Rescuing -Specific Cytolytic T from Activation-Induced Cell Death, by SP600125, an Inhibitor of JNK: Implications in This information is current as of September 24, 2021. Shikhar Mehrotra, Arvind Chhabra, Subhasis Chattopadhyay, David I. Dorsky, Nitya G. Chakraborty and Bijay Mukherji

J Immunol 2004; 173:6017-6024; ; Downloaded from doi: 10.4049/jimmunol.173.10.6017 http://www.jimmunol.org/content/173/10/6017 http://www.jimmunol.org/ References This article cites 44 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/173/10/6017.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Rescuing Melanoma Epitope-Specific Cytolytic T Lymphocytes from Activation-Induced Cell Death, by SP600125, an Inhibitor of JNK: Implications in Cancer Immunotherapy1

Shikhar Mehrotra, Arvind Chhabra, Subhasis Chattopadhyay, David I. Dorsky, Nitya G. Chakraborty, and Bijay Mukherji2

Activation-induced cell death (AICD) as well as programmed cell death (PCD) serve to control the expansion of activated T cells to limit untoward side effects of continued effector responses by T cells and to maintain homeostasis. AICD of T cells in tumor immunotherapy can be counterproductive particularly if the activated T cells undergo apoptotic death after the very first sec- ondary encounter of the specific epitope. We examined the extent to which tumor epitope-specific CTLs that are activated and expanded in an in vitro-matured -based primary stimulation protocol undergo AICD following their first secondary Downloaded from encounter of the cognate epitope. Using the MART-127–35 epitope as a prototype vaccine epitope, we also examined whether these

CTLs could be rescued from AICD. Our results demonstrate that a substantial fraction of MART-127–35 epitope-specific primary CTLs undergo AICD upon the very first secondary encounter of the cognate epitope. The AICD in these CTLs is neither caspase dependent nor is it triggered by the extrinsic death signaling pathways (Fas, TNFR, etc.). These CTLs, interestingly, could be rescued from AICD by the JNK inhibitor, SP600125. We also found that SP600125 interferes with their IFN-␥ response but does not block their cytolytic function. The rescued CTLs, however, regain their capacity to synthesize IFN-␥ if continued in culture http://www.jimmunol.org/ without the inhibitor. These observations have implications in tumor immunotherapy and in further studies for regulation of AICD in CTLs. The Journal of Immunology, 2004, 173: 6017–6024.

rogrammed cell death (PCD)3 and activation-induced cell normal healthy hosts harbor precursor CTLs for such “self” death (AICD) in T cells are important physiologic pro- . Ex vivo stimulation of T cells as well as in vivo immu- P cesses that prevent untoward side effects of a continued nization with such self or tumor-associated Ags (TAA) and uncontrolled -mediated effector response as well as lead to the activation and expansion of the Ag-specific CTLs (5, 6). maintain homeostasis (1–3). Both processes involve apoptotic de- These TAA-specific T cells are also susceptible to AICD. To our

