Published online before print October 24, 2006
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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
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Key Words: JNK • AICD • CTL
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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.
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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-
-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- (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 (
)
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 [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).
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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.
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(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.
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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- 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.
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-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- 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- 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)
.
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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--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.
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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- response by the effector cells. Significantly reduced IFN- 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.
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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.
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in CTL undergoing AICD 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
].
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Figure 6. Effect on cell surface markers and . (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 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.
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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. |
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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-, 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- 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 [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-, TNF-
, and IL-1ß among others [35
, 36
]. Of interest, following rescue from AICD with SP600125, the rescued CTL regain their IFN- 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.
Received July 28, 2006; revised September 22, 2006; accepted September 25, 2006.
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J. Exp. Med. 179,1109-11183 domain mutants of MHC class I/peptide complex Immunity 14,591-602[CrossRef][Medline] ß T lymphocytes for apoptosis Nature 353,858-861[CrossRef][Medline]This article has been cited by other articles:
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  H. Norell, T. Martins da Palma, A. Lesher, N. Kaur, M. Mehrotra, O. S. Naga, N. Spivey, S. Olafimihan, N. G. Chakraborty, C. Voelkel-Johnson, et al. Inhibition of Superoxide Generation upon T-Cell Receptor Engagement Rescues Mart-127-35-Reactive T Cells from Activation-Induced Cell Death Cancer Res., August 1, 2009; 69(15): 6282 - 6289. [Abstract] [Full Text] [PDF]
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