Regulation of lck degradation and refractory state in CD8+ cytotoxic T lymphocytes

Abstract

After specific activation, CD8+ cytotoxic T lymphocytes (CTLs) enter a refractory state termed activation-induced nonresponsiveness (AINR) that is characterized by the inability of T cells to respond to a secondary stimulus. Here, we show that T cell receptor triggering results in rapid degradation of the src-family protein kinase lck through a mechanism that is proteasome- and lysosome-independent, sensitive to cysteine protease inhibitors, and distinct from the pathways involved in degradation of ZAP-70 kinase or zeta-chain of the CD3 complex. Pharmacologic blockade of lck degradation, as well as transfection of refractory cells with an lck expression vector, increased responsiveness of CTLs to repeated antigenic challenge. The development or maintenance of AINR was not affected by exogenously added IL-2, whereas IL-15 or IFN-alpha restored both lck expression and responsiveness of preactivated CTLs. Our results suggest that lck degradation plays an important role in the development of AINR in human CTLs and that this condition can be reverted by pharmacologic agents or lymphokines that prevent lck degradation or induce its expression.

Fig. 1.

TCR triggering induces lck down-regulation in activated CD8⁺ T cells. (A) C1R/A11 cells were pulsed with the AVF or IVT peptide (1 × 10^-7 M), irradiated, and used to stimulate the indicated polyclonal CTL cultures. Expression of lck in CTL lysates was determined by immunoblotting with polyclonal lck-specific Abs. Shown is one representative of five experiments. (B) Expression of lck in CAR CTLs stimulated with the JAC-B2 LCL or L5 LCL (control). The latter carries mutations in the AVF and IVT epitopes that prevent their presentation at the cell surface (42). Shown is one representative of three experiments. (C) T cell blasts were generated from peripheral blood mononuclear cells of five healthy blood donors by PHA activation. On day 7 of culturing in IL-2 medium, >95% of cells in every culture were CD3⁺/CD8⁺ as determined by FACS analyses. On the indicated day of culture, cells were stimulated with beads conjugated with anti-CD3 Abs for 24 h and lysed in sample buffer, and lck expression was tested by immunoblotting and quantified as described in Materials and Methods.

Fig. 2.

Kinetics of lck down-regulation in IVT-specific CTL. BK289 CTLs were activated with IVT-pulsed C1R/A11 cells for the indicated periods of time. Viable cells were counted, harvested, and lysed in SDS sample buffer. (A) Results of one representative immunoblotting experiment with lck-specific Ab. (B) Densitometry of lck-specific bands revealed in lysates of activated CTLs was performed as described above, and band intensities were expressed as the percentage relative to that of controls. Shown is one representative of five experiments.

Fig. 3.

AID of lck is proteasome- and lysosome-independent but sensitive to inhibition by MG132. BK bulk CTLs were activated by IVT-pulsed APCs for 4 h in the absence (control) or presence of the indicated protease inhibitors. (A) Immunoblotting of lysates of BK289 cells with ubiquitin-, lck-, p53-, or actin-specific Abs. (B) Effect of epoxomycin on the steady-state levels of lck and p53. Intensities of lck- and p53-specific bands in lysates of cells incubated in the presence of epoxomycin were quantified and expressed as the percentage relative to control. Shown are the means ± SD of three experiments. (C) Effects of epoxomycin on AID of lck or p53 were analyzed and expressed as described in B. Shown are the means ± SD of three experiments. (D) Effect of MG132 on lck degradation assessed at the indicated concentrations. Cells activated by peptide-pulsed APCs in the presence of leupeptin were used as an additional control. (E) Quantification of the effect of MG132 at a concentration of 100 μM. Shown is one representative of three experiments in which samples of cells treated with lactacystin were also included. (F) Control or activated CTLs were cultured in the presence or absence of NH₄Cl. Immunoblotting of total cell lysates with lck-, IL-15Rα-, or actin-specific Abs. Shown is one representative of five experiments.

Fig. 4.

The calpain inhibitor Z-LL-H blocks AID of lck. BK bulk CTLs were activated by IVT-pulsed APCs for 4 h in the absence (control) or presence of Z-LL-H at the indicated concentrations. (A) Effect Z-LL-H on the AID of lck, ZAP-70, or ζ-chain tested by immunoblotting with the relevant Abs. (B) Intensities of lck, ZAP-70, or ζ-chain-specific bands expressed as percentage expression relative to band intensities in samples of unstimulated CTLs. Shown are the means ± SD of five experiments performed with lck-specific Abs and one representative of two experiments performed with ZAP-70- and ζ-chain-specific Abs.

Fig. 5.

Responsiveness of CTLs correlates with lck expression, and treatment with Z-LL-H prevents the development of AINR in specific CTLs. (A) BK bulk CTLs were stimulated during the indicated periods of time with C1R/A11 cells preincubated with the indicated synthetic peptides. The expression of lck was accessed by immunoblotting and quantified as described above. (B) CTLs preactivated by using the indicated peptides were restimulated with IVT-pulsed APCs after 48 h, and their proliferation was evaluated by a [³H]thymidine incorporation assay. Shown are the results of one representative of two comparable experiments. (C 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 20 μM Z-LL-H. Lck expression in these cells was evaluated by immunoblotting. Shown are data of one representative experiment. (D) Cells were then activated by IVT-peptide-pulsed APCs, and their proliferation was evaluated by a [³H]thymidine incorporation assay. The results are expressed as the percentage relative to thymidine incorporation in control cultures not stimulated by anti-CD3 and cultured either with or without Z-LL-H. Shown are the means ± SD of three experiments.

Fig. 6.

Transfection of pcDNA3.1-lck into refractory CTLs enhances their capacity to produce IFN-γ in response to specific stimulation. Refractory CAR bulk CTLs were transfected with pcDNA3.1, pcDNA3.1-lck, or pcDNA3.1-EGFP plasmids and restimulated 18 h after transfection with IVT-pulsed APCs. The secretion of IFN-γ in control and transfected cells was assessed by intracellular staining with the specific APC-conjugated Ab and FACS analysis. Monitoring of green fluorescence (FL1) was used to determine the general efficiency of transfection based on the percentage of green cells in pcDNA3.1-EGFP-transfected culture that varied between 7% and 10% in different experiments (data not shown). The increase of IFN-γ-positive cells observed after pcDNA3.1-lck transfection corresponded with the expected values calculated from the percentage of positive cells in nonrefractory CTL cultures and general transfection efficiency.

Fig. 7.

IL-15 and IFN-α increase lck expression after specific activation and restore responsiveness in preactivated CTLs. (A) The BK bulk CTLs were activated with peptide-pulsed APCs and cultured for 48 h in standard medium alone (control) or with the addition of exogenous IL-2, IL-7, IL-15, or IFN-α. The expression of lck was analyzed as described above. (B) Capacity of CTLs to proliferate in response to the secondary challenge as evaluated by a [³H]thymidine incorporation assay. The results are expressed as the percentage relative to thymidine incorporation of control CTLs, which were not preactivated with peptide-pulsed APCs and cultured in the absence (control) or presence of the indicated lymphokine. Shown are the means ± SD of four experiments.