letion of T cells. PCD entails the deletion of the expanded T cell knowledge, neither the extent of AICD in these self-but-TAA re- by guest on September 24, 2021 population usually during the contraction phase of the response. active primary CTLs nor the feasibility of rescuing them from AICD, in contrast, involves the apoptotic deletion of a significant AICD has been carefully examined. We have recently shown that fraction of the activated population after an effector response. In Melan-A/MART-127–35 epitope-specific CTLs expanded in an in AICD, the effector function and the death are, paradoxically, triggered vitro dendritic cell (DC)-based stimulation protocol undergo apo- by TCR-driven signaling (i.e., activation induced). In any event, both ptotic death on repetitive stimulation by immature as well as by processes (i.e., PCD and AICD) are designed to serve useful purposes fully activated DCs (7). Using the Melan-A/MART-127–35 epitope by limiting the expansion of activated T cells. AICD in activated T (8, 9) as a prototype self but melanoma-associated Ag, we studied cells in tumor immunotherapy, however, can be counterproductive the extent of AICD in MART-127–35 epitope-specific primary particularly if the activated T cells undergo apoptotic death after CTLs and examined whether it could be prevented. In this study, the very first encounter of the specific epitope. we show that a large fraction of MART-127–35 epitope-specific Lately, much interest has been generated in ther- CTLs indeed undergo AICD upon the very first secondary encoun- apy with specific peptides, Ags, DNA, etc. Most of these ter of the cognate epitope. The AICD in these CTLs is neither immunogens are “self” Ag (4), yet many cancer patients as well as caspase dependent nor is it triggered by the engagement of extrin- sic death receptors. We also show that the JNK inhibitor, SP600125, can rescue a significant fraction of them from death. In Division of Hematology/, Department of Medicine, University of Connect- the process of rescuing, the JNK inhibitor interferes with their icut Health Center, Farmington, CT 06030 capacity to produce IFN-␥ but does not interfere with their cyto- Received for publication June 24, 2004. Accepted for publication September 1, 2004. lytic function. Of further interest, the rescued MART-127–35 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance epitope-specific CTLs, when continued in culture in IL-15 and with 18 U.S.C. Section 1734 solely to indicate this fact. without the inhibitor, regain capacity to synthesize IFN-␥. These 1 The work was supported by Public Health Service Grants CA 61398, CA 83130, and observations, therefore, have implications in cancer immunother- CA 88059. apeutic strategies and in further studies of AICD in CTLs. 2 Address correspondence and reprint requests to Dr. Bijay Mukherji, Division of Hematology/Oncology, Department of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: Materials and Methods [email protected] Study population 3 Abbreviations used in this paper: PCD, programmed cell death; AICD, activation- induced cell death; TAA, tumor-associated Ag; DC, dendritic cell; CM, complete The study population consisted of HLA-A2-positive melanoma patients or ⌬␺ media; PARP, poly(ADP-ribose) polymerase; m, mitochondrial transmembrane healthy donors. The participants were included in this study with informed potential. consent.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 6018 RESCUING CTLs FROM AICD

Culture medium and reagents Assay for AICD induction

The MART-127–35 (AAGIGILTV) and MAGE-3271–279 (FLWG To test whether or not the epitope-specific primary CTLs undergo AICD, PRALV) were purchased from Multiple Peptide Systems (San Diego, CA) the activated CTLs were exposed to peptide (1 ␮g/ml) loaded T2 cells ␤ while 2-microglobulin was purchased from Sigma-Aldrich (St. Louis, (E:T ϭ 100) at different time points of the cultures. Thereafter, the evi- MO). Culture medium consisted of IMDM (Invitrogen Life Technologies, dence of was determined by flow cytometry with triple color

