Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul 5;9(1):2618.
doi: 10.1038/s41467-018-05050-6.

A Window of Opportunity for Cooperativity in the T Cell Receptor

Affiliations
Free PMC article

A Window of Opportunity for Cooperativity in the T Cell Receptor

N Martin-Blanco et al. Nat Commun. .
Free PMC article

Abstract

The T-cell antigen receptor (TCR) is pre-organised in oligomers, known as nanoclusters. Nanoclusters could provide a framework for inter-TCR cooperativity upon peptide antigen-major histocompatibility complex (pMHC) binding. Here we have used soluble pMHC oligomers in search for cooperativity effects along the plasma membrane plane. We find that initial binding events favour subsequent pMHC binding to additional TCRs, during a narrow temporal window. This behaviour can be explained by a 3-state model of TCR transition from Resting to Active, to a final Inhibited state. By disrupting nanoclusters and hampering the Active conformation, we show that TCR cooperativity is consistent with TCR nanoclusters adopting the Active state in a coordinated manner. Preferential binding of pMHC to the Active TCR at the immunological synapse suggests that there is a transient time frame for signal amplification in the TCR, allowing the T cells to keep track of antigen quantity and binding time.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Optimum time for Active conformation of the TCR at sub-saturating concentrations of the pMHC ligand. a Cartoon of the Resting and ligand-bound Active conformations of the TCR. Bivalent or multivalent ligation of two or more TCRs by its pMHC ligand results in an outside-in transmission of a conformational change detected in the cytoplasmic tails of the CD3 subunits. One of these changes consists in exposing an epitope in the proline-rich region of CD3ε for mAb APA1/1 binding. b OT-1 CD8 + T cells were incubated at 37 °C with the indicated concentrations of APC-labelled OVAp tetramer for 24 h. After stimulation, cells were stained with anti-CD69 and analyzed by cytometry. The mean fluorescence intensity (MFI) for CD69 staining is shown as red circles and that of tetramer binding as blue diamonds. c OT-1 CD8 + T cells were incubated on ice with the indicated concentrations of APC-labelled OVAp tetramer for 40 min. MFI was calculated by flow cytometry. d OT-1 CD8 + T cells were incubated at different times at 0 °C with 1 nM or 200 nM of OVAp-APC tetramer and subsequently fixed, permeabilized and stained with the APA1/1 mAb prior to flow cytometry analysis. MFI for APA1/1 staining is shown as red circles and that of tetramer binding as blue diamonds. e OT-1 CD8 + T cells were incubated at different times at 37 °C with 1 nM OVAp-APC tetramer and stained with the APA1/1 mAb as in Fig. 1d. f Histogram overlay for APA1/1 expression in OT-1 and HY CD8 + T cells not incubated (blue lines) or incubated for 2 min (red line) or 5 min (brown line) with 1 nM OVAp-APC tetramer at 0 °C. All data shown in Fig. 1 represent the mean ± s.d. of triplicate datasets; *p < 0.05; **p < 0.005; ***p < 0.0005 (two-tail unpaired t-test)
Fig. 2
Fig. 2
TCRs are in transit between three different states of conformations. a Cartoon of the Resting, Active and Inhibited states. Blue and red arrows suggest a possible movement of the αβ ectodomains. Adoption of the three states is also reflected by movements of the cytoplasmic tails of the CD3 subunits. One of these movements results in exposure of the APA1/1 epitope in CD3ε. b A mathematical model with just two states (Resting and Active) cannot explain the optimum time point for the Active conformation shown in Fig. 1d, e. Rather, Active conformation reaches a plateau of indefinite duration. c Summary of reactions included in the model. The letters R, A and I stand for the states Resting, Active and Inhibited. A nanocluster of three TCRs and a trimeric pMHC ligand were chosen for modelling. The numerical values of the parameters and an explanatory description of the states are summarised in Methods. The plus symbols stand for either empty or non-crosslinked bound ligand used for conciseness. d Model to explain how ligation of two or more TCRs by pMHC results in stabilisation of the Active conformation in the entire TCR nanocluster. Adoption of the Active conformation by unbound TCRs facilitates their binding to additional pMHC ligands. e Numerical integration of the 3-state model after addition of ligand. The right panel shows the existence of an optimum of the Active conformation (red circles), even though binding has not yet reached saturation. This behaviour reflects the experimental results in Fig. 1d–e. f Prediction of the existence of cooperative effects on pMHC ligand binding derived from the mathematical model. Preincubation of TCR nanoclusters with free ligand at different times displays an optimum cooperation at the time when the maximal number of TCR nanoclusters in the Active conformation is found (Figs. 1d, e and 2e)
Fig. 3
Fig. 3
Binding of a first pMHC tetramer ligand at sub-saturating concentrations transiently favours binding of a second pMHC tetramer ligand. a OT-1 T CD8 + T cells were preincubated at 0 °C with 1 nM OVAp-APC tetramer for the indicated times and subsequently incubated with 5 nM of OVAp-PE tetramer for five additional minutes. Time 0 indicates binding of OVAp-PE tetramer in the absence of preincubation with OVAp-APC tetramer. Cells were fixed and stained for CD8 before analysis by flow cytometry and calculation of the MFI for OVAp-PE and OVAp-APC. Histograms for OVAp-APC tetramers and OVAp-PE tetramer fluorescence intensity are shown on the left panels. The MFI values for OVAp-APC tetramers and OVAp-PE tetramer are represented on the right panels. b CD8 + T cells from double transgenic female OT-1xHY mice were preincubated on ice with 1 nM OVAp-APC tetramer for the indicated times and subsequently incubated with 20 nM of HYp-PE tetramer for five additional minutes. Cells were fixed and stained for CD8 before analysis by flow cytometry to generate MFI values for HYp-PE and OVAp-APC binding. All data shown in Fig. 3 represent the mean ± s.d. of triplicate datasets; *p < 0.05; **p < 0.005; ****p < 0.00005 (2-tail unpaired t-test)
Fig. 4
Fig. 4
