Proc. Natl. Acad. Sci. USA Vol. 91, pp. 11571-11575, November 1994 Immunology A null mutation in the perforin gene impairs cytolytic T lymphocyte- and natural killer cell-mediated cytotoxicity (gene targeg/ceillmedhad cotoi) BENTE LOWIN*, FRIEDRICH BEERMANN*t, ANDREA SCHMIDT*t, AND JORG TSCHOPP** *Institute of Biochemistry, University of Lausanne, and tSwiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland Communicated by Hans J. Maller-Eberhard, July 15, 1994 (receivedfor review April 12, 1994)

ABSTRACT Lymphocytmedted cytotoxicity has been Numerous experiments have been performed to assess the proposed to consist ofthe polariz secretion ofgranule-stored role of granules and in particular the role of perforin in performn leading to target-cell lysis. Nevertheless, perform- cytotoxicity. For example, perforin antisense oligonucleo- independent pathways were polated to explain the cytolytic tides partially suppressed CTL-mediated cytolysis (18), and activity of apparently perfoin-free lymphocytes and the DNA the expression ofperforin intogranules ofmast cells rendered degradation found in dying target cells. To evaluate the role of these cells hemolytic (19). Although these data have sug- perforin, we used gene targetg in embryonic stem cells to gested a crucial role ofperforin in cell killing, direct evidence produce mice lacking perform. Mce homozygous for the is still lacking. dsrupted gene have no perforin mRNA. The mice are healthy. Activation and granzyme A secretion of perforin-free cytolytic T cells are unaltered. The killing activity of cytolytic T cells as MATERIAL AND METHODS well as natural killer (NK) cells, however, is i ed but not Cloning ofthe Mouse Perforin Gene and C truction ofthe abolished. Approximately one-third of the killlng activity re- Targeting Vector. A 35-kb fragment containing the perforin mains when lysis of3T3 fibroblast targets and the apoptotic cell gene was isolated by screening a cosmid library derived from death of YAC-1 NK targets are analyzed. We conclude that DNA of NIH 3T3 mouse fibroblasts (Stratagene). A Pst perforin is a crucial effector molecule in T cell- and NK I-fragment containing exon 2 and part of exon 3 was sub- cell-mediated cytolysis. However, alternative perforin- cloned into pBluescript SK(+) (Stratagene). The targeting independent lytic mhanisms also exist. construct was made by insertion of a neomycin-resistance (neo) gene (pMClneopolyA; Stratagene) into a single Sph I The mechanisms of cytolytic T lymphocyte (CTL)- and site of the third exon, for positive selection. The herpes natural killer (NK) cell-mediated lysis have been studied simplex virus thymidine kinase gene was placed into pBlue- extensively in recent years. One proposed mechanism, script SK(+) at the 5' end ofthe perforin sequence, allowing known as the granule-exocytosis model, consists of the negative selection. polarized secretion of granule-stored molecules by the lym- Elecroporation and Selection ofEmbryonic Stem (ES) Cells. phocytes, after the formation of CTL-target cell conjugates. D3 ES cells (a gift of Frank Hilberg, Bender, Vienna) were Isolated granules are cytotoxic on their own (1, 2), indicating routinely grown on virradiated (4'50Srad) neor primary em- that they harbor effector proteins responsible for cell killing. bryonic fibroblasts isolated from the MTKneo3 transgenic Contained within the granules are perforin, a pore-forming, mouse line (received from C. Ovitt, European Molecular membranolytic protein (3, 4); granzymes, a family of serine Biology Laboratory, Heidelberg) in Dulbecco's modified esterases (5); calreticulin, a Ca2+-binding protein (6); TIA-1, Eagle's medium (DMEM; GIBCO) supplemented with 15% an RNA-binding protein (7); and proteoglycans (8). fetal bovine serum, nonessential amino acids (GIBCO), 0.1 Perforin is the only protein which is cytolytic in its isolated mM 2-mercaptoethanol (Sigma), and leukemia inhibitory form. After granule exocytosis, perforin monomers are re- factor (ESGRO, 103 units/ml; GIBCO). Electroporation with leased into the conjugate juncture and, in the presence of 20 gg of linearized (Kpn I-cut) targeting vector was per- Ca2+, bind to the target cell membrane and polymerize into formed with a single pulse at 500 pF/280 V by a Gene Pulser pore-like transmembrane channels, leading to lysis (9). Nev- (Bio-Rad) electroporator. After 24 hr, cells were transferred ertheless, perforin does not induce DNA degradation that is into selection medium containing G418 (300 pg/ml, dry observed during lymphocyte attack (10, 11). Recent data powder; GIBCO) and 2 pM ganciclovir and were incubated suggest that apoptosis may be induced by a combination of for 10-12 days. Doubly resistant colonies were picked into perforn and granzymes and/or TIA-1 by as yet unknown phosphate-buffered saline, trypsinized, and analyzed by mechanisms (7, 12, 13). PCR. The following oligonucleotide primers were used to Some investigators have questioned whetherperforin is the identify the rearranged perforin locus: primer 1 (5'-TGG- chiefeffector molecule ofCTL-induced killing, since most of GCA-GCA-GTC-CTG-GTT-GGT-GAC-CTT-3'), comple- the evidence in favor of the granule-exocytosis model was mentary to genomic DNA that lies downstream of the per- obtained with CTL lines cultured in vitro in the presence of forin sequence contained within the targeting vector, and high levels of interleukin 2 (for a debate, see ref. 14). Some primer 2 (5'-ATT-CGC-AGC-GCA-TCG-CCT-TCT-ATC- CTL clones analyzed killed target cells in the absence ofCa2+ GCC-3'), complementary to sequences at the 3' terminus of (15, 16), yet the lytic activity of perforin and successful the neo gene. exocytosis of granular proteins are strictly Ca2+-dependent. Southern Blot Analysis. Genomic DNA (15 jug) was di- Therefore, alternative, Ca2+-independent pathways were gested with EcoRV (Boehringer) for Southern blot analysis. proposed, such as those involving the Fas receptor (17). The 620-bp Sph I-Pst I (PCR I) fragment from the perforin

The publication costs ofthis article were defrayed in part by page charge Abbreviations: CTL, cytolytic T lymphocyte; NK, natural killer; ES payment. This article must therefore be hereby marked "advertisement" cell, embryonic stem cell. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 11571 Downloaded by guest on September 29, 2021 11572 Immunology: Lowin et al. Proc. Nati. Acad. Sci. USA 91 (1994) gene, residing within the targeting vector, and the 512-bp Pst code for the protein. To disrupt the perforin gene by homol- I-EcoRV (SB I) fragment, downstream of the targeting ogous recombination, a targeting construct was made by construct, were used as probes. subcloning a 3.4-kb Pst I fragment containing part of exon 2 PCR Analysis. RNA was isolated from a 5-day mixed and the complete exon 3 (Fig. 1A). Exon 3 was interrupted by lymphocyte culture (107 cells) (21). cDNA was prepared with insertion of a neo cassette, thereby interrupting the putative the cDNA Cycle kit (Invitrogen), and perforin-specific mes- transmembrane domain of perforin that is thought to consist sage was amplified by PCR (940C for 1 min, 1 min at 600C, and of two amphipathic a-helices spanning amino acid residues 3 min at 720C for 30 cycles) using primers for exon 2 167-221 (5'-GTG-GCC-GCA-GCC-AAG-GTC-CAC-TC-3') and exon (29). Mutations in the analogous region of comple- 3 (5'-TGG-GCA-GCA-GTC-CTG-GTT-GGT-GAC-CTT-3'); ment component C9 were shown to lead to a nonfunctional