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HLA-E-dependent presentation of Mtb-derived antigen to human CD8+ T cells.

Heinzel AS, Grotzke JE, Lines RA, Lewinsohn DA, McNabb AL, Streblow DN, Braud VM, Grieser HJ, Belisle JT, Lewinsohn DM - J. Exp. Med. (2002)

Bottom Line: In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule.This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen.Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary & Critical Care Medicine, Portland VA Medical Center, Oregon Health Sciences University, Portland, OR 97201, USA.

ABSTRACT
Previous studies in mice and humans have suggested an important role for CD8+ T cells in host defense to Mtb. Recently, we have described human, Mtb-specific CD8+ cells that are neither HLA-A, B, or C nor group 1 CD1 restricted, and have found that these cells comprise the dominant CD8+ T cell response in latently infected individuals. In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule. This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen. Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.

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Mtb-derived antigen in Mtb/DC-conditioned media. (A) To assay the activity of the supernatant, DCs were seeded at 5 × 104 cells per well in 96-well, flat-bottomed plates in 50 μl of media. 5 × 104 T cells in 50 μl of media and 100 μl of conditioned media were then added, and IFN-γ assayed by ELISA after 18 h incubation at 37°C. Results represent the mean of duplicate determinations. Error bars denote the SEM. *P < 0.05, where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Control DC Supe). This experiment is representative of three such experiments. (B) Where indicated, Mtb/DC-conditioned media was treated with either lipase (3,000 U/ml L-8525, Sigma-Aldrich), DNase (2 U/ml D5793, Sigma-Aldrich), or proteinase K (0.2 mg/ml P-0390, Sigma-Aldrich). After overnight incubation, PMSF (60 μg/ml; Sigma-Aldrich) was added to all samples to inactivate the proteinase K. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote a significant loss of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (No Treatment). This experiment is representative of two such experiments. (C) To determine whether or not proteasomal function is required for the generation of the peptide antigen, DCs were incubated with lactacystin (40 μM; E.J. Corey; Harvard Biolabs, Harvard, MA) added either before or 18 h after the addition of Mtb. T cell–dependent IFN-γ release was assessed as described in A. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote significant inhibition of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Mtb/DC Supe). This experiment is representative of three such experiments.
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fig1: Mtb-derived antigen in Mtb/DC-conditioned media. (A) To assay the activity of the supernatant, DCs were seeded at 5 × 104 cells per well in 96-well, flat-bottomed plates in 50 μl of media. 5 × 104 T cells in 50 μl of media and 100 μl of conditioned media were then added, and IFN-γ assayed by ELISA after 18 h incubation at 37°C. Results represent the mean of duplicate determinations. Error bars denote the SEM. *P < 0.05, where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Control DC Supe). This experiment is representative of three such experiments. (B) Where indicated, Mtb/DC-conditioned media was treated with either lipase (3,000 U/ml L-8525, Sigma-Aldrich), DNase (2 U/ml D5793, Sigma-Aldrich), or proteinase K (0.2 mg/ml P-0390, Sigma-Aldrich). After overnight incubation, PMSF (60 μg/ml; Sigma-Aldrich) was added to all samples to inactivate the proteinase K. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote a significant loss of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (No Treatment). This experiment is representative of two such experiments. (C) To determine whether or not proteasomal function is required for the generation of the peptide antigen, DCs were incubated with lactacystin (40 μM; E.J. Corey; Harvard Biolabs, Harvard, MA) added either before or 18 h after the addition of Mtb. T cell–dependent IFN-γ release was assessed as described in A. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote significant inhibition of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Mtb/DC Supe). This experiment is representative of three such experiments.

Mentions: To test this hypothesis, human DCs were infected overnight with Mtb (H37Rv; MOI = 50), and the resulting supernatants twice filtered through a 0.2-μ filter to remove viable bacteria. These supernatants were then used to sensitize fresh DCs, and reactivity of the nonclassical CD8+ clones assessed by IFN-γ release. Mtb/DC-conditioned media was able to elicit T cell–dependent cytokine release (Fig. 1 a).


HLA-E-dependent presentation of Mtb-derived antigen to human CD8+ T cells.

