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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 antibody on antigen-induced tyrosine  phosphorylation of TCR subunits. (a) 3C6 T cells (1 × 107) were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM) for 10  min in the presence of increasing concentrations of anti-CD4 mAb (RM  4.5) or anti-class II mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton X-100, and TCR subunits were immunoprecipitated  using a mAb against mouse CD3ε (500A2). Immunoprecipitates were immunoblotted using a mAb against phosphotyrosine (4G10). (b) Optical  density of the pp21, pp23, and the pZAP-70 signals from three independent experiments was measured using an imaging densitometer, and the  pp23/pp21 and pZAP-70/pp21 ratios displayed. (c) 3C6 T cells (1 × 107)  were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM)  for 10 min in the presence of anti-CD4 mAb (RM 4.5) or anti-class II  mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton  X-100, and TCR subunits were immunoprecipitated using a mAb against  mouse ZAP-70. Immunoprecipitates were immunoblotted using a mAb  against phosphotyrosine (4G10).
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Figure 3: Effect of anti-CD4 antibody on antigen-induced tyrosine phosphorylation of TCR subunits. (a) 3C6 T cells (1 × 107) were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM) for 10 min in the presence of increasing concentrations of anti-CD4 mAb (RM 4.5) or anti-class II mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton X-100, and TCR subunits were immunoprecipitated using a mAb against mouse CD3ε (500A2). Immunoprecipitates were immunoblotted using a mAb against phosphotyrosine (4G10). (b) Optical density of the pp21, pp23, and the pZAP-70 signals from three independent experiments was measured using an imaging densitometer, and the pp23/pp21 and pZAP-70/pp21 ratios displayed. (c) 3C6 T cells (1 × 107) were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM) for 10 min in the presence of anti-CD4 mAb (RM 4.5) or anti-class II mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton X-100, and TCR subunits were immunoprecipitated using a mAb against mouse ZAP-70. Immunoprecipitates were immunoblotted using a mAb against phosphotyrosine (4G10).

Mentions: To determine whether this functional switch in response to one resembling variant ligand stimulation also reflects a corresponding change in TCR signaling, we examined TCR subunit tyrosine phosphorylation in T cells responding to wild-type ligand in the presence of CD4 blockade. At concentrations of anti-CD4 affecting cytokine production, the early TCR-dependent signaling response clearly changes from one typical of agonist to one close to that characteristic of partial agonists (preferential accumulation of the p21 tyrosine phosphorylated form of TCR-ζ, with less phosphorylated CD3ε, and no detectable phosphorylated ZAP-70) (Fig. 3 a). This shift is unlikely to reflect active signaling following antibody interaction with the CD4 molecule because we used soluble deaggregated antibodies (41), the L cell APC do not express Fc receptors, and no significant tyrosine phosphorylation of TCR subunits is observed when antigen is omitted from cultures containing anti-CD4, T cells, and APC (data not shown). The effect of anti-class II mAb is different from that of the anti-CD4 and similar to what has been reported previously for changes in antigenic peptide concentration (1, 2, 16), namely, a uniform decrease in phosphorylation of all TCR subunits or the associated ZAP-70. These results seen using anti-CD4 versus anticlass II antibodies were confirmed by quantitative densitometric analysis of the pp21, pp23, and pZAP-70 bands from three different experiments (Fig. 3 b). The ratio between pp23 and pp21 is markedly decreased with higher concentrations of mAb against CD4, whereas this ratio remains stable for samples treated with anti-class II MHC. In addition, the ratio between pZAP-70 and pp21 falls to zero in those samples with higher concentrations of anti-CD4 mAb. Nevertheless, as reported previously (2, 3), ZAP-70 is recruited to the TCR complex upon TCR engagement of ligand even if anti-CD4 antibody is present and no phosphorylated ZAP-70 is observed (Fig. 3 c).


