Allosteric inhibition of the T cell receptor by a designed membrane ligand
Figures
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Peptide inhibitor of T cell receptor (PITCR) reduces phosphorylation of the ζ chain in response to OKT3.
(A) Cartoon that illustrates TCR proximal downstream signaling. The plasma membrane is shown as a horizontal bar, and phosphorylation sites are shown as yellow dots. ECD: extracellular domain; TMD: transmembrane domain; ICD: intracellular domain of TCR. (B) Jurkat cells were treated with PITCR, followed by stimulation with OKT3. Lysates were analyzed by immunoblot to detect TCR phosphorylation of ζ (pY142 and pY83). Total ζ levels were assessed and no change was observed. Data are representative of at least five independent experiments. (C) Quantification of phosphorylation at both tyrosine residues in the presence of OKT3, normalized to data in the absence of PITCR (based on data from Figure 1—figure supplement 2). Error bars are the SD. p-Values were calculated using a two-tailed Mann-Whitney test.
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Figure 1—source data 1
Original western blots.
- https://cdn.elifesciences.org/articles/82861/elife-82861-fig1-data1-v1.zip
![](https://iiif.elifesciences.org/lax:82861%2Felife-82861-fig1-figsupp1-v1.tif/full/617,/0/default.jpg)
The secondary structures of PITCR and PITCRG41P at a physiological pH and an acidic pH.
(A) Top, the partial amino acid sequence from human CD3ζ comprising a small segment of the extracellular domain, the transmembrane domain (underlined), and a small portion of intracellular domain. Middle, the amino acid sequence of peptide inhibitor of T cell receptor (PITCR) peptide. Bottom, the amino acid sequence of PITCRG41P. Introduced residues are highlighted in green. Residue numbers are labeled in human CD3ζ sequence. Circular dichroism spectra for PITCR and PITCRG41P in different conditions: 10 mM sodium phosphate buffer at pH 7.4 (B), and in the presence of vesicles of 16:0–18:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleolyl-glycero-3-phosphocholine (POPS/POPC) (1/9) at pH 7.4 (C) and at pH 5.0. (D) Each spectrum is the mean of three independent experiments. (E) Representative circular dichroism pH titration curve of PITCR in the presence of 16:0–18:1 POPS/POPC (1/9) liposomes. Data are calculated by the difference of molar ellipticity ([θ]) between 222 and 260 nm. Data are representative of two independent experiments. pK CD is 5.87±0.03 (mean ± SD).
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Quantification of phosphorylation of ζ (pY142) (A) and ζ (pY83) (B) after OKT3 stimulation.
Band intensities were normalized to ζ (total). Each dot pair represents one independent experiment. p-Values were calculated using a two-tailed paired t-test.
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Peptide inhibitor of T cell receptor (PITCR) reduces phosphorylation of TCR proximal signaling proteins after activation.
(A) Immunoblot analysis of lysates to detect phosphorylation of Zap70 (pY319 and pY493), LAT (pY132 and pY191), SLP76 (pY128), and PLCγ1 (pY783). Total protein levels of Zap70, LAT, and the housekeeping protein β-actin were assessed, revealing no changes. Data are representative of at least five independent experiments. (B) Quantification of phosphorylation in the presence of OKT3, normalized to data in the absence of PITCR (based on data from Figure 2—figure supplement 1). Error bars are the SD. p-Values were calculated using a two-tailed Mann-Whitney test.
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Figure 2—source data 1
Quantification results of multiple TCR proximal phophorylated proteins.
- https://cdn.elifesciences.org/articles/82861/elife-82861-fig2-data1-v1.zip
![](https://iiif.elifesciences.org/lax:82861%2Felife-82861-fig2-figsupp1-v1.tif/full/617,/0/default.jpg)
Quantification of Zap70 (pY319), Zap70 (pY493), LAT (pY132), LAT (pY191), SLP76 (pY128), and PLCγ1 (pY783) in response to OKT3 stimulation.
Zap70 (pY319) and Zap 70 (pY493) were normalized with Zap70 (total). LAT (pY132) and LAT (pY191) were normalized with LAT (total). SLP76 (pY128) and PLCγ1 (pY783) were normalized with β-actin. Each dot pair represents one independent experiment. All p-values except PLCγ1 (pY783) were calculated using a two-tailed paired t-test. p-Value for PLCγ1 (pY783) was calculated using a two-tailed Wilcoxon matched-pairs signed-rank test because the p-value for the F test to compare variance was 0.0494.
