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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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Biochemical and functional consequences of anti-CD3–fos,  anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun bivalent cross-linking.  Tyrosine phosphorylation in cloned T cells after CD3 cross-linking,  CD3/CD4 cocross-linking, or CD4 cross-linking. T cells (1 × 107 per  sample) were stimulated with the 10 μg/ml of antibody in 100 μl of medium for 10 min. Cells were then lysed and a portion of the lysate used  for immunoprecipitation with an antiserum against ZAP-70. Both cell lysates (a) and ZAP-70 immunoprecipitates (b) were electrophoresed and  immunoblotted using anti-phosphotyrosine antibody. (c) Cell proliferation and IL-2 production by T cells stimulated with soluble anti-CD3– fos, anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun antibodies.
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Figure 5: Biochemical and functional consequences of anti-CD3–fos, anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun bivalent cross-linking. Tyrosine phosphorylation in cloned T cells after CD3 cross-linking, CD3/CD4 cocross-linking, or CD4 cross-linking. T cells (1 × 107 per sample) were stimulated with the 10 μg/ml of antibody in 100 μl of medium for 10 min. Cells were then lysed and a portion of the lysate used for immunoprecipitation with an antiserum against ZAP-70. Both cell lysates (a) and ZAP-70 immunoprecipitates (b) were electrophoresed and immunoblotted using anti-phosphotyrosine antibody. (c) Cell proliferation and IL-2 production by T cells stimulated with soluble anti-CD3– fos, anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun antibodies.

Mentions: The differences in TCR-mediated signaling and response seen upon TCR engagement with peptide–MHC ligands with or without adequate CD4 coengagement are also observed when activation of T cells with heterobivalent antibodies with specificity for both CD3 and CD4 (anti-CD3–fos × anti-CD4–jun) or with bivalent antibody specific for CD3 alone (anti-CD3–fos) are compared. Immunoblotting of proteins in lysates of T cells exposed to anti-CD3–fos × anti-CD4–jun antibody shows the presence of ligand-induced tyrosine-phosphorylated species of approximately 36, 38, 42–44, 50, 60, 70, 80, 90, 120, 140, and 150 kDa. In contrast, immunoblotting of lysates of T cells exposed to antiCD3–fos antibody only shows induction of tyrosine-phosphorylated 44, 60, 90, and 120 kDa species, the latter being of variable magnitude (Fig. 5 a). When analyzed by blotting of anti-ZAP-70 immunoprecipitates, coengagement of CD3 and CD4 results in a clear induction of tyrosine-phosphorylated p21 and p23 TCR-ζ, CD3ε, and ZAP-70, similar to the pattern seen with agonist peptide–MHC class II ligands (1, 2, 16). Activation of T cells with CD3 engagement alone instead induces a pattern of TCR-associated phosphoproteins resembling that seen using partial agonists/antagonists, with pp21 TCR-ζ predominating, a very low amount of pp23 TCR-ζ, and no detectable phosphorylated ZAP-70 (Fig. 5 b). In some, but not other experiments, a limited amount of pp21 TCR-ζ was formed in response to anti-CD4–jun alone (data not shown), as reported previously (24). Functionally, substantial cell proliferation and IL-2 production is seen upon stimulation with antiCD3–fos × anti-CD4–jun, but not using anti-CD3–fos only or anti-CD4–jun only (Fig. 5 c), in agreement with previous observations that IL-2 production most closely tracks pp23 TCR-ζ accumulation and/or phosphorylation of ZAP-70 (1, 2).


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)

Biochemical and functional consequences of anti-CD3–fos,  anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun bivalent cross-linking.  Tyrosine phosphorylation in cloned T cells after CD3 cross-linking,  CD3/CD4 cocross-linking, or CD4 cross-linking. T cells (1 × 107 per  sample) were stimulated with the 10 μg/ml of antibody in 100 μl of medium for 10 min. Cells were then lysed and a portion of the lysate used  for immunoprecipitation with an antiserum against ZAP-70. Both cell lysates (a) and ZAP-70 immunoprecipitates (b) were electrophoresed and  immunoblotted using anti-phosphotyrosine antibody. (c) Cell proliferation and IL-2 production by T cells stimulated with soluble anti-CD3– fos, anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196122&req=5

Figure 5: Biochemical and functional consequences of anti-CD3–fos, anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun bivalent cross-linking. Tyrosine phosphorylation in cloned T cells after CD3 cross-linking, CD3/CD4 cocross-linking, or CD4 cross-linking. T cells (1 × 107 per sample) were stimulated with the 10 μg/ml of antibody in 100 μl of medium for 10 min. Cells were then lysed and a portion of the lysate used for immunoprecipitation with an antiserum against ZAP-70. Both cell lysates (a) and ZAP-70 immunoprecipitates (b) were electrophoresed and immunoblotted using anti-phosphotyrosine antibody. (c) Cell proliferation and IL-2 production by T cells stimulated with soluble anti-CD3– fos, anti-CD4–jun, or anti-CD3–fos × anti-CD4–jun antibodies.
Mentions: The differences in TCR-mediated signaling and response seen upon TCR engagement with peptide–MHC ligands with or without adequate CD4 coengagement are also observed when activation of T cells with heterobivalent antibodies with specificity for both CD3 and CD4 (anti-CD3–fos × anti-CD4–jun) or with bivalent antibody specific for CD3 alone (anti-CD3–fos) are compared. Immunoblotting of proteins in lysates of T cells exposed to anti-CD3–fos × anti-CD4–jun antibody shows the presence of ligand-induced tyrosine-phosphorylated species of approximately 36, 38, 42–44, 50, 60, 70, 80, 90, 120, 140, and 150 kDa. In contrast, immunoblotting of lysates of T cells exposed to antiCD3–fos antibody only shows induction of tyrosine-phosphorylated 44, 60, 90, and 120 kDa species, the latter being of variable magnitude (Fig. 5 a). When analyzed by blotting of anti-ZAP-70 immunoprecipitates, coengagement of CD3 and CD4 results in a clear induction of tyrosine-phosphorylated p21 and p23 TCR-ζ, CD3ε, and ZAP-70, similar to the pattern seen with agonist peptide–MHC class II ligands (1, 2, 16). Activation of T cells with CD3 engagement alone instead induces a pattern of TCR-associated phosphoproteins resembling that seen using partial agonists/antagonists, with pp21 TCR-ζ predominating, a very low amount of pp23 TCR-ζ, and no detectable phosphorylated ZAP-70 (Fig. 5 b). In some, but not other experiments, a limited amount of pp21 TCR-ζ was formed in response to anti-CD4–jun alone (data not shown), as reported previously (24). Functionally, substantial cell proliferation and IL-2 production is seen upon stimulation with antiCD3–fos × anti-CD4–jun, but not using anti-CD3–fos only or anti-CD4–jun only (Fig. 5 c), in agreement with previous observations that IL-2 production most closely tracks pp23 TCR-ζ accumulation and/or phosphorylation of ZAP-70 (1, 2).

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