Grand Island, NY) supplemented with 10% FBS (Gemini Bioproducts, staining (CD8, MART-127–35/HLA-A2 tetramer, and annexin V) at differ- Calabasas, CA), 0.55 mM L-arginine, 0.24 mM L-asparagine (both from ent time points (4–18 h). Experiments were conducted in triplicate wells Invitrogen Life Technologies), 1.5 mM L-glutamine (Sigma-Aldrich), 50 and significance was calculated by one-way ANOVA using Sigma Stat U/ml penicillin, and 50 ␮g/ml streptomycin (both from Abbott Laborato- statistical software (Chicago, IL). ries, North Chicago, IL). This will be referred to as complete media (CM). To evaluate the effect of various agents in modulating AICD, the CTLs The TAP-deficient line, T2, was a gift of P. Cresswell (Yale University, were preincubated with various compounds at optimal concentration for 45 New Haven, CT). Recombinant human GM-CSF was purchased from Im- min at 37°C and then exposed to T2 cells alone or loaded with peptide. The munex (Seattle, WA). Recombinant human IL rhIL-4, rhIL-2, rhIFN-␥ was optimal dose used in the experiments shown in this paper was determined purchased from R&D Systems (Minneapolis, MN). LPS from Escherichia by using these compounds at different concentrations in the preliminary coli 055:B5 was purchased from Sigma-Aldrich. An Annexin V kit to track experiments (data not shown). the early apoptotic cells for exposure of phosphatidylserine was purchased from BD Pharmingen (San Jose, CA). MART-127–35 (EAGIGILTV) tet- ramer labeled with PE with and without FITC-labeled anti-CD8 was pur- chased from Beckman Coulter (Fullerton, CA). Fluorochrome-labeled Results mAbs to CD25, CD27, CD28, CD95, CD95L, 4-1BB, 4-1BBL, OX-40 We selected donors (healthy donors as well as melanoma patients) were purchased from BD Biosciences (San Jose, CA). Inhibitors for var- who harbor MART-127–35 epitope-specific CTLs in relatively high ious kinase pathways, as SB203580 for p38 kinase, SP600125 for JNK, Downloaded from PD98059 for ERK were purchased from BIOMOL (Plymouth Meeting, frequencies and whose CTL precursors could be easily activated PA). Pan caspase inhibitor, human Fas/Fc chimera, human TNF- RI/Fc (7). Because IL-2 has been implicated in apoptosis in activated T chimera, human TRAIL-RI/Fc chimera, human TRAIL-RII/Fc chimera, cells (12), we looked for an alternative support in the in ␥ and human IFN- RI/Fc chimeric were purchased from R&D vitro CTL generation protocol. We found that rIL-15 supports the Systems. activation and expansion of the epitope-specific CTLs in the ma- ture DC-based epitope presentation system. IL-15 not only sup-

Generation of DCs from peripheral blood http://www.jimmunol.org/ ports the primary activation process, but also supports survival of The procedure for generating myeloid DCs from peripheral blood mono- the activated T cells in an Ag-independent manner (13). IL-15R␣ cyte was followed. Briefly, circulating monocyte were isolated by2had- is not down-regulated after T cell activation (14) and IL-15 has an herence of Ficoll-Hypaque density gradient-cut PBMC as previously described (7). The adherent cells were cultured in CM with 1000 U/ml important role in T cell memory generation, as such, in T cell GM-CSF and 500 U/ml IL-4 for 3–5 days to obtain a population of im- survival (15). Therefore, we conducted all our experiments in the mature DCs. Maturation of immature DCs was done by first priming in presence of IL-15. The MART-1 epitope-specific CTLs were ␥ 27–35 IFN- (1000 U/ml) for 2 h and then priming in the presence of 100 readily activated and expanded when they were stimulated by pep- ng/ml LPS. tide-pulsed matured DCs in the presence of IL-15, in vitro. Fig. 1A

shows an example of the expansion of the MART-1 epitope- by guest on September 24, 2021 IFN-␥ response assay 27–35 specific CTLs–derived from a normal healthy individual and a ␥ IFN- response assay for the effector cells has been described previously melanoma patient. The expanded MART-127–35 epitope-specific (10). Briefly, the effector cells were cocultured with the peptide-pulsed (1 cells also exhibited IFN-␥ response in an epitope-specific manner ␮g/ml) T2 cells. After 4–16 h, culture supernatants were harvested and IFN-␥ was measured by ELISA as per manufacturer’s protocol (R&D (Fig. 1B). Systems). The fate of the MART-127–35 epitope-specific CTLs upon sec- ondary encounter of the cognate epitope (i.e., whether following FACS analysis effector function, they survive or die?) was then examined. The CTLs and the peptide-pulsed T2 cells were cocultured in CM with- The procedure for phenotypic analyses and for determining the number of epitope-specific cells with tetramer staining by flow cytometry has been out any cytokine and 4 h later the evidence of early death was described (7). To determine tetramer and annexin V-positive cells, the examined by annexin V staining. As shown in Fig. 2, although the effector cells were stained with CD8, MART-127–35/HLA-A2 tetramer, and CTLs were fully functional in IFN-␥ assay (Fig. 2A), a large frac- annexin V. The stained cells were then analyzed for single-positive vs tion of these activated CTLs became annexin V positive at 4 h double-positive populations in flow cytometry using a FACSCalibur and CellQuest software (BD Biosciences). (Fig. 2B). When the cocultures were continued in IL-15 for 5 days and the number of epitope-specific CTLs was counted, only 20– Microcytoxicty assay 30% of the starting population could be recovered from the cocul- ture of the CTLs with the cognate target. An example for loss of The chromium release microcytotoxicity assay has been previously de- the epitope-specific CTLs following effector function is shown in scribed (11). Fig. 2C. Thus, ϳ50% of the Ag-specific population showed early

