[Cancer Research 61, 3092-3095, April 1, 2001]
© 2001 American Association for Cancer Research
Inhibition of Caspases Maintains the Antineoplastic Function of 
T Cells Repeatedly Challenged with Lymphoma Cells1
Marina Ferrarini2,
Giuseppe Consogno,
Patrizia Rovere,
Clara Sciorati,
Lorenzo Dagna,
Davide Resta,
Claudio Rugarli and
Angelo A. Manfredi
Laboratory of Tumor Immunology and Cancer Immunotherapy and Gene Therapy Program, Department of Medicine [M. F., G. C., P. R., L. D., D. R., C. R., A. A. M.], and Department of Neuroscience [C. S.], Hospital San Raffaele Scientific Institute, 20132 Milano, Italy, and Vita-Salute San Raffaele University, 20132 Milano, Italy [C. R.]
 |
ABSTRACT
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T lymphocytes recognizing tumor antigens eventually undergo anergy or Fas-mediated death. V
9/V
2+ T cells recognize poorly characterized ligand moieties on human B-cell lymphomas. Here we show that T cells, a model for the study of activation-induced apoptosis, activate on repeated in vitro antigen-recognition caspase 3 and 8 and dramatically down-regulate their cytotoxic and secretory function. Caspase hindrance enhanced T cell survival and sustained the killing of neoplastic cells and the release of IFN- and tumor necrosis factor 
. Caspases of tumor-specific T cells represent a candidate target to complement adoptive immunotherapy strategies.
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INTRODUCTION
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Most tumors growing in vivo hinder the function of immune cells, and in particular of T cells: they avoid T cell activation or, once activated, circumvent their function. In particular, the exposure to persisting tumor cell antigens determines the "serial triggering" of the TcRs3
(1)
. In the absence of costimulation, which neoplastic cells are unlikely to provide, serial triggering of the TcR confers susceptibility to Fas-mediated death (2
, 3) . The interaction between Fas and its physiological ligand, expressed by tumors or activated lymphocytes, recruits caspases 3 and 8 of T cells (4)
. Caspase activation either determines apoptosis of chronically stimulated T cells or contributes to their functional inactivation: the 
chain of the TcR is required for TcR signaling and caspase 3 activation ensuing tumor cell recognition determines chain selective cleavage (4)
. Conversely, pharmacological blockade of caspases prevents the death of tumor-specific T cells (4
, 5)
. To our knowledge, little is known on the fate and the residual function of T cells rescued from apoptosis by caspase inhibition (6)
.
Human V9/V2+ T cells recognize and kill Daudi lymphoma cells via a TcR-dependent pathway (7)
. The TcR-mediated recognition of soluble phosphate moieties has been involved in their sentinel function against transformed and infected cells (7)
. It has been recently exploited to generate an antiplasma cells activity in vitro and in multiple-myeloma patients (8)
. Antigen recognition results, in chronically activated but not in recently activated cells, in the enforcement of the apoptotic program, mediated via the Fas-Fas ligand interaction and requiring the integrity of the caspase enzyme machinery (9
, 10)
.
In this study we show that the pharmacological blockade of caspases sustains the viability and the antineoplastic function of T cells undergoing repeated tumor antigen recognition.
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MATERIALS AND METHODS
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Cells.
V9/2+ T-cell clones were derived as described previously (9)
and propagated by cyclic restimulation (every 4–6 weeks) with irradiated allogeneic mononuclear cells and phytohemagglutinin A (5 µg/ml) in the presence of recombinant IL-2 (50 units/ml; EuroCetus, Amsterdam). All of the cells expressed the V9 TcR, as assessed by staining with the TiA mAb (kindly provided by T. Hercend). Unless otherwise indicated, cells were used 3–4 weeks after restimulation, when they were fully sensitive to activation-induced apoptosis (9, 10, 11)
.
Cytotoxicity and Proliferation.
Cytolytic activity of T cells was evaluated in a 4-h 51Cr release assay (9)
. 51Cr-labeled Daudi lymphoma or control cells (EBV-transformed human lymphoblastoid cell line, human leukemia MOLT-4 cells and human small cell lung cancer N592 cells; 103 cells/sample in triplicate) were used as targets at serial E:T ratios. Specific lysis was calculated as (average experimental cpm - average spontaneous cpm)/(average maximum spontaneous cpm - average spontaneous cpm) x 100. When indicated, T cells were preincubated with mitomycin-C-treated Daudi cells (1:1 ratio) for 18 h before the assay.
