Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling
- University of California, San Francisco, United States
Figures
Figure 1 with 4 supplements
Strategy for testing kinetic proofreading with optogenetic tools.
(A) Conventional methods of mutating the pMHC to alter the binding half-life also change the binding interface, which changes several parameters at once. By contrast, optogenetic control allows …
Figure 1—figure supplement 1
Light-based control of T cell signaling is durable for hours.
(A) Clonal Jurkat cells expressing both the Zdk-CAR and DAG reporter were exposed to periodic pulses of blue-light (blue bars near the x-axis) for five hours while on SLBs functionalized with LOV2. …
Figure 1—figure supplement 2
Cells spread in response to blue-light illumination.
In addition to increasing DAG production, antigen stimulation leads to increased cell spreading in T cells. We observe similar cell spreading following optogenetic stimulation of the CAR. Cell area …
Figure 1—figure supplement 3
Colocalization of ligand binding and downstream signaling.
Higher magnification TIRF images show how ligand binding to the CAR spatially relates to downstream signaling reporters. Jurkat cells expressing the CAR and either a ZAP70-mCherry or DAG reporter …
Figure 1—figure supplement 4
Absolute quantification of cell surface TCRs and CARs.
The number of cell surface exposed TCRs and CARs was measured using beads with known antibody binding capacities and flow cytometry. (A) To ensure measurements were made at saturating antibody …
Figure 2 with 3 supplements
Blue light intensity titrates binding half-life, CAR occupancy and DAG levels.
(A) In vitro measurements of blue light intensity-based control of LOV2-Zdk binding half-life. SLBs functionalized with LOV2 were combined with soluble, dye-labeled Zdk. After washing out free Zdk, …
Figure 2—figure supplement 1
Purification of LOV2 and Zdk1.
(A) Schematic of LOV2 construct expressed in E. coli. Arrow indicates TEV protease cleavage site. The Avitag is biotinylated by BirA, which was co-expressed in the cells. KCK tag used for maleimide …
Figure 2—figure supplement 2
Calculating DAG levels.
(A) Mean TIRF561 pixel intensities within the cell mask are plotted over time. TIRF561 signals at steady state (green circles) are calculated as the average of frames taken during the last minute of …
Figure 2—figure supplement 3
Calculating CAR occupancy.
(A) RICM images were used to construct a cell mask and a local background mask. When used to mask the TIRF488 images, the fluorescence within the cell mask is the sum of freely diffusing LOV2 and …
Figure 3 with 2 supplements
Binding half-life, not receptor occupancy, dominates CAR signaling.
(A) A cell exposed to a high LOV2 density but a short binding half-life can have the same receptor occupancy as a cell exposed to a low LOV2 density but a long binding half-life. (B) At constant …
Figure 3—figure supplement 1
Long LOV2 binding half-lives signal better than short binding half-lives, even at equal receptor occupancy.
(A) Single cell measurements were binned together over narrow ranges of CAR occupancy, and DAG levels were plotted as a function of LOV2 binding half-life. Long LOV2 binding half-lives lead to …
Figure 3—figure supplement 2
DAG levels are most strongly correlated with ligand binding half-life.
DAG levels from cell stimulated with the same intensities of blue light but different LOV2 concentrations are plotted as a function of receptor occupancy or ligand binding half-life. Each black line …
Figure 4 with 3 supplements
A kinetic proofreading model best explains T cell signaling.
(A) Models for how CAR occupancy and binding half-life affect T cell signaling in the presence of moderate (left) or no kinetic proofreading (middle). To facilitate visualization, single cell …
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Figure 4—source data 1
This spreadsheet contains all the single cell data used in this study.
It includes measurements of receptor occupancy, ligand binding half-life and cell signaling (either DAG levels or ZAP70 recruitment).
- https://doi.org/10.7554/eLife.42498.025
Figure 4—figure supplement 1
DAG normalization method only has a minor effect on the calculated degree of proofreading.
Regardless of normalization method, DAG levels are strongly influenced by LOV binding half-life and produce similar degrees of proofreading (n). Heat maps of DAG signaling were generated by fitting …
Figure 4—figure supplement 2
Justification for DAG normalization.
(A) Different concentrations of the LOV2 ligand on the SLB drive very different CAR occupancies, as expected. CAR occupancy was mostly linear in response to changing half-lives, suggesting that we …
Figure 4—figure supplement 3
ZAP70 recruitment does not show evidence of kinetic proofreading.
Unlike DAG levels, ZAP70 recruitment is dominated by CAR occupancy and is relatively unaffected by the LOV2 binding half-life. Because the ZAP70 reporter is not compatible with the anti-β2 …
Scheme 1
Zdk dissociation from LOV2.
https://doi.org/10.7554/eLife.42498.027
Scheme 2
Kinetic proofreading steps.
https://doi.org/10.7554/eLife.42498.028Videos
Video 1
LOV2 reversibly binds the CAR.
