Transient protein accumulation at the center of the T cell antigen-presenting cell interface drives efficient IL-2 secretion
- University of Bristol, United Kingdom
- Carnegie Mellon University, United States
- University of Texas Southwestern Medical Center, United States
- National Human Genome Research Institute, National Institutes of Health, United States
- Albert Ludwig University of Freiburg, Germany
Figures
Figure 1 with 4 supplements
CD28 and Itk regulate IL-2 secretion and signaling organization.
(A) In vitro primed 5C.C7 T cells, wild type or Itk-deficient (‘Itk ko’), were activated by CH27 APCs and the indicated concentration of MCC peptide in the absence or presence of 10 µg/ml anti-CD80 …
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Figure 1—source data 1
Sensors used in Figure 1C,D.
Publications describing the sensors used in Figure 1C,D and representative 5C.C7 T cell imaging data are given.
- https://cdn.elifesciences.org/articles/45789/elife-45789-fig1-data1-v2.pdf
Figure 1—figure supplement 1
The generation of IL-2 mRNA is focused on the first 6 hr after cell couple formation.
(A) In vitro primed 5C.C7 T cells were activated by CH27 APCs and 10 µM of MCC peptide in the absence or presence of 10 µg/ml anti-CD80 plus anti-CD86 (‘full stimulus’ or ‘costimulation blockade’). …
Figure 1—figure supplement 2
Sensor accumulation at the T cell:APC interface under full stimulus conditions.
(A) The panel graphically represents the six categories used to classify spatiotemporal sensor distribution as underpinned by defined cell biological structures at the T cell:APC interface (Roybal …
Figure 1—figure supplement 3
Sensor accumulation at the T cell:APC interface under costimulation blocked conditions.
The graphs display the percentage of cell couples that displayed accumulation of the indicated sensor (Figure 1—source data 1) with the indicated patterns (Figure 1—figure supplement 2A) relative to …
Figure 1—figure supplement 4
Sensor accumulation at the T cell:APC interface in the absence of Itk.
The graphs display the percentage of cell couples that displayed accumulation of the indicated sensor (Figure 1—source data 1) with the indicated patterns (Figure 1—figure supplement 2A) relative to …
Figure 2 with 2 supplements
LAT localization and activation is regulated by costimulation and Itk.
(A) An interaction of a LAT-GFP-transduced 5C.C7 T cell with a CH27 APC (10 μM MCC) is shown at the indicated time points (in minutes) relative to the time of formation of a tight cell couple. …
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Figure 2—source data 1
Statistical significance of differences in LAT accumulation under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test.
No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance.
- https://cdn.elifesciences.org/articles/45789/elife-45789-fig2-data1-v2.pdf
Figure 2—figure supplement 1
Representative phospho-LAT Y191 western blots.
(A) Wild type or Itk-deficient 5C.C7 T cells were activated with CH27 B cells and 10 µm MCC in the presence or absence of 10 µg/ml anti-CD80 plus anti-CD86. At the given time points (in minutes) T …
Figure 2—video 1
Representative interactions of 5C. C7 T cells retrovirally transduced to express the indicated GFP fusion proteins with CH27 B cell lymphoma APCs and 10 μM MCC peptide in the presence or absence of 10 µg/ml anti-CD80 plus anti-CD86 (‘costimulation blockade’) are shown in Figure 2—Video 1, Figure 4—Videos 1–3, Figure 7—Videos 1–3 and Figure 8—Video 1.
DIC images are shown on the top, with matching top-down, maximum projections of 3D sensor fluorescence data on the bottom. The sensor fluorescence intensity is displayed in a rainbow-like, …
Figure 3 with 3 supplements
The cSMAC consists of multiple smaller complexes and is associated with extensive membrane undulations.
(A) Two representative STED midplane images are given of 5C.C7 T cells activated by CH27 APCs (10 µm MCC) for 4.5 min and stained with α-LAT pY191. Staining fluorescence intensity is given in …
Figure 3—figure supplement 1
CLEM work flow.
Wild type 5C.C7 T cells expressing LAT-GFP were imaged upon activation with CH27 APCs and 10 µm MCC on a finder grid using DIC and spinning disk confocal fluorescence microscopy. The left and middle …
Figure 3—video 1
Representative EM tomograms are shown in Figure 3—Videos 1 and 2.
Reconstructed Z-sections are first shown without tracing and subsequently with the T cell and APC plasma membranes at the interface traced in blue and red, respectively. The 5C.C7 T cell in Figure …
Figure 3—video 2
The video is displayed similar to Figure 3—Video 1.
