DC function as antigen-presenting cells. Four volunteers were studied to assess the relative importance of priming and cross-presentation in the early stages of S. Typhi infection. Results indicate different levels of cytokine production in the various volunteers. Whilst in 2 of 4 volunteers higher induction of cytokine production was observed following DC co-cultured with S. Typhi-infected blasts (Figs. 6 and 7), a third volunteer showed similar levels when PBMC were co-cultured DC exposed to live S. Typhi or with S. Typhi-infected blasts (data not shown), and in the 4th volunteer the cytokine production was higher in cultures of PBMC with DC infected with live S. Typhi (data not shown). Therefore it is likely that both mechanisms, cross- and direct-priming by DC, might be involved in stimulating cytokine production from PBMC.


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#65 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 and TNF-α and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their 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. During sample acquisition, routinely 300,000-500,000 events were collected in the forward and side scatter (FS/SS) lymphocyte gate. This large number of gated lymphocyte events was necessary to ensure that a sufficient number of positive cells for a defined subset would be collected for each tube analyzed. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691582&req=5

pone-0005879-g007: DC priming of S. Typhi-specific T cell responses.PBMC from volunteer CVD4000#65 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 and TNF-α and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their 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. During sample acquisition, routinely 300,000-500,000 events were collected in the forward and side scatter (FS/SS) lymphocyte gate. This large number of gated lymphocyte events was necessary to ensure that a sufficient number of positive cells for a defined subset would be collected for each tube analyzed. 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.
Mentions: The effect of Salmonella on DC maturation and cytokine production described above, as well as their capacity to induce apoptosis in the infected cells, prompted us to investigate the ability of Salmonella antigen-loaded DC to prime naïve human T cells. Previous studies have shown that by exposing DC to antigen and then to naïve T cells in vitro, it is possible to mimic the process of antigen presentation in vivo [37], [38]. We used this approach to evaluate the ability of DC to either directly (e.g., priming upon uptake and processing of Salmonella) or indirectly (e.g., by cross-priming through a bystander mechanism) present Salmonella antigens to naïve T cells. This was studied by co-culturing ex vivo PBMC from individuals non-exposed to S. Typhi (naïve subjects) with DC that had been pulsed with live or heat-killed S. Typhi at 10∶1 MOI (priming), or pulsed with uninfected or S. Typhi-infected blasts at a 1∶1 blast to DC ratio (cross-priming). After 30 hours of incubation, the S. Typhi specificity of primed PBMC was evaluated by their ability to secrete IL-2, IFN-γ and TNF-α detected as measured by intracellular staining. To avoid infection of DC by live bacteria from dying infected-blasts, blasts were harvested, washed 3 times with RMPI containing gentamicin (100 µg/ml) and incubated for 1 hour at 37°C to kill and remove extracellular bacteria before being added to PBMC. Moreover, to assess the pre-existing background from cross-reactive effector T cells, a control consisting of PBMC co-cultured with the S. Typhi-infected blasts in the absence of DC was also included. Although PBMC in the presence of S. Typhi-infected blasts alone (in the absence of DC) produced moderate levels of cytokines, these levels were considerably lower than those observed in PBMC co-cultured with DC pre-mixed with S. Typhi-infected blasts (Figs. 6 and 7). Thus, these results clearly indicate that DC function as antigen-presenting cells. Four volunteers were studied to assess the relative importance of priming and cross-presentation in the early stages of S. Typhi infection. Results indicate different levels of cytokine production in the various volunteers. Whilst in 2 of 4 volunteers higher induction of cytokine production was observed following DC co-cultured with S. Typhi-infected blasts (Figs. 6 and 7), a third volunteer showed similar levels when PBMC were co-cultured DC exposed to live S. Typhi or with S. Typhi-infected blasts (data not shown), and in the 4th volunteer the cytokine production was higher in cultures of PBMC with DC infected with live S. Typhi (data not shown). Therefore it is likely that both mechanisms, cross- and direct-priming by DC, might be involved in stimulating cytokine production from PBMC.

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