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Listeriolysin o is strongly immunogenic independently of its cytotoxic activity.

Carrero JA, Vivanco-Cid H, Unanue ER - PLoS ONE (2012)

Bottom Line: Mutations of two key tryptophan residues reduced LLO toxicity by 10-100-fold but had no effect on its presentation to CD4 T cells.LLO was also immunogenic after in vivo administration into mice.Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, United States of America. jacarrer@wustl.edu

ABSTRACT
The presentation of microbial protein antigens by Major Histocompatibility Complex (MHC) molecules is essential for the development of acquired immunity to infections. However, most biochemical studies of antigen processing and presentation deal with a few relatively inert non-microbial model antigens. The bacterial pore-forming toxin listeriolysin O (LLO) is paradoxical in that it is cytotoxic at nanomolar concentrations as well as being the source of dominant CD4 and CD8 T cell epitopes following infection with Listeria monocytogenes. Here, we examined the relationship of LLO toxicity to its antigenicity and immunogenicity. LLO offered to antigen presenting cells (APC) as a soluble protein, was presented to CD4 T cells at picomolar to femtomolar concentrations- doses 3000-7000-fold lower than free peptide. This presentation required a dose of LLO below the cytotoxic level. Mutations of two key tryptophan residues reduced LLO toxicity by 10-100-fold but had no effect on its presentation to CD4 T cells. Thus there was a clear dissociation between the cytotoxic properties of LLO and its very high antigenicity. Presentation of LLO to CD8 T cells was not as robust as that seen in CD4 T cells, but still occurred in the nanomolar range. APC rapidly bound and internalized LLO, then disrupted endosomal compartments within 4 hours of treatment, allowing endosomal contents to access the cytosol. LLO was also immunogenic after in vivo administration into mice. Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells.

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Binding, uptake and catabolism of radiolabeled LLO.A. BMM (106) were treated with ∼150 ng of 125I-LLO or HEL for 30 min. on ice. The amount of CPM bound to cells or found in the supernatant was measured and a percent of total bound to cells was calculated. B.125I-LLO, LLOW492A, or HEL (∼50 ng) were given to BMM for 15, 30, or 60 min. and the amount of incorporation of radioactivity was measured. Values are plotted as a percent of total input radioactivity that was taken by the cells. C–D. BMM (106) were treated with 50 ng 125I labeled LLOWT (C) or LLOW492A (D) for the indicated times. Then, TCA precipitation was performed and the amount of total radioactivity present in the precipitate (ppt) or supernatant (sol) was measured. The ppt fraction contains large polypeptides and represents the non-catabolized fraction and the sol fraction contains small peptides and free amino acids and represents the catabolized fraction. Similar uptake and catabolism results were obtained with BMDC. Each experiment in (A–D) is representative of at least two independent experiments performed in triplicate. E–F. BMM (105) were incubated with LLO(190–201 peptide), LLOWT, or LLOW492A at the indicated antigen concentrations for 15 (E), 30 (F), 60 (G) or 120 (H) min. BMM were fixed with PFA, quenched with lysine, washed and then LLO specific 57-3 T cell hybridoma (5×104) was added for ∼16 hr. T cell activation was determined by CTLL-2 assay. Experiment is representative of 2 independent experiments performed in triplicate. Similar results were obtained with BMDC.
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pone-0032310-g005: Binding, uptake and catabolism of radiolabeled LLO.A. BMM (106) were treated with ∼150 ng of 125I-LLO or HEL for 30 min. on ice. The amount of CPM bound to cells or found in the supernatant was measured and a percent of total bound to cells was calculated. B.125I-LLO, LLOW492A, or HEL (∼50 ng) were given to BMM for 15, 30, or 60 min. and the amount of incorporation of radioactivity was measured. Values are plotted as a percent of total input radioactivity that was taken by the cells. C–D. BMM (106) were treated with 50 ng 125I labeled LLOWT (C) or LLOW492A (D) for the indicated times. Then, TCA precipitation was performed and the amount of total radioactivity present in the precipitate (ppt) or supernatant (sol) was measured. The ppt fraction contains large polypeptides and represents the non-catabolized fraction and the sol fraction contains small peptides and free amino acids and represents the catabolized fraction. Similar uptake and catabolism results were obtained with BMDC. Each experiment in (A–D) is representative of at least two independent experiments performed in triplicate. E–F. BMM (105) were incubated with LLO(190–201 peptide), LLOWT, or LLOW492A at the indicated antigen concentrations for 15 (E), 30 (F), 60 (G) or 120 (H) min. BMM were fixed with PFA, quenched with lysine, washed and then LLO specific 57-3 T cell hybridoma (5×104) was added for ∼16 hr. T cell activation was determined by CTLL-2 assay. Experiment is representative of 2 independent experiments performed in triplicate. Similar results were obtained with BMDC.

