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Updated: May 31, 2026
Retroviral Transduction of T-cell Receptors in Mouse T-cells
Published on: October 22, 2010
In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase
Catherine B Carbone1,2,3, Nadja Kern1,2, Ricardo A Fernandes4,5,6
1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158.
Abstract:
T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.
Insights
T cell receptor (TCR) signaling involves CD45 phosphatase exclusion from pMHC complexes. This spatial segregation, driven by binding energy, regulates T cell activation and can be modulated by CD45 isoforms and PD-1/PD-L1 interactions.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- T cell activation relies on T cell receptor (TCR) signaling, initiated by peptide-MHC (pMHC) binding.
- The kinetic segregation hypothesis posits that exclusion of the CD45 phosphatase from TCR-pMHC complexes favors TCR phosphorylation.
- Understanding the physical basis of this spatial partitioning is crucial for deciphering T cell signaling dynamics.
Purpose of the Study:
- To reconstitute and investigate the segregation of CD45 from receptor-ligand pairs using purified proteins on model membranes.
- To determine the role of protein size, binding affinity, and specific receptor-ligand interactions in CD45 exclusion.
- To explore the implications of CD45 isoform size and PD-1/PD-L1 interactions in T cell signaling regulation.
Main Methods:
- Reconstitution of receptor-ligand interactions on model membranes using purified proteins (FRB-FKBP system).
- Quantitative analysis of CD45 exclusion dynamics using different CD45 isoforms (RABC and R0).
- Investigation of CD45 exclusion by TCR-pMHC and PD-1/PD-L1 interactions at varying binding affinities (Kd).
Main Results:
- CD45 exclusion from model receptor-ligand complexes was successfully reconstituted.
- The larger CD45 RABC isoform showed greater exclusion (∼50%) than the smaller R0 isoform (∼20%).
- TCR-pMHC interactions with Kds ≤15 µM and PD-1/PD-L1 binding induced CD45 exclusion, demonstrating the role of binding energy.
Conclusions:
- The binding energy of physiological receptor-ligand pairs is sufficient to drive spatial organization at membrane interfaces.
- CD45 isoform size differences can modulate the efficiency of CD45 exclusion, potentially regulating signaling thresholds in distinct T cell populations.
- CD45 exclusion is a general mechanism applicable to both activating TCR-pMHC and inhibitory PD-1/PD-L1 interactions, highlighting its broad significance in T cell regulation.
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