ϩ evidence of death at 4 h (Fig. 2B) and a much larger fraction Activation of CD8 T cells by DC-based presentation of eventually died following secondary encounter of the cognate epitopes, in vitro epitope for the very first time. The basic procedure for peptide-loaded DC-based in vitro activation and We then examined the mechanism of AICD on these CTLs and ϩ expansion of epitope-specific CD8 T cells has been described (7). Briefly, also examined whether these CTLs could be rescued from AICD Ficoll-Hypaque gradient separated blood mononuclear cells were purified for CD8ϩ T cells (routinely exceeding 90%) by a Dynal magnetic bead by interfering with the death signaling pathway(s). We examined isolation kit (Dynal, Oslo, Norway) and cocultured with autologous DCs the effect of interfering with the external death signal receptors ␮ ␮ ␤ pulsed with relevant peptides (100 g/ml) and 5 g/ml 2-microglobulin (such as Fas, TNFR, TRAIL, etc.) on AICD. We also examined ϩ at a CD8 T cell to DC ratio of 100. For our current purpose, because IL-2 whether any of the MAPK inhibitors could block the AICD in has been known to facilitate apoptosis in activated T cells (12), the cocul- these CTLs because MAPKs have been implicated in the negative tures were conducted in the presence of rhIL-15 (10 ng/ml). Before setting up cocultures, the DCs were irradiated to 3000 rad. The activated CTLs selection of developing thymocytes (16, 17). As shown in Fig. 3A, were also maintained in culture in IL-15. the death of these CTLs was not caspase dependent, as the pan The Journal of Immunology 6019

FIGURE 1. An example of activation and expansion

of MART-127–35 epitope-specific CTLs in the in vitro CTL generation culture. A, Number of HLA-A2/

MART127–35 tetramer-positive cells from a normal do- nor (top), and from a melanoma patient (bottom). B, IFN-␥ response by the corresponding population (M3 ϭ ϭ MAGE-3271–279,M1 MART-127–35). Downloaded from

caspase inhibitor, z-VAD-fmk did not prevent apoptosis. The ap- the IFN-␥ response by these CTLs (Fig. 3B) but did not affect their optosis was not also affected by the blockade of the common ex- cytotoxic function (Fig. 3C) suggesting that the cytolytic machin- trinsic death signal receptors such as Fas, TNFR, or TRAIL. How- ery and the IFN-␥ response pathways are differently regulated in ever, we found that while the p38 and ERK inhibitors had no these CTLs. Induction of AICD upon secondary exposure to the http://www.jimmunol.org/ effect, the JNK inhibitor, SP600125, rescued a significant fraction cognate epitope in the primary CTLs and the protective effect of of the CTLs from AICD. Interestingly, SP600125 also inhibited SP600125 from AICD were observed in CTLs generated from two by guest on September 24, 2021