The 5C.C7 T cell in Figure 3—Video 2 is transduced with LAT V3-GFP and responds to a full stimulus upon costimulation blockade. It displayed central LAT-GFP accumulation at the time of fixation.
Figure 4 with 4 supplements
LAT localization can be controlled by fusion with protein domains with a strong interface localization preference.
(A) A schematic representation of LAT-GFP is given (top) with LAT accumulation data under full stimulus conditions (bottom, from Figure 2B) as a reference for the rest of the figure. (B) On top …
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Figure 4—source data 1
Statistical significance of differences in accumulation of spatially targeted as compared to non-targeted LAT under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test.
No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance.
- https://cdn.elifesciences.org/articles/45789/elife-45789-fig4-data1-v2.pdf
Figure 4—figure supplement 1
Patterning of isolated targeting domains and interface recruitment of LAT-GFP and spatially targeted version thereof.
(A) The graphs give the percentage of cell couples that displayed accumulation of the isolated targeting domains, LAT V3, LAT Vav, and LAT PLCδPH as indicated, with the indicated patterns as in Figur…
Figure 4—video 1
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 4—Video 1 is transduced with LAT V3-GFP and responds to a full stimulus upon costimulation blockade. Cell coupling occurs in frame 4 (2s indicated video time).
Figure 4—video 2
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 4—Video 2 is transduced with LAT Vav-GFP and responds to a full stimulus upon costimulation blockade. Cell coupling occurs in frame 5 (2s indicated video time).
Figure 4—video 3
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 4—Video 3 is transduced with LAT PLCδ PH-GFP and responds to a full stimulus upon costimulation blockade. Cell coupling occurs in frame 5 (2s indicated video time).
Figure 5 with 1 supplement
Restoration of LAT centrality enhances IL-2 mRNA generation.
(A) Wild type or Itk-deficient (‘Itk ko’) 5C.C7 T cells expressing LAT-GFP or a spatially targeted variant thereof as indicated were activated by CH27 APCs (10 µM MCC) in the absence or presence of …
Figure 5—figure supplement 1
IL2 mRNA amounts upon expression of the isolated targeting domains.
Wild type or Itk-deficient (‘Itk ko’) 5C.C7 T cells were transduced to express the isolated targeting domains, LAT V3, LAT Vav, and LAT PLCδPH as indicated, and reactivated by CH27 APCs (10 µM MCC …
Figure 6
Restoration of LAT centrality only modestly affects centrality of other signaling intermediates.
Wild type and Itk-deficient 5C.C7 T cells were transduced to express LAT V3 together with the indicated GFP-tagged signaling intermediate and activated with CH27 APCs (10 µM MCC) in the absence or …
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Figure 6—source data 1
Statistical significance of differences in accumulation of Grb2, Lck and Vav1 in the presence as compared to absence of LATV3 under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test.
No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance.
- https://cdn.elifesciences.org/articles/45789/elife-45789-fig6-data1-v2.pdf
Figure 7 with 3 supplements
SLP-76 localization is regulated by costimulation and Itk and restoration of early SLP-76 centrality enhances IL-2 mRNA generation.
(A) An interaction of a SLP-76-GFP-transduced 5C.C7 T cell with a CH27 APC (10 μM MCC) is shown at the indicated time points (in minutes) relative to the time of formation of a tight cell couple as …
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Figure 7—source data 1
Statistical significance of differences in SLP-76 accumulation and in accumulation of spatially targeted compared to non-targeted SLP-76 under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test.
No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance.
- https://cdn.elifesciences.org/articles/45789/elife-45789-fig7-data1-v2.pdf
Figure 7—video 1
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 7—Video 1 is transduced with SLP-76-GFP and responds to a full stimulus. Cell coupling occurs in frame 4 (2s indicated video time).
Figure 7—video 2
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 7—Video 2 is transduced with SLP-76 V3-GFP and responds to a full stimulus upon costimulation blockade. Cell coupling occurs in frame 4 (2s indicated video time).
Figure 7—video 3
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 7—Video 3 is transduced with SLP-76 Vav-GFP and responds to a full stimulus upon costimulation blockade. Cell coupling occurs in frame 5 (2s indicated video time).
Figure 8 with 3 supplements
Grb2 localization is regulated by costimulation and Itk and doesn’t regulate IL-2 mRNA generation.
(A) An interaction of a Grb2-GFP-transduced 5C.C7 T cell with a CH27 APC (10 μM MCC) is shown at the indicated time points (in minutes) relative to the time of formation of a tight cell couple as in …
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Figure 8—source data 1
Statistical significance of differences in Grb2 accumulation and in accumulation of spatially targeted as compared to non-targeted Grb2 under different T cell activation conditions is given for the indicated patterns as determined by proportion’s z-test.
