Rescuing Melanoma Epitope-Specific Cytolytic T Lymphocytes from Activation-Induced Cell Death, by SP600125, an Inhibitor of JNK: Implications in Cancer This information is current as Immunotherapy 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 dendritic cell-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- epitopes. 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 peptides or tumor-associated Ags (TAA) and uncontrolled T cell-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 cancer vaccine ther- CTLs indeed undergo AICD upon the very first secondary encoun- apy with specific peptides, protein 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/Oncology, 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 peptide (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 apoptosis 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).