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Peptide inhibitor of T cell receptor (PITCR) does not reduce phosphorylation of Lck after OKT3 activation.
(A) Immunoblot analysis of Lck (pY394 and pY505), Lck (total), and the housekeeping protein β-actin. Data are representative of four independent experiments. (B) and (C) show normalized quantifications of phosphorylation of tyrosine at the positions of 394 and 505 of Lck after TCR activation. Data in the presence of PITCR was normalized to data in the absence of PITCR in response to OKT3 activation. Error bars are the SD. p-Values were calculated using a two-tailed Mann-Whitney test. (D) and (E) show quantification of phosphorylation of Lck at pY394 and pY505 after the OKT3 stimulation. Band intensities were normalized to Lck (total). Each dot pair represents one independent experiment. p-Value was calculated using a two-tailed paired t-test.
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Figure 2—figure supplement 2—source data 1
Quantification results of phosphorylated Lck in presence of PITCR.
- https://cdn.elifesciences.org/articles/82861/elife-82861-fig2-figsupp2-data1-v1.zip
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Peptide inhibitor of T cell receptor (PITCR) reduces the TCR intracellular calcium response.
Jurkat cells were stained with the fluorescent dye Indo-1 AM, followed by treatment with PITCR (A), pHLIP as a negative control (B), or the variant PITCRG41P (C) and stimulated with OKT3. Ionomycin was applied as a positive calcium control. The Indo-1 ratio was calculated from fluorescence at 405 nm (calcium-bound) divided by 475 nm (calcium-free). Data are representative of three independent experiments. Each independent experiment includes at least four technical replicates. Error bars are the SEM. (D) Quantification of the maximum Indo-1 increase after OKT3 activation, normalized to no peptide treatment. Error bars are the SD. p-Values were calculated from a Kruskal-Wallis test with Dunn’s multiple comparisons test.
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Peptide inhibitor of T cell receptor (PITCR) reduces intracellular calcium responses for PITCR (A), but not for pHLIP (B) or PITCRG41P (C).
Quantification of the magnitude of Indo-1 ratio between OKT3 peak and baseline. Each dot pair represents one independent experiment. Each independent experiment (n=3) includes at least four technical replicates. p-Values were calculated using two-tailed paired Student’s t-test.
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Peptide inhibitor of T cell receptor (PITCR) reduces CD69 expression after T cell activation by antigen-presenting cell (APC).
(A) Cartoon showing T cell interaction with APC. (B) A live cell microscopy image that depicts engineered Jurkat-OT1+ TCR-CD8+ T cells interacting with T2Kb APC pre-incubated with the peptide antigen ovalbumin (OVA). (C) Jurkat-OT1+ TCR-CD8+ T cells were treated with PITCR or pHLIP (as a negative control), and then incubated with T2Kb cells at different concentrations of OVA, followed by CD69 flow cytometry analysis. The upregulation of CD69 is representative of four independent experiments. Each independent experiment includes two technical replicates. Error bars are the SD. (D) Quantification of CD69-positive cells at [OVA]=1 μM for PITCR (red) and negative control pHLIP (green). Each dot pair represents one independent experiment. p-Values were calculated from two-tailed paired t-test.
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Peptide inhibitor of T cell receptor (PITCR) co-localizes with TCR.
(A) PITCR680 and CD3ɛ co-localization was studied in the presence and absence of OKT3. Scale bars = 10 μm. Representative areas with co-localization at the plasma membrane (top) and cytoplasm (bottom) were zoomed-in, where scale bars are 0.5 μm and 1 μm, respectively. Confocal images are representative of three independent experiments. (B) and (C) show graphic profile curves (dotted yellow lines) plotted across the zoom-in regions of interest (ROI) in +OKT3 and –OKT3, respectively. Magenta lines denote PITCR, and green lines denote CD3ɛ. Overlap indicates co-localization. (D) Quantification of co-localization by Mander’s coefficient (M1), corresponding to the fraction of PITCR that overlaps with CD3ɛ. Error bars indicate SD. p-Value was calculated from two-tailed unpaired t-test.