␥ FIGURE 2. Effector function and the fate of MART-127–35 epitope-specific CTLs upon secondary encounter of the cognate epitope. A, IFN- response ϭ ϭ by the effector cells. (M3 MAGE-3271–279,M1 MART-127–35). B, Evidence of early death in the Ag-specific effector cells after secondary encounter ϩ ϩ ϩ of the epitope. Top panels, Number of MART-127–35 tetramer CD8 T cells; bottom panels, MART-127–35 tetramer /annexin V populations. The numbers at the top of the bottom panels represent the mean Ϯ SE of three replicate samples while superscript letters a, b, c, and d indicate that an increase in the ϩ ϩ Ͻ MART-127–35 tetramer /annexin V cells in a, when compared with that in b, c, and d, was significant with p 0. 001 (one-way ANOVA). C, Survival ␮ of the MART-127–35-specific population after secondary encounter of the epitope. The effector cells were coincubated with peptide (1 g/ml) pulsed irradiated T cells (CTL:T2 ϭ 100). The cultures were washed 18 h later and were then maintained in IL-15-containing medium for 5 days. The recovery of the Ag-specific CTLs was determined by counting the number of viable cells (trypan blue-negative population) and by factoring in the number of the Ag-specific population (percent tetramerϩ population). The difference in the percent tetramerϩ CTLs rescued after 5 days postsecondary stimulation with p Յ 0.001 (one-way ANOVA). A and B represent one of five ,ء MART-1 peptide-loaded T2 cells, when compared with other groups, was significant at separate experiments. C represents one of three separate experiments. 6020 RESCUING CTLs FROM AICD Downloaded from

FIGURE 3. Effect of external death receptor blockade and MAPK inhibitors on AICD in the epitope-specific CTLs upon secondary encounter of the cognate epitope. The CTLs were preincubated at optimal concentration for 45 min at 37°C with pan caspase inhibitor -z-VAD-fmk (100 ␮M); human Fas/Fc chimera, human TNF- RI/Fc chimera, human TRAIL-RI/Fc chimera, human TRAIL-RII/Fc chimera, and human IFN-␥ RI/Fc chimeric proteins (10 ␮g/ml); p38 kinase inhibitor SB203580, JNK inhibitor SP600125 JNK, ERK inhibitor PD98059 (25 ␮M). Then the pretreated as well as untreated CTLs were http://www.jimmunol.org/ incubated with T2 cells either alone or loaded with peptide. Four hours after secondary exposure, cells were stained for determining A, the number of tetramerϩ cells of the CD8 T cells (top panels) and the number of tetramerϩ/annexin Vϩ and tetramerϩ/annexinϪ populations (bottom panels). The numbers on the top margin of the bottom panels represent tetramerϩ/annexinϪ from the mean Ϯ SE of three replicate samples while superscript letters a, b, c, and d indicate that the increase in the number of tetramerϩ/annexin VϪ population in a, when compared with that in b, c, and d, was significant with p Ͻ 0.001 (one-way ␥ ϭ ϭ ␥ ANOVA). B, Effect of the MAPK inhibitors on IFN- response by the effector cells. (M3 MAGE-3271–279,M1 MART-127–35). The reduction in IFN- Յ ϭ ϭ synthesis in a, when compared with b, c, and d, was significant at p 0.001 by one-way ANOVA. M3 MAGE-3271–279,M1 MART-127–35. C, Effect of ϭ ϭ the MAPK inhibitor on cytotoxic response by the effector cells. (M3 MAGE-3271–279,M1 MART-127–35). The difference in the percent-specific lysis in the Յ ء MART-1 peptide-loaded T2 cells was significant ( , p 0.001 by Student’s t test) only when compared with that of MAGE-3271–279 peptide-loaded T2 cells at all E:T ratios (E:T). A represents one of four separate experiments while B and C represent one of two separate experiments. by guest on September 24, 2021

normal donors and two melanoma patients (collective data not blocked the PARP cleavage. We also confirmed that SP600125 shown). abrogates c-jun activation (Fig. 4B). The caspase-independent death in these primary CTLs upon We examined the status of the mitochondrial membrane poten- TCR engagement was confirmed as poly(ADP-ribose) polymerase tial in the CTLs during AICD. As shown in Fig. 5A, the CTLs (PARP) was found to be uncleaved (Fig. 4A). Of note, PARP was surprisingly exhibited hyperpolarization of the mitochondrial ⌬␺ cleaved in staurosporine-treated Jurkat cells and z-VAD-fmk transmembrane potential ( m) (18, 19) while the apoptotic Jurkat

