Complement Component C9 (Cytotoxic Lymphocyte/Pore-Forming Proteins/Cytotoxic Mechanisms/Primary Structure) DAVID M

Home , Perforin

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 247-251, January 1989 Immunology Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9 (cytotoxic lymphocyte/pore-forming proteins/cytotoxic mechanisms/primary structure) DAVID M. LOWREY*, TONY AEBISCHERt, KRISTIN OLSEN*, MATHIAS LICHTENHELD*, FABIO Ruppt, HANS HENGARTNERt, AND ECKHARD R. PODACK** *Department of Microbiology and Immunology (R-138), University of Miami, School of Medicine, P.O. Box 016960, Miami, FL 33101; and tDepartment for Experimental Pathology, University of Zurich, Zurich, Switzerland Communicated by Herman N. Eisen, September 14, 1988

ABSTRACT The nucleotide sequence coding for the cyto- share primary sequence homology (sequence for C6 is not toxic T-lymphocyte (CTL) protein perforin 1 (P1) has been available) (22, 23). determined and the corresponding protein sequence has been Here we report definitive information on the sequence derived. Murine CTL cDNA libraries contained in the vector homology between C9 and P1§ and provide evidence for a Agtll were screened by using a monospecific antiserum to role of P1 in the cytolytic process of lymphocytes. purified P1. Three recombinant phages were isolated and their cDNA inserts were sequenced. The derived protein sequence MATERIALS AND METHODS contains 554 amino acids and displays, as expected, consider- Materials. Restriction enzymes, the large (Klenow) fragment the comple- of DNA polymerase, and T4 DNA polymerase were obtained able homology with certain functional domains in from Boehringer Mannheim. Deoxyribonucleotides and ment components C9, C8a, C8fi, and C7. The identity of P1 dideoxyribonucleotides were purchased from Pharmacia. The cDNA clones was verified by prokaryotic expression and the 17-nucleotide universal primer for M13 sequencing was ob- reactivities of antisera produced to the expressed proteins. In tained from Collaborative Research and Bio-Rad. 5-[a- addition, antisera were produced to two synthetic peptides [35S]thio]dATP (400 Ci/mmol, 1 Ci = 37 GBq) as well as located in the center and C-terminal portions ofP1. AU antisera [a-32P]dCTP (3000 Ci/mmol) were obtained from Amersham. reacted with purified P1. In Northern blot analyses, P1 cDNA Oligonucleotides used as primers for DNA sequencing were probes recognized a 2.9-kilobase mRNA only in CTL. Perforin synthesized on a DuPont Coder 300 DNA synthesizer using the mRNA was found in all cloned CTL and in all mixed lympho- phosphoramidite methodology. pEX plasmids 1-3 and host cyte reactions that gave rise to cytotoxic cells. Perforin mRNA Escherichia coli strain pop 2136 were kindly provided by K. K. was also detected in virus-specific CTL that had been generated Stanley (EMBL, Heidelberg). The Agt1l cDNA libraries 3F9 in vivo and isolated from liver tissue of mice infected with and AD9 were made from CTL clones as described (24). A CTL lymphocytic choriomeningitis virus. The cell-specific expres- cDNA library from a hapten-specific clone (25) was kindly sion ofperforin is consistent with its postulated role in cytolysis. provided by Pamela Ohashi (University of Toronto). Antibody Screening. Monospecific rabbit antiserum to murine Cytotoxic T lymphocytes (CTL) are of primary importance P1 (12) was absorbed with detergent lysates of E. coli Y1090 control virus infection (1, 2). before being used for screening (26). From 100,000 recombinant for the immunological of plaques, a single positive phage was plaque purified in four Together with natural killer (NK) cells, CTL may play a role subsequent platings. Longer cDNA clones of 1.8 and 2.9 in tumor surveillance (3), in transplantation rejection (4), and kilobases (kb) were obtained by using the 600-base-pair (bp) in graft-versus-host reactions (5). insert of the original clone as a hybridization probe. The mechanism oflymphocyte-mediated cytolysis appears DNA Sequencing. Phage cDNA inserts were excised with to involve more than one pathway ultimately leading to the EcoRI and subcloned in the pTZ18U plasmid (Bio-Rad). destruction ofthe target cell membrane and nucleus (6-8). An Sequencing was performed by using the "shotgun" protocol early event in target cell lysis by CTL and NK cells is the and dideoxynucleotide chain termination method exactly as vectorial secretion onto target cell membranes of cytolytic described (27). The 500 bp at the 5' end and 1100 bp at the 