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Originally published online as doi:10.1189/jlb.0706479 on October 24, 2006

Published online before print October 24, 2006
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(Journal of Leukocyte Biology. 2007;81:539-547.)
© 2007 by Society for Leukocyte Biology

Inhibition of c-Jun N-terminal kinase rescues influenza epitope-specific human cytolytic T lymphocytes from activation-induced cell death

Shikhar Mehrotra1, Arvind Chhabra, Upendra Hegde, Nitya G. Chakraborty and Bijay Mukherji2

Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA

2 Correspondence: University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA. E-mail: mukherji{at}nso2.uchc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytolytic T lymphocytes (CTL) play an important role in defense against viral infections. Following clonal expansion and effector functions, a vast majority of the antigen-specific CTL undergoes programmed cell death to maintain homeostasis. We have shown earlier that melanoma epitope-specific CTL are quite sensitive to activation-induced cell death (AICD) even on the secondary encounter of the antigen. Excessive sensitivity of viral antigen-specific CTL to AICD, however, would be counterproductive. It might be argued that although CTL for a "self" epitope might be more prone to AICD for maintaining self-tolerance, viral antigen-specific CTL are likely to be less sensitive to AICD. We show here that influenza matrix protein-derived MP58–66 epitope-specific CTL, activated in vitro and bearing a memory phenotype, are just as sensitive to AICD. The AICD in these CTL is not blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone or by soluble Ig-Fc chimeras of the death receptors [Fas, TNF receptor (TNF-R), TRAIL-RI, TRAIL-RII]. However, the MP58–66-specific CTL can be rescued from AICD by the c-jun-N-terminal kinase (JNK) inhibitor SP600125. These results have implications for immunotherapeutic intervention in rescuing viral epitope-specific CTL from AICD.

Key Words: JNK • AICD • CTL


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Control of lymphocyte function and proliferation is crucial to the homeostatic balance that exists in the immune system [1 ]. CD8+ cytolytic T lymphocytes (CTL) are activated after encountering APC carrying their specific peptide antigen. The immune system then returns to homeostasis through apoptosis of this vast majority of antigen-specific effector cells. Most self-reactive T cells are centrally deleted, but a small percentage enters the periphery, where other tolerance mechanisms control homeostasis. Thus, self-reactive T cells that escape central deletion are peripherally deleted so as to maintain "self-tolerance" [2 ].

We have shown recently that primary CTL activated and expanded against a "self", but a melanoma-associated epitope such as (MART-1)27–35 underwent activation-induced cell death (AICD) after a secondary encounter of their epitope in a caspase-independent mechanism and that their death could be prevented by the c-jun-N-terminal (JNK) inhibitor SP600125 [3 ]. As these were self-reactive CTL, it is possible that as a mechanism for tolerance induction against self, they might be preferentially sensitive to AICD. As CTL play an important role in the clearance or control of viral and other infectious agents, it is likely that CTL, specific for a foreign epitope, are less sensitive to AICD after a secondary encounter of antigen. However, the relative sensitivity to AICD of CTL specific for self and nonself epitope has not been studied carefully. We therefore examined the sensitivity of CTL against a nonself and dangerous epitope influenza matrix protein (MP)58–66 to AICD in vitro. We found that MP58–66-specific CTL, which were activated in vitro and bore memory phenotype, were just as sensitive to AICD. These MP58–66-specific CTL also underwent AICD in a caspase-independent manner, and some of these mature CTL were rescued from AICD by the JNK inhibitor SP600125. These observations extend our understanding of AICD in CTL, which might allow the development of strategies to protect them from cell death during viral infection and to maintain an effective internal immune surveillance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study population
The study population consisted of HLA-A2-positive, healthy donors. The participants were included in this study with informed consent.