the expected amplification product is 900 bp. Control TIA-1 protein (30). A herpes simplex virus thymidine kinase gene message was amplified in the same reaction mixture with was placed 3' of the perforin sequence in order to allow the primer 1 (5'-CTC-CAG-CTC-CAA-AGA-GTA-C-3') and use of a positive/negative selection strategy (31). primer 2 (5'-CGG-GAT-CCT-CAC-TGG-GTT-TCA-TAC- Detection of Targeted Clones and Generation of Perform 3'); the size of the amplification product is 500 bp. -/- Mice. Successfully targeted ES cell clones were first Northern Blot Analysis. This was performed on Genescreen identified by PCR and then confirmed by Southern blotting membranes (NEN) according to the manufacturer's protocol, using EcoRI- and EcoRV-digested ES cell DNA and an using as probe the random primer-labeled BstXI fragment internal (PCR I) and external (SB I) probe (data not shown). containing the entire perforin open reading frame. Of349 doubly resistant clones screened, two were positive in Generation of Chhmeric Mice. Approximately 10-15 ES the PCR screen and showed an additional band in the cells were injected into C57BL/6J blastocysts. Injected blas- Southern blot analysis, indicative of targeted disruption of tocysts (10-20) were transferred to the uteri of pseudopreg- the perforin gene. None of these clones had additional nant NMRI females at 2.5 days postcoitum. Germline chi- random integrations of the targeting vector. meras were identified by the presence of agouti coat color in The two ES cell clones with the disrupted perforin gene the F1 progeny. were injected into C57BL/6J blastocysts and the embryos Cytotoxicity Assay of CTL. Alloreactive CTLs were gener- were reimplanted into NMRI foster mothers. One clone gave ated in a 5-day mixed lymphocyte culture (22). Responder rise to chimeric animals. One male chimera transmitted the spleen cells (2.5 x 106 per ml) were from adult (5- to 6-week- disrupted allele to all offspring. Males and females hetero- old) mice with the normal perforin gene (+/+), and mice zygous for the perforin mutation were then crossed to pro- heterozygous (+/-) and homozygous (-/-) for the disrupted perforin gene. The mice were ofH-2b haplotype (129/C57BL/ A 6J). Stimulators were irradiated spleen cells (2.5 x 106 per ml, EXON 1 EXON 2 EXON 3 3000 rads; 1 rad = 0.01 Gy) from DBA/2 (H-2d) mice. Target Pst.ISoh Eco RV cells were haplotype-matched P815 mastocytoma cells (H-2d) and 3T3.A31 fibroblasts (H-2d). EL-4 cells (H-2b) were used to c...... 'RVm measure major EcoRV fragment - 3.4 kb---- histocompatibility complex (MHC)-unre- PCR stricted cytotoxicity. Cellular cytotoxicity and DNA degrada- SBI tion were assayed as described (23): P815 cells and EL-4 cells (106 cells in DMEM with 5% fetal bovine serum) were first 13 5 7 kb labeled for 1 hr with 4 uCi (148 kBq) of mI-Urd (Amersham) in a final volume of 200 jd. The cells were then incubated in the presence of100 AGCiofsodium [51Cr~chromate (Amersham) P~~~~~~~~~~~~~t~~~~ for an additional hour. 3T3.A31 fibroblasts were labeled with 51Cr (2 uCi per well) in 96-well plates for 12-16 hr (24). =3~~~~~~EPc f-- Isolation of NK Cells and Cytotoxicity Test. To activate NK cells in vivo, mice were inoculated intraperitoneally with poly(IC) (100 pug in phosphate-buffered saline; Sigma) 16 hr prior to the experiment (20). For the cytotoxicity assay, EcoRV fragment- -45b - spleens were excised and homogenized, and cells were purified over a Ficoll (Pharmacia) gradient in order to elim- inate red blood cells. The cell suspension was then washed B three times in DMEM with 5% fetal bovine serum and tested, as described for CTLs, in a standard 4-hr 51Cr/125I-release assay using YAC-1 tumor cells (American Type Culture Collection) as specific targets. 