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Impaired Cytolytic Activity in Calreticulin-Deficient CTLs Simonetta Sipione, Catherine Ewen, Irene Shostak, Marek Michalak and R. Chris Bleackley This information is current as of September 28, 2021. J Immunol 2005; 174:3212-3219; ; doi: 10.4049/jimmunol.174.6.3212 http://www.jimmunol.org/content/174/6/3212 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Impaired Cytolytic Activity in Calreticulin-Deﬁcient CTLs1

Simonetta Sipione,* Catherine Ewen,† Irene Shostak,* Marek Michalak,* and R. Chris Bleackley2*

Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated .killing, we generated CTL lines from crt؉/؉ and crt؊/؊ mice expressing a constitutively active form of calcineurin in the heart ,Crt؊/؊ CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt؊/؊ cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt؊/؊ CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is Downloaded from dispensable for the cytolytic activity of granzymes and perforin, but it is required for efﬁcient CTL-target cell interaction and for the formation of the death synapse. The Journal of Immunology, 2005, 174: 3212–3219.

ytotoxic T lymphocytes and NK cells play a major role observation that a 60-kDa protein present in the granules consis- in the response mounted by the immune system tently coeluted with perforin (15). This protein was identified as ϩ C against virally infected and tumor cells. Killing of tar- calreticulin, an endoplasmic reticulum (ER)3 chaperone and Ca2 - http://www.jimmunol.org/ get cells by CTLs and NK cells predominantly occurs through binding protein (16, 17). Later it was shown that calreticulin in- two major pathways: apoptosis induced by Fas-Fas ligand in- teracts with perforin through its P domain, which has lectin-like teraction and/or cell death induced by the components of the activity (18) and high Ca2ϩ-binding affinity (19), and, to a lesser lytic granules (1, 2). Lytic granules are specialized lysosomes extent, through its N domain. Interaction between the two proteins containing a pore-forming protein, perforin, and a family of occurs only in the absence of Ca2ϩ (20). Calreticulin was found to serine proteases collectively named granzymes (3). Upon rec- inhibit the lytic activity of preparations of purified perforin on ognition of target cells, the contents of the lytic granules are erythrocytes (21, 22). The inhibitory action, however, was not me- released into the synapse formed between CTL and target cells, diated by either the N or P domain, but, rather, by the calreticulin by guest on September 28, 2021 ultimately leading to destruction of the target (4–6). The mech- C domain, a highly acidic region that binds Ca2ϩ with high ca- anism by which granzymes gain access to the cytosol of target pacity and low affinity (19). Although the nature of the inhibitory cells is still unknown. Granzyme B can enter target cells role of calreticulin was not investigated in detail, it was proposed through receptor-mediated endocytosis (7, 8), but once inside that by binding to the cell membrane, calreticulin could provide the cell, it is unable to escape the endocytic compartment to protection against osmotic lysis and perforin polymerization trigger apoptosis without the presence of perforin or other lytic within the phospholipid bilayer. It was also speculated that such a agents (9–11). mechanism could protect CTLs from their own perforin during the The major role played by perforin in granule-mediated cytotox- killing of target cells (21). This hypothesis has never been tested, icity became evident after studies in perforin knockout mice in and the possibility that exogenously added calreticulin may block which target cells could not be killed by the granule-mediated perforin activity in a nonspecific fashion, simply chelating Ca2ϩ pathway (12–14). Initial attempts to purify perforin from the cy- ions in the medium, has never been experimentally excluded. The totoxic granules of lymphokine-activated killer cells lead to the data on calreticulin interaction with perforin, along with studies indicating that calreticulin expression is up-regulated during T cell activation (23), suggested that the ER chaperone may play a role Departments of *Biochemistry and †Medical Microbiology and Immunology, Uni- in degranulation and CTL-induced killing. Unfortunately, the lack versity of Alberta, Edmonton, Canada of a proper cell model has hampered assessment of calreticulin Received for publication September 7, 2004. Accepted for publication December function in CTLs. 17, 2004. Calreticulin knockout (crtϪ/Ϫ) mice have been generated (24), 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 but they die at the embryonic stage due to impaired heart devel- Ϫ/Ϫ with 18 U.S.C. Section 1734 solely to indicate this fact. opment, making it impossible to study mature crt CTLs and Ϫ/Ϫ 1 This work was supported by grants from the Canadian Institute of Health Research NK cells. The nature of the cardiac defect observed in crt mice and the National Cancer Institute of Canada. R.C.B. is a Canadian Institute of Health was found to reside in the failure to mobilize ER Ca2ϩ stores and Research Distinguished Scientist, a Medical Scientist of the Alberta Heritage Foun- dation for Medical Research, a Howard Hughes International Scholar, and a Canada to activate the serine/threonine phosphatase calcineurin (25). By Research Chair. M.M. is a Canadian Institute of Health Research Senior Scientist and crossing a transgenic mouse expressing a cardiac-specific, consti- a Medical Scientist of the Alberta Heritage Foundation for Medical Research. S.S. tutively active form of calcineurin (26) with crtϩ/Ϫ mice, viable was supported by a research fellowship from the Alberta Heritage Foundation for Medical Research. 2 Address correspondence and reprint requests to Dr. R. Chris Bleackley, Department 3 Abbreviations used in this paper: ER, endoplasmic reticulum; BLT, N-␣-benzyloxy- of Biochemistry, 460 Medical Sciences Building, University of Alberta, Edmonton, carbonyl-L-lysine thiobenzyl ester; CMA, concanamycin A; LDH, lactic dehydroge- Alberta, Canada T6G 2H7. E-mail address: [email protected] nase; crtϩ/ϩ, calreticulin wild type; crtϪ/Ϫ, calreticulin knockout.