FIGURE 4. Evidence for a caspase-independent pathway for apoptosis in MART-1-specific primary CTLs. A, The CTLs were preincubated at optimal concentration for 45 min at 37°C with p38 kinase inhibitor SB203580 and JNK inhibitor SP600125 (25 ␮M). The pretreated as well as untreated CTLs were then incubated with T2 cells either alone or loaded with peptide. Six hours after secondary exposure cells were used for Western blot analysis for PARP cleavage (top panel). Jurkat T cells either preincubated with pan caspase inhibitor -z-VAD-fmk (100 ␮M) or untreated were exposed with staurosporine (1 ␮M) to establish the PARP cleavage alongside. B, Jurkat T cells were stimulated with PMA (10 ng/ml)/ionomycin (0.5 ␮M) for the indicated time both in the presence and absence of JNK inhibitor SP600125 JNK (25 ␮M). Immunoprecipitation was done to ascertain the blockade of c-jun phosphorylation/activation in the presence of SP600125, as seen in the blot. The Journal of Immunology 6021 Downloaded from http://www.jimmunol.org/

⌬␺ FIGURE 5. Effect of Ag-specific secondary stimulation on mitochondrial transmembrane potential ( m), pro- and antiapoptotic protein, and expression of cell surface molecules. A, Differential effect on membrane polarization in MART-1 primary CTLs (top panels) vs Jurkat T cells (bottom panels). CTLs were incubated with T2 cells either alone or loaded with peptide. After overnight secondary exposure, cells were stained with MART-1 tetramer/CD8 and ϩ DiOC6. The histogram represents the fluorescence of DiOC6 in the gated Ag-specific tetramer population (top panels). Jurkat T cells were also analyzed for modulation of membrane potential after TCR-driven stimulation (bottom panels). Values represent the mean Ϯ SD of fluorescence intensity triplicates p Ͻ 0.001 (one-way ANOVA). B, Left panel, Western blots for anti- and proapoptotic proteins using the whole by guest on September 24, 2021 ,ء .from one of three separate experiments CTL population following secondary stimulation in the presence or absence of the MAPK inhibitors. Right panel, The densitometric analysis of the relevant Bcl family proteins normalized to ␤-actin controls using OPTIMAS version 5.2 software (Bioscan, Bothell, WA). C, Expression of different cell surface receptors for survival or death signal in the Ag-specific population upon secondary stimulation. Values in the upper left corner represent the geometric mean of the fluorescence intensity for that particular marker when stimulated with T2/MAGE-3271–279 while those on the upper right corner represent the geometric mean of the fluorescence intensity when stimulated with T2/MART-127–35.

cells exhibited hypopolarization of the mitochondrial mem- the JNK inhibitor SP600125. Bim–a pro-apoptotic protein–was brane. Hyperpolarization has been associated with the apoptotic markedly up-regulated (or released from sequestered sites) by the process in peripheral blood lymphocytes and has also been CTLs undergoing AICD. Of interest, the CTLs up-regulated shown to occur independently from activation of caspases (20, 4-1BB (10- to 20-fold increase of the geometric mean intensity of 21). Further, hyperpolarization of mitochondrial membrane po- fluorescence) and CD25 (3- to 6-fold increase of the geometric tential has been shown to be a reversible stage in the mitochon- mean intensity of fluorescence) upon secondary encounter of the drial death decision process while hypopolarization usually rep- Ag (Fig. 5C). resents a point of irreversible commitment to death (18, 19). Finally, we examined the functional status of SP600125-rescued Interestingly, SP600125 (JNK inhibitor) as well as SB203580 CTLs upon continuous culture. After overnight exposure of the (p38 inhibitor) decreased the level of hyperpolarization induced CTLs to the cognate epitope in the presence or absence of by the restimulation with the cognate peptide (data not shown) SP600125, the CTLs were washed and then maintained in contin- suggesting that SP600125-mediated rescue is not specifically uous culture in IL-15 containing CM without the inhibitor. Five mediated through the modulation of mitochondrial membrane days after, the number of viable MART-1 tetramer-positive potential. 27–35 ␥ The status of Bcl family pro- and antiapoptotic proteins and cells were determined and their function was tested in the IFN- costimulation through certain TCRs (CD28, 4-1BB, OX-40, etc.) response assay. Fig. 6A shows that only a small fraction of the influence the survival of activated T cells (22). As such, we starting population could be recovered from the cognate epitope- examined the expression levels of the anti- and proapoptotic Bcl exposed culture in the absence of the JNK inhibitor. A larger frac- family proteins and these receptors in the CTLs in a condition that tion of the starting epitope-specific population was recovered from the coculture that was started in the presence of the JNK inhibitor, induces AICD. As shown in Fig. 5B, while Bcl-2 and Bcl-xL were nonselectively up-regulated by SB203580 and SP600125, only SP600125. Remarkably, the rescued CTLs regained their capacity phosphorylated Bcl-2 and Mcl-1 were selectively up-regulated by to synthesize IFN-␥ (Fig. 6B). 6022 RESCUING CTLs FROM AICD

