T cell receptor (TCR) clustering in the immunological synapse integrates TCR and costimulatory signaling in selected T cells

Abstract

During T cell activation, T cell receptors (TCR) cluster at the center of the T cell/antigen-presenting cell interface forming a key component of the immunological synapse. The function of this TCR clustering is still unresolved. A comprehensive search for such a function yielded a very limited and specific result. A micrometer-scale receptor clustering integrated the TCR and CD28 signals required for IL-2 secretion in primary 5C.C7 T cells, a low-affinity/avidity TCR system. 5C.C7 TCR signaling itself was not affected. In addition, central TCR accumulation was not required for any T cell effector function tested in three other TCR transgenic models. Central TCR accumulation thus had a specific role in signaling integration in low-affinity T cells.

Fig. 1.

Central TCR accumulation associates with IL-2 secretion in 5C.C7 T cells. (A) Central TCR and B7-2 accumulation and effector functions in 5C.C7/I-E^k-GFP transfected A20 B cell lymphoma APC interactions under the indicated conditions are shown as normalized data. Values for 10 μM MCC are set to 100%, and values for no agonist peptide are set to 0%. All other values are scaled accordingly. Raw data are either published (TCR accumulation) (6) or given in Fig. 7 (B7 accumulation) or Table 1 (all other data). WASP C-ter, 200 nM of the tat Wiscott–Aldrich syndrome protein C-terminal domain; Jasp, 0.5 μM Jasplakinolide; anti-ICAM and anti-B7′, 10 μg/ml blocking antibodies against ICAM-1 and B7-1/B7-2, respectively. (B) Normalized data for primary DO11.10/A20 APC interactions are shown similarly. Raw data are given in Table 1 and Fig. 2.

Fig. 2.

Central TCR accumulation is rapid and requires high concentrations of agonist peptide. (A) The percent of T cell/APC couples with central TCR accumulation in at least one of the four time points analyzed (1′, 3′, 5′, and 7′–15′; central, black bars); any other accumulation phenotype (other, gray bars) or no accumulation (none, unfilled bars) are given for different TCR transgenic T cells as indicated in the first column. Fig. 9 provides a kinetic analysis of the same data. TCR accumulation was determined by using EL-4 target cell transfection with H2-D^b-GFP or T cell transduction with TCR-ζ/GFP or ZAP-70 double SH2-GFP (2SH2) as indicated in the second column at the given concentration of peptide (third column). The ZAP-70 double SH2 domain-GFP protein is further characterized in Supporting Text and Fig. 11, which is published as supporting information on the PNAS web site. Imaging probes are discussed in Supporting Text. Agonist peptides were used throughout with the exception of the A4Y partial P14 agonist and the addition of 10 μg/ml blocking B7-1/B7-2 antibodies (B7) to one set of DO11.10 samples. n, cells from at least three independent experiments were analyzed per condition. (B) An interaction of a P14 T cell that has been transduced with ZAP-70 double SH2–GFP (Lower) with an EL4 APC incubated with 10 μM p33 peptide (Upper) is shown at the indicated time points relative to tight cell couple formation (t = 0:00) in still images derived from Movie 1, which is published as supporting information on the PNAS web site. (Upper) Bright field images are displayed. (Lower) Matching projections of the three-dimensional ZAP-70 double SH2 domain–GFP fluorescence data in a false color scale (increasing from purple to red and white).

Fig. 3.

IL-2 secretion and Akt phosphorylation are particularly costimulation-dependent in 5C.C7 T cells. (A) The dependence of IL-2 secretion (Fig. 5) on costimulation in 5C.C7/I-E^k-GFP transfected A20 versus DO11.10/A20 interactions in the presence of the indicated concentrations of agonist peptide is shown. Blockade of ICAM-1 and B7–1/B7–2 with 10 μg/ml antibody is indicated. IL-2 secretion was normalized (the value for 10 μM agonist peptide = 100%). Two to five independent experiments were performed, and averages plus standard deviations are given. *, a significant (P < 0.05) difference between buffer only and anti-B7. (B) Akt S473 phosphorylation after 30 min of T cell/APC interactions (Fig. 8B) is shown similar to A. In C, correlation coefficients (R² values) of normalized Akt phosphorylation against central TCR accumulation and IL-2 data as determined by linear regression (similar to Fig. 6) are listed.

Fig. 4.

Central TCR accumulation associates with sustained actin accumulation but not with critical effector functions in P14 and HY T cells. (A and B) Central TCR accumulation and effector functions in primary T cell/EL4 APC interactions (P14 T cells in A and HY T cells in B) under the indicated agonist peptide concentrations are shown as normalized data similar to Fig. 1. Raw data are given in Table 1 and Fig. 2. (C) The percent of actin-GFP-transduced P14/EL4 cell couples showing actin accumulation at the T cell/APC interface at >40% of the cellular background fluorescence intensity at the indicated time after cell couple formation in the presence of the given gp33 agonist peptide concentrations are displayed. Differences between values at 10 μM versus 1 μM and 100 nM gp33 peptide at 4′ and 5′ are significant (P ≤ 0.001). Between 22 and 36 cell couples from at least two independent experiments were analyzed per condition.