Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity

Abstract

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4+ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.

Figure 1. Characterization of the Protein and Lipid Bilayer Components of Artificial APCs

(A) Schematic of the interaction between the T cell and the NTA-Ni lipid bilayer-based artificial APC. (B) The expanded area of the POPC bilayer doped with 0.2 mol% fluorescent DOPE-NBD (green). The scale bar represents 10 μm. (C) Fluorescence recovery of a photobleached area (r = 4 μm) on the POPC/1mol% DOPE-NBD bilayer. (D) Immobilized agonist, but not endogenous pMHC, stimulates AD10 T cells to produce IL2. The 96-well ELISA plates were coated with IEk proteins at the indicated concentrations. AD10 CD4⁺ T cells were assayed for IL2 production.

Figure 2. Very Low Numbers of Monomeric Agonist IEk Anchored on a Lipid Bilayer Are Sufficient to Trigger T Cell Calcium Flux, Independent of Endogenous IEk

(A) IEk-MCC mixed with GFP-HisTag, IEk-ER60, or IEk-HSP70 at the indicated ratios was anchored on a lipid bilayer containing 5 mol% DOGS-NTA-Ni. Increase in the intracellular calcium level in AD10 TCR transgenic CD4⁺ T cells labeled with fura-2 is indicated by an increase of the 340 nm/380 nm ratio in the pseudocolor images. Bilayers with GFP-HisTag or endogenous IEk only, without IEk-MCC, served as controls. (B) Percentage of AD10 T cells with at least two times increased intracellular calcium levels in response to IEk-MCC alone or mixed with GFP-HisTag, IEk-ER60, or IEk-HSP70 on NTA-Ni lipid bilayers at the indicated ratios. (C) Same as (B), except that the average peak calcium level is shown as the 340 nm/380 nm ratio. The 340 nm/380 nm ratios of T cells stimulated with “No MCC” are average ratios of T cells interacting with lipid bilayers anchored with only GFP-HisTag or endogenous IEks. (D) Change in AD10 T cell intracellular calcium levels over time after contacting NTA-Ni bilayers anchored with IEk-MCC mixed with IEk-ER60 or IEk-HSP70 at a 1:1,000 ratio. Data were compiled from 17 AD10 T cells in each group.

Figure 3. Very Low Numbers of Agonist IEk Anchored on a Fixed Surface Induce AD10 T Cell IL2 Production, Independent of Endogenous or Null IEk, or IAk

(A) Bio-IEk-MCC, bio-IEk-ER60, bio-IEk-HSP70, or bio-IEk-99A at the indicated concentrations were added to 96-well plates precoated with streptavidin and incubated for 18 h at 37 °C. AD10 CD4⁺ T cells were added to the wells and stimulated for 7 h prior to IL2 assay. (B) The level of IEk proteins on streptavidin plates coated as in (A) was determined by ELISA using anti-IEk 14-4-4s antibody followed by HRP-conjugated goat anti-mouse antibody. ABTS substrate color development was monitored at 405 nm. (C) Streptavidin plates coated with bio-IEk-MCC at the indicated concentrations were subsequently saturated with 10 μg/ml bio-IEk-ER60, bio-IEk-HSP70, bio-IEk-99A, or buffer for 1 h at room temperature before being used to stimulate AD10 T cells. (D) The 96-well ELISA plates were coated with bio-IEk-MCC at the indicated concentrations overnight at 4 °C. The wells were then saturated with 10 μg/ml BSA, IEk-ER60, IEk-HSP70, IEk-99A, or IAk-CA for 4 h at room temperature. Stimulation of AD10 and measurement of IL2 measurements were done as described in (A). (E and F) Same as (C) and (D), respectively, except that 5C.C7 cells were used.

Figure 4. On Real APCs, Endogenous Peptides Do Not Affect T Cell Activation by Agonist Peptides

CH27 cells were cultured with 1 mM β2m-bio, ER60-bio, or ER60scrbl-bio peptides for 20 h at 37 °C. After washing, cells were incubated with MCC-FITC peptides at the indicated concentrations for 30 min before washing. (A) The level of MCC-FITC on CH27 cells after pulsing, as measured by flow cytometry. (B) AD10 T cells were mixed with CH27 cells pulsed with MCC-FITC at the indicated concentrations and IL2 production was measured by intracellular staining after 7 h. Data are representative of three independent experiments.

Figure 5. TCR Triggering by Monomeric Agonist pMHC Requires T Cell Adhesion to the Bilayer and Intact Actin Cytoskeletal Function

(A and B) Lipid bilayers containing 5 mol% DOPE-biotin were anchored with monovalent (IEk-MCC)-SA. The bilayers were then used to stimulate T cells directly (A) or were further anchored with (ICAM-1)₂-SA (B) before adding T cells. Bilayers anchored with only (ICAM-1-bio)₂-SA did not induce T cell calcium flux. (C and D) A layer of CH27 cells pulsed for 1 h with 1 μM MCC-FITC was attached on cover glasses coated with poly-l-lysine. Fura-2 AM–pulsed AD10 T cells treated with 10 μM cytochalasin D for 1 h (D), or untreated AD10 T cells (C) were introduced, and calcium flux was monitored. (E) Similar to (C), except that 10 μM cytochalasin D (or DMSO as control) was introduced after T cell calcium flux reached peak levels.

Figure 6. T Cells Respond to Agonist pMHC in a Sublinear Fashion

AD10 T cell calcium responses to IEk-MCC diluted by IEk-HSP70 on lipid bilayers in Figure 2B and AD10 T cell IL2 responses to bio-IEk-MCC on streptavidin plates in Figure 3A are plotted as power functions of the calculated number of IEk-MCC per cell. T cell responses are shown as the ratio of responding to nonresponding cells. Also shown are theoretical curves of linear and second-degree function responses.

Figure 7. The Receptor Deformation Model of TCR Triggering

(A) Interaction of pMHC and TCRs at the membrane–membrane contact site of the T cell/APC interface facilitated by adhesion molecules. The interaction between agonist pMHC and TCR per se does not initiate TCR signaling. (B) Forces generated from the cytoskeleton, which drive T cell morphological changes and active T cell movement relative to the APC, detach the T cell plasma membrane from the APC. Part of this force is delivered to the TCR/CD3 complex via pMHC-TCR binding. Binding between agonist pMHC and TCR is strong enough to deliver a force that deforms the TCR/CD3 complex to a conformation capable of initiating an activation signal. Weak binding between TCR and self pMHC breaks before such a force can be delivered. This binding could, however, deliver a weak force that causes minor receptor deformation, leading to a survival signal. The detaching force might also deform the MHC molecule to a conformation that binds coreceptors with higher affinity.