FIGURE 6. Survival of the Ag-specific CTLs following secondary encounter of the epitope in the presence of SP600125 and recovery of function in the SP600125-rescued CTLs. Effector cells were cocultured with peptide-pulsed T2 cells in the presence or absence of the kinase inhibitors (25 ␮M). The cultures were washed 18 h later and then recultured in IL-15-containing medium. On day 5, the numbers of the viable Ag-specific CTLs were determined. A, Top panels, The forward and side scattering of the total population on day 5 and the number of viable cells that could be recovered and gated for analysis. ϩ ϩ The number (11%) of MART-127–35 tetramer /CD8 population exposed to MART-127–35 peptide-pulsed T2 cells (T2/M1) is a reflection of loss of ϩ ϩ epitope-specific CTLs from AICD. Bottom panels, The percentage of MART-127–35 tetramer /CD8 cells recovered 5 days after coculture of the CTLs ␥ ϭ ϭ with peptide-pulsed T2 cells in the presence or absence of SP600125. B, IFN- response by the recovered populations (M3 MAGE-3271–279,M1 Downloaded from MART-127–35).

␥ Discussion in apoptosis and has also been shown to interfere with IFN- re- http://www.jimmunol.org/ Ashwell et al. (1) first noted growth arrest in T cell hybridoma sponse but not with in CTLs (32, 33). A connection upon TCR-mediated stimulation. Shi et al. (2) thereafter showed between 4-1BB-mediated signaling and activation of the JNK that TCR- driven signaling leads to death of the cells and coined pathway has also been suggested (34). Second, the MART- the term AICD. In physiology, T cell responses follow three phas- 127–35 epitope-specific CTLs generated in the in vitro DC-based es: amplification, contraction, and memory generation. During the epitope presentation protocol in the presence of IL-15 and contraction phase of a primary T cell response, the majority of the maintained in IL-15 were just as susceptible to AICD as the Ag-specific T cells undergo apoptotic death (23) as a form of PCD CTLs that were generated and maintained in IL-2 (collective primarily to maintain homeostasis (24). It has been thought for data not shown). Third, the results clearly reveal that SP600125 some time that the apoptotic signal is initiated through the Fas and is capable of protecting these CTLs from AICD (Fig. 3). Be- by guest on September 24, 2021 TNF receptors and that the cell death is mediated by the eventual cause SP600125 functions as an inhibitor of the JNK, the data activation of an executioner caspase (25, 26). A universal role of suggest a role for JNK in the apoptotic process. Fourth, while the Fas and TNF family receptor-driven signaling for T cell apo- SP600125 negatively regulates the IFN-␥ responsiveness of the ptosis has, however, been controversial (27) and both caspase- CTLs, it does not affect their cytotoxic function, suggesting that dependent and caspase-independent deaths in T cells have been the two functional pathways in CTLs are differently regulated. described (28). Further, it is now increasingly apparent that an Finally, and most importantly, the rescued CTLs regained intrinsic cell death pathway triggers AICD in CTLs without in- IFN-␥ responsiveness in continued culture after the inhibitor volving the extrinsic death receptors such as Fas and/or TNF fam- being washed off. ily receptors (29, 30). Although further studies will be needed to The JNK is a member of the MAPK family of signaling pro- establish the mechanism underlying apoptosis in the epitope-spe- teins. JNK has been associated with a variety of biological pro- cific primary CTLs in our system, our data lend support to the cesses–most notably in inflammation and transformation (35). latter view. All three MAPKs (i.e., p38, JNK, and ERK) have been found to The results are also noteworthy for a number of other reasons. have different roles in T cell biology such as in proliferation, First, the data show that a large fraction of the epitope-specific effector function, survival as well as death (16, 17, 36–40). primary CTLs generated, in vitro, are indeed susceptible to AICD Different isoforms of JNK have been found to have divergent ϩ ϩ from their very first re-encounter of the Ag even during the roles in CD4 and CD8 T cells (17). For example, JNK1 is amplification phase (Fig. 2). It should be mentioned that the needed for the expression of IL-2R␣ (CD25) upon activation