4.5 kb T-Cell Activation. The activation of T cells during the - 3.4 kb mixed lymphocyte reaction was followed by measuring the concentration of interferon y in the supernatant of 2.5 x 107 responder cells, after 5 days of culture (25). FIG. 1. Gene targeting ofthe perforin locus. (A) Structure ofthe Degranulation of CTIs. Ninety-six-well plates (Dynatech) murine perforn locus, perforn targeting vector, and homologous- were coated with rat anti-mouse CD3 monoclonal antibody recombination allele. Translated exon sequences are depicted as 17A2 (26). Subsequently, activated lymphocytes from a stippled boxes, and untranslated sequences as open boxes. Introns mixed lymphocyte reaction were added (10W cells per well) in are represented as bold lines. Probes used for Southern blots are PCR serum-free medium and incubated for 4 hr at 37rC. The I, the internal probe, and SB I, the external probe. Genomic amount of secreted granzyme A was measured by the BLT- restriction fiagments detected by Southern blotting are indicated by esterase assay (27). a double-arrowed line. Primer sites for PCR are represented as arrowheads. Hatched boxes represent the neomycin-resistance gene and herpes simplex virus thymidine kinase gene. (B) Representative RESULTS Southern blot analysis of progeny of +/- x +/- mating. Tail- biopsy genomic DNA was digested with EcoRV and probed with the Targeting Strategy. The perforin gene contains three exons SB I figment. The wild-type allele is 3.4 kb, and the homologous- spanning a region of -1.6 kb (28). Only the last two exons recombination allele shows the predicted 4.5-kb fragment. Downloaded by guest on September 29, 2021 Immunology: Lowin et al. Proc. Nati. Acad. Sci. USA 91 (1994) 11573 duce homozygotes. A representative Southern blot of tail 4000 - genomic DNA from a heterozygote x heterozygote mating is -T shown in Fig. 1B. Heterozygous mutant mice as well as homozygotes appeared to be healthy. 3000 - Lack of Perftrm Trans ts In Homozygous Mutants. To :-z El +1+ evaluate perforin expression, spleen cells of homozygotes I- 2000 - (perforin -/-) and control mice (perforin +/+ and +1-) 2z EL +1- were isolated and stimulated with v-irradiated DBA/2- derived splenocytes and the perforin RNA was analyzed by 1000 - PCRafter 5 days. A perforin-specific amplification product of -900 bp was detected in perforin +/+ and +/- mice, whereas no transcript was discernible in spleen cells of -/- 0- animals (Fig. 2A). The TIA-1 RNA was amplified at similar levels in all samples, indicating that the lack of perforin FIG. 3. Unaltered interferon y production in perforin-free lym- message in -/- cells was not due to defective cDNA phocytes in mixed lymphocyte culture. Interferon v (IFN-y) con- synthesis. centration [units (U)/ml] was determined in supernatants of spleen When the splenic RNA was subjected to Northern blot cells (H-2b) derived from perforin +/+, +/-, and -/- mice 5 days after activation by irradiated spleen cells ofDBA/2 (H-2d) mice. The analysis, no 2.9-kb perforin message was detectable in cells interferon y production in the absence of a stimulatory signal is from -/- animals (Fig. 2B). Moreover, a reduced amount of shown with the open bar. perforin message was observed in cells from heterozygous animals. wild-type mice, a slightly lower activity was consistently Cytotoxicity of T Lympbocytes. To investigate the role of noticed with the +/- lymphocytes (forexample, 63% versus perforin in CTL-mediated cytotoxicity, spleen cells derived 43% lysis at a 10:1 killer-to-target ratio). In contrast, cells from -/-, +/-, and +/+ mice (H-2b) were activated in a derived from homozygous -/- mice did not develop a bulk mixed lymphocyte culture with irradiated allogeneic