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

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.

Figure 1

The IL-2 response of 3C6 T cells to mutant I-E^k 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-E^k) 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-E^k, 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.

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-E^k. T cells (5 × 10⁴ per well) were stimulated with I-E^kexpressing 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%.

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-E^k-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-E^k-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).

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-E^k-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-E^k-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).

Figure 4

TK.G4 T cell responses to cognate peptide bound to wildtype MHC class II molecules or to mutant MHC class II molecules unable to bind CD4. (a) Proliferative response of TK.G4 cells stimulated with Swmyo(102–118) + wild-type I-A^d or Swmyo(102–118) + I-A^d mutated at the primary CD4 binding site. (b) Tyrosine phosphorylation analysis of cell lysates (top) and anti-CD3ε (bottom) immunoprecipitates from TK.G4 cells stimulated under the same conditions.

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.

Figure 6

Anergy induction by stimulation with wild-type ligand in the presence of anti-CD4 antibody. (a) A.E7 T cells were incubated with I-E^k-expressing L cells without or with PCC(81–104) (100 nM) and antiCD4 mAb (1:200 dilution of supernatant) for 24 h. T cells were then isolated and rested for 7 d. At this point, T cells were restimulated with I-E^k-transfected L cells and PCC(81–104) (1 μM) for 24 h and IL-2 production was measured by ELISA. (b) Effect of variation in the concentration of anti-CD4 mAb in the primary culture on the extent of anergy induction. A. E7 cells were treated as in (a), but using various concentrations of anti-CD4 antibody (1:200, open squares; 1:2,000, closed diamonds; 1:20,000, half-filled squares; 1:200,000, half-filled diamonds; no antiCD4, closed triangles). After rest, each of these cell populations was restimulated as in (a) and IL-2 in the medium measured after 24 h by ELISA.