![](https://iiif.elifesciences.org/lax:82861%2Felife-82861-fig5-figsupp1-v1.tif/full/617,/0/default.jpg)
Peptide inhibitor of T cell receptor (PITCR) co-localizes with TCR.
(A) PITCR and CD3ɛ co-localization images and nuclear staining (DAPI) are shown. Confocal images are representative of three independent experiments. Scale bars = 10 μm. (B) Quantification of PITCR and CD3ɛ co-localization calculating Pearson’s r value. Each dot of panel B denotes one technical replicate from three independent experiments. N=19–21. Error bars indicate SD. p-Value was calculated using a two-tailed unpaired t-test. (C) Jurkat cells were treated with control followed by an anti-CD3ɛ immunofluorescent staining. Confocal images are representative of three independent experiments. Scale bars = 10 μm.
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Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) spectra of NEC-peptide inhibitor of T cell receptor (PITCR).
(A), PITCR conjugated with dylight680 (B), and PITCR conjugated with AZ555 (C). The theoretical MW of NEC-PITCR is 3812.31. The MW of dylight 680 and AZ555 are 972 Da and 969.12 Da, respectively.
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Co-localization of peptide inhibitor of T cell receptor (PITCR) and the TCR-pMHC complex in primary murine CD4+ T cells.
(A) Images of plasma-membrane-localized PITCR555 and TCR-pMHC complex in a representative T cell adhering to supported lipid bilayer functionalized with pMHC (19–23 molecules/µm2, 9% labeled with Atto-647N) and ICAM-1 (~20 molecules/µm2). TCR-pMHC complex was selectively visualized with a long exposure time (500 ms). PITCR exhibited localization at central supramolecular activation cluster (c-SMAC) together with the TCR-pMHC complex. (B) Vehicle control showed no signal at the PITCR channel. Panel labels correspond to those in A.
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The NFAT dose-response curve of primary murine T cells is unaffected by peptide inhibitor of T cell receptor (PITCR).
AND-TCR primary murine CD4+ T cells expressing NFAT-mCherry reporter protein were stimulated by supported lipid bilayers functionalized with varied density of pMHC (moth cytochrome c [MCC] peptide 100% labeled with Atto-647N) and ICAM-1 (~20 molecules/µm2). Cells were defined as activated if the NFAT-mCherry signal in the nucleus was greater than the signal in the cytoplasm, as determined by epifluorescence imaging. The fraction of activated cells is indistinguishable between cells treated with or without PITCR at all pMHC densities. Error bars denote SEM.
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Peptide inhibitor of T cell receptor (PITCR) interacts with TCR.
Jurkat cells were treated with PITCR-680. TCR nanodiscs were immunoprecipitated with the monoclonal antibody (mAb) anti-CD3 (UCHT1 clone), or with IgG as a negative control. Fluorescent detection of PITCR-680 after co-immunoprecipitation (Co-IP) or run in the gel directly as a positive control (right side panel). Below are shown immunoblot analysis of Co-IP samples and whole lysates to probe CD3ɛ, TCRβ, and CD3ζ. β-Actin was a loading control. Data are representative of at least three independent experiments.
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Figure 7—source data 1
This data contains the PITCR immunoprecipitation results.
- https://cdn.elifesciences.org/articles/82861/elife-82861-fig7-data1-v1.zip
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Peptide inhibitor of T cell receptor (PITCR) weakens the interaction of the ζ chain with the rest of the complex after TCR activation.
(A) Immunoblot analysis of immunoprecipitated samples and whole lysate samples solubilized with DDM. Data are representative of at least three independent experiments. (B) Quantification of ζ/β2 and ζ/ε after OKT3 stimulation. (C) ζ/β2 and ζ/ε in PITCR-treated OKT3 samples, normalized to OKT3 stimulation. The β2 subunit was studied since it is the most abundant β subunit at the plasma membrane. Error bars are SD. p-Values were calculated from two-tailed Mann-Whitney test.
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Figure 8—source data 1
Quantification of immunoprecipitation results in presence of PITCR with/without OKT3 stimulations.
