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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 transgenic mice develop increased numbers of CD8+ cells that are M3 restricted and LemA specific. (A) Thymus, spleen, and lymph node cells from nontransgenic (D7Tg−) and D7 transgenic (D7Tg+) B6 animals were stained with FITC–anti-CD8 and APC–M3-LemA tetramers. Percentages of D7 TCR+ cells are indicated from a representative experiment. (B) Splenocytes from D7+TCRα2/− mice were incubated at 5 × 106/ml with 1 μM LemA peptide for 3 d. Ficoll-purified effector cells were tested for cytolytic activity against L929, B10.CAS2 fibroblasts, and an M3wt transfectant (TR8.4a) in the presence (black bars) or absence (hatched bars) of 1 μM LemA peptide at an E/T ratio of 10:1.
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Figure 1: D7 transgenic mice develop increased numbers of CD8+ cells that are M3 restricted and LemA specific. (A) Thymus, spleen, and lymph node cells from nontransgenic (D7Tg−) and D7 transgenic (D7Tg+) B6 animals were stained with FITC–anti-CD8 and APC–M3-LemA tetramers. Percentages of D7 TCR+ cells are indicated from a representative experiment. (B) Splenocytes from D7+TCRα2/− mice were incubated at 5 × 106/ml with 1 μM LemA peptide for 3 d. Ficoll-purified effector cells were tested for cytolytic activity against L929, B10.CAS2 fibroblasts, and an M3wt transfectant (TR8.4a) in the presence (black bars) or absence (hatched bars) of 1 μM LemA peptide at an E/T ratio of 10:1.

Mentions: The D7 hybridoma generated from CTL clone CN.8 was chosen for the construction of TCR transgenic mice 2528. This CTL is CD8+ and specific for a listerial peptide, LemA (f-MIGWII), in the context of M3, and can confer protection from infection by adoptive transfer. Leader and J-C intron primers were designed and used to clone the entire coding sequence (Vα10.2JαD7 and Vβ5.2Jβ2.2) from D7 hybridoma DNA into the TCR cassette vectors 33. Three lines of D7 transgenic mice were generated and bred onto the B6 background. Lymphocytes isolated from thymus, spleen, and lymph nodes of D7 transgenic mice were stained with FITC–anti-CD8, PE–anti-CD4, and APC–M3-LemA tetramer to examine the development of D7+ T cells. FACS® analysis showed that D7+ T cells (M3-LemA tetramer positive) are highly enriched in the CD8 lineage (Fig. 1 A). Compared with nontransgenic controls, the transgenic mice showed an increase in percentage of D7+CD8+ cells (4-fold in thymus, 20-fold in spleen, and 18-fold in lymph node).


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 transgenic mice develop increased numbers of CD8+ cells that are M3 restricted and LemA specific. (A) Thymus, spleen, and lymph node cells from nontransgenic (D7Tg−) and D7 transgenic (D7Tg+) B6 animals were stained with FITC–anti-CD8 and APC–M3-LemA tetramers. Percentages of D7 TCR+ cells are indicated from a representative experiment. (B) Splenocytes from D7+TCRα2/− mice were incubated at 5 × 106/ml with 1 μM LemA peptide for 3 d. Ficoll-purified effector cells were tested for cytolytic activity against L929, B10.CAS2 fibroblasts, and an M3wt transfectant (TR8.4a) in the presence (black bars) or absence (hatched bars) of 1 μM LemA peptide at an E/T ratio of 10:1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195718&req=5

Figure 1: D7 transgenic mice develop increased numbers of CD8+ cells that are M3 restricted and LemA specific. (A) Thymus, spleen, and lymph node cells from nontransgenic (D7Tg−) and D7 transgenic (D7Tg+) B6 animals were stained with FITC–anti-CD8 and APC–M3-LemA tetramers. Percentages of D7 TCR+ cells are indicated from a representative experiment. (B) Splenocytes from D7+TCRα2/− mice were incubated at 5 × 106/ml with 1 μM LemA peptide for 3 d. Ficoll-purified effector cells were tested for cytolytic activity against L929, B10.CAS2 fibroblasts, and an M3wt transfectant (TR8.4a) in the presence (black bars) or absence (hatched bars) of 1 μM LemA peptide at an E/T ratio of 10:1.
Mentions: The D7 hybridoma generated from CTL clone CN.8 was chosen for the construction of TCR transgenic mice 2528. This CTL is CD8+ and specific for a listerial peptide, LemA (f-MIGWII), in the context of M3, and can confer protection from infection by adoptive transfer. Leader and J-C intron primers were designed and used to clone the entire coding sequence (Vα10.2JαD7 and Vβ5.2Jβ2.2) from D7 hybridoma DNA into the TCR cassette vectors 33. Three lines of D7 transgenic mice were generated and bred onto the B6 background. Lymphocytes isolated from thymus, spleen, and lymph nodes of D7 transgenic mice were stained with FITC–anti-CD8, PE–anti-CD4, and APC–M3-LemA tetramer to examine the development of D7+ T cells. FACS® analysis showed that D7+ T cells (M3-LemA tetramer positive) are highly enriched in the CD8 lineage (Fig. 1 A). Compared with nontransgenic controls, the transgenic mice showed an increase in percentage of D7+CD8+ cells (4-fold in thymus, 20-fold in spleen, and 18-fold in lymph node).

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