A multimeric pMHC ligand is required to promote cooperative pMHC binding. a CD8 + OT-1 T cells were preincubated at 0 °C with 100 nM biotinylated OVAp monomer for the indicated times and subsequently incubated with 5 nM of OVAp-APC tetramer for five additional minutes. Time 0 indicates binding of OVAp-APC tetramer in the absence of preincubation with OVAp monomer. Cells were fixed and stained with streptavidin-PE in order to detect binding of the monomer. Plots represent the MFI values for OVAp-PE monomer and OVAp-APC tetramer binding. b CD8 + OT-1 T cells were incubated at 0 °C with the indicated concentrations of biotinylated OVAp monomer for 6 min and subsequently incubated with 5 nM of OVAp-PE tetramer for 5 additional minutes. Cells were then fixed and stained with streptavidin-APC in order to detect the monomer. Plots represent the MFI values for OVAp-APC monomer and OVAp-PE tetramer binding. c CD8 + OT-1 T cells were preincubated at 0 °C with 1 nM OVAp-APC tetramer for the indicated times and subsequently incubated with 1.5 μM of biotinylated OVAp monomer (detected with streptavidin-PE) for 5 additional minutes. Time 0 indicates binding of OVAp monomer in the absence of preincubation with OVAp-APC tetramer. Cells were fixed and stained with streptavidin-PE in order to detect binding of the monomer. Plots represent the MFI values for OVAp-PE monomer and OVAp-APC tetramer binding. d CD8 + OT-1 T cells were preincubated or not at 0 °C with 1 nM of OVAp-PE tetramer for 6 min prior to incubation with the indicated concentrations of biotinylated OVAp monomer for five additional minutes. Cells were fixed and stained with streptavidin-APC in order to detect binding of the monomer. Plots represent the MFI values for OVAp-APC monomer and OVAp-PE tetramer binding. All data shown in Fig. 4 represent the mean ± s.d. of triplicate datasets; * p < 0.05; **p < 0.005; ***p < 0.0005 (2-tail unpaired t-test)
Fig. 5
Fig. 5
Ligand binding cooperativity depends on TCR nanoclusters adopting the Active conformation. a OT-1 T cells were incubated with 10 mM methyl-β-cyclodextrin (MβCD) or vehicle for 30 min at 37 °C, cooled to 0 °C and preincubated with 1 nM OVAp-APC tetramer for the indicated times, then incubated with 5 nM OVAp-PE for 5 additional min. Cells were fixed and stained with the anti-TCRβ antibody H57 to assess TCR levels. Left panel: TCR expression at each time point of preincubation with OVAp-APC for non-treated and MβCD-treated cells. Middle panel: OVAp-APC tetramer binding at different times of preincubation. Right panel: OVAp-PE tetramer binding during 5 min as a function of preincubation time with the OVAp-APC tetramer. b Cartoon of a TCR indicating the position of the Cys80 residue mutated to glycine in the extracellular domain of CD3ε. c CD8 + OT-1 T cells expressing wild type or C80G mutant CD3ε were incubated at 0 °C with the indicated concentrations of OVAp-PE tetramer for 5 min. Cells were fixed and stained with the anti-CD3 antibody 2C11 to assess TCR expression (left panel). Tetramer binding data (right panel) was obtained from WT and CD3ε C80G cell populations, and from a WT subpopulation gated for TCR expression similar to CD3ε C80G cells (WTnorm, left panel). d CD8 + OT-1 T cells expressing wild type or C80G CD3ε were preincubated at 0 °C with 1 nM OVAp-APC tetramer for the indicated times and with 5 nM OVAp-PE for 5 additional minutes. Cells were fixed and stained with the anti-TCRβ antibody H57 to normalise TCR levels. Left panel: OVAp-APC tetramer binding at different times of preincubation. Right panel: OVAp-PE tetramer binding during 5 min for all preincubation times in WT C80G OT-1 cells. OVAp-APC and OVAp-PE tetramer binding is compared between CD3ε C80G mutant cells and CD3ε WT cells normalised for equal TCR expression as in c. All data shown in Fig. 5 represent the mean ± s.d. of triplicate datasets; *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.00005 (2-tail unpaired t-test)
Fig. 6
Fig. 6
Ligand binding induces changes in the proximity or orientation of TCRs within nanoclusters. a Schematic of the experimental set-up of Fig. 6b. FRET changes were detected upon simultaneous binding of PE- and APC-labelled OVAp tetramers. b CD8 + OT-1 T cells were incubated on ice with 5 nM each of OVAp-PE and OVAp-APC tetramers for the indicated times and fixed. FRET intensity and efficiency (left and right panels) were measured by flow cytometry. For FRET controls, OT-1 T cells were incubated with 5 nM of the OVAp-PE tetramer for the indicated times and, after fixation, cells were further incubated for 50 min with either APC-labelled anti-CD3 (positive control, C + ) or APC-labelled anti-CD27 (negative control, C-) at saturating concentrations. Arrows indicate the time points at which an upward tendency of the FRET slope was noticed. c Schematic of the experimental set-up in df. FRET changes were detected in CD8 + OT-1 T cells that bound PE-labelled OVAp tetramer and APC-labelled anti-Vβ5 antibody. d–f CD8 + OT-1 or OT-1xHY T cells pretreated or not with 10 mM MβCD for 30 min at 37 °C and subsequently were incubated at 0 °C with 1 nM unlabelled OVAp tetramer for the indicated times. After fixation, they were incubated for 50 min with anti-Vα2-PE and anti-Vβ5-APC (d), anti-Vβ5-PE and anti-Vβ5-APC (e) or anti-T3.70-PE and Vα2-APC (f). For all antibody combinations, a negative control is included using the donor antibody and anti-CD27-APC (C-). FRET efficiency is calculated as described in the Methods section and represented versus time of preincubation with tetramer. All data shown in Fig. 6 represent the mean ± s.d. of triplicate datasets; *p < 0.05 (2-tail unpaired t-test)
Fig. 7
Fig. 7
The sites of enhanced tetramer binding co-localise with the Active TCR at the periphery of the immunological synapse. a CD8 + OT-1 T cells were treated with 20 µM PP2 or vehicle for 30 min at 37 °C and then preincubated for the indicates times at 37 °C in the presence of mouse bone-marrow-derived dendritic cells (mDCs) loaded with OVA peptide. Subsequently, cells were incubated with 5 nM OVAp-PE for 5 min. Afterwards, cells were fixed and stained with anti-CD8 and anti-H57 antibodies to quantify TCR expression. b CD8 + T cells from OT-1 CD3ζ-GFP mice were purified by negative selection and incubated for 8 min at 37 °C in the presence of mDCs attached to a coverslip and pre-loaded with OVA peptide. Subsequently, cells were incubated with 20 nM OVAp-APC for 5 min. Before visualisation by confocal microscopy, cells were fixed, permeabilized and stained with APA1-1 (top panel). Scale bar represents 1 μm. Line scan drawn along the synaptic zone to show relative fluorescence intensity of CD3ζ, APA1-1 and OVAp-tetramer (middle left panel). Relative fluorescence intensities of CD3ζ, APA1-1 and OVAp-tetramer are represented versus distance (middle centre). Ratios between the relative fluorescence intensity of APA1-1 and CD3ζ or OVAp-tetramer and CD3ζ are represented versus distance (middle right). Pearson correlation coefficients between the tetramer and APA1-1, the tetramer and CD3ζ, or APA1-1 and CD3ζ intensity distributions (bottom panel) are for n = 27 immunological synapses