Proliferation on treatment with the TiA mAb was evaluated by [3H]thymidine incorporation. The contribution of the Fas/Fas ligand interaction was evaluated with the Fas blocking IgG ZB4 mAb (100 ng/ml; Immunotech, Marseille, France).
Collection of Supernatants and Cytokine Measurement.
Cytokine secretion was evaluated as described previously (12)
. Briefly, supernatants were retrieved after TcR cross-linking, centrifuged at 13.000 rpm for 10 min to remove debris and stored at -30°C. TNF-, IFN-, and IL-8 concentrations were detected by ELISA (R&D Systems, Minneapolis, MN). Assays were performed according to the manufacturer’s instructions. When indicated, T cells were preincubated with mitomycin-C-treated Daudi cells (1:1 ratio) or with the soluble phospho-antigen IPP (Sigma, St. Louis, MO: final concentration 35 µM) for 18 h before the supernatant collection.
Apoptosis.
DNA content was assessed by staining in PBS, containing 50 µg/ml propidium iodide, 100 µg/ml RNase A, 0.01% NP40 (all reagents were from Sigma), at 37°C and analyzed by flow cytometry (FACStarPlus; Becton Dickinson, Sunnyvale, CA; Refs. 9
, 10
).
Caspase Activation and Blockade.
Caspase 3 and 8 activity on TcR activation was evaluated by fluorimetry, as described previously (11)
. Briefly, 2 x 106 cells/sample were either left untreated or preincubated with caspase inhibitors (see below). The TcR was then activated using phospho-antigens (IPP, final concentration 35 µM) or anti-TcR antibodies (TiA ascites, used at a 1:5000 dilution). Cells were rinsed and lysed in a 25 mM HEPES buffer (pH 7.5), containing EDTA (5 mM), EGTA (1 mM), MgCl2 (5 mM), DTT (5 mM), CHAPS (1%), pepstatin (10 µg/ml), leupeptin (10 µg/ml) and phenylmethyl sulfonyl fluoride (1 mM; all of the reagents were from Sigma). Lysates were cleared and stored at -80°C. Protein content was assayed by the bicinchonic acid procedure (Pierce, Rockford, IL). Lysates were incubated at 37°C in a 25 mM HEPES (pH 7.5) buffer containing sucrose (10%), CHAPS (0.1%), and DTT (1 mM), supplemented with the Ac-DEVD-7 amc (50 µM) or IETD-afc (25 µM) fluorogenic substrates. The fluorescence increase following the cleavage of the amc or afc moieties was monitored for 10 min and quantified in a LS50 Perkin-Elmer fluorimeter. Standard curves using increasing fluorogene concentrations were run in parallel. When indicated, T cells were preincubated for 60 min at 37°C with the caspase inhibitors acetyl-Tyr-Val-Ala-Asp chloromethylketone and acetyl-Asp-Glu-Val-Asp aldehyde (100 µM final concentration, Calbiochem-Novabiochem, La Jolla-CA) before assessment of caspase activity or of cytolytic or secretory functions on antigen recognition (see above).
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RESULTS AND DISCUSSION
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Active and adoptive immunotherapy often fail, even in the majority of initially responsive cancer patients, to prompt long-term complete remissions (13)
. The ability of growing tumors to escape immune responses relies on independent mechanisms, which include loss of antigens, loss of MHC molecules, and induction of anergy or death of tumor-specific T cells (14
, 15)
. Persisting neoplasms chronically restimulate tumor-specific T cells. Repeated TcR activation causes sensitivity to Fas-mediated apoptosis (2
, 3
, 16)
, and vaccinations requiring multiple recalls are more successful in Fas-deficient animals (17)
. The activation of T-cell caspases 3 and 8 as a consequence of the chronic recognition of tumor antigens plays a major role (4)
; accordingly, the pharmacological blockade of caspases efficiently prevents the tumor-induced T-cell-assisted suicide (4
, 5)
.
