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The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus.

Chiu NM, Wang B, Kerksiek KM, Kurlander R, Pamer EG, Wang CR - J. Exp. Med. (1999)

Bottom Line: Positive selection was rescued in TAP(-/-) lobes by nonagonist mitochondrial and bacterial peptides, whereas LemA and a partial agonist variant caused negative selection.Thus, M3-restricted CD8(+) T cells are positively and negatively selected by M3, with no contribution from the more abundant class Ia molecules.These results demonstrate that class Ib molecules can function in thymic education like class Ia molecules, despite limited ligand diversity and low levels of expression.

View Article: PubMed Central - PubMed

Affiliation: Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunology, Department of Pathology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
The major histocompatibility complex (MHC) class Ib molecule H2-M3 binds N-formylated peptides from mitochondria and bacteria. To explore the role of M3 expression and peptide supply in positive and negative selection, we generated transgenic mice expressing an M3-restricted TCR-alpha/beta from a CD8(+) T cell hybridoma (D7) specific for a listerial peptide (LemA). Development of M3-restricted transgenic T cells is impaired in both beta2-microglobulin-deficient and transporter associated with antigen processing (TAP)-deficient mice, but is not diminished by changes in the H-2 haplotype. Maturation of M3/LemA-specific CD8(+) single positive cells in fetal thymic organ culture was sensitive to M3 expression levels as determined by antibody blocking and use of the castaneus mutant allele of M3. Positive selection was rescued in TAP(-/-) lobes by nonagonist mitochondrial and bacterial peptides, whereas LemA and a partial agonist variant caused negative selection. Thus, M3-restricted CD8(+) T cells are positively and negatively selected by M3, with no contribution from the more abundant class Ia molecules. These results demonstrate that class Ib molecules can function in thymic education like class Ia molecules, despite limited ligand diversity and low levels of expression.

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D7+CD8sp development is peptide dependent. (A) Thymic lobes from D7+TAP−/−TCRα2/− mice were harvested on gestational day 16.5. One lobe from each animal was incubated with 20 μM of the indicated N-formylated peptide from mitochondria (ND1 and COI) or L. monocytogenes (Fr38). The matching lobe was incubated with an appropriate concentration of solvent alone. After 10 d of culture, thymocytes were stained with CyChrome–anti-CD4, PE–anti-CD8α, and FITC–anti-Vβ5. All CD8sp thymocytes were Vβ5 positive. The plots are representative of experiments from several animals, with the percentage of CD8sp thymocytes indicated. (B) Relative increase in CD8sp cells was calculated for each pair of lobes as follows: % of CD8sp cells in the peptide-treated lobe/% of CD8sp cells in the control lobe. Circles represent pairs of thymic lobes, and the horizontal bar indicates the mean value. For ND1, n = 14; for COI, n = 20; for Fr38, n = 16; and for ND4, n = 6. No significant differences in the total number of thymocytes were observed between peptide-treated lobes and controls. (C) Increase in functional development in peptide treated lobes was calculated in a limiting dilution CTL development assay. After 10 d of culture as above, four or five lobes were pooled, and thymocytes were diluted in series ranging from 10,000 to 3 cells per well and incubated with B6 stimulators, IL-2, and LemA peptide. On day 7, 51Cr-labeled, LemA-coated RMA cells were used as targets for CTL assay. Percent increase in CTL precursor (CTL p) frequency is calculated as follows: [(number of positive wells from peptide-treated lobes/number of positive wells from the control lobes) − 1] × 100. Wells were considered positive if the total counts released were 3 SD above the spontaneous release value.
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Figure 6: D7+CD8sp development is peptide dependent. (A) Thymic lobes from D7+TAP−/−TCRα2/− mice were harvested on gestational day 16.5. One lobe from each animal was incubated with 20 μM of the indicated N-formylated peptide from mitochondria (ND1 and COI) or L. monocytogenes (Fr38). The matching lobe was incubated with an appropriate concentration of solvent alone. After 10 d of culture, thymocytes were stained with CyChrome–anti-CD4, PE–anti-CD8α, and FITC–anti-Vβ5. All CD8sp thymocytes were Vβ5 positive. The plots are representative of experiments from several animals, with the percentage of CD8sp thymocytes indicated. (B) Relative increase in CD8sp cells was calculated for each pair of lobes as follows: % of CD8sp cells in the peptide-treated lobe/% of CD8sp cells in the control lobe. Circles represent pairs of thymic lobes, and the horizontal bar indicates the mean value. For ND1, n = 14; for COI, n = 20; for Fr38, n = 16; and for ND4, n = 6. No significant differences in the total number of thymocytes were observed between peptide-treated lobes and controls. (C) Increase in functional development in peptide treated lobes was calculated in a limiting dilution CTL development assay. After 10 d of culture as above, four or five lobes were pooled, and thymocytes were diluted in series ranging from 10,000 to 3 cells per well and incubated with B6 stimulators, IL-2, and LemA peptide. On day 7, 51Cr-labeled, LemA-coated RMA cells were used as targets for CTL assay. Percent increase in CTL precursor (CTL p) frequency is calculated as follows: [(number of positive wells from peptide-treated lobes/number of positive wells from the control lobes) − 1] × 100. Wells were considered positive if the total counts released were 3 SD above the spontaneous release value.