Ϫ Ϫ crt / transgenic mice were obtained. In these animals, normal anti-mouse CD3⑀ Ab (clone 145-2C11; 1/500 dilution of hybridoma su- cardiac development was restored, and embryonic lethality was pernatant) before performing the killing assay. Cells were then centrifuged, suppressed (25). Because the expression of active calcineurin oc- resuspended in RHFM medium, counted, and incubated with the target cells. curred only in heart tissue, noncardiac phenotypes specifically due to ablation of the calreticulin gene were not corrected (25). There- Flow cytometry fore, this mouse represents an excellent model to study calreticulin CTL were washed with PBS and fixed in 2% paraformaldehyde in PBS for functions in systems other than heart. To specifically investigate 15 min at room temperature. After three washes in PBS, cells were per- the role of calreticulin in CTL lytic function, we generated CTL meabilized for 5 min at room temperature with 0.3% saponin in PBS con- lines from splenocytes derived from these mice. In this study we taining 1% FCS. PE-conjugated anti-granzyme B mAb (clone CLB-GB12; show that in the absence of calreticulin, CTL cytotoxicity is im- PeliCluster) was added to each sample at a final concentration of 5 ␮g/ml paired. Because calreticulin ablation does not seem to affect com- and incubated at room temperature for 30 min. Cells were washed three times with PBS containing 1% FCS and analyzed with a FACScan flow ponents of the lytic machinery, the ability of CTLs to degranulate, cytometer equipped with CellQuest software (BD Biosciences). or their survival during degranulation and attack of target cells, but does affect the efficiency of effector/target conjugate formation, we Granzyme B enzymatic assay propose that calreticulin may be involved in the mechanisms un- Cell lysates were prepared by resuspending CTL in 20 mM Tris-HCl (pH derlying target recognition by CTLs and/or formation of the death 7.2), 150 mM NaCl, and 1% Triton X-100 at a concentration of 107 synapse. cells/ml and incubating for 30 min on ice. Lysates were freeze-thawed once, centrifuged at 10,000 rpm for 7 min to remove cell debris, and stored Ϫ

