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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.

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Effect of anti-CD4 and anti-MHC class II mAbs on IL-2 and  IL-3 production by 3C6 T cells responding to the wild-type ligand  PCC(88–104)–I-Ek. T cells (5 × 104 per well) were stimulated with I-Ekexpressing L cells and increasing concentrations of PCC(88–104) for 24 h  in the absence or the presence of the indicated concentrations of antiCD4 or anti-class II mAb. Supernatants were then collected and IL-2  (open circles) and IL-3 (closed triangles) measured by ELISA. Results are  expressed as the percent of cytokine produced considering the maximal  cytokine measured in each experiment in the absence of blocking antibody as 100%.
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Figure 2: Effect of anti-CD4 and anti-MHC class II mAbs on IL-2 and IL-3 production by 3C6 T cells responding to the wild-type ligand PCC(88–104)–I-Ek. T cells (5 × 104 per well) were stimulated with I-Ekexpressing L cells and increasing concentrations of PCC(88–104) for 24 h in the absence or the presence of the indicated concentrations of antiCD4 or anti-class II mAb. Supernatants were then collected and IL-2 (open circles) and IL-3 (closed triangles) measured by ELISA. Results are expressed as the percent of cytokine produced considering the maximal cytokine measured in each experiment in the absence of blocking antibody as 100%.

Mentions: 3C6 cells were exposed to PCC(88–104)–I-Ek complexes (wild-type ligand) on APC in the presence of increasing concentrations of a blocking mAb specific for CD4 (RM4.5). This was compared with stimulation of the cells in the presence of a blocking anti-class II mAb specific for I-E (14-4-4S), to limit ligand availability by a means other than peptide titration as had been done previously (2) and shown here in Fig. 2. Addition of anti-CD4 inhibits both IL-2 and IL-3 secretion, but IL-2 release is disproportionately sensitive to the antibody. Thus, any given fractional decrease in IL-2 production requires less anti-CD4 than a similar decrease in IL-3 production, and IL-2 secretion is abolished using much less anti-CD4 than is necessary to achieve the same effect on IL-3. In contrast, anti-class II MHC antibody inhibits IL-2 and IL-3 production to a similar extent. The results obtained using anti-CD4 resemble those seen upon simultaneous exposure of 3C6 to both agonist and antagonist, under which conditions IL-2 production is preferentially inhibited (7). If PCC(81–104) peptide is used to stimulate 3C6 instead of PCC(88–104), production of IL-2 requires less TCR occupancy than production of IL-3, as indicated by the fact that 50% maximal response for IL-2 production is reached at a lower concentration of peptide than is required for 50% maximal IL-3 production (7). Strikingly, under these conditions, exposure to antiCD4 mAb leads to an inversion in the IL-2 and IL-3 dose– response relationship, with relatively greater fractional IL-3 production than IL-2 production at each point in the dose– response (data not shown). Thus, blockade of CD4 leads to a functional response similar to that seen upon engagement of the 3C6 TCR with a variant ligand, and given the distinct results obtained with anti-class II antibody (Fig. 2), this effect cannot be explained by a simple decrease in occupancy of the TCR.


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)

Effect of anti-CD4 and anti-MHC class II mAbs on IL-2 and  IL-3 production by 3C6 T cells responding to the wild-type ligand  PCC(88–104)–I-Ek. T cells (5 × 104 per well) were stimulated with I-Ekexpressing L cells and increasing concentrations of PCC(88–104) for 24 h  in the absence or the presence of the indicated concentrations of antiCD4 or anti-class II mAb. Supernatants were then collected and IL-2  (open circles) and IL-3 (closed triangles) measured by ELISA. Results are  expressed as the percent of cytokine produced considering the maximal  cytokine measured in each experiment in the absence of blocking antibody as 100%.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Effect of anti-CD4 and anti-MHC class II mAbs on IL-2 and IL-3 production by 3C6 T cells responding to the wild-type ligand PCC(88–104)–I-Ek. T cells (5 × 104 per well) were stimulated with I-Ekexpressing L cells and increasing concentrations of PCC(88–104) for 24 h in the absence or the presence of the indicated concentrations of antiCD4 or anti-class II mAb. Supernatants were then collected and IL-2 (open circles) and IL-3 (closed triangles) measured by ELISA. Results are expressed as the percent of cytokine produced considering the maximal cytokine measured in each experiment in the absence of blocking antibody as 100%.
Mentions: 3C6 cells were exposed to PCC(88–104)–I-Ek complexes (wild-type ligand) on APC in the presence of increasing concentrations of a blocking mAb specific for CD4 (RM4.5). This was compared with stimulation of the cells in the presence of a blocking anti-class II mAb specific for I-E (14-4-4S), to limit ligand availability by a means other than peptide titration as had been done previously (2) and shown here in Fig. 2. Addition of anti-CD4 inhibits both IL-2 and IL-3 secretion, but IL-2 release is disproportionately sensitive to the antibody. Thus, any given fractional decrease in IL-2 production requires less anti-CD4 than a similar decrease in IL-3 production, and IL-2 secretion is abolished using much less anti-CD4 than is necessary to achieve the same effect on IL-3. In contrast, anti-class II MHC antibody inhibits IL-2 and IL-3 production to a similar extent. The results obtained using anti-CD4 resemble those seen upon simultaneous exposure of 3C6 to both agonist and antagonist, under which conditions IL-2 production is preferentially inhibited (7). If PCC(81–104) peptide is used to stimulate 3C6 instead of PCC(88–104), production of IL-2 requires less TCR occupancy than production of IL-3, as indicated by the fact that 50% maximal response for IL-2 production is reached at a lower concentration of peptide than is required for 50% maximal IL-3 production (7). Strikingly, under these conditions, exposure to antiCD4 mAb leads to an inversion in the IL-2 and IL-3 dose– response relationship, with relatively greater fractional IL-3 production than IL-2 production at each point in the dose– response (data not shown). Thus, blockade of CD4 leads to a functional response similar to that seen upon engagement of the 3C6 TCR with a variant ligand, and given the distinct results obtained with anti-class II antibody (Fig. 2), this effect cannot be explained by a simple decrease in occupancy of the TCR.

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