significant CTL response against these target cells. Only at a DBA/2 (H-2d) spleen cells for 5 days. The allo response and large excess ofcytolytic cells overtarget cells did some ofthe activation of perforin-lacking T cells were normal, since the cells lose the 51Cr label. This killing activity was above the production of interferon y was identical in all cultures (Fig. spontaneous lysis observed with the irrelevant EL-4 target 3) and the T-cell subset distribution was not altered (data not (H-2b). shown). Moreover, no difference in the distribution of lym- During target cell lysis by lymphocytes, plasma membrane phocyte populations upon allogeneic stimulation was ob- impairment is preceded by the characteristic signs of apop- served (data not shown). tosis-i.e., DNA condensation and fragmentation. These The cytotoxic activity of the responder cells 5 days post- latter events are undetectable in a 4-br 51Cr release assay but stimulation was assessed in a standard 51Cr release assay require the determination ofradioactively labeled DNA firag- against H-2d alloantigen-carrying P815 mastocytoma cells ments released into the cytoplasm of the target cell. After 4 (Fig. 4A). Although the cytolytic activity ofT cells from +/- hr, complete DNA fragmentation was detected in targets mice was comparable to the effector activity of cells from attackedbyT cells derived from +/+ and +/- animals, even A at low killer-to-target ratios (Fig. 4A). Again, +/- cells were +1+ +1- i- weaker effector cells than the +/+ lymphocytes. In spite of the low 51Cr release observed with perforin -/- cells, fragmentation ofthe genomic DNA was clearly detected and reached levels up to 50% at a 100:1 effector-to-target cellratio 900 bp- - Perforin (10%1 in the 51Cr assay). 500 bp- - TIA-1 Fibroblasts are known to be more resistant to CTL- mediated cytolysis than the highly susceptible P815 cells (32). Moreover, DNA ladder formation, one of the hallmarks of apoptosis, is not detectable in these target cells. When B lymphocytes of +/+ and +/- mice were added, 60% of3T3 cells succumbed to this attack after a 7-hr incubation at a I 100:1 excess of killer cells (Fig. 4B). Perforin-free lympho- I I cytes were only 3 times less efficient in lysing the fibroblasts.

- Perforin Cytotoxit of NK Cels. NK cells, which constitutively produce perforin, are believed to utilize a killing mechanism similar to CTLs. We therefore analyzed the effect of the defect of perforin on NK cell cytotoxic capacity. The 51Cr release from YAC-1 cells, the typical NK target, was mea- sured by using poly(I{-stimulated NK effector cells. NK cells isolated from normal (+/+) mice exhibited 2-fold higher lytic activity than NK cells derived from heterozygous +/- mice (Fig. 4C). In NK cells lacking perforin, the effector FIG. 2. Absence of perforin message in lymphocytes derived activity was reduced almost to background levels. As in from paforin -/- mice. (A) PCR analysis of spleen cells isolated CTLs, the apoptosis-inducing activity of NK cells was di- from +/+, +/-, and -/- mice and allogeneically activated for 5 minished only 3-fold in perforin -/- cells as reflected by the days. The cDNA was amplified with perforin-specific primers, considerable amount of DNA fagments recovered in the resulting in a 900-bp product. The 500-bp band corresponds to the YAC-1 cytoplasm (Fig. 4C). amplification product from the T-cell specific TIA-1 message, which was included for control purposes. (B) Northern blot analysis of the Exocytoss of Cytotoxic Granules. Perforin is stored in activated spleen cells. Ten micrograms of total RNA was loaded in cytoplasmic granules together with other proteins-in par- each case. The band migrating at -1.4 kb is nonspecific but dem- ticular, granzyme proteases. We could not exclude that the onstrates equal loading of RNA. diminished cytotoxicity was due merely to an inefficient Downloaded by guest on September 29, 2021 11574 Immunology: Lowin et al. Proc. Nadl. Acad. Sci. USA 91 (1994) A B C 1001 60 YAC-1 +/+ k75 - . +1- r. 40- -/- +/. ._. -*- -I- U.= 40 50- /y ~~~-4-* /. Q 9 30- '.) EL4 +/+ $- 20- A 25- mn 10- 0- 0 L al9 * I - 3 10 30 100 3 10 30 100 18.75 37.5 75 150 E : T ratio E : T ratio E : T ratio 125- 60- YAC-1