No entry indicates p>0.05. 0.000 indicates p<0.0005. Gray scale is used to visualize the level of significance.
- https://cdn.elifesciences.org/articles/45789/elife-45789-fig8-data1-v2.pdf
Figure 8—video 1
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 8—Video 1 is transduced with Grb2-GFP and responds to a full stimulus. Cell coupling occurs in frame 5 (2s indicated video time).
Figure 8—video 2
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 8—Video 2 is transduced with Grb2 V3-GFP and responds to a full stimulus upon costimulation blockade. Cell coupling occurs in frame 4 (2s indicated video time).
Figure 8—video 3
The video is displayed similar to Figure 2—Video 1.
The 5C.C7 T cell in Figure 8—Video 3 is transduced with Grb2 Vav-GFP and responds to a full stimulus upon costimulation blockade. Cell coupling occurs in frame 5 (2s indicated video time). A second …
Author response image 1
Representative anti-phospho Src staining experiment.
Given are 3 5C.C7/LAT-Vav-GFP T cell CH27 B cell APC couples after one minute of interaction under full stimulus conditions as fixed and stained with anti-phospho Src 419-Alexa 568 and imaged by …
Author response image 2
anti-phospho LAT Western blot with no peptide control.
5C.C7 T cells were activated with CH27 B cell APCs for the indicated time in minutes under full stimulus (‘wt’) and costimulation blocked (‘αB7’) conditions, lysed and blotted with an anti-LAT pY191 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | 5C.C7 TCR transgenic | Davis lab, Stanford (Seder et al., 1992) | RRID:MGI:3799371 | |
| Genetic reagent (M. musculus) | 5C.C7 TCR transgenic, Itk ko | This paper | generated by crossing 5C.C7 TCR transgenic with Itk-deficient B6 mice (Schaeffer et al., 1999) (RRID:MGI:4356470). | |
| Cell line (Mus musculus) | CH27 | Davis lab, Stanford | RRID:CVCL_7178 | |
| Cell line (Homo sapiens) | Phoenix E | Nolan lab, Stanford | RRID:SCR_003163 | |
| Antibody | Anti - LAT pY191 (Rabbit polyclonal) | Cell Signaling | Cat#3584, RRID:AB_2157728 | WB (1:1000), Immunostaining (1:100) |
| Antibody | Anti-LAT (Rabbit polyclonal) | Cell Signaling | Cat#9166, RRID:AB_2283298 | WB (1:1000), Immunostaining (1:50) |
| Antibody | Anti-rabbit IgG, Alexa Fluor 488 (Donkey polyclonal) | Molecular Probes | Cat#R37118, 1:1000, RRID:AB_2556546 | Immunostaining (1:1000) |
| Antibody | Anti - GAPDH Clone 14C10 (Rabbit monoclonal) | Cell Signaling | Cat#2118, RRID:AB_561053 | WB (1:1000) |
| Antibody | Anti- alpha Tubulin Clone DM1A (Mouse monoclonal) | Thermo Fisher Scientific | Cat#62204, RRID:AB_1965960 | WB (1:1000) |
| Antibody | Anti – CD80 Clone16–10-A1 (Armenian Hamster monoclonal) | BD Pharmingen | Cat#553736 | Blocking at 10 µg/ml |
| Antibody | Anti – CD86 Clone GL1 (Rat monoclonal) | BD Pharmingen | Cat#553689 | Blocking at 10 µg/ml |
| Peptide, recombinant protein | MCC | Davis lab, Stanford | Sequence: ANERADLIAYLKQATK | |
| Commercial assay or kit | RNeasy Micro Kit | Qiagen | Cat#74004 | |
| Commercial assay or kit | AMV First-Strand cDNA synthesis kit | Invitrogen | Cat#12328032 | |
| Commercial assay or kit | SYBR Green PCR master mix | Life Technologies | Cat#4344463 | |
| Commercial assay or kit | IL-2 OptEIA ELISA | BD Biosciences | Cat#555148 | |
| Sequence-based reagent | qPCR Oligonucleotides, IL-2 | This paper | AGCTGTTGATGGACCTA and CGCAGAGGT CCAAGTTCAT | |
| Software, algorithm | Metamorph image analysis software | Molecular Devices | RRID:SCR_002368 | |
| Software, algorithm | Amira image analysis software | VSG | RRID:SCR_007353 |