Culture medium and reagents
MP58–66 (GILGFVFTL) and MART-127–35 (AAGIGILTV) peptides were purchased from MP Systems (San Diego, CA). Culture medium was IMDM (Gibco-BRL, Grand Island, NY). Recombinant cytokines were purchased from R&D Systems (Minneapolis, MN). MHC Class I pentamers were obtained from ProImmune (Oxford, UK). Fluorochrome-labeled mAb and Annexin V were purchased from BD Biosciences (San Jose, CA). Inhibitors for various kinase pathways, such as SB203580 for p38 kinase, SP600125 for JNK, and PD098059 for Erk, were purchased from EMD Biosciences (La Jolla, CA). Pan-caspase inhibitor, human Fas/Fc, TNF receptor I (TNF-RI)/Fc, TRAIL-RI/Fc, TRAIL-RII/Fc, and IFN-{gamma}-RI/Fc chimeric proteins were purchased from R&D Systems. Fluorogenic caspase detection kit was purchased from Oncogene (San Diego, CA).

Generation of dendritic cells (DC) from peripheral blood monocyte
The procedure for generating myeloid DC from peripheral blood monocyte has been published [4 ]. Briefly, circulating monocytes were isolated by 2 h adherence of Ficoll-Hypaque density gradient-cut PBMC. The adherent cells were cultured in conditioned medium with 1000 U/ml GM-CSF and 500 U/ml IL-4 for 3–5 days to obtain a population of immature DC (iDC). Maturation of iDC was done by first priming in IFN-{gamma} (1000 U/ml) for 2 h and then culturing in the presence of 100 ng/ml LPS.

Activation of CD8+ T cells by DC-based presentation of epitopes in vitro
The procedure for peptide-loaded, DC-based in vitro activation and expansion of epitope-specific CD8+ T cells was as described [5 ]. Briefly, Ficoll-Hypaque gradient-separated blood mononuclear cells were purified for CD8+ T cells (routinely exceeding 90%) by Dynal magnetic bead isolation kit (Invitrogen, Grand Island, NY) and cocultured with autologous DC pulsed with relevant peptides (100 µg/ml) and 5 µg/ml ß2-microglobulin at a CD8+ T cell:DC ratio of 100:1. Prior to setting up cocultures, the DC were irradiated to 3000 rad. The activated CTL were maintained in media containing IL-15 (10 ng/ml).

Assay for AICD induction
Activated CTL were preincubated with inhibitors at a predetermined optimal concentration for 45 min at 37ºC and then exposed to the MHC Class I pentamer reagent (ProImmune) for induction of apoptosis [6 ]. The pentamer contains five MHC-peptide complexes, which are pentamerized by a self-assembling coiled-coil domain. All five MHC-peptide complexes are held facing in the same direction, similar to a bouquet of flowers. Therefore, all five MHC-peptide complexes are available for binding to TCRs, resulting in an interaction with high avidity. Apoptosis was determined by flow cytometry with triple-color staining (CD8, MP58–66/HLA-A2 pentamer, and Annexin V) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

Measurement of mitochondrial membrane potential ({Delta}{psi})
{Delta}{psi} was estimated by staining with 20 nM DiOC6 (Molecular Probes, Junction City, OR), a cationic, lipophilic dye, for 15 min at 37°C in the dark before flow cytometry (excitation, 488 nm; emission, 525 nm; recorded in FL-1). Fluorescence of DiOC6 is oxidation-independent and correlates with {Delta}{psi} [7 ].

Microcytoxicity assay
The chromium release microcytotoxicity assay has been described previously [8 ].

Western blot
Briefly, protein extractions were performed in radioimmunoprecipitation assay buffer; samples were separated on 15% SDS polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Beford, MA). The primary antibodies (anti-JNK, anti-pJNK, anti-c-Jun, anti-p-c-Jun) used for Western blot analysis and all the secondary antibodies used were obtained and purchased from Santa Cruz Biotechnologies (CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotype of activated MP58–66-specific CTL
We used HLA-A2+ healthy donors as well as melanoma patients who harbor MART-127–35 and MP58–66 epitope-specific CTL precursors in relatively high frequencies to generate MART-127–35- and MP58–66-specific CTL for analysis of their relative sensitivity to AICD. As IL-2 has been shown to sensitize activated T cells to apoptosis [9 ], we used recombinant IL-15 to support the activation and expansion of the epitope-specific CTL [10 ]. Figure 1A shows an example of expansion of MART-127–35 self and MP58–66 nonself epitope-specific CTL when they were stimulated with same amount of peptide pulsed on autologous mature DC (mDC) from the same individual. Priming with same amount of peptide pulsed on autologous mDC showed that the expansion obtained for MP58–66 antigen-specific CTL was more pronounced than for MART-127–35 (Fig. 1A) . Figure 1B shows the phenotype of the activated CTL. The MP58–66-specific CTL were CD45RAloCD45ROhiCD62Llo (upper right quadrant in Fig. 1Bi ), indicating a memory phenotype [11 ]. In contrast, the MART-127–35-specific CTL from the same individual were CD45RAhiCD45ROloCD62Lhi (Fig. 1Bii) . It is interesting that the antigen-nonspecific CD8+ cells in MP58–66-specific culture also had a CD45RAhiCD45ROloCD62Lhi phenotype (lower right quadrant in Fig. 1Bi ), similar to MART-127–35. Figure 1C shows that the expanded epitope-specific CTL stain positively for perforin, granzyme A, granzyme B, and CD107a.