100- P815 Q -* +1+ -- +/1 40- -.- -75- +1- 4. // U-r. -0-1 30- W- U'WI // 20- C-q 25- 10-

0 3 10 30 100 18.75 37.5 75 150 E : T ratio E : T ratio

FIG. 4. Lack ofCTl and NK cytotoxic activity. (A) The CTL activity ofthe responder cells after 5 days of stimulati in a mixed lymphocyte culture was assessed in a standard 4-hr 51Cr-release assay against P815 cells (Upper) and in a 4-hr 125I-Urd DNA fgmentation/release assay (Lower). (B) As in A, but 3T3 fibroblasts (H-2d) were used as target cells. 51Cr release was assessed after 7 hr. (C) Cytotoxicity of NK cells. Splenic NK cells were added to YAC-1 cells, and 51Cr and '"I-Urd release was determined after 4 hr. E, effector; T, target. granule fusion and/or exocytosis event. Perforin comprises of alternative, perforin-independent, mechanisms has been high-affinity lipid binding sites and may act as a fusogen of suggested. We decided to use the gene knockout technology membranes, as has been demonstrated for the structural in ES cells to generate perforin-deficient mice to unambigu- homologue complement component C9 (33). Upon incuba- ously evaluate the contribution of perform. A replacement tion with immobilized anti-CD3 antibody, cytotoxic granules vector was devised which disrupts the putative transmem- fuse with the plasma membrane and release their contents brane domain ofperforin, making it unlikely that a truncated into the supernatant, where the secreted proteins can be protein will display lytic activity. Indeed, no perforin 2.9-kb detected by immunological or enzymatic means (34). Fig. 5 message and 900-bp PCR amplification product were de- shows that comparable amounts of granzyme A are released tected in activated cells of perforin -/- mice. from +/+, +/-, and -/- T cells. Our results now provide direct evidence for a crucial role of perforin in lymphocyte-mediated cytotoxicity. For P815 target cells, at least a 30-fold higher ratio of killer to target DISCUSSION cells was required to achieve the same degree oflysis as with Although perforin has been shown to participate, at least in T cells derived from +/- or +/+ mice. The difference was part, in CTL- and NK cell-mediated cell killing, the existence less pronounced when fibroblasts were used as targets, where only a 3-fold reduction of the cytotoxic activity was ob- 40 - served. T cells from heterozygous +/- mice were consistently less

C active than the wild-type T cells, indicating that in primay ., 30- cultures oflymphocytes perforin represents a limiting factor. *5 This is in agreement with an antisense oligonucleotide study '._ l +/+ (18), where a 2-fold reduction of perforin directly correlated , 20- EJ +1- with a decrease in cytolytic activity. Almost identical were cn I/. results obtained with NK cells, thus U) providing evidence that the two effector cells use similar if X 10- not identical killing mechanisms. Again, the 51Cr-releasing activity was almost frag- m entirely abolished, whereas DNA mentation in target cells was only partly impaired. 