Mentions: The uptake and catabolism of LLO was examined in order to relate it to its antigenicity. LLO was labeled with 125Iodine and its binding, uptake, and catabolism by APC was evaluated. Radiolabeling did not affect the hemolytic activity of LLOWT, LLOW492A, or LLOWW (data not shown). All LLO preparations bound to BMM incubated on ice, at the levels shown in Figure 5A, ranging from 2–4% of input cpm. In contrast the proteins HEL-125I bound to BMM at ∼0.297% of input. As noted the mutation of tryptophans reduced, but did not prevent LLO from binding to cells. LLO-125I was internalized rapidly by BMM (Fig. 5B) and BMDC (data not shown) following incubation at 37°C. About 40–50% of total input LLO was found as cell-associated protein within 30 minutes. There was ∼15–20% reduction in total internalization of LLOW492A (Fig. 5B) or LLOWW (not shown) compared to LLOWT. In contrast, HEL-125I was internalized at ∼0.8% of total input protein. LLOWT was rapidly catabolized, with ∼20% of total 125I counts found in the TCA soluble fraction of the cell culture media within 1 hour of treatment (Fig. 5C). By 24 hr., >80% of total 125I counts were found in the soluble fraction. Similar rates of catabolism were detected with LLOW492A (Fig. 5D) and LLOWW (not shown).


Listeriolysin o is strongly immunogenic independently of its cytotoxic activity.

Carrero JA, Vivanco-Cid H, Unanue ER - PLoS ONE (2012)

Binding, uptake and catabolism of radiolabeled LLO.A. BMM (106) were treated with ∼150 ng of 125I-LLO or HEL for 30 min. on ice. The amount of CPM bound to cells or found in the supernatant was measured and a percent of total bound to cells was calculated. B.125I-LLO, LLOW492A, or HEL (∼50 ng) were given to BMM for 15, 30, or 60 min. and the amount of incorporation of radioactivity was measured. Values are plotted as a percent of total input radioactivity that was taken by the cells. C–D. BMM (106) were treated with 50 ng 125I labeled LLOWT (C) or LLOW492A (D) for the indicated times. Then, TCA precipitation was performed and the amount of total radioactivity present in the precipitate (ppt) or supernatant (sol) was measured. The ppt fraction contains large polypeptides and represents the non-catabolized fraction and the sol fraction contains small peptides and free amino acids and represents the catabolized fraction. Similar uptake and catabolism results were obtained with BMDC. Each experiment in (A–D) is representative of at least two independent experiments performed in triplicate. E–F. BMM (105) were incubated with LLO(190–201 peptide), LLOWT, or LLOW492A at the indicated antigen concentrations for 15 (E), 30 (F), 60 (G) or 120 (H) min. BMM were fixed with PFA, quenched with lysine, washed and then LLO specific 57-3 T cell hybridoma (5×104) was added for ∼16 hr. T cell activation was determined by CTLL-2 assay. Experiment is representative of 2 independent experiments performed in triplicate. Similar results were obtained with BMDC.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3293810&req=5