- https://cdn.elifesciences.org/articles/82861/elife-82861-fig8-data1-v1.zip
![](https://iiif.elifesciences.org/lax:82861%2Felife-82861-fig8-figsupp1-v1.tif/full/617,/0/default.jpg)
Quantification of DDM immunoprecipitation results for the following conditions.
(A) OKT3 stimulation, (B) OKT3 stimulation in the presence or absence of peptide inhibitor of T cell receptor (PITCR), and (C) PITCR incubation without stimulation. Error bars are SD. p-Values were calculated with a two-tailed Mann-Whitney test.
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AlphaFold-Multimer (AlFoM) model for peptide binding to T cell receptor (TCR).
(A) Side view of the model that shows the TCR bound to peptide inhibitor of TCR (PITCR) (red). The N-terminus of PITCR is at the top. TCR subunits are colored as shown in the legend. (B) Bottom (cytoplasmic) view of the isolated TCR is shown as a reference in the top left. The two zeta chains are labeled ZA -ζ(εδ)- and ZB -ζ(εγ)-. AlFoM models are shown for TCR in association with PITCR (rank 2) and the negative control peptides PITCRG41P (rank 2), pHLIP (rank 1), and TYPE7 (rank 1), all in red. The extracellular domains are shown semi-transparently. The leucine zipper appended to the ζ chains to constrain dimer formation (see methods) is not shown in A or B.
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Figure 9—source data 1
Interactions and CD3ζ chain displacements predicted by AlphaFold-Multimer.
(A) Table listing residues in peptide inhibitor of T cell receptor (PITCR) and PITCRG41P that closely interact with one or both zeta chains and the corresponding interaction distance. Zeta chains are labeled A and B as in Figure 9B. (B) Table listing the maximum displacement distance for α-carbons induced in zeta chain by the indicated peptide, measured by superimposition of the TCRα chain in the AlphaFold model of TCR alone and the model of TCR associated with each peptide. The root-mean-square deviation (RMSD) value for the zeta chains is also listed, calculated by superimposition of the TCRα chain in the AlphaFold model of TCR alone and the model of TCR associated with each peptide. Note that both zeta chains are displaced to a greater extent by PITCR than by the other peptides.
- https://cdn.elifesciences.org/articles/82861/elife-82861-fig9-data1-v1.pdf
![](https://iiif.elifesciences.org/lax:82861%2Felife-82861-fig9-figsupp1-v1.tif/full/617,/0/default.jpg)
Comparison between cryoEM structure and AlphaFold-Multimer prediction of T cell receptor (TCR).
(A) Side views are shown for experimentally determined structure of the human TCR (PDB 6JXR) and the highest ranked AlphaFold-Multimer prediction (rank 1). The extracellular domain is shown at the top. In the AlphaFold-Multimer model, we show in black a segment of the leucine zipper appended to the cytoplasmic termini of the zeta subunits to ensure dimer formation between these chains. The transmembrane (TM) helices are indicated. The color scheme for TCR subunits is shown at the bottom. (B) Overlay between the cryoEM structure (blue) and the prediction (red).
![](https://iiif.elifesciences.org/lax:82861%2Felife-82861-fig9-figsupp2-v1.tif/full/617,/0/default.jpg)
Comparison of AlphaFold-Multimer model of T cell receptor (TCR) associated with peptide inhibitor of TCR (PITCR) or PITCRG41P.
(A) PITCR (red) and PITCRG41P (gray) peptide bound to TCR are shown in side view. Overlay between the TCR associated with PITCR (orange) and PITCRG41P (yellow) are shown. Zeta chains of the TCR associated with PITCR and PITCRG41P are shown in blue and purple, respectively. The same view with the rest of TCR invisible is shown to highlight the position of the zeta chains or peptides alone. (B) Bottom (cytoplasmic) view of the overlay in panel A, with the extracellular domains semi-transparent. The leucine zipper is not shown in A or B.
Videos
Real-time imaging of peptide inhibitor of T cell receptor (PITCR) and pMHC in a T cell adhering to supported bilayer.
Left: RICM, center: PITCR555, right: pMHC. Cell footprint is shown as cyan line. Scale bar: 10 µm.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/82861/elife-82861-mdarchecklist1-v1.docx
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Source data 1
This source data contains all original plot data.
- https://cdn.elifesciences.org/articles/82861/elife-82861-data1-v1.zip