0- The involvement of perforin in the cytotoxic process may be explained in several ways. The simplest interpretation is FiG. 5. Exocytosis of cytotoxic granules. Granzyme A estero- lytic activity in the supernatant of spleen cells allogeneically acti- that the absence of perforin results in the lack of transmem- brane pores vated for 5 days was determined after a 4-hr incubation with in the target cell membrane. Incorporation of immobilized anti-CD3 antibodies. Granzyme A activity (percent of isolated perforin into membranes induces massive ion con- total granzyme A activity) was detected using the lysine S-benzyl ductance (35). Since, in perforin-lacking CTLs, no toxic ester(BLT) substrate. The total amount ofgranzyme A activity ofthe lethal massive Ca2+ influx will ensue, target cells resist the various mixed lymphocyte cultures was similar (±15%). attack ofthe killer cells. However, other explanations cannot Downloaded by guest on September 29, 2021 Immunology: Lowin et al. Proc. Natd. Acad. Sci. USA 91 (1994) 11575 be excluded a priori. Not only may the absence alter the 6. Dupuis, M., Schaerer, E., Krause, K. H. & Tschopp, J. (1993) biogenesis of the secretory granules so that they become J. Exp. Med. 177, 1-7. 7. Tian, Q., Streuli, M., Saito, H., Schlossman, S. F. & Ander- incompetent for the storage of other proteins, but perforin son, P. (1991) Cell 67, 629-639. might play the role of a fusogen akin to viral proteins, thus 8. MacDermott, R. P., Schmidt, R. E., Caulfield, J. P., Hein, A., allowing the fusion ofgranule and cell membrane. Moreover, Bartley, G. T., Ritz, J., Schlossman, S. F., Austen, K. F. & the absence of perforin may lead to a defective activation of Stevens, R. L. (1985) J. Exp. Med. 162, 1771-1787. T cells, including altered cytokine release. The absence ofall 9. Tschopp, J. & Nabholz, M. (1990) Annu. Rev. Immunol. 8, these cellular activities would result in an impaired release of 279-302. 10. Gromkowski, S. H., Brown, T. C., Masson, D. & Tschopp, J. granule harbored proteins or potentially lytic cytokines and (1988) J. Immunol. 141, 774-778. thus to apparent nonactive killer cells. This supposition 11. Duke, R. C., Persechini, P. M., Chang, S., Liu, C. C., Cohen, seems unlikely, however. Our results show (i) a normal J. J. & Young, J. D. (1989) J. Exp. Med. 170, 1451-1456. activation of T lymphocytes as reflected by unchanged in- 12. Shi, L., Kam, C. M., Powers, J. C., Aebersold, R. & Green- terferon y secretion and (ii) an unaffected T-cell receptor- berg, A. H. (1992) J. Exp. Med. 176, 1521-1529. induced exocytosis of granzyme A. 13. Shiver, J. W., Su, L. & Henkart, P. A. (1992) Cell 71, 315-322. 14. Krahenbuhl, 0. & Tschopp, J. (1991) Immunol. Today 12, CTLs are known to efficiently induce apoptosis in target 399-402. cells. DNA degradation, an indicator of ongoing apoptosis, 15. Ostergaard, H. L., Kane, K. P., Mescher, M. F. & Clark, was impaired as well in target cells attacked by perforin-free W. R. (1987) Nature (London) 330, 71-72. cells, although not to the same extent as the plasma mem- 16. Trenn, G., Takayama, H. & Sitkovsky, M. V. (1987) Nature brane lysis. In fact, with P815 cells, only a 3-fold reduction (London) 330, 72-74. in DNA degradation was seen with perforin-negative T cells, 17. Rouvier, E., Luciani, M. F. & Golstein, P. (1993) J. Exp. Med. 177, 195-200. in spite of the 30-fold higher resistance in plasma membrane 18. Acha-Orbea, H., Scarpellino, L., Hertig, S., Dupuis, M. & lysis. Almost identical results were obtained with NK effec- Tschopp, J. (1990) EMBO J. 9, 3815-3819. tor cells. This indicates that other, perforin-independent 19. Shiver, J. W. & Henkart, P. A. (1991) Cell 64, 1175-1181. mechanisms are operational which cause the slow apoptotic 20. Morelli, L., Lusignan, Y. & Lemieux, S. (1992) Cell. Immunol. death. Since granzymes are not cytotoxic in the absence of 141, 148-160. the Fas-based which is known to 21. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, perforin, cytotoxic pathway 156-159. lead to apoptotic cell death (36, 37) is the most likely 22. Maryanski, J. L., MacDonald, H. R. & Cerottini, J. C. (1980) candidate. J. Immunol. 124, 42-48. Perforin message has been shown to be highly increased in 23. Kruisbek, A. M. (1992) in Current Topics in Immunology, ed. T cells at the site of lesions in various disease models, Coico, R. (Wiley Interscience, New York), Vol. 1, pp. 3121- including murine choriomenengitis, herpesvirus infection, 3126. nonobese diabetic mice, myocarditis, rejection, and 24. Ucker, D. S., Obermiller, P. S., Eckhart, W., Apgar, J. R., graft Berger, N. A. & Meyers, J. (1992) Mol. Cell. Biol. 12, 3060- rheumatoid arthritis. It will therefore be interesting to ana- 3069. lyze these in vivo models in perforin knockout mice to 25. Muller, I., Kropf, P., Etges, R. J. & Louis, J. A. (1993) Infect. evaluate the contribution of the pore-forming protein. Immun. 61, 3730-3738. Note. The analysis ofanother perforn-deficient mouse line 26. Miescher, G. C., Schreyer, M. & MacDonald, H. R. (1989) has been reported by Kagi et al. (38), providing convincing Immunol. Lett. 23, 113-118. evidence that perforn -/- mice failed to clear lymphocytic 27. Takayama, H., Trenn, G. & Sitkovsky, M. V. (1987) J. Immu- choriomenengitis virus. However, KWgi et al. did not observe nol. Methods 104, 183-190. residual NK cells and 28. Trapani, J. A., Kwon, B. S., Kozak, C. A., Chintamaneni, C., lysis of target fibroblasts by perforin- Young, J. D. & Dupont, B. (1990) J. Exp. Med. 171, 545-557. free CTLs and concluded that perforin accounted for 100 of 29. Peitsch, M. C., Amiguet, P., Guy, R., Brunner, J., Maizel, J. J. the lytic activity against most target cells. The reasons forthis & Tschopp, J. (1990) Mol. Immunol. 27, 589-602. discrepancy are unclear. 30. Dupuis, M., Peitsch, M. C., Hamnann, U., Stanley, K. K. & Tschopp, J. (1993) Mol. Immunol. 30, 95-100. We gratefully acknowledge the technical assistance of C. Matt- 31. Thomas, K. R. & Capecchi, M. R. (1987) Cell 51, 503-512. man, P. Allegrini, and E. Burnier. The advice and help ofDrs. Michel 32. Sellins, K. S. & Cohen, J. J. (1991) J. Immunol. 147, 795-803. Aguet (University of Zdrich), Frank Hilberg (Bender, Vienna), and 33. Tschopp, J., Podack, E. R. & MOller-Eberhard, H. (1982) Edith Hummler (University of Lausanne) were essential for the Nature (London) 298, 534-538. establishment ofthe knockout technique. The helpful comments and 34. Takayama, H., Trenn, G., Humphrey, J. W., Bluestone, J. A., suggestions of Drs. J. Mariansky, R. MacDonald, and H. Acha- Henkart, P. A. & Sitkovsky, M. V. (1987) J. Immunol. 136, Orbea at our research center are gratefully acknowledged. This work 566-569. was supported by grants from the Swiss National Science Founda- 35. Young, J. E., Nathan, C. F., Podack, E. R., Palladino, M. A. tion. & Cohn, Z. A. (1986) Proc. Natd. Acad. Sci. USA 83,150-154. 36. Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Moller, P., 1. Millard, P. J., Henkart, M. P. & Reynolds, C. W. (1984) J. Falk, W., Debatin, K. M. & Krammer, P. H. (1989) Science Immunol. 132, 3197-3204. 245, 301-305. 2. Podack, E. R. (1985) Immunol. Today 6, 21-27. 37. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, 3. Masson, D. & Tschopp, J. (1985) J. Biol. Chem. 260, 9069- S., Sameshima, M., Hase, A., Seto, Y. & Nagata, S. (1991) Cell 9072. 66, 233-243. 4. Podack, E. R., Young, J. E. & Cohn, Z. A. (1985) Proc. Natd. 38. Kigi, D., Lederman, B., Bfirki, K., Seiler, P., Odermatt, B., Acad. Sci. USA 82, 8629-8633. Olsen, K. J., Podack, E. R., Zinkernagel, R. M. & Hengartner, 5. Jenne, D. E. & Tschopp, J. (1988) Immunol. Rev. 103, 53-71. H. (1994) Nature (London) 369, 31-37. Downloaded by guest on September 29, 2021