Time course showing the photoreversible binding of LOV2-Alexa488 to cells expressing the Zdk-CAR in the presence or absence of strong blue light. Images taken in TIRF with a 488 nm laser. Because …
Video 2
LOV2 quickly unbinds the CAR.
Time course showing that LOV2 binds and unbinds the CAR on the order of seconds in response to blue light. To track LOV2 binding in real time, LOV2 was labeled with Cy3 (instead of Alexa 488 which …
Video 3
DAG signaling is photoreversible.
Time course showing optogenetic control CAR signaling, as measured by the photoreversible accumulation of DAG in the presence or absence of strong blue light. Cells are the same as in Video 1, …
Video 4
DAG signaling is LOV2-dependent.
Time course showing that in the absence of LOV2, the cells fail to exhibit blue light-induced changes in DAG accumulation. Jurkat cells expressing both the Zdk-CAR and DAG reporter were passively …
Video 5
Light-based titration of cell spreading.
Time course showing RICM images of cells exposed to intermediate blue-light intensities. The mean response of cell spreading is plotted.
Video 6
Light-based titration of CAR occupancy.
Time course showing the quantification of receptor occupancy (via LOV2 localization) in response to intermediate blue-light intensities. Because the 488 nm laser strongly activates LOV2, LOV2 …
Video 7
Light-based titration of DAG signaling.
Time course showing the quantification of DAG levels in response to intermediate blue-light intensities. Cells are the same as in Video 5, imaged in TIRF with a 561 nm laser. The mean response of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | C1-Halo | PMID: 17629516 | Dr. Mark M Davis (Stanford University) | |
| Recombinant DNA reagent | ZAP70-mCherry | PMID: 23840928 | Dr. Jay Groves (UC Berkeley) | |
| Recombinant DNA reagent | LOV2 (V529N) | PMID: 27427858, PMID: 18604202 | Dr. Klaus Hahn (UNC Chapel Hill) | |
| Recombinant DNA reagent | Zdk1 (purified) | PMID: 27427858 | Dr. Klaus Hahn (UNC Chapel Hill) | |
| Recombinant DNA reagent | Zdk-CAR | This paper and PMID: 1705867 | Zdk1 was fused to the N-terminus of an existing CD8 CAR, provided by Dr. Art Weiss (UCSF) | |
| Cell line (H. sapiens, male) | Jurkat | PMID: 6327821 | RRID: CVCL_0367 | Dr. Art Weiss (UCSF) |
| Cell line (H. sapiens, male) | Jurkat expressing Zdk-CAR and C1- Halo reporter | This paper. | A clonal Jurkat line expressing the Zdk-CAR and C1-Halo reporter made via lentiviral transduction. | |
| Cell line (H. sapiens, male) | Jurkat expressing Zdk-CAR and ZAP70 -mCherry reporter | This paper. | A clonal Jurkat line expressing the Zdk-CAR and ZAP70-mCherry reporter made via lentiviral transduction. | |
| Antibody | Mouse anti-human B2 microglobulin | BioLegend | Cat. #: 316308 RRID: AB_493689 | Cells labeled at 0.5 ug/ml in growth media. |
| Chemical compound, drug | PP2 | abcam | Cat. #: ab120308 | Used at 10 uM |
| Chemical compound, drug | Halo dye (JF549) | PMID: 25599551 | Dr. Luke Lavis (Janelia Research Campus) | |
| Chemical compound, drug | Alexa Fluor 488 C5 Maleimide | ThermoFisher Scientific | Cat. #: A10254 | |
| Chemical compound, drug | Sulfo-Cyanine3 maleimide | Lumiprobe | Cat. #: 11380 | |
| Chemical compound, drug | POPC | Avanti Polar Lipids | Cat. #: 850457C | |
| Chemical compound, drug | PEG-PE | Avanti Polar Lipids | Cat. #: 880230C | |
| Chemical compound, drug | biotinyl CAP PE | Avanti Polar Lipids | Cat. #: 870277X |
Table 1
Plasmids use in this study.
The plasmid name, the expressed protein, and a brief description of the construct are given. The entire coding regions of all constructs were verified by Sanger Sequencing. Plasmids and detailed …
| Plasmid | Expressed protein | Backbone | Description |
|---|---|---|---|
| pDT326 | DAG reporter | pHR (James and Vale, 2012) | C1 domains mPKCθ (aa 45–166)-HaloTag |
| pDT481 | Zdk1 | pETM11-SUMO3 | 10xHis-TEV-SUMO3-KCK-SpyCatcher-GS linker-Zdk1 |
| pDT523 | ZAP70 reporter | pHR | hZAP70-mCherry |
| pDT537 | Zdk-CAR | pHR (James and Vale, 2012) | IgK ss-HA tag-Zdk1-GS linker-hCD8α (aa 22–208)-mCD3ζ (aa 52–164)-tagBFP |
| pDT552 | LOV2 | pETM11-SUMO3 | 10xHis-TEV-AviTag-KCK-LOV2 V529N |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.42498.029
