Limits...
The efficiency of CD4 recruitment to ligand-engaged TCR controls the agonist/partial agonist properties of peptide-MHC molecule ligands.

Madrenas J, Chau LA, Smith J, Bluestone JA, Germain RN - J. Exp. Med. (1997)

Bottom Line: Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern.These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR-ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide-MHC molecule ligands.Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment ofimmunological tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of Western Ontario, London, Canada.

ABSTRACT
One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation ofligand from an engaged TCR and (b) recruitment oflck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide-MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-zeta, reduced tyrosine phosphorylation of CD3epsilon, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR-ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide-MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment ofimmunological tolerance.

Show MeSH

Related in: MedlinePlus

The IL-2 response of 3C6 T cells to mutant I-Ek is selectively antagonized by the addition of PCC(81–104). (a) Tyrosine phosphorylation analysis of proteins in CD3ε immunoprecipitates from 3C6  cells stimulated with increasing concentrations of agonist (PCC[81–104]– I-Ek) or antagonist (PCC[81–104]–mutant I-E) ligands. T cells were stimulated for 10 min with APC plus PCC(81–104) and CD3ε immunoprecipitates (9 × 106 cell equivalents/lane) were electrophoresed and  immunoblotted with an anti-phosphotyrosine antibody. (b) 3C6 T cells (5 ×  104) were stimulated with mitomycin C–treated, wild-type I-Ek, or mutant I-E-transfected L cells and PCC(81–104) (100 μM). Production of  IL-2 and IL-3 was measured in 24-h supernatants by ELISA. Crosshatched bars show IL-2 and closed bars show IL-3.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196122&req=5

Figure 1: The IL-2 response of 3C6 T cells to mutant I-Ek is selectively antagonized by the addition of PCC(81–104). (a) Tyrosine phosphorylation analysis of proteins in CD3ε immunoprecipitates from 3C6 cells stimulated with increasing concentrations of agonist (PCC[81–104]– I-Ek) or antagonist (PCC[81–104]–mutant I-E) ligands. T cells were stimulated for 10 min with APC plus PCC(81–104) and CD3ε immunoprecipitates (9 × 106 cell equivalents/lane) were electrophoresed and immunoblotted with an anti-phosphotyrosine antibody. (b) 3C6 T cells (5 × 104) were stimulated with mitomycin C–treated, wild-type I-Ek, or mutant I-E-transfected L cells and PCC(81–104) (100 μM). Production of IL-2 and IL-3 was measured in 24-h supernatants by ELISA. Crosshatched bars show IL-2 and closed bars show IL-3.

Mentions: We have previously reported that 3C6 proliferates and secretes both IL-2 and IL-3 in response to a mutant I-Ek molecule agonist expressed on transfected L cell APC (7). Addition of PCC(81–104) to the culture results in the formation of an altered TCR ligand consisting of the PCC peptide and the mutant I-E molecule (7). The pattern of protein tyrosine phosphorylation in anti-CD3ε immunoprecipitates is different for 3C6 cells exposed to an agonist consisting of wild-type I-Ek and PCC(81-104) or to this peptide–mutant MHC class II combination (2). The former is characterized by the presence of similar levels of p21 and p23 tyrosine-phosphorylated forms of TCR-ζ, tyrosine-phosphorylated CD3ε, and kinase-active tyrosinephosphorylated ZAP-70. In contrast, the latter leads to the predominant presence of the p21 tyrosine phosphorylated form of TCR-ζ, little or no tyrosine phosphorylated CD3ε, and the presence of ZAP-70 that is neither detectably phosphorylated nor kinase active, as reported previously (2) and shown here (Fig. 1 a). This PCC peptide–mutant I-E molecule ligand selectively antagonizes IL-2 production, while also acting as a partial agonist capable of modestly stimulating IL-3 production (7) (Fig. 1 b).


The efficiency of CD4 recruitment to ligand-engaged TCR controls the agonist/partial agonist properties of peptide-MHC molecule ligands.

Madrenas J, Chau LA, Smith J, Bluestone JA, Germain RN - J. Exp. Med. (1997)

The IL-2 response of 3C6 T cells to mutant I-Ek is selectively antagonized by the addition of PCC(81–104). (a) Tyrosine phosphorylation analysis of proteins in CD3ε immunoprecipitates from 3C6  cells stimulated with increasing concentrations of agonist (PCC[81–104]– I-Ek) or antagonist (PCC[81–104]–mutant I-E) ligands. T cells were stimulated for 10 min with APC plus PCC(81–104) and CD3ε immunoprecipitates (9 × 106 cell equivalents/lane) were electrophoresed and  immunoblotted with an anti-phosphotyrosine antibody. (b) 3C6 T cells (5 ×  104) were stimulated with mitomycin C–treated, wild-type I-Ek, or mutant I-E-transfected L cells and PCC(81–104) (100 μM). Production of  IL-2 and IL-3 was measured in 24-h supernatants by ELISA. Crosshatched bars show IL-2 and closed bars show IL-3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196122&req=5

Figure 1: The IL-2 response of 3C6 T cells to mutant I-Ek is selectively antagonized by the addition of PCC(81–104). (a) Tyrosine phosphorylation analysis of proteins in CD3ε immunoprecipitates from 3C6 cells stimulated with increasing concentrations of agonist (PCC[81–104]– I-Ek) or antagonist (PCC[81–104]–mutant I-E) ligands. T cells were stimulated for 10 min with APC plus PCC(81–104) and CD3ε immunoprecipitates (9 × 106 cell equivalents/lane) were electrophoresed and immunoblotted with an anti-phosphotyrosine antibody. (b) 3C6 T cells (5 × 104) were stimulated with mitomycin C–treated, wild-type I-Ek, or mutant I-E-transfected L cells and PCC(81–104) (100 μM). Production of IL-2 and IL-3 was measured in 24-h supernatants by ELISA. Crosshatched bars show IL-2 and closed bars show IL-3.
Mentions: We have previously reported that 3C6 proliferates and secretes both IL-2 and IL-3 in response to a mutant I-Ek molecule agonist expressed on transfected L cell APC (7). Addition of PCC(81–104) to the culture results in the formation of an altered TCR ligand consisting of the PCC peptide and the mutant I-E molecule (7). The pattern of protein tyrosine phosphorylation in anti-CD3ε immunoprecipitates is different for 3C6 cells exposed to an agonist consisting of wild-type I-Ek and PCC(81-104) or to this peptide–mutant MHC class II combination (2). The former is characterized by the presence of similar levels of p21 and p23 tyrosine-phosphorylated forms of TCR-ζ, tyrosine-phosphorylated CD3ε, and kinase-active tyrosinephosphorylated ZAP-70. In contrast, the latter leads to the predominant presence of the p21 tyrosine phosphorylated form of TCR-ζ, little or no tyrosine phosphorylated CD3ε, and the presence of ZAP-70 that is neither detectably phosphorylated nor kinase active, as reported previously (2) and shown here (Fig. 1 a). This PCC peptide–mutant I-E molecule ligand selectively antagonizes IL-2 production, while also acting as a partial agonist capable of modestly stimulating IL-3 production (7) (Fig. 1 b).

Bottom Line: Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern.These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR-ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide-MHC molecule ligands.Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment ofimmunological tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of Western Ontario, London, Canada.

ABSTRACT
One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation ofligand from an engaged TCR and (b) recruitment oflck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide-MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-zeta, reduced tyrosine phosphorylation of CD3epsilon, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR-ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide-MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment ofimmunological tolerance.

Show MeSH
Related in: MedlinePlus