In this study we took advantage of a well-characterized model of T-cell apoptosis induced by repeated tumor antigen recognition (9)
and exploited the blockade of caspases to elevate the antineoplastic activity of human T cells. We relied on T cells, which specifically recognize uncharacterized moieties on B-cell lymphomas (7)
. TcR-specific mAbs or synthetic phospho-antigens mimic in vitro the ensuing functional activation. T cells kill Daudi lymphoma cells on antigen recognition in vitro. In contrast, they ignore human EBV-transformed lymphoblastoid cells, leukemia MOLT-4 cells, and small cell lung cancer N592 cells (Fig. 1A
; see also Ref. 7
).
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Fig. 1. T cells kill Daudi lymphoma cells and secrete cytokines on TcR activation. In A, T cells kill Daudi cells ( ) but not relevant controls (LCL,  ; Molt-4 leukemia cells,  ; N592 small cell lung carcinoma,  ) in standard 4-h 51Cr release assays. Results are expressed as percentage of specific lysis (Y axis) at the given cell:Daudi ratio (X axis) and are from one experiment that is representative of five. In B, recently activated Fas-resistant T lymphocytes incorporate [3H]thymidine (cpm x 10-3, Y axis) when challenged with different dilutions of anti-TcR TiA mAb (1:5000 and 1:500, X axis). Results are expressed as mean ± SD of triplicates and are from one experiment that is representative of three. In C, the proliferation of chronically activated Fas-sensitive T lymphocytes abates on TcR activation with the anti-TcR TiA mAb (1:5000 final dilution) and is partially rescued by the Fas-blocking ZB4 mAb (see "Materials and Methods" for details). Results are from one experiment that is representative of two. In D–F, chronically activated Fas-sensitive T lymphocytes secrete IFN- (D), TNF- (E), and IL-8 (F) on TcR activation with the anti-TcR TiA mAb (1:5000 final dilution).
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Recently activated cells proliferated on TcR engagement (Fig. 1B)
. In contrast, the proliferation of chronically activated T cells abates on TcR engagement (Fig. 1C)
. Chronically activated T cells are sensitive to Fas-mediated apoptosis (9, 10, 11)
. Accordingly, the blockade of the Fas/Fas ligand interaction with the ZB4 mAb partially rescued their proliferation (Fig. 1C)
. Chronically and recently activated cells killed their target with the same efficiency (not shown). Furthermore, on TcR engagement, they secreted substantial amounts of IFN-, TNF-, and IL-8 (Fig. 1 D–F)
, i.e., cytokines involved both in the direct killing of tumor cells and in the associated inflammation and recruitment of effector leukocytes (18)
.
The TcR engagement of chronically activated cells recruits caspase 3 and 8 (Fig. 2, A and B)
, as described for Fas-sensitive lymphocytes coincubated with head and neck squamous carcinoma cells (4)
. Caspase 3 activity of T cells increased from 13.9 pmol of fluorogen/min/mg of cell lysate to 53 and 50.38 pmol after treatment with anti-TcR mAbs or with soluble phospho-antigens, respectively. In the same conditions, caspase 8 activity increased from 3.5 pmol to 7.41 and 7.07 pmol, respectively. The caspase enzyme activities of T cells abated after treatment with cell-permeant synthetic acetyl-Tyr-Val-Ala-Asp chloromethylketone and acetyl-Asp-Glu-Val-Asp aldehyde inhibitors (Fig. 2, A and B)
: caspase 3 activity of cells treated with anti-TcR mAbs dropped to 2.4 pmol and caspase 3 activity of IPP-stimulated cells to 1.3 pmol. Caspase 8 activity dropped to 2.16 and 1.96 pmol, respectively. In the presence of caspase inhibitors, TcR engagement did not result in the appearance of cells with a subdiploid DNA content (Fig. 2, C–E)
. Therefore, in agreement with previous reports, caspase blockade prevents the TcR-mediated apoptosis of specific T cells (4
, 5) .
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Fig. 2. TcR activation determines caspase 3 and 8 activation and apoptosis. Caspase 3 (A) and caspase 8 (B) activity was assessed at 120 min after TcR activation with the anti-TcR TiA mAb (1:5000 final dilution; left panels, open columns) or with the IPP phospho-antigen (right panels, open columns) by fluorimetry, as described in the "Materials and Methods" section. Values, expressed as picomoles of fluorogen/min/mg of cell lysate, are reported on the Y axis. Enzyme activity abated in the presence of acetyl-Tyr-Val-Ala-Asp chloromethylketone and acetyl-Asp-Glu-Val-Asp aldehyde caspase inhibitors (C.I., filled columns). Results are from one experiment that is representative of two. The DNA content of untreated T cells (C), of cells that underwent T-cell activation on 18-h treatment with the IPP phospho-antigen, either in the absence (D) or in the presence (E) of caspase inhibitors was evaluated by flow cytometry. The number of events is reported on the Y axis. Cells with an apparent hypodiploid DNA content (M1 region) were 5.9, 25, and 8.3% in C, D, and E, respectively. Results are from one experiment representative of three.