Similar articles

See all similar articles

Cited by 1 article

  • T Cell Reprogramming Against Cancer.
    Katz SG, Rabinovich PM. Katz SG, et al. Methods Mol Biol. 2020;2097:3-44. doi: 10.1007/978-1-0716-0203-4_1. Methods Mol Biol. 2020. PMID: 31776916 Free PMC article.

References

    1. Karjalainen K. High sensitivity, low affinity—paradox of T-cell receptor recognition. Curr. Opin. Immunol. 1994;6:9–12. doi: 10.1016/0952-7915(94)90027-2. - DOI - PubMed
    1. Alarcon B, Reth M, Schamel W. Preface to special issue on nanoscale membrane organisations. Biochim Biophys. Acta. 2015;1853:765–766. doi: 10.1016/j.bbamcr.2015.02.003. - DOI - PubMed
    1. Lillemeier BF, et al. TCR and Lat are expressed on separate protein islands on T-cell membranes and concatenate during activation. Nat. Immunol. 2009;11:90–96. doi: 10.1038/ni.1832. - DOI - PMC - PubMed
    1. Schamel WW, Alarcon B. Organization of the resting TCR in nanoscale oligomers. Immunol. Rev. 2013;251:13–20. doi: 10.1111/imr.12019. - DOI - PubMed
    1. Schamel WW, et al. Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response. J. Exp. Med. 2005;202:493–503. doi: 10.1084/jem.20042155. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback