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Selection of antigen-specific T cells by a single IEk peptide combination.

Liu CP, Parker D, Kappler J, Marrack P - J. Exp. Med. (1997)

Bottom Line: Some of the TCRs on the selected T cells were related to those on cells from normal mice and some were not.IEk bound to the Hb variant, on the other hand, did not select any T cells which could react with IEk + MCC.These results demonstrate that although positive selection is a partially degenerate event, the sequence of the peptide involved in positive selection controls the selected repertoire.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

ABSTRACT
In normal mice, major histocompatibility complex (MHC) proteins are bound to many different peptides, derived from the proteins of their host. In the thymus, the diversity of this collection of MHC + peptide ligands allows thymocytes bearing many different T cell receptors (TCRs) to mature by low avidity reactions between the MHC + peptide ligands and the thymocyte TCRs. To investigate this problem, the selection of T cells specific for a well-studied combination of MHC + peptide, IEk + moth cytochrome c 88-103 (MCC), was investigated. Mice were created that expressed IEk bound to a single peptide, either a variant of MCC in which a critical TCR contact residue, 99K, was changed to A, or a variant of a mouse hemoglobin 64-76 (Hb) peptide, 72A. IEk bound to the MCC variant caused the clonal deletion of some T cells specific for the IEk + MCC ligand; nevertheless, it also positively selected many T cells that could react with this ligand. Some of the TCRs on the selected T cells were related to those on cells from normal mice and some were not. IEk bound to the Hb variant, on the other hand, did not select any T cells which could react with IEk + MCC. These results demonstrate that although positive selection is a partially degenerate event, the sequence of the peptide involved in positive selection controls the selected repertoire.

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(A) IEk expression on transgenic thymus cells. Large cells from the thymuses of 72ATg and 99ATg and control B10.BR and Tg− mice were  prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of thymus cells from the various mice overlaid on the  stained background control of Tg− thymus cells. (B) IEk expression on transgenic spleen cells. Spleen cells from 72ATg, 99ATg, IEwtTg, and IEwtTg Ii+  mice and from control B10.BR and Tg− animals were prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of the spleen cells from the various types of mice overlaid on the stained background control of Tg− spleen cells is shown. For both A and B, the results are representative of at least three independent experiments.
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Figure 1: (A) IEk expression on transgenic thymus cells. Large cells from the thymuses of 72ATg and 99ATg and control B10.BR and Tg− mice were prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of thymus cells from the various mice overlaid on the stained background control of Tg− thymus cells. (B) IEk expression on transgenic spleen cells. Spleen cells from 72ATg, 99ATg, IEwtTg, and IEwtTg Ii+ mice and from control B10.BR and Tg− animals were prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of the spleen cells from the various types of mice overlaid on the stained background control of Tg− spleen cells is shown. For both A and B, the results are representative of at least three independent experiments.

Mentions: Chimeric and normal mice were primed with MCC in complete Freund's adjuvant in the base of the tail. T cell hybridomas were prepared from these animals as previously described (28). In brief, lymph node cells were harvested from the draining nodes of the immunization 7 d later. The cells were incubated for 4 d with MCC and then live cells were purified and cultured for 3 more d with saturating amounts of IL-2. The live cells were then fused to an α−β− variant of BW5147, BWα−β− (28). Hybridomas were assayed for their ability to react with IEk in the presence or absence of added MCC. Presenting cells were B10.BR spleen cells which express IEk at high levels, or cells from IEwt transgenic mice, which express IEk at low levels (Fig. 1). All hybridomas used for further analysis expressed high levels of TCR and CD4.


Selection of antigen-specific T cells by a single IEk peptide combination.

Liu CP, Parker D, Kappler J, Marrack P - J. Exp. Med. (1997)

(A) IEk expression on transgenic thymus cells. Large cells from the thymuses of 72ATg and 99ATg and control B10.BR and Tg− mice were  prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of thymus cells from the various mice overlaid on the  stained background control of Tg− thymus cells. (B) IEk expression on transgenic spleen cells. Spleen cells from 72ATg, 99ATg, IEwtTg, and IEwtTg Ii+  mice and from control B10.BR and Tg− animals were prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of the spleen cells from the various types of mice overlaid on the stained background control of Tg− spleen cells is shown. For both A and B, the results are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199109&req=5

Figure 1: (A) IEk expression on transgenic thymus cells. Large cells from the thymuses of 72ATg and 99ATg and control B10.BR and Tg− mice were prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of thymus cells from the various mice overlaid on the stained background control of Tg− thymus cells. (B) IEk expression on transgenic spleen cells. Spleen cells from 72ATg, 99ATg, IEwtTg, and IEwtTg Ii+ mice and from control B10.BR and Tg− animals were prepared and stained with anti-IEk as described in Materials and Methods. Here, the staining profile of the spleen cells from the various types of mice overlaid on the stained background control of Tg− spleen cells is shown. For both A and B, the results are representative of at least three independent experiments.
Mentions: Chimeric and normal mice were primed with MCC in complete Freund's adjuvant in the base of the tail. T cell hybridomas were prepared from these animals as previously described (28). In brief, lymph node cells were harvested from the draining nodes of the immunization 7 d later. The cells were incubated for 4 d with MCC and then live cells were purified and cultured for 3 more d with saturating amounts of IL-2. The live cells were then fused to an α−β− variant of BW5147, BWα−β− (28). Hybridomas were assayed for their ability to react with IEk in the presence or absence of added MCC. Presenting cells were B10.BR spleen cells which express IEk at high levels, or cells from IEwt transgenic mice, which express IEk at low levels (Fig. 1). All hybridomas used for further analysis expressed high levels of TCR and CD4.

Bottom Line: Some of the TCRs on the selected T cells were related to those on cells from normal mice and some were not.IEk bound to the Hb variant, on the other hand, did not select any T cells which could react with IEk + MCC.These results demonstrate that although positive selection is a partially degenerate event, the sequence of the peptide involved in positive selection controls the selected repertoire.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

ABSTRACT
In normal mice, major histocompatibility complex (MHC) proteins are bound to many different peptides, derived from the proteins of their host. In the thymus, the diversity of this collection of MHC + peptide ligands allows thymocytes bearing many different T cell receptors (TCRs) to mature by low avidity reactions between the MHC + peptide ligands and the thymocyte TCRs. To investigate this problem, the selection of T cells specific for a well-studied combination of MHC + peptide, IEk + moth cytochrome c 88-103 (MCC), was investigated. Mice were created that expressed IEk bound to a single peptide, either a variant of MCC in which a critical TCR contact residue, 99K, was changed to A, or a variant of a mouse hemoglobin 64-76 (Hb) peptide, 72A. IEk bound to the MCC variant caused the clonal deletion of some T cells specific for the IEk + MCC ligand; nevertheless, it also positively selected many T cells that could react with this ligand. Some of the TCRs on the selected T cells were related to those on cells from normal mice and some were not. IEk bound to the Hb variant, on the other hand, did not select any T cells which could react with IEk + MCC. These results demonstrate that although positive selection is a partially degenerate event, the sequence of the peptide involved in positive selection controls the selected repertoire.

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