Regulation of lck degradation and refractory state in CD8 + cytotoxic T lymphocytes. Michael Uhlin, Maria G. Masucci, and Victor Levitsky * PNAS | June 28, 2005 | vol. 102 | no. 26 | 9264-9269. Date : 10/30/07 Name : Park Jong Chan. Abstract.
Michael Uhlin, Maria G. Masucci, and Victor Levitsky*
PNAS | June 28, 2005 | vol. 102 | no. 26 | 9264-9269
Date : 10/30/07
Name : Park Jong Chan
- Cell lines, CTL cultures and Clones
EA/BK/ CAR: TCR structure of the IVT-peptide-specific
from EBV (Epstein–Barr virus)
JAC-B2 : processes and presents the two peptides(IVT and AVT) endogenously.
- Analysis of protein expression by immunoblotting
- Inhibition of the proteasome and other protease.
Epoxomycin(300nM), MG132(50-100uM), Leupeptin(100uM)
- FACS analysis
- Plasmid preparation and Transfection of CTLs
pcDNA3.1 , pcDNA3.1-lck
- Analysis of T Cell Activation and Death.
Peptide treatment (1*10-7 M)
Stimulation with APC
Fig. 1. TCR triggering induces lck down-regulation in activated CD8+ T cells.
2. Specific Activation of CTLs Induces Rapid and Persistent Down-Regulationof Lck Expression.
Fig. 2. Kinetics of lck down-regulation in IVT-specific CTL
Fig. 3. AID of lck is proteasome- and lysosome-independent but sensitive to inhibition by MG132
Fig. 3. Inhibitors. AID of lck is proteasome- and lysosome-independent but sensitive to inhibition by MG132
(D) Effect of MG132 on lck degradation assessed at the indicated concentrations.
(E) Quantification of the effect of MG132 at a concentration of 100 µM
(F) Control or activated CTLs were cultured in the presence or absence of NH4Cl. Immunoblotting of total cell lysates with lck-, IL-15Ra -, or actin-specific Abs.
Is Lck Degradation necessary for Efficient T Cell Activation???
[ One possible interpretation ]
lck degradation is required for efficient TCR signal transduction and T cell activation.
The effect of Z-LL-H on the capacity of BK CTLs to kill C1RA11 cells and produce IL-2 or IFN-r in response to stimulation with IVT pulsed APCs.
Result : None of these parameters of T cell activation was negatively affected by Z-LL-H.
A comparable decrease of cell recovery was observed in BK bulk CTL cultures activated by IVT-pulsed APCs in the presence or absence of Z-LL-H, whereas the release of IFN-r was slightly increassed in the presence of the inhibitor.
Lck Degradation Is Not a Prerequisite for Efficient T Cell Activation.
Development of AINR in CTLs.
Wild type : IVT
Partial agonistic variants :
Y5 : F to Y substitution
A8 : I to A substitution
Wild type : 34% to 25% decrease
Fig. 5. Responsiveness of CTLs correlates with lck expression, and treatment with Z-LL-H prevents the development of AINR in specific CTLs.
* A strong correlation was observed between the level of lck down-regulation induced by a given peptide and the extent to which responsiveness of the CTLs was inhibited.
( in theC and D) BK bulk CTLs were activated for 2 h on plastic plates with absorbed CD3-specific Ab, transferred to a clean plate, and cultured for 48 h either in the absence or presence of 20uM Z-LL-H. Lck expression in these cells was evaluated by immunoblotting.
(D) Cells were then activated by IVT-peptide pulsed APCs, and their proliferation was evaluated by a [3H]thymidine incorporation assay.
* The presence of L-ZZ-H during the first CTL triggering inhibited lck degradation as well as the development of AINR in these cells.
Fig. 6. Transfection of pcDNA3.1-lck into refractory CTLs enhances their capacity to produce IFN-r in response to specific stimulation
* First triggering -> Transfection with pcDNA3.1-lck or control pcDNA3.1 plasmid-> Overnight culture -> Re-stimulation -> measuring the IFN-r
6. IFN- and IL-15 Reconstitute Lck Expression and Abrogate the Development
of AINR in Specific CTLs.