at 80°C until used. Granzyme B activity in supernatant and total lysates Downloaded from Materials and Methods was assayed as previously reported (30). Briefly, aliquots of incubation Cells and reagents medium or cell lysates were dispensed into 96-well, flat-bottom tissue culture plates (Nalge Nunc International). Enzymatic reaction was conducted Mouse mastocytoma P815 cells, mouse lymphocytic leukemia L1210 cells, at 37°C in 50 mM HEPES (pH 7.5), 10% (w/v) sucrose, 0.05% (w/v) and mouse lymphoma EL4 cells were obtained from American Type Cul- CHAPS, and 5 mM DTT containing 200 ␮M acetyl-Ile-Glu-Pro-Asp-pa- ture Collection and were maintained in RPMI 1640 medium supplemented ranitroanilide (Kamiya Biomedical) as substrate. Hydrolysis of acetyl-Ile- with 20 mM HEPES, 50 U/ml penicillin, 50 ␮g/ml streptomycin, 1 mM Glu-Pro-Asp-paranitroanilide was measured at 405 nm using a Multiskan sodium pyruvate (Invitrogen Life Technologies), 0.1 mM 2-ME (Sigma- Ascent spectrophotometer (Thermo Lab-system). The absorbance of a http://www.jimmunol.org/ Aldrich), and 10% heat-inactivated FBS (HyClone; RHFM medium). blank containing no proteins was subtracted from each sample. Granzyme A/granzyme B double-knockout mice were generated in our laboratory by crossing granzyme A knockout (27) with granzyme B knock- N-␣-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) esterase out mice (28) (The Jackson Laboratory) and subsequently inbreeding F1 animals. CTL lines were generated from these mice as described below. activity assay The hybridoma 145-2C11 producing anti-CD3⑀ Ab was obtained from Serine esterase activity in total cell lysates and medium was measured American Type Culture Collection. All reagents were purchased from Sig- spectrophotometrically in 96-well, flat-bottom tissue culture plates. Enzy- ma-Aldrich unless otherwise specified. matic reaction was conducted for 30 min at 37°C in PBS containing 0.2 Generation of the crtϪ/Ϫ CTL line mM BLT and 0.2 mM dithiobis-2-nitrobenzoic acid. Absorbance at 405 nm was read with a Multiskan Ascent spectrophotometer. The absorbance of CrtϪ/Ϫ mice expressing a cardiac-specific constitutively active form of total cell lysate from granzyme A/granzyme B double-knockout CTLs was by guest on September 28, 2021 calcineurin have been previously described (25). Briefly, homozygote mice subtracted from sample readings. Serine esterase activity was calculated ⑀ ϭ bearing the activated calcineurin transgene (FVB/N strain) (26) were based on the extinction coefficient of dithiobis-2-nitrobenzoic acid ( 412 crossed with crtϩ/Ϫ mice (C57BL/6J strain) (24). Mice of the resulting 13,600). progeny were interbred to generate crtϩ/ϩ and crtϪ/Ϫ mice expressing activated calcineurin in the heart. CTL lines were generated from these mice Immunoblotting by coculture of isolated splenocytes with irradiated (2500 rad) BALB/c splenocytes (1:1 ratio) in RHFM medium supplemented with 80 U/ml CTL were washed in PBS, counted, and resuspended in SDS sample buffer 7 rhIL-2 (Chiron) for 3 days. Irradiated stimulators were removed by Ficoll- at a concentration of 10 cells/ml. Forty microliters of sample was resolved Paque (Amersham Biosciences) centrifugation, and CTLs were maintained by electrophoresis on a 12% SDS-polyacrylamide gel under reducing con- in RHFM medium supplemented with 80 U/ml rhIL-2 for 3 additional days ditions. Proteins were transferred to a polyvinylidene difluoride membrane. before further stimulation. Subsequent stimulations with irradiated The membrane was blocked for 1 h with 5% (w/v) milk/0.1% Tween 20 in BALB/c splenocytes were performed weekly at a ratio of 1:14 (CTLs: TBS, then incubated overnight at 4°C with rabbit anti-perforin Ab (1/1000; stimulators). CTLs that had been exposed to 4–13 cycles of stimulation Torrey Pines Biolabs). The secondary Ab was HRP-conjugated goat anti- were used for the experiments, from 3 to 6 days after stimulation. rabbit IgG (Bio-Rad). Visualization of the immunoblot was performed by ECL Plus (Amersham Biosciences). Killing assays Immunocytochemistry CTL cytolytic activity was evaluated as previously described (29), measuring membrane damage (51Cr release) and DNA fragmentation ([3H]thy- Cells were washed and resuspended at a concentration of 107 cells/ml in midine release) induced in target cells. Briefly, allogeneic target cells (106) PBS. Fifty microliters of the cell suspension was spotted onto silane-treated ␮ 51 ␮ 3 were labeled with 100 Ci of Na2 CrO4 or 1 Ci/ml [ H]thymidine, Hystobond slides (Marienfeld Laboratory Glassware) and incubated at washed, and incubated at 37°C with effector CTLs at the indicated E:T cell 37°C for 10 min. PBS was gently withdrawn, and adherent CTL were fixed ratio in 96-well plates. Where indicated, Con A was added to the samples with 2% paraformaldehyde for 30 min at room temperature. After three at the final concentration of 2 ␮g/ml. To block perforin activity and to washes with PBS, cells were permeabilized with 0.1% Triton X-100 for 3 assess the contribution of Fas-mediated killing, CTL were preincubated for min and blocked for 1 h with PBS containing 5% donkey serum. Incubation 2 h with 500 nM concanamycin A (CMA; Sigma-Aldrich), then added to with the primary Abs was performed for1hatroom temperature. The the target cells in the presence of the inhibitor. 51Cr release in the medium following Abs were used: mouse monoclonal anti-granzyme B (clone was measured 4 h later using a 1470 Wizard automatic gamma counter CLB-GB12; 1/50), rabbit anti-perforin (1/50), mouse monoclonal anti- (Wallac). DNA fragmentation was measured 2 h after incubation with perforin (provided by Dr. G. Griffiths, University College London, London, CTL. Cells were lysed with 1% Triton X-100 in 10 mM Tris and 1 mM U.K., and goat anti-lysosomal-associated membrane protein-1), and goat EDTA, pH 8.0, and were centrifuged at 10,000 rpm in a microcentrifuge at anti-LAMP-1 (1/20; Santa Cruz Biotechnology). Secondary Abs were: 4°C. [3H]Thymidine release in the supernatant was measured using an donkey anti-rabbit Alexa 555, donkey anti-mouse Alexa 488 and Alexa LS7800 beta counter (Beckman Coulter). Maximum [3H]thymidine release 594, and donkey anti-goat Alexa 488, all used at 1/400 dilution, except for was determined by lysis of target cells with 2% SDS in 0.2 M NaOH. The Alexa 594-conjugated Abs, which were used at 1/200 dilution (Molecular percent membrane lysis and DNA fragmentation were calculated as: [(sam- Probes). Slides were mounted with ProLong (Molecular Probes) and ana- ple cpm Ϫ spontaneous cpm)/(maximum cpm Ϫ spontaneous cpm)] ϫ lyzed with an LSM510 laser scanning confocal microscope mounted on a 100. In some experiments CTL were preincubated for 4 h with soluble Zeiss Axiovert 100M microscope. 3214 IMPAIRED CYTOTOXIC ACTIVITY IN CALRETICULIN-DEFICIENT CTLs