pone-0032310-g005: Binding, uptake and catabolism of radiolabeled LLO.A. BMM (106) were treated with ∼150 ng of 125I-LLO or HEL for 30 min. on ice. The amount of CPM bound to cells or found in the supernatant was measured and a percent of total bound to cells was calculated. B.125I-LLO, LLOW492A, or HEL (∼50 ng) were given to BMM for 15, 30, or 60 min. and the amount of incorporation of radioactivity was measured. Values are plotted as a percent of total input radioactivity that was taken by the cells. C–D. BMM (106) were treated with 50 ng 125I labeled LLOWT (C) or LLOW492A (D) for the indicated times. Then, TCA precipitation was performed and the amount of total radioactivity present in the precipitate (ppt) or supernatant (sol) was measured. The ppt fraction contains large polypeptides and represents the non-catabolized fraction and the sol fraction contains small peptides and free amino acids and represents the catabolized fraction. Similar uptake and catabolism results were obtained with BMDC. Each experiment in (A–D) is representative of at least two independent experiments performed in triplicate. E–F. BMM (105) were incubated with LLO(190–201 peptide), LLOWT, or LLOW492A at the indicated antigen concentrations for 15 (E), 30 (F), 60 (G) or 120 (H) min. BMM were fixed with PFA, quenched with lysine, washed and then LLO specific 57-3 T cell hybridoma (5×104) was added for ∼16 hr. T cell activation was determined by CTLL-2 assay. Experiment is representative of 2 independent experiments performed in triplicate. Similar results were obtained with BMDC.
Mentions: The uptake and catabolism of LLO was examined in order to relate it to its antigenicity. LLO was labeled with 125Iodine and its binding, uptake, and catabolism by APC was evaluated. Radiolabeling did not affect the hemolytic activity of LLOWT, LLOW492A, or LLOWW (data not shown). All LLO preparations bound to BMM incubated on ice, at the levels shown in Figure 5A, ranging from 2–4% of input cpm. In contrast the proteins HEL-125I bound to BMM at ∼0.297% of input. As noted the mutation of tryptophans reduced, but did not prevent LLO from binding to cells. LLO-125I was internalized rapidly by BMM (Fig. 5B) and BMDC (data not shown) following incubation at 37°C. About 40–50% of total input LLO was found as cell-associated protein within 30 minutes. There was ∼15–20% reduction in total internalization of LLOW492A (Fig. 5B) or LLOWW (not shown) compared to LLOWT. In contrast, HEL-125I was internalized at ∼0.8% of total input protein. LLOWT was rapidly catabolized, with ∼20% of total 125I counts found in the TCA soluble fraction of the cell culture media within 1 hour of treatment (Fig. 5C). By 24 hr., >80% of total 125I counts were found in the soluble fraction. Similar rates of catabolism were detected with LLOW492A (Fig. 5D) and LLOWW (not shown).

Bottom Line: Mutations of two key tryptophan residues reduced LLO toxicity by 10-100-fold but had no effect on its presentation to CD4 T cells.LLO was also immunogenic after in vivo administration into mice.Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, United States of America. jacarrer@wustl.edu

ABSTRACT
The presentation of microbial protein antigens by Major Histocompatibility Complex (MHC) molecules is essential for the development of acquired immunity to infections. However, most biochemical studies of antigen processing and presentation deal with a few relatively inert non-microbial model antigens. The bacterial pore-forming toxin listeriolysin O (LLO) is paradoxical in that it is cytotoxic at nanomolar concentrations as well as being the source of dominant CD4 and CD8 T cell epitopes following infection with Listeria monocytogenes. Here, we examined the relationship of LLO toxicity to its antigenicity and immunogenicity. LLO offered to antigen presenting cells (APC) as a soluble protein, was presented to CD4 T cells at picomolar to femtomolar concentrations- doses 3000-7000-fold lower than free peptide. This presentation required a dose of LLO below the cytotoxic level. Mutations of two key tryptophan residues reduced LLO toxicity by 10-100-fold but had no effect on its presentation to CD4 T cells. Thus there was a clear dissociation between the cytotoxic properties of LLO and its very high antigenicity. Presentation of LLO to CD8 T cells was not as robust as that seen in CD4 T cells, but still occurred in the nanomolar range. APC rapidly bound and internalized LLO, then disrupted endosomal compartments within 4 hours of treatment, allowing endosomal contents to access the cytosol. LLO was also immunogenic after in vivo administration into mice. Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells.

Show MeSH
Related in: MedlinePlus