Heinzel AS, Grotzke JE, Lines RA, Lewinsohn DA, McNabb AL, Streblow DN, Braud VM, Grieser HJ, Belisle JT, Lewinsohn DM - J. Exp. Med. (2002)

Mtb-derived antigen in Mtb/DC-conditioned media. (A) To assay the activity of the supernatant, DCs were seeded at 5 × 104 cells per well in 96-well, flat-bottomed plates in 50 μl of media. 5 × 104 T cells in 50 μl of media and 100 μl of conditioned media were then added, and IFN-γ assayed by ELISA after 18 h incubation at 37°C. Results represent the mean of duplicate determinations. Error bars denote the SEM. *P < 0.05, where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Control DC Supe). This experiment is representative of three such experiments. (B) Where indicated, Mtb/DC-conditioned media was treated with either lipase (3,000 U/ml L-8525, Sigma-Aldrich), DNase (2 U/ml D5793, Sigma-Aldrich), or proteinase K (0.2 mg/ml P-0390, Sigma-Aldrich). After overnight incubation, PMSF (60 μg/ml; Sigma-Aldrich) was added to all samples to inactivate the proteinase K. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote a significant loss of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (No Treatment). This experiment is representative of two such experiments. (C) To determine whether or not proteasomal function is required for the generation of the peptide antigen, DCs were incubated with lactacystin (40 μM; E.J. Corey; Harvard Biolabs, Harvard, MA) added either before or 18 h after the addition of Mtb. T cell–dependent IFN-γ release was assessed as described in A. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote significant inhibition of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Mtb/DC Supe). This experiment is representative of three such experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194265&req=5

fig1: Mtb-derived antigen in Mtb/DC-conditioned media. (A) To assay the activity of the supernatant, DCs were seeded at 5 × 104 cells per well in 96-well, flat-bottomed plates in 50 μl of media. 5 × 104 T cells in 50 μl of media and 100 μl of conditioned media were then added, and IFN-γ assayed by ELISA after 18 h incubation at 37°C. Results represent the mean of duplicate determinations. Error bars denote the SEM. *P < 0.05, where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Control DC Supe). This experiment is representative of three such experiments. (B) Where indicated, Mtb/DC-conditioned media was treated with either lipase (3,000 U/ml L-8525, Sigma-Aldrich), DNase (2 U/ml D5793, Sigma-Aldrich), or proteinase K (0.2 mg/ml P-0390, Sigma-Aldrich). After overnight incubation, PMSF (60 μg/ml; Sigma-Aldrich) was added to all samples to inactivate the proteinase K. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote a significant loss of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (No Treatment). This experiment is representative of two such experiments. (C) To determine whether or not proteasomal function is required for the generation of the peptide antigen, DCs were incubated with lactacystin (40 μM; E.J. Corey; Harvard Biolabs, Harvard, MA) added either before or 18 h after the addition of Mtb. T cell–dependent IFN-γ release was assessed as described in A. Results represent the mean of duplicate determinations. Error bars denote the SEM. Asterisks denote significant inhibition of antigenic activity (P < 0.05) where the “Dunnett's test” has been used to compare each experimental mean with the control mean (T plus Mtb/DC Supe). This experiment is representative of three such experiments.
Mentions: To test this hypothesis, human DCs were infected overnight with Mtb (H37Rv; MOI = 50), and the resulting supernatants twice filtered through a 0.2-μ filter to remove viable bacteria. These supernatants were then used to sensitize fresh DCs, and reactivity of the nonclassical CD8+ clones assessed by IFN-γ release. Mtb/DC-conditioned media was able to elicit T cell–dependent cytokine release (Fig. 1 a).

Bottom Line: In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule.This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen.Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary & Critical Care Medicine, Portland VA Medical Center, Oregon Health Sciences University, Portland, OR 97201, USA.

ABSTRACT
Previous studies in mice and humans have suggested an important role for CD8+ T cells in host defense to Mtb. Recently, we have described human, Mtb-specific CD8+ cells that are neither HLA-A, B, or C nor group 1 CD1 restricted, and have found that these cells comprise the dominant CD8+ T cell response in latently infected individuals. In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule. This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen. Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.

Show MeSH
Related in: MedlinePlus