Mentions: Because of the ability of these N-formylated peptides to stabilize surface expression of M3 on thymic stromal cells, we analyzed the role of each peptide in positive selection. Fig. 6 A shows representative staining of thymocytes developed in FTOC of D7+TCR-α2/−TAP−/− thymic lobes incubated with or without 20 μM peptide for 10 d. Although each peptide was able to stabilize surface expression of M3, the increase in positive selection of CD8sp thymocytes varied between peptides. Fig. 6 B displays the percentage increase in CD8sp thymocytes between pairs of lobes from each animal. The average increase in CD8sp cells was approximately twofold for ND1 (1.85 ± 0.65, n = 14) and COI (1.78 ± 0.60, n = 20), and approximately threefold for Fr38 (2.86 ± 1.15, n = 16). ND4 is a mitochondrial peptide that binds to M3 weakly and is very inefficient at stabilization of M3 on the cell surface. Incubation with ND4 caused no increase in positive selection of transgenic CD8sp thymocytes (1.05 ± 0.31, n = 6).


The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus.

Chiu NM, Wang B, Kerksiek KM, Kurlander R, Pamer EG, Wang CR - J. Exp. Med. (1999)

D7+CD8sp development is peptide dependent. (A) Thymic lobes from D7+TAP−/−TCRα2/− mice were harvested on gestational day 16.5. One lobe from each animal was incubated with 20 μM of the indicated N-formylated peptide from mitochondria (ND1 and COI) or L. monocytogenes (Fr38). The matching lobe was incubated with an appropriate concentration of solvent alone. After 10 d of culture, thymocytes were stained with CyChrome–anti-CD4, PE–anti-CD8α, and FITC–anti-Vβ5. All CD8sp thymocytes were Vβ5 positive. The plots are representative of experiments from several animals, with the percentage of CD8sp thymocytes indicated. (B) Relative increase in CD8sp cells was calculated for each pair of lobes as follows: % of CD8sp cells in the peptide-treated lobe/% of CD8sp cells in the control lobe. Circles represent pairs of thymic lobes, and the horizontal bar indicates the mean value. For ND1, n = 14; for COI, n = 20; for Fr38, n = 16; and for ND4, n = 6. No significant differences in the total number of thymocytes were observed between peptide-treated lobes and controls. (C) Increase in functional development in peptide treated lobes was calculated in a limiting dilution CTL development assay. After 10 d of culture as above, four or five lobes were pooled, and thymocytes were diluted in series ranging from 10,000 to 3 cells per well and incubated with B6 stimulators, IL-2, and LemA peptide. On day 7, 51Cr-labeled, LemA-coated RMA cells were used as targets for CTL assay. Percent increase in CTL precursor (CTL p) frequency is calculated as follows: [(number of positive wells from peptide-treated lobes/number of positive wells from the control lobes) − 1] × 100. Wells were considered positive if the total counts released were 3 SD above the spontaneous release value.
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Related In: Results  -  Collection

