Regulation of lck degradation and refractory state in CD8+ cytotoxic T lymphocytes -- Uhlin et al. 102 (26): 9264 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Uhlin et al. 10.1073/pnas.0406333102.

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Supporting Figure 8
Supporting Figure 9
Supporting Figure 10

Supporting Figure 8

Fig. 8. Preactivated CD8⁺ lymphocytes from T cell receptor (TCR)-transgenic mice down-regulate lck in response to ex vivo stimulation. TCR318 mice transgenic for a TCR, specific for a peptide from the LCMV glycoprotein 1 (amino acids 33-41), were immunized with 100 mg of the specific synthetic peptide in incomplete Freund’s adjuvant. Nine days after immunization, splenocytes were either left untreated (lane 1) or restimulated with the peptide at a final concentration of 1 × 10^–9 M (lane 2) or 1 × 10^–8 M (lane 3) at day 2 of culture and collected after overnight incubation, and lck expression was assessed by immunoblotting.

Supporting Figure 9 Fig. 9.
Calpeptin does not prevent activation induced degradation of lck. BK bulk cytotoxic T lymphocytes (CTLs), activated by IVT-pulsed APCs for 4 h in the absence or presence of calpeptin, were assessed for lck expression by immunoblotting. Similar results were obtained with cells collected after overnight incubation (data not shown).

Supporting Figure 10 Fig. 10.
Inhibition of lck degradation by Z-LL-H does not interfere with T cell activation and effector functions. The capacity of BK bulk CTLs to kill target cells, produce lymphokines, or undergo activation-induced cell death has been tested in the absence or presence of 20 mM Z-LL-H. (A) C1R/A11 cells were pulsed with the indicated concentrations of the IVT peptide and tested for killing by the CTLs in a standard 4-h ⁵¹Cr-release assay at an effector-to-target (E:T) ratio of 10:1. Shown are means ± SD of percentage of specific lysis obtained in three independent experiments. Specific lysis of target cells without the peptide was always <10%. (B–D) The CTLs were cocultured for 24 h with irradiated, IVT-peptide (1 × 10^–7 M) pulsed or unpulsed C1R/A11 cells at an E:T ratio of 10:1. The amount of IFN-g (B) or IL-2 (C) in the culture supernatants of CTLs was measured by ELISA, and the recovery of viable cells (D) was estimated by using trypan blue staining.