Figure 1
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  		Figure 1. Expansion of epitope-specific CTL and phenotypic characterization. (A) An illustration for expansion of MP58–66 and MART-127–35 epitope-specific CTL before and after priming with cognate peptide-pulsed, autologous mDC from the same individual. (B) Phenotypic expression of cell surface markers in (i) MP58–66 and (ii) MART-127–35 epitope-specific CTL. Black histograms represent mean fluorescence intensity (MFI) for the marker on the cells gated from the adjacent quadrant, and gray represents isotype. (C) Intracellular staining of MP58–66-specific CTL for various molecules. Data from one of four separate experiments with similar results are presented.

 
Sensitivity of MP58–66-specific CTL to AICD
We evaluated the effect of TCR ligation on the activated MART-127–35- and MP58–66-specific cells by exposing them to HLA-A*0201/ELAGIGILTVG and HLA-A*0201/GILGFVFTL pentamers, respectively. As shown in Figure 2A , ligation of CTL with its cognate pentamer induced apoptosis in a dose-dependent manner, as measured by an increase in Annexin V staining. In contrast, the control pentamer and cognate-free peptide epitope had no effect. Thus, the cell death was antigen-specific and consistent with the view that apoptosis in the activated CTL is a result of continued signals conveyed via the TCR. Although MP58–66-specific CTL became Annexin V-positive 4 h after exposure to relatively low concentration (2.5 pg/ml) of the cognate pentamer, supernatant collected after overnight incubation from a parallel culture with the same CTL secreted IFN-{gamma} (Fig. 2B) . This shows that CTL, while or after performing the effector function, get sensitized to undergo AICD. When the cells were cultured in the presence of IL-15, only 20% of the starting population could be recovered 48 h after a secondary encounter with the cognate epitope (Fig. 2C) . Thus, the MP58–66-specific population showed early evidence of death at 4 h, and a larger fraction eventually died following the first secondary encounter of the cognate epitope.


Figure 2
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  		Figure 2. Sensitivity of MP58–66 CTL to AICD. (A) MP58–66 and MART-127–35 epitope-specific CTL were exposed to the MHC Class I cognate pentamer reagent at different doses and stained after 4 h with Annexin V. Numbers in the upper right quadrant represent epitope+ Annexin V+ cells. (B) IFN-{gamma} secretion by MP58–66-specific CTL upon exposure to MP58–66 epitope in a dose-dependent manner. (C) Total number of MP58–66-specific CTL when continued in culture for 3 days after engagement of TCR with MP58–66 pentamer (MP, MP58–66; M1, MART-127–35). Data are presented from one of three separate experiments with similar results.