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Figure 6: D7+CD8sp development is peptide dependent. (A) Thymic lobes from D7+TAP−/−TCRα2/− mice were harvested on gestational day 16.5. One lobe from each animal was incubated with 20 μM of the indicated N-formylated peptide from mitochondria (ND1 and COI) or L. monocytogenes (Fr38). The matching lobe was incubated with an appropriate concentration of solvent alone. After 10 d of culture, thymocytes were stained with CyChrome–anti-CD4, PE–anti-CD8α, and FITC–anti-Vβ5. All CD8sp thymocytes were Vβ5 positive. The plots are representative of experiments from several animals, with the percentage of CD8sp thymocytes indicated. (B) Relative increase in CD8sp cells was calculated for each pair of lobes as follows: % of CD8sp cells in the peptide-treated lobe/% of CD8sp cells in the control lobe. Circles represent pairs of thymic lobes, and the horizontal bar indicates the mean value. For ND1, n = 14; for COI, n = 20; for Fr38, n = 16; and for ND4, n = 6. No significant differences in the total number of thymocytes were observed between peptide-treated lobes and controls. (C) Increase in functional development in peptide treated lobes was calculated in a limiting dilution CTL development assay. After 10 d of culture as above, four or five lobes were pooled, and thymocytes were diluted in series ranging from 10,000 to 3 cells per well and incubated with B6 stimulators, IL-2, and LemA peptide. On day 7, 51Cr-labeled, LemA-coated RMA cells were used as targets for CTL assay. Percent increase in CTL precursor (CTL p) frequency is calculated as follows: [(number of positive wells from peptide-treated lobes/number of positive wells from the control lobes) − 1] × 100. Wells were considered positive if the total counts released were 3 SD above the spontaneous release value.
Mentions: Because of the ability of these N-formylated peptides to stabilize surface expression of M3 on thymic stromal cells, we analyzed the role of each peptide in positive selection. Fig. 6 A shows representative staining of thymocytes developed in FTOC of D7+TCR-α2/−TAP−/− thymic lobes incubated with or without 20 μM peptide for 10 d. Although each peptide was able to stabilize surface expression of M3, the increase in positive selection of CD8sp thymocytes varied between peptides. Fig. 6 B displays the percentage increase in CD8sp thymocytes between pairs of lobes from each animal. The average increase in CD8sp cells was approximately twofold for ND1 (1.85 ± 0.65, n = 14) and COI (1.78 ± 0.60, n = 20), and approximately threefold for Fr38 (2.86 ± 1.15, n = 16). ND4 is a mitochondrial peptide that binds to M3 weakly and is very inefficient at stabilization of M3 on the cell surface. Incubation with ND4 caused no increase in positive selection of transgenic CD8sp thymocytes (1.05 ± 0.31, n = 6).

Bottom Line: Positive selection was rescued in TAP(-/-) lobes by nonagonist mitochondrial and bacterial peptides, whereas LemA and a partial agonist variant caused negative selection.Thus, M3-restricted CD8(+) T cells are positively and negatively selected by M3, with no contribution from the more abundant class Ia molecules.These results demonstrate that class Ib molecules can function in thymic education like class Ia molecules, despite limited ligand diversity and low levels of expression.

View Article: PubMed Central - PubMed

Affiliation: Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunology, Department of Pathology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
The major histocompatibility complex (MHC) class Ib molecule H2-M3 binds N-formylated peptides from mitochondria and bacteria. To explore the role of M3 expression and peptide supply in positive and negative selection, we generated transgenic mice expressing an M3-restricted TCR-alpha/beta from a CD8(+) T cell hybridoma (D7) specific for a listerial peptide (LemA). Development of M3-restricted transgenic T cells is impaired in both beta2-microglobulin-deficient and transporter associated with antigen processing (TAP)-deficient mice, but is not diminished by changes in the H-2 haplotype. Maturation of M3/LemA-specific CD8(+) single positive cells in fetal thymic organ culture was sensitive to M3 expression levels as determined by antibody blocking and use of the castaneus mutant allele of M3. Positive selection was rescued in TAP(-/-) lobes by nonagonist mitochondrial and bacterial peptides, whereas LemA and a partial agonist variant caused negative selection. Thus, M3-restricted CD8(+) T cells are positively and negatively selected by M3, with no contribution from the more abundant class Ia molecules. These results demonstrate that class Ib molecules can function in thymic education like class Ia molecules, despite limited ligand diversity and low levels of expression.

Show MeSH
Related in: MedlinePlus