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Peptide-independent recognition by alloreactive cytotoxic T lymphocytes (CTL).

Smith PA, Brunmark A, Jackson MR, Potter TA - J. Exp. Med. (1997)

Bottom Line: Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2(b)) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules.Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2-incompatible skin grafts.These findings provide evidence that not all allorecognition is peptide dependent.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206-2761, USA.

ABSTRACT
We have isolated several H-2K(b)-alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2K(b). These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2K(b) molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2K(b) on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2(b)) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2K(bm8) molecule. These findings suggested that the clones recognized determinants on H-2K(b) that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2K(b) produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2K(b) molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2-incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent.

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(A) Reactivity of alloreactive anti H-2Kb hybridoma 34.9  with non-transfected T2 and T2 cells transfected with H-2Kb. (B) Reactivity of hybridoma 34.9 to immobilized soluble H-2Kb treated with either PBS at pH 7.4 (middle) or 100 μM sodium acetate/acetic acid buffer  at pH 3.0 (right). In both panels, activation was determined by a colorimetric assay with ONPG as the β-galactosidase substrate. Absorbance was  read at 405 nM.
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Figure 9: (A) Reactivity of alloreactive anti H-2Kb hybridoma 34.9 with non-transfected T2 and T2 cells transfected with H-2Kb. (B) Reactivity of hybridoma 34.9 to immobilized soluble H-2Kb treated with either PBS at pH 7.4 (middle) or 100 μM sodium acetate/acetic acid buffer at pH 3.0 (right). In both panels, activation was determined by a colorimetric assay with ONPG as the β-galactosidase substrate. Absorbance was read at 405 nM.

Mentions: An expression system using Drosophila melanogaster cell lines has been used to produce quantities of soluble H-2Kb molecule (39). The H-2Kb isolated from this system are mostly devoid of peptides (39, 40), although some of the H-2Kb molecules contain a peptide derived from the yeast extract used as a supplement in the culture media. This yeast-derived peptide has low affinity for H-2Kb and does not possess the motif found in peptides which bind to H-2Kb with high affinity. To assay the ability of the anti–H-2Kb alloreactive T cells to recognize the H-2Kb produced by Drosophila melanogaster cells, T cell hybridomas were produced. The fusion partner used was a derivative of the BW5147 line transfected with a β-galactosidase reporter gene inserted behind the IL-2 promoter (33). Upon engagement of the TCR, hybridomas prepared using this fusion partner produce β-galactosidase which can be detected by a colormatic assay. Hybridomas were prepared from clones 34 and 59, and both reacted readily with T2 cells transfected with H-2Kb (data for hybridoma 34.9, derived from clone no. 34, shown in Fig. 9 A) and not with T2 cells transfected with the H-2Ld molecule (data not shown).


Peptide-independent recognition by alloreactive cytotoxic T lymphocytes (CTL).

Smith PA, Brunmark A, Jackson MR, Potter TA - J. Exp. Med. (1997)

(A) Reactivity of alloreactive anti H-2Kb hybridoma 34.9  with non-transfected T2 and T2 cells transfected with H-2Kb. (B) Reactivity of hybridoma 34.9 to immobilized soluble H-2Kb treated with either PBS at pH 7.4 (middle) or 100 μM sodium acetate/acetic acid buffer  at pH 3.0 (right). In both panels, activation was determined by a colorimetric assay with ONPG as the β-galactosidase substrate. Absorbance was  read at 405 nM.
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Related In: Results  -  Collection

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Figure 9: (A) Reactivity of alloreactive anti H-2Kb hybridoma 34.9 with non-transfected T2 and T2 cells transfected with H-2Kb. (B) Reactivity of hybridoma 34.9 to immobilized soluble H-2Kb treated with either PBS at pH 7.4 (middle) or 100 μM sodium acetate/acetic acid buffer at pH 3.0 (right). In both panels, activation was determined by a colorimetric assay with ONPG as the β-galactosidase substrate. Absorbance was read at 405 nM.
Mentions: An expression system using Drosophila melanogaster cell lines has been used to produce quantities of soluble H-2Kb molecule (39). The H-2Kb isolated from this system are mostly devoid of peptides (39, 40), although some of the H-2Kb molecules contain a peptide derived from the yeast extract used as a supplement in the culture media. This yeast-derived peptide has low affinity for H-2Kb and does not possess the motif found in peptides which bind to H-2Kb with high affinity. To assay the ability of the anti–H-2Kb alloreactive T cells to recognize the H-2Kb produced by Drosophila melanogaster cells, T cell hybridomas were produced. The fusion partner used was a derivative of the BW5147 line transfected with a β-galactosidase reporter gene inserted behind the IL-2 promoter (33). Upon engagement of the TCR, hybridomas prepared using this fusion partner produce β-galactosidase which can be detected by a colormatic assay. Hybridomas were prepared from clones 34 and 59, and both reacted readily with T2 cells transfected with H-2Kb (data for hybridoma 34.9, derived from clone no. 34, shown in Fig. 9 A) and not with T2 cells transfected with the H-2Ld molecule (data not shown).

Bottom Line: Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2(b)) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules.Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2-incompatible skin grafts.These findings provide evidence that not all allorecognition is peptide dependent.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206-2761, USA.

ABSTRACT
We have isolated several H-2K(b)-alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2K(b). These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2K(b) molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2K(b) on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2(b)) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2K(bm8) molecule. These findings suggested that the clones recognized determinants on H-2K(b) that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2K(b) produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2K(b) molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2-incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent.

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