Induction of granule exocytosis by immobilized anti-CD3⑀ Ab Because the expression of the transgene was under control of the ␣ Ninety-six-well, U-bottom plates were coated overnight at 4°C with anti- cardiac specific -myosin H chain promoter, we expected the CD3⑀ mAb (clone 145-2C11; BD Biosciences) at the indicated dilutions in transgene to be silent in the CTL lines. Western blotting analysis 0.1 M bicarbonate buffer, pH 9.6. The following day, wells were washed confirmed that the transgene was not expressed at any time in the four times with cold PBS and blocked for 30 min at room temperature with CTLs (data not shown). ␮ 2% FCS in PBS. Blocking solution was then replaced with 100 l/well The cytolytic potential of the crtϩ/ϩ and crtϪ/Ϫ CTLs was as- CTL suspension in phenol-free RPMI 1640 medium containing 2% FCS (5 ϫ 106 cells/ml). CTL were allowed to degranulate at 37°C for 5 h. After sessed in in vitro killing assays in which the two CTL lines were centrifugation at 1200 rpm for 5 min in an Allegra 6R centrifuge (Beckman challenged with allogeneic target cells, the mouse mastocytoma Coulter), 50 ␮l of supernatant was transferred to a 96-well, flat-bottom cell line P815 and the lymphocytic leukemia L1210 line (Fig. 1). plate for detection of granzyme B, BLT esterase, or lactic dehydrogenase Compared with wild-type cells, crtϪ/Ϫ CTLs showed reduced cy- (LDH) enzymatic activity. totoxicity toward target cells at all E:T cell ratios tested (shown in LDH Fig. 1A for L1210 cells; killing of P815 targets occurred with a LDH released by cells into the medium (50 ␮l) was measured at room similar trend). Both membrane lysis (Fig. 1B) and DNA fragmen- temperature in a reaction volume of 200 ␮l and in the presence of 0.4 mM tation (Fig. 1C) in target cells exposed to the CTL attack were sodium pyruvate and 0.2 mM NADH. The kinetics of the enzymatic reac- measured. In all cases the cytotoxic activity of crtϪ/Ϫ CTLs was tion were monitored spectrophotometrically, and the rate of absorbance significantly reduced with respect to that of wild-type cells. The decrement at 334 nm over 2 min was used to calculate LDH activity in the difference was limited to the perforin-dependent killing mecha- samples. Total LDH activity was measured in fresh cell lysates and used to derive the percentage of LDH released into the medium. nism, rather then Fas-mediated killing, because in the presence of CMA (31) cytotoxicity was greatly reduced, with no significant Downloaded from Analysis of conjugate formation differences between the residual killing activities of the crtϩ/ϩ Ϫ Ϫ CTL were labeled with 2 ␮M Orange Cell Tracker (Molecular Probes), and and crt / CTLs. P815 cells were labeled with 1 ␮M Green CellTracker (Molecular Probes) It has been previously demonstrated that the level of granzyme according to the manufacturer’s instructions. Labeled CTLs were left rest- B activity released by CTLs into the medium during incubation ingfor3hat37°C in regular medium or in the presence of soluble anti- CD3⑀ Abs (1/500), then washed in cold phenol-free HBSS. After resus- with target cells strictly correlates to the levels of degranulation

pension in cold phenol-free HBSS containing 5% FCS, CTL and target and killing (30). As expected, the amount of granzyme B released http://www.jimmunol.org/ cells were mixed at the ratio of 1:3 and centrifuged at 1000 rpm in an by crtϪ/Ϫ CTL during incubation with target cells was 50 Ϯ 23% Allegra 6R centrifuge (Beckman Coulter) for 3 min at 4°C to facilitate ( p ϭ 0.0032) lower than the amount secreted by wild-type cells. In cell-cell contact. Cells were then incubated at 37°C for the indicated time light of these data, three possible explanations could be considered to allow conjugate formation. At the end of the incubation, samples were Ϫ/Ϫ gently vortexed for 2 s and fixed by addition of an equal volume of 4% for the reduced cytotoxicity of crt CTLs: 1) CTL death during paraformaldehyde solution. Analysis of double-labeled cell conjugates was the killing assay, 2) lower expression of granzymes and perforin, performed with a FACScan flow cytometer. or 3) inefficient target-induced degranulation. Statistical analysis CrtϪ/Ϫ CTLs are viable during the attack of target cells

