T-cell antagonism by short half-life pMHC ligands can be mediated by an efficient trapping of T-cell polarization toward the APC

Abstract

T-cell activation results from productive T-cell receptor (TCR) engagement by a cognate peptide-MHC (pMHC) complex on the antigen presenting cell (APC) surface, a process leading to the polarization of the T-cell secretory machinery toward the APC interface. We have previously shown that the half-life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters determining T-cell activation. However, whether the half-life of the TCR/pMHC interaction can modulate the efficiency of T-cell secretory machinery polarization toward an APC still remains unclear. Here, by using altered peptide ligands conferring different half-lives to the TCR/pMHC interaction, we have tested how this parameter can control T-cell polarization. We observed that only TCR/pMHC interactions with intermediate half-lives can promote the assembly of synapses that lead to T-cell activation. Strikingly, intermediate half-life interactions can be competed out by short half-life interactions, which can efficiently promote T-cell polarization and antagonize T-cell activation that was induced by activating intermediate half-life interactions. However, short TCR/pMHC interactions fail at promoting phosphorylation of signaling molecules at the T-cell-APC contact interface, which are needed for T-cell activation. Our data suggest that although intermediate half-life pMHC ligands promote assembly of activating synapses, this process can be inhibited by short half-life antagonistic pMHC ligands, which promote the assembly of non activating synapses.

Fig. 1.

Intermediate half-lives of TCR/pMHC interaction are required to induce efficient T-cell activation and effector function. pOVA APLs–pulsed DCs were cocultured with OT-I T cells, and IL-2 secretion (A) and TCR internalization (B) were determined. Activated OT-I T cells were cocultured with pOVA APLs–pulsed DCs cells, and CD69 expression (C) and IFN-γ secretion (D) were determined. Graphs represent average of three to five independent experiments; error bars represent SE.

Fig. 2.

DCs loaded with short half-life pMHC ligands can antagonize T-cell activation induced by DCs loaded with intermediate half-life ligands. OT-I T cells were simultaneously stimulated with DCs pulsed with pOVA and DCs pulsed with pOVA variants, and then CD69 upregulation and IL-2 release were determined. Graphs show means of three independent experiments; error bars represent SE.

Fig. 3.

T cells polarize toward DCs presenting pMHC ligands that bind the TCR either with short or intermediate half-lives. Peptide-pulsed DCs were stained with BODIPY 630 (blue) or CMTMR orange (red) and cocultured with OT-I T cells stained for Golgi apparatus (green). Golgi apparatus polarization was determined by time-lapse video microscopy. Data shown are representative sequences of snapshot images for pOVA-pulsed DCs vs. unpulsed or pOVA APL-pulsed DCs, derived from one experiment out of five. Snapshots on A, B, C, D, E, and F correspond to Movies S1, S3, S5, S6, S8, and S10, respectively. (G) Percentage of T cell–DC conjugates with T-cell Golgi apparatus polarized toward the indicated peptide-pulsed DC.

Fig. 4.

DCs loaded with antagonist ligands can compete for T-cell polarization with DCs loaded with agonist ligands. Peptide-pulsed DCs were stained with BODIPY 630 (blue) or CMTMR orange (red) and cocultured with OT-I T cells stained for Golgi apparatus (green). Triple conjugates (T cells interacting at the same time with two different types of DCs) were searched, and the polarization toward each one of the different DCs was quantified.

Fig. 5.

Significant accumulation of tyrosine-phosphorylated molecules at the IS requires intermediate half-life for the TCR/pMHC interaction. (A) Distribution of pTyr (blue) was determined by confocal microscopy on T-cell conjugates with DCs (red), either unpulsed or pulsed with pOVA APLs. Arrowheads indicate pTyr accumulation at the DC–T-cell interface. pTyr accumulation at the IS was quantified by using a linescan tool from Image ProPlus software. OS and CS represent fluorescence intensity at opposite site and contact site of T cell–DC synapse, respectively, as described by the diagram. Representative data from one of three experiments are shown. (B) Percentage of conjugates with pTyr accumulation for each pOVA APL-pulsed DC of all of the conjugates analyzed in three experiments. **P < 0.01, ANOVA two-way test.

Fig. 6.

T-cell cross-antagonism requires T-cell Golgi polarization. Control (A) or nocodazole-treated (B) OT-I T cells were simultaneously stimulated with DCs pulsed with pOVA and DCs pulsed with pOVA variants, and then CD69 upregulation was determined. Graphs show means of three independent experiments; error bars represent SE.