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 antibody on antigen-induced tyrosine  phosphorylation of TCR subunits. (a) 3C6 T cells (1 × 107) were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM) for 10  min in the presence of increasing concentrations of anti-CD4 mAb (RM  4.5) or anti-class II mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton X-100, and TCR subunits were immunoprecipitated  using a mAb against mouse CD3ε (500A2). Immunoprecipitates were immunoblotted using a mAb against phosphotyrosine (4G10). (b) Optical  density of the pp21, pp23, and the pZAP-70 signals from three independent experiments was measured using an imaging densitometer, and the  pp23/pp21 and pZAP-70/pp21 ratios displayed. (c) 3C6 T cells (1 × 107)  were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM)  for 10 min in the presence of anti-CD4 mAb (RM 4.5) or anti-class II  mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton  X-100, and TCR subunits were immunoprecipitated using a mAb against  mouse ZAP-70. Immunoprecipitates were immunoblotted using a mAb  against phosphotyrosine (4G10).
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Figure 3: Effect of anti-CD4 antibody on antigen-induced tyrosine phosphorylation of TCR subunits. (a) 3C6 T cells (1 × 107) were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM) for 10 min in the presence of increasing concentrations of anti-CD4 mAb (RM 4.5) or anti-class II mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton X-100, and TCR subunits were immunoprecipitated using a mAb against mouse CD3ε (500A2). Immunoprecipitates were immunoblotted using a mAb against phosphotyrosine (4G10). (b) Optical density of the pp21, pp23, and the pZAP-70 signals from three independent experiments was measured using an imaging densitometer, and the pp23/pp21 and pZAP-70/pp21 ratios displayed. (c) 3C6 T cells (1 × 107) were stimulated with I-Ek-transfected L cells and PCC(88–104) (100 μM) for 10 min in the presence of anti-CD4 mAb (RM 4.5) or anti-class II mAb (14-4-4S). Cell were lysed with lysis buffer containing 1% Triton X-100, and TCR subunits were immunoprecipitated using a mAb against mouse ZAP-70. Immunoprecipitates were immunoblotted using a mAb against phosphotyrosine (4G10).
Mentions: To determine whether this functional switch in response to one resembling variant ligand stimulation also reflects a corresponding change in TCR signaling, we examined TCR subunit tyrosine phosphorylation in T cells responding to wild-type ligand in the presence of CD4 blockade. At concentrations of anti-CD4 affecting cytokine production, the early TCR-dependent signaling response clearly changes from one typical of agonist to one close to that characteristic of partial agonists (preferential accumulation of the p21 tyrosine phosphorylated form of TCR-ζ, with less phosphorylated CD3ε, and no detectable phosphorylated ZAP-70) (Fig. 3 a). This shift is unlikely to reflect active signaling following antibody interaction with the CD4 molecule because we used soluble deaggregated antibodies (41), the L cell APC do not express Fc receptors, and no significant tyrosine phosphorylation of TCR subunits is observed when antigen is omitted from cultures containing anti-CD4, T cells, and APC (data not shown). The effect of anti-class II mAb is different from that of the anti-CD4 and similar to what has been reported previously for changes in antigenic peptide concentration (1, 2, 16), namely, a uniform decrease in phosphorylation of all TCR subunits or the associated ZAP-70. These results seen using anti-CD4 versus anticlass II antibodies were confirmed by quantitative densitometric analysis of the pp21, pp23, and pZAP-70 bands from three different experiments (Fig. 3 b). The ratio between pp23 and pp21 is markedly decreased with higher concentrations of mAb against CD4, whereas this ratio remains stable for samples treated with anti-class II MHC. In addition, the ratio between pZAP-70 and pp21 falls to zero in those samples with higher concentrations of anti-CD4 mAb. Nevertheless, as reported previously (2, 3), ZAP-70 is recruited to the TCR complex upon TCR engagement of ligand even if anti-CD4 antibody is present and no phosphorylated ZAP-70 is observed (Fig. 3 c).

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