Data were analyzed by two-tailed Student’s t test for paired samples. A by guest on September 28, 2021 value of p Ͻ 0.05 was considered signiﬁcant. Because it has been proposed that calreticulin may have a protective role on CTLs against their own perforin (21), we investigated Results Ϫ/Ϫ Ϫ/Ϫ whether the lower level of killing observed with crt CTLs Crt CTLs exert reduced cytotoxic activity on target cells could be attributed to CTL death during incubation with target ϩ/ϩ Ϫ/Ϫ 51 Crt and crt CTL lines were generated from transgenic mice cells. Labeling of CTLs with Na2 CrO4 allowed us to monitor expressing a constitutively active form of calcineurin in the heart. CTL death during the killing assay. No difference in CTL survival

FIGURE 1. Impaired cytotoxicity in crtϪ/Ϫ CTLs. Spontaneous release in the absence of effector cells was subtracted from each sample. Where indicated, CTLs were preincubated with 500 nM CMA to block perforin- dependent cell death. A, Effect of increasing E:T cell ratio on the killing of L1210 cells. B, Membrane lysis induced in target cells exposed to CTLs. C, DNA fragmentation in target cells exposed to CTLs. The E:T cell ratio was 2:1 for the killing of P815 cells and 5:1 for the killing of L1210 cells. The data shown for killing in control conditions represent the mean Ϯ SD of ﬁve to eight experiments, each performed in triplicate. The data on CMA inhibition are the mean Ϯ SD of three experiments. D, CTL death during the killing assay. ␮ 51 CTLs were labeled with 100 Ci of Na2 CrO4 before incubation with target cells. The speciﬁc 51Cr release measures the percentage of CTL death during 4-h incubation with or without (sp) target cells. Data represent the mean Ϯ SD of two independent experiments, each ;p Յ 0.002 ,ءء ;p Յ 0.0002 ,ءءء .performed in triplicate .p Յ 0.03 ,ء The Journal of Immunology 3215 was observed (Fig. 1D). CTL death was as low as 6–7% for both crtϩ/ϩ and crtϪ/Ϫ cells and was comparable to the level of death observed in the absence of target cells (spontaneous death), indicating that CTLs were viable during the assay.

CrtϪ/Ϫ CTLs have normal granzyme content and express high levels of perforin To test the second hypothesis, we measured granzyme and perforin expression in crtϪ/Ϫ CTLs. As shown in Fig. 2, granzyme B expression, as measured by FACS staining, was not signiﬁcantly different between crtϩ/ϩ and crtϪ/Ϫ cells (Fig. 2A). Moreover, the enzymatic activity of granzyme B was comparable, suggesting that in the absence of calreticulin, granzyme B can still fold properly without loss of activity (Fig. 2B). Crtϩ/ϩ and crtϪ/Ϫ CTLs also contained the same amount of serine esterase (granzyme A) activity (Fig. 2C). In contrast, levels of perforin expression were higher in crtϪ/Ϫ CTLs than in wild-type cells (Fig. 2D). No differences in granzyme B and perforin intracellular localization were evident by confocal microscopy, as assessed by colocalization with lysosom- Downloaded from al-associated membrane protein-1, a lysosomal marker also present in lytic granules (32) (Fig. 3).

CrtϪ/Ϫ CTLs degranulate more efﬁciently than crtϩ/ϩ cells when stimulated with immobilized anti-CD3⑀ Ab

To investigate whether impaired transduction of the signal from http://www.jimmunol.org/ the TCR-CD3 complex could account for the reduced cytotoxicity observed in the absence of calreticulin, we triggered activation of the TCR-CD3 complex and exocytosis of lytic granules by exposing CTL to immobilized anti-CD3⑀ Ab (33, 34). In these experi- by guest on September 28, 2021

FIGURE 3. Confocal microscopy on crtϩ/ϩ and crtϪ/Ϫ CTLs. ϩ/ϩ, crtϩ/ϩ CTLs; Ϫ/Ϫ, crtϪ/Ϫ CTLs. The images shown are representative of three experiments.