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We then evaluated whether the blockade of caspases influenced the function of T cells repeatedly challenged with tumor antigens. To this aim, we incubated T cells with nonproliferating mitomycin-C-treated Daudi lymphoma cells. Eighteen h later, we rechallenged them with 51Cr-labeled "fresh" lymphoma cells in a 4-h cytotoxicity assay. Fig. 3B
shows that preincubated cells lost the ability to kill Daudi cells. This was not attributable to cell death occurring during the overnight coincubation, because the basal rate of cell death was only slightly increased in preincubated versus nonpreincubated lymphocytes (not shown). Furthermore, the rechallenge with phospho-antigens after overnight TcR engagement dramatically reduced the secretion of TNF- and IFN- (Fig. 3, C and D)
by T cells. Preincubation with Daudi cells also induced a drop in the ability to secrete cytokines (not shown).
The blockade of the caspase machinery of cells at the first encounter with the antigen increased neither their cytotoxic activity nor their ability to secrete cytokines (Table 1)
, in agreement with the results reported with melanoma-specific CD8+ T cells derived from tumor-infiltrating lymphocytes (5)
.
Caspase inhibitors rescued the loss of function of T cells at the second challenge with the antigen; cells with a poised caspase machinery maintained the ability to kill Daudi cells and to secrete IFN- and TNF- when rechallenged with relevant antigens (Table 1)
. In some experiments (e.g., see the representative example depicted in Fig. 3
) the first antigenic challenge was prolonged or particularly efficient at abrogating cytokine release. The concentration of TNF- in the supernatant dropped from 520 to 18 pg/ml, whereas IFN- dropped from 2297 to 650 pg/ml. In these conditions, caspase blockade rescued IFN- secretion (from 650 to 2349 pg/ml) but was less efficient in maintaining the ability to release TNF- (from 18 to 75 pg/ml). The molecular basis of the different sensitivity of T-cell-derived cytokines to caspase blockade needs further elucidation. For example, we used peptide inhibitors endowed with a broad specificity, which includes caspase 1. Caspase 1- and caspase 11-deficient mice bear a selective defect in the processing and exports of proinflammatory cytokines, including TNF- (6)
.
All together, our findings suggest that the activation of caspases of T cells that are repeatedly challenged with tumor cells limits their response, possibly impinging on the success of vaccination procedures. Successful viruses exploit caspase-blocking genes to protect their reservoir and ensure their replication in infected cells (19)
. The genetic manipulation of tumor-specific T lymphocytes may sustain their function and prove valuable for the success of adoptive immunotherapy strategies.
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ACKNOWLEDGMENTS
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We thank Celine De Nadai (HRS-DIBIT, Milan, Italy) for the kind help with the assessment of caspase activity, Valérie S. Zimmermann and Emilio Clementi (HRS-DIBIT, Milan, Italy) for helpful discussions, and Thierry Hercend (Institut Gustave-Roussy, Villejúif, France) for the kind gift of the TiA mAb.
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FOOTNOTES
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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 with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Italian Association for Cancer Research (AIRC), by the Istituto Superiore di Sanità (AIDS-related Infections Project) and by the Ministero dell’Università e Ricerca Scientifica e Tecnologica (MURST; cofinanziamento 1999). 
2 To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology, H San Raffaele Scientific Institute, via Olgettina 60, 20132 Milano, Italy. Phone: 39-02-2643-2612; Fax: 39-02-2643-2611; E-mail: m.ferrarini{at}hsr.it
3 The abbreviations used are: TcR, T-cell receptor; mAb, monoclonal antibody; TNF, tumor necrosis factor; IL, interleukin; IPP, isopentenyl PPi; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
Received 9/14/00.
Accepted 1/26/01.
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ABSTRACT
INTRODUCTION
). Preincubation was carried out using equal numbers of