MART-127–35 epitope-specific populations expanded more than and for proliferation. As such, jnk-1-null mice exhibit marked 1000-fold in IL-15 without a second round of stimulation and then reduction in the expansion of Ag-driven CD8ϩ T cells (39). senesced around day 50. These CTLs, interestingly, exhibited pro- JNK2 down-regulates IL-2 production in CD8ϩ T cells (39, 40). pensity to undergo AICD as early as on day 9 (collective data not JNK has also been implicated in negative selection of thymo- shown). This, therefore, suggests that the apoptotic program in this cytes (17, 40). Both JNK1 and JNK2 have been found to have in vitro CTL activation system is turned on quite early and lends stage-dependent roles in T cell development. For example, support to the recent observations that the apoptotic program in while JNK1 has been associated with anti-CD3-induced apo- viral Ag-specific CTLs gets activated substantially before the con- ptosis of double-positive thymocytes, jnk2-null double-positive traction phase, in vivo (31). Of interest, stimulation of the CTLs thymocytes are resistant to anti-CD3-induced apoptosis al- with the cognate peptide that induced apoptosis paradoxically up- though they are sensitive to apoptosis induced by dexametha- regulated the expressions of IL-2R␣ (CD25) and 4-1BB (CD137) sone, anti-Fas Ab, or UV radiation (38, 39). Thus, immature substantially (Fig. 5C). Although these molecules are associated thymocytes seem to need one form of JNK or another for un- with T cell growth and survival, 4-1BB has lately been implicated dergoing receptor-driven apoptosis but mature T cells need The Journal of Immunology 6023

JNK1 for proliferation and IL-2 synthesis, and they do not seem 5. Parmiani, G., C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, to need JNK2 for activation-induced apoptosis (39–40). Al- and A. Anichini. 2002. with peptide based vaccines: what have we achieved: where are we going? J. Natl. Cancer Inst. 94:805. though these observations suggest a role for JNK in apoptosis in 6. Mukherji, B., N. G. Chakraborty, S. Yamasaki, T. Okino, H. Yamase, J. R. Sporn, immature thymocytes but not in mature T cells, the results of S. K. Kurtzman, M. T. Ergin, J. Ozols, and J. Meehan. 1995. Induction of anti- gen-specific cytolytic T cells in situ in human melanoma by immunization with our study reveal that JNK may be an important player in apo- synthetic peptide-pulsed autologous presenting cells. Proc. Natl. Acad. ptotic death in CTLs in the periphery. A careful examination of Sci. USA 92:8078. this type of AICD in CTLs with specificity for a “self” vs a 7. Mehrotra, S., R. 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