 
Mechanism of AICD in MP58–66-specific CTL
We then examined if these CTL could be rescued from AICD by interfering with the death signaling pathway(s). We blocked the external death receptors (DRs) using a predetermined, optimal dose of human Fc-chimeric proteins for Fas, TNF, TRAIL, and IFN-{gamma}-R and exposed the cells to the cognate epitope. In line with our earlier observation with MART-127–35-specific CTL [3 ], we found that a blockade of these DRs did not inhibit AICD (Fig. 3 ). Histograms show mean fluorescence values of Annexin V expression on epitope-specific and nonspecific cell populations. We also examined if the AICD in these CTL was caspase-dependent and found that pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-fmk) did not prevent apoptosis (Fig. 4A ). As our earlier observation with MART-127–35 epitope-specific CTL revealed a protective effect of JNK inhibitor SP600125 [3 ], we examined if JNK inhibition could also prevent AICD of MP58–66-specific CTL. It is interesting that SP600125 rescued a significant fraction of the CTL from AICD (Fig. 4A) . However, inhibitors of p38 kinase (SB203580) and Erk (PD098590) failed to prevent them from AICD (Fig. 4A) . As expected, SP600125 also inhibited the IFN-{gamma} release by these CTL (Fig. 4B) , but in agreement with our previous observation with MART-127–35-specific CTL, the cytotoxic function of MP58–66-specific CTL was also not affected (Fig. 4C) , indicating that the cytolytic machinery and the IFN-{gamma} response pathways might be regulated differently in CTL. As would be expected, a large fraction of the starting MP58–66-specific population could be recovered from the coculture 5 days after it was exposed to the pentamer in the presence of the JNK inhibitor SP600125 (Fig. 4D) .


Figure 3
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  		Figure 3. Effect of external DR blockade on AICD in MP58–66-specific CTL upon secondary encounter of the cognate epitope. The CTL were preincubated for 45 min at 37°C with predetermined optimal concentrations of human Fas/Fc, TNF-RI/Fc, TRAIL-RI/Fc, TRAIL-RII/Fc, and IFN-{gamma}-RI/Fc chimeric proteins (10 µg/ml). The pretreated as well as untreated CTL were exposed to the HLA-A*0201/GILGFVTL pentamer for induction of AICD. Four hours after secondary exposure, cells were stained with Annexin V. Histograms in each quadrant represent the MFI of Annexin V on MP58–66-specific CTL (right quadrant) and an epitope-negative bystander population (left quadrant). Data from one of the two separate experiments are shown.

 

Figure 4
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  		Figure 4. Rescue of MP58–66 epitope-specific CTL from AICD. (A) MP58–66 CTL were preincubated for 45 min at 37°C at optimal concentrations of pan-caspase inhibitor z-VAD-fmk (100 µM), p38 kinase inhibitor SB203580, JNK inhibitor SP600125 JNK, and Erk inhibitor PD098059 (25 µM) before being exposed to the HLA-A*0201/GILGFVTL pentamer for induction of AICD. Four hour later, cells were stained to determine the numbers of MP58–66 epitope+/Annexin V+ cells. Experiments carried out in replicates showed that SP600125-pretreated CTL had significantly less incidence of apoptosis as compared with other treated groups (P<0.5; Student’s t-test). Data from one of seven different experiments with similar results are shown. (B) Effect of SP600125 on IFN-{gamma} response by the effector cells. Significantly reduced IFN-{gamma} secretion was found from restimulated CTL, which were pretreated with SP600125 (*, P<0.1; Student’s t-test). (C) Effect of SP600125 on cytotoxic response by the effector cells. The difference in the percent-specific lysis of the peptide-loaded T2 cells was significant (*, P≤0.05; Student’s t-test) only when compared with that of MAGE-3271–279 (M3) control peptide-loaded T2 cells. (D) Survival of SP600125-pretreated, MP58–66-specific CTL following a secondary encounter of the epitope. After CTL were cocultured with the cognate pentamer for 4 h in the presence or absence of the JNK inhibitor (25 µM), the cells were washed and recultured in IL-15 containing medium. On Day 5, the numbers of the viable antigen-specific CTL were determined.