mental conditions, the crtϪ/Ϫ CTLs degranulated and released granzyme B and BLT esterase activity efficiently (Fig. 4). Al- though at a saturating concentration (10 ␮g/ml) of immobilized Ϫ/Ϫ u Ab, the release of granzyme B and BLT esterases was comparable FIGURE 2. Expression of granzymes and perforin in crt CTL. , ϩ/ϩ Ϫ/Ϫ Ϫ/Ϫ crtϩ/ϩ CTLs; f, crtϪ/Ϫ CTLs. A, FACS analysis of CTLs stained with in crt and crt CTLs, at lower Ab concentrations crt cells PE-conjugated anti-granzyme B mAb. Data are expressed as the percentage secreted even more granzyme B than wild-type cells (Fig. 4A). CTL of mean fluorescence intensity over crtϩ/ϩ CTLs and represent the mean Ϯ death during the5hofincubation was Ͻ7% of the total cell number, SD of a total of nine observations performed in two independent experi- as assessed by measuring LDH release into the medium, with no ments. B, Granzyme B enzymatic activity in total cell lysates was measured significant difference between cell lines or between control (no Ab) as described in Materials and Methods and is expressed as percentage over and stimulated samples (data not shown). Therefore, the higher se- ϩ ϩ crt / CTLs. Results were calculated from the mean values of seven in- cretion of granzymes by crtϪ/Ϫ cells was due to specific triggering of dependent experiments, each performed in triplicate. C, Serine esterase TCR-CD3 signaling by immobilized anti-CD3 Ab. activity was measured in total cell lysates. Values represent the average Ϯ SD granzyme A activity after subtracting the nonspecific esterase activity Effector/target conjugate formation is impaired in crtϪ/Ϫ CTLs, of granzyme A/B knockout CTLs. Mean values were calculated from two and cytotoxicity is restored in conditions that favor cell independent experiments, each performed in triplicate. D, Western blot for adhesion perforin performed on total cell lysates. The equivalent of 4 ϫ 106 cells was loaded in each lane. A nonspecific immunoreactive band was used as Impaired cytotoxicity may result from defective recognition and/or Ϫ Ϫ loading control. Data are representative of three experiments. adhesion to target cells. Indeed, we found that crt / CTLs were 3216 IMPAIRED CYTOTOXIC ACTIVITY IN CALRETICULIN-DEFICIENT CTLs Downloaded from

FIGURE 5. Conjugate formation between effector and target cells. Red- labeled CTLs and green-labeled P815 cells (see Materials and Methods) were incubated together in a ratio of 1:3 (E:T), then ﬁxed and analyzed by FACS. Data represent the average percentage Ϯ SD of CTLs engaged in the formation of conjugates at each of the indicated time points under basal

conditions (A) and after prestimulation of CTLs with soluble anti-CD3⑀ http://www.jimmunol.org/ p ϭ ,ء .Abs (B). Experiments were performed twice, each time in triplicate 0.0002 for 5 min point; p Ͻ 0.006 for 10 and 15 min.

to the increased ability to degranulate upon recognition of target cells. Similar results were obtained when the killing assay was performed in the presence of Con A (Fig. 7A). Con A is known to FIGURE 4. CrtϪ/Ϫ CTL degranulation in response to immobilized anti- induce lectin-dependent, cell-mediated cytotoxicity, a process in by guest on September 28, 2021 CD3⑀ Ab. A, Cells (5 ϫ 105) were seeded onto immobilized anti-CD3⑀ Ab which target cells are killed by CTLs in an Ag-independent fash- at the indicated concentrations and incubated at 37°C for 5 h. Degranula- ion. This is achieved through the bridging action of Con A that tion was assessed by measuring granzyme B released in the incubation binds to glycoproteins on the surface of both effectors and target Ϯ medium as described in Materials and Methods. Data are the mean SD cells and at the same time cross-links the TCR on the CTL mem- of four independent experiments, each performed in triplicate. The inset Ϫ Ϫ brane (36–40). In the presence of Con A, crt / CTL cytotoxicity shows the linearity of a mouse recombinant granzyme B standard curve in the range of absorbance used for the evaluation of granzyme B degranu- increased signiﬁcantly, allowing for the killing of allogeneic tar- lation. B, BLT esterase activity released in the incubation medium under gets (P815) at levels close to those observed with wild-type CTLs. the same conditions as those described in A. Total intracellular BLT es- Moreover, Ag-independent cytotoxicity toward syngeneic target ϩ ϩ Ϫ Ϫ terase activity was measured on cell lysates and was used to derive the cells (EL-4) was observed for both crt / and crt / CTLs percentage release. (Fig. 7B).

Discussion Previous studies have identified calreticulin, an ER-resident chap- less efficient than wild-type cells in forming conjugates with target erone and a Ca2ϩ-binding protein, as a component of CTL and NK cells (Fig. 5A). If reduced conjugate formation (or stability) was cell lytic granules (15). Upon activation of CTLs, calreticulin gene the cause of the low levels of killing observed with crtϪ/Ϫ CTLs, expression is up-regulated (23), and the protein is targeted to lytic we expected to see increased cytotoxicity after treatments and in granules (20) and the plasma membrane (41). Although several conditions that enhance cell adhesion. Therefore, we prestimulated studies have reported on functions played by the protein outside CTLs with soluble anti-CD3 Abs before washing and exposing the ER (reviewed in Refs. 16 and 17), there is no information on them to target cells. Soluble anti-CD3 Abs do not induce CTL its role in the lytic granules and CTL physiology. In this regard, degranulation (34), but are known to activate adhesion of CTLs to our study provides the first direct evidence of calreticulin involve- substrates and target cells (35). As expected, antiCD3-stimulated ment in the cytotoxic activity of CTLs. Because the calreticulin crtϪ/Ϫ CTLs became indistinguishable from wild-type cells with knockout is embryonically lethal (24), we generated a CTL line respect to the number of conjugates formed (Fig. 5B) and the abil- from crtϪ/Ϫ mice that were able to survive due to the ectopic ity to kill target cells (Fig. 6, A and B). The restored cytotoxicity expression of active calcineurin in the heart (25). The transgenic was mainly perforin-dependent, because killing was greatly re- constitutively active form of calcineurin was not expressed in tis- duced (from Ͼ80 to 17%) by blocking perforin activity with CMA sues other than heart, allowing for the specific analysis of calreti- (data not shown). The levels of granzyme B released into the me- culin deficiency in CTLs. The crtϪ/Ϫ CTLs were healthy, provid- dium correlated with the killing of target cells (Fig. 6C), further ing evidence that calreticulin function is dispensable during demonstrating that restoration of the crtϪ/Ϫ CTL activity was due development and maturation of CTLs. Our study also indicates that The Journal of Immunology 3217 Downloaded from