 
Caspase-independent AICD in CTL undergoing AICD
To confirm our observation that AICD in of MP58–66-specifc CTL is caspase-independent and JNK-dependent and to further determine if JNK inhibitor SP600125 can inhibit apoptosis in any other cell type, we examined apoptosis in Jurkat T cells using staurosporine (STS), which has been shown earlier to induce capsase-dependent death [12 ]. Results in Figure 5A confirm that STS-induced apoptosis in the Jurkat cell is caspase-dependent and is thus inhibited by z-VAD-fmk, and none of the MAPK inhibitors, including SP600125, rescues Jurkat T cells from this kind of apoptosis. This supports the view that cell death varies with different cell type and stimulation being provided. Figure 5B shows inhibition of c-jun phosphorylation at different doses of SP600125 in Jurkat cells activated using PMA and ionomycin, which are known positive inducers of JNK activity. Earlier studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for JNK activation [13 ]. This issue was readdressed by Rivas et al. [14 ], who showed that CD28 is not required for JNK activation in murine T cells. This study showed that JNK activity was readily induced in CD28–/–/2C TCR transgenic/RAG2–/– T cells stimulated with anti-CD3 mAb or with Ld/peptide dimers. We also found that stimulation of freshly purified human CD8 T cells with anti-CD3/28 antibody resulted in increased expression of JNK, which was inhibited by its inhibitor SP600125 (data not shown). A similar observation was made when we did flow cytometric evaluation of the antigen-specific population after it was stimulated with MHC Class I multimers (Fig. 5C) . Further, we also measured the activation of caspases in these CTL undergoing AICD using cell-permeable z-VAD-fmk-FITC as per the manufacturer’s protocol. This reagent binds to activated caspases and thus, caspase activity could be measured by flow cytometry. MP59–68-specific CTL, when restimulated with MP59–68 pentamer, did not show any increase in caspase activity, whereas the same CTL, when stimulated with STS, resulted in activation of caspases (Fig. 5Di) . Similar results were obtained when Jurkat T cells were also treated with STS, a known positive control, showing an enormous increase in caspase activity, which was blocked by using z-VAD-fmk (Fig. 5Dii) .


Figure 5
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  		Figure 5. Caspase-independent and JNK-dependent AICD in MP58–66 CTL. (A) SP600125 does not affect STS-mediated, caspase-dependent apoptosis in Jurkat T cells, which were preincubated for 45 min at 37°C at optimal concentrations of pan-caspase inhibitor z-VAD-fmk (100 µM), p38 kinase inhibitor SB203580, JNK inhibitor SP600125 JNK, and Erk inhibitor PD098059 (25 µM) before being exposed to 1 µM STS for inducing apoptosis. Four hours later, cells were stained with Annexin V. Histograms represent the MFI of Annexin V (open black) and control isotype (solid gray). (B) Western blot showing JNK activation (c-Jun phosphorylation) and effect of SP600125 on c-Jun phosphorylation in (i) Jurkat T cells activated with PMA and ionomycin and (ii) in MP58–66 CTL undergoing AICD (C, CTL control; M3, restimulated with MAGE-3271–279 control epitope; MP, restimulated with MP58–66 epitope; MP + SP, restimulated with MP58–66 epitope after pretreatment with JNK inhibitor SP600125). (C) JNK activation as measured by c-Jun phosphorylation in MP58–66-specific, gated CTL, as measured by flow cytometry after AICD induction. Percent phosphorylation was calculated using MFI values of the depicted groups over control CTL alone. (D, i) Caspase activation as measured using cell-permeable FITC-z-VAD-fmk, which irreversibly binds to activated caspases in the apoptotic cells. Histogram represents MFI of the gated MP58–66 CTL undergoing AICD in the presence of MP58–66 epitope and the same CTL undergoing apoptosis with STS, a known activator of caspases. (D, ii) Caspase activation in Jurkat T cell undergoing apoptosis on treatment with STS.

 
Effect on surface marker expression and {Delta}{psi} in CTL undergoing AICD
We then examined the expression of molecules related to apoptosis in these restimulated CTL by FACS analysis. As demonstrated in Figure 6A and 6B , expression of CD28, CD134 (or OX-40), CD95 (Fas), and CD95L remained unaffected, whereas the CTL undergoing AICD had an up-regulation of CD25 (i.e., IL-2-R) and CD69. Up-regulation of these activation markers CD25 and CD69 on cognate epitope-exposed CTL leaves them in a highly activated state and probably makes them sensitive to AICD. IL-2/IL-2-R are known to be involved in peripheral tolerance through the elimination of self-reactive T cells through AICD [15 ]. Another interesting observation was a marked up-regulation of CD137 (or 4-1BB) in CTL exposed to the cognate epitope, which was inhibited specifically by SP600125 (Fig. 6B) . As 4-1BB has been implicated lately in Fas-independent apoptosis and has also been shown to interfere with the IFN-{gamma} response but not with cytotoxicity in CTL [16 , 17 ], this observation adds strength to our argument for the involvement of the JNK pathway in AICD of mature CTL. A connection between 4-1BB-mediated signaling and activation of the JNK kinase pathway has also been suggested [18 ].


Figure 6
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  		Figure 6. Effect on cell surface markers and {Delta}{psi}. (A) Expression of different cell surface receptors for survival or death signal in an antigen-specific population upon secondary stimulation. Histogram represents expression for that particular marker on a MP58–66-specific, gated population when stimulated with cognate MP58–66 pentamer (red) and control pentamer (green). Gray-filled histogram represents control, unstimulated CTL. PD-1, Programmed death 1. (B) Modulation of CD25 and CD137 expression by SP600125. Expression of CD25 and CD137 analyzed on CTL undergoing AICD by TCR re-engagement was blocked specifically by SP600125. (C) Effect on {Delta}{psi} upon AICD induction. Histogram represents MFI values for DiOC6 on MP58–66-specific, gated cells 4 h after treatments shown. (*, P<0.5; Student’s t-test). Data are presented from one of three separate experiments with similar results.

 
We also examined the effect of TCR ligation and SP600125 on MP58–66-specific CTL, as most apoptotic signals converge on mitochondria, and the consequence of reduced mitochondrial potential is membrane permeability leading to cell death [19 ]. We found that SP600125 interfered specifically with the {Delta}{psi} secretion, which occurred upon AICD after TCR re-engagement of the MP58–66-specific CTL (Fig. 6C) . The induction of apoptosis following exposure to epitope was also observed in another primary CTL expanded from the same individual against the MART-127–35 eptitope, as has been shown by us earlier [3 ], suggesting that this kind of apoptosis could be a general phenomenon of activated CTL.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The engagement of TCR with peptide/MHC complexes is an important event in the initiation of the T cell response, and signaling via the TCR can have a number of outcomes, ranging from activation/proliferation to anergy or apoptosis. Most T cell responses contract after a rapid expansion phase through programmed cell death (PCD). It is believed that PCD is a genetically imprinted mechanism to maintain homeostasis [20 ]. It is interesting that activated T cells also die after re-encountering antigen through AICD [21 ]. It is not clear if AICD contributes to homeostasis, but from the viewpoint of the host’s protection against infection, AICD could be counterproductive, as virulent infections can be lengthened as a result of the deletion of antigen-specific CTL through clonal exhaustion, which is thought to reflect antigen-dependent apoptosis of CTL [22 ]. Thus, AICD is somewhat paradoxical.

The relationship among T cell activation, the nature of the apoptotic signal, and the mechanism of death is poorly understood. By definition, the signal for AICD originates from TCR ligation, hence, the term "activation-induced". It is believed that AICD is driven by signals from DRs and that the cell death takes place in a caspase-dependent manner [23 ]. However, it is now increasingly apparent that AICD in CTL can be triggered by an intrinsic cell death pathway, which does not involve extrinsic DRs such as Fas and/or TNF family receptors [24 ]. It has been proposed recently that the contraction phase of the virus-specific T cell response is unlikely to require caspase-dependent PCD and that contraction may be mediated by an alternative, caspase-independent pathway(s) [25 ]. A systematic evaluation of virus-specific T cell responses using different regimens of pan-caspase inhibitor zVAD administration in vivo was attempted to increase the memory pool by reducing CD8+ T cell death. However, the results of this study [25 ] showed that there exists a caspase-dependent death pathway in freshly isolated, virus-specific T cells and a caspase-independent pathway during the contraction phase of the response. This second mechanism of the CTL deletion remains poorly understood.

Similar to our earlier observation that human melanoma epitope-specific CTL undergo AICD in a caspase-independent manner [3 ], we show here that human CTL, which are specific for the influenza MP58–66, also underwent apoptosis after TCR ligation but not through Fas, TNF, IFN-{gamma}, or TRAIL-R. The death of these CTL is not caspase-dependent, as the pan-caspase inhibitor z-VAD-fmk does not prevent apoptosis. However, JNK inhibitor SP600125 rescues the CTL from AICD. JNK is a member of the MAPK family, which is involved in signal transduction of apoptosis as well as cell growth and differentiation [26 ]. The possible role of a JNK pathway in apoptosis signaling has been demonstrated by several studies using a JNK-deficient mouse. JNK2 was shown to be required for apoptosis of immature thymocytes induced by an anti-CD3 antibody [27 ]. Studies using various in vitro experimental systems have provided strong evidence that the JNK signal transduction cascade mediates neuronal apoptosis [28 ]. Prolonged JNK activation has also been demonstrated in various apoptotic models [29 ]. Taken together, our data are consistent with the recent idea that the contraction phase of the virus-specific CD8+ T cell response is independent of caspases [25 ] and support a role for JNK in AICD of nonself epitope-specific human CTL.