ϩ/ϩ FIGURE 7. Killing assay in the presence of Con A. u, crt CTLs; f, http://www.jimmunol.org/ crtϪ/Ϫ cells. A, CTL cytotoxicity in the presence of Con A is expressed as the percentage of killing (DNA fragmentation) of target cells (P815) with respect to the level of killing induced by crtϩ/ϩ CTLs. CTRL, assay performed under control conditions; Con A, assay performed in the presence of Con A. B, Killing of syngeneic target cells (EL4). Where indicated, Con A was added to the incubation medium. Fas-mediated killing was assessed by preincubation of CTLs with 500 nM CMA (CMA ϩ). Data represent the FIGURE 6. Killing assays after prestimulation with anti-CD3⑀ Ab. percentage of target cells killed (DNA fragmentation). Values are the CTLs were exposed to soluble anti-CD3⑀ Ab (␣CD3 ϩ) for 4 h, washed, mean Ϯ SD of two independent experiments, each performed in triplicate. by guest on September 28, 2021 .p ϭ 0.00002 ,ء and then incubated with target cells (E:T cell ratio, 2:1 for P815 and 5:1 for L1210 cells). u, crtϩ/ϩ CTLs; f, crtϪ/Ϫ cells. A, DNA fragmentation in target cells. Data are expressed as the percentage of cytotoxicity with respect to the levels of killing induced by crtϩ/ϩ CTLs. Values are the p ϭ 0.01; from lack of protein folding quality control in the absence of the ,ء .mean Ϯ SD of six experiments, each performed in triplicate -p ϭ 0.0008. B, Membrane lysis in target cells. Data are expressed as the ER chaperone calreticulin, with consequent accumulation of mis ,ءء ϩ/ϩ percentage of cytotoxicity with respect to the killing induced by crt folded proteins that would otherwise be degraded. Alternatively, Ϯ CTLs. Values are the mean SD of five experiments, each performed in higher gene expression or increased protein half-life (or decreased p Ͻ 0.02. C, Levels of degranulation (measured by granzyme ,ء .triplicate protein turnover) could account for the increased perforin content B activity released in the medium) during the killing of P815 cells. CTLs Ϫ/Ϫ were preincubated with anti-CD3⑀ Ab (␣CD3 ϩ) or with regular medium in the crt CTLs. Whether all the perforin molecules produced Ϫ/Ϫ (␣CD3 Ϫ) before exposing them to target cells. Data are expressed as the by crt CTLs were correctly folded is not known. However, we percentage of granzyme B released over the amount released from crtϩ/ϩ believe that at least an amount of active perforin comparable with Ϫ Ϫ CTLs. Values are the mean Ϯ SD of four experiments, each performed in the wild-type content must have been present in the crt / CTLs, p ϭ 0.00006. otherwise we would not have been able to restore cytotoxicity by ,ء .triplicate treatment with aCD3 or Con A. The data on perforin are intriguing, and they suggest that the lack of calreticulin may generate a mul- calreticulin is not necessary to protect CTLs from lytic activity of tifaceted phenotype in CTLs. However, the higher levels of the stored perforin, a hypothesis that was proposed when the ER chap- pore-forming protein in the crtϪ/Ϫ CTLs did not seem to have an erone was first identified in the CTL granules in association with impact on CTL viability or their cytolytic function. perforin (15). It has been proposed that calreticulin may be involved in sorting The most striking phenotype of crtϪ/Ϫ CTLs was their markedly granule components, perforin in particular, to the lytic granules reduced cytotoxicity compared with wild-type cells. Levels of kill- (20). However, our data argue against such a hypothesis, because ing correlated with levels of granzyme B exocytosis during incu- intracellular localization of perforin and granzyme B in crtϪ/Ϫ bation with target cells, indicating that the nature of the problem CTLs appeared to be similar to that in wild-type cells. could reside in the amount of degranulation occurring in response Calreticulin has been shown to block the lysis of erythrocytes to the target cells, rather than ineffectiveness of the lytic material induced by perforin (21, 22). Consequently, it was proposed that per se. Indeed, we found that expression, activity, and intracellular calreticulin might bind to the CTL surface upon degranulation, localization of granzymes were comparable in wild-type and preventing polymerization of perforin within the CTL plasma crtϪ/Ϫ cells. Interestingly, perforin levels were substantially higher membrane and consequent lysis of the effector cells. If that hy- in the crtϪ/Ϫ CTLs. Several hypotheses could be formulated to pothesis were correct, one would have expected to observe crtϪ/Ϫ explain these data. Higher protein content may result, for example, CTL lysis during the killing of target cells and overall reduced 3218 IMPAIRED CYTOTOXIC ACTIVITY IN CALRETICULIN-DEFICIENT CTLs target killing. However, when we measured crtϪ/Ϫ CTL lysis dur- calreticulin deficiency may alter the integrins and/or throm- ing incubation with target cells, we did not find any specific in- bospondin-mediated adhesive properties of CTLs. crease in CTL death relative to wild-type cells or with respect to In conclusion, we have shown that despite functional lytic the levels of cell death spontaneously occurring in CTL popula- machinery, cytotoxicity is impaired in calreticulin-deficient tions in the absence of targets. Similar results were obtained when CTLs, probably due to inefficient effector-target conjugate for- CTL lysis was measured during immobilized anti-CD3 Ab-in- mation. Such a deficit could have important implications in the duced degranulation. These data argue against a protective role of regulation of the immune response in vivo. In light of our data, calreticulin on CTLs. Other molecules must be involved in CTL it is tempting to speculate that calreticulin deficiency could re- protection, such as the recently identified cathepsin B (42). sult in reduced or delayed clearance of infected and transformed Because crtϪ/Ϫ CTLs secreted less granzyme B than wild-type cells. At the same time, it could severely affect lymphocyte CTLs in response to stimulation by target cells, we investigated the homeostasis and result, as do several genetic defects impeding hypothesis that aberrant transduction of the signal downstream of degranulation of CTLs or inactivating perforin (60, 61), in he- the TCR-CD3 complex could result in impaired degranulation. mophagocytic-like syndromes or autoimmune diseases. Inter- When we triggered granule exocytosis by incubation with immo- estingly, autoantibodies against calreticulin have been found in bilized anti-CD3 Abs, thus bypassing the need for recognition and several autoimmune conditions (62–67). contact with target cells, crtϪ/Ϫ CTLs responded better than wild- type cells, showing a lower threshold for maximal induction of Acknowledgments degranulation. Granule exocytosis is triggered in CTLs by initial ϩ We thank Dr. Gillian Griffiths for the gift of the mouse monoclonal anti-

2 Downloaded from release of Ca from intracellular stores, followed by sustained perforin Ab. We also thank Drs. Hanna Ostergaard and Ing Swie Goping 2ϩ 2ϩ influx of extracellular Ca through a store-operated Ca channel for helpful discussion. (reviewed in Ref. 43). In our study calreticulin was dispensable for induction of degranulation when this was triggered by CD3 cross- Disclosures linking. In line with our observations are data showing that over- The authors have no financial conflict of interest. expression of calreticulin decreases store-operated Ca2ϩ influx 2ϩ through a mechanism that seems to be independent from its Ca - References http://www.jimmunol.org/ buffering activity (44). 1. Lowin, B., M. Hahne, C. Mattmann, and J. Tschopp. 1994. Cytolytic T-cell The remaining possible explanation for the reduced cytotoxic cytotoxicity is mediated through perforin and Fas lytic pathways. Nature activity of crtϪ/Ϫ CTLs is impaired recognition and/or adhesion to 370:650. Ϫ/Ϫ 2. 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