Of further interest, MP58–66-specific CTL undergoing AICD up-regulated expression of CD25 and CD137 (or 4-1BB), and this up-regulation was blocked by SP600125 pretreatment. Up-regulation of CD25 on cognate epitope-exposed CTL leaves them in a highly activated state and probably makes them sensitive to AICD. Lately, 4-1BB has been implicated in apoptosis and has been shown to interfere with the IFN-{gamma} secretion but not with the cytotoxic function of CD8+ T cells [17 ]. A connection between 4-1BB-mediated signaling and activation of the JNK kinase pathway has also been suggested [18 ]. We also found that SP600125 specifically interfered with the {Delta}{psi} [19 ], which occurred upon AICD after TCR re-engagement of the MP58–66-specific CTL. Given that death in CD8+ T cells is turning out to be driven mostly by the internal pathway of apoptosis and results from mitochondrial dysfunction—release of free radical intermediates, stress [30 ]—and as JNK has a role in stress-induced activation of the death pathway [31 ], a role for JNK in AICD of antigen-specific CD8+ CTL can be envisioned.

As is known, viral epitope-specific CTL are preferentially eliminated during disease progression [32 ], and continuous activation of an initially expanded, specific CD8+ T cell has been implicated for its rapid disappearance [33 ]. A better understanding of the mechanisms by which virus-specific CTL are deleted during viral infection would be useful in vaccine development [34 ], and vaccine efficacy might be improved by increasing the size of the CTL poll as well as by preventing premature AICD.

Our data suggest that the lifespan of viral epitope-specific CTL might be prolonged during virus infection with a suitable JNK inhibitor [35 ]. Admittedly, SP600125 might not be ideal for the purpose, as it interferes with the secretion of proinflammatory cytokines such as IFN-{gamma}, TNF-{alpha}, and IL-1ß among others [35 , 36 ]. Of interest, following rescue from AICD with SP600125, the rescued CTL regain their IFN-{gamma} synthetic capacity after the drug is washed off [3 ]. Further, SP600125 does not affect cytolytic function of the CTL [3 ]. Although careful studies will be needed to establish the therapeutic value of the JNK inhibitor, in general, and SP600125, in particular, our observations have translational implications.


   ACKNOWLEDGEMENTS
 
The work was supported by Public Health Service Grants CA88059 and CA117254 to B. M. We thank Drs. Linda Cauley and Kamal Khanna in the Department of Immunology, University of Connecticut School of Medicine for their critical reading and suggestions in preparation of this manuscript.


   FOOTNOTES
 
1 Current address: Division of Surgery, Medical University of South Carolina, Hollings Cancer Center, 86 Jonathan Lucas Street, Suite 512, Charleston, SC 29425, USA. Back

Received July 28, 2006; revised September 22, 2006; accepted September 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lenardo, M. J. (2003) Molecular regulation of T lymphocyte homeostasis in the healthy and diseased immune system Immunol. Res. 27,387-398[CrossRef][Medline]
  2. Zheng, L., Boehme, S. A., Critchfield, J. M., Zuniga-Pflucker, J. C., Freedman, M., Lenardo, M. J. (1994) Immunological tolerance by antigen-induced apoptosis of mature T lymphocytes Adv. Exp. Med. Biol. 365,81-89[Medline]
  3. Mehrotra, S., Chhabra, A., Chattopadhyay, S., Dorsky, D. I., Chakraborty, N. G., Mukherji, B. (2004) Rescuing melanoma epitope-specific cytolytic T lymphocytes from activation-induced cell death, by SP600125, an inhibitor of JNK: implications in cancer immunotherapy J. Immunol. 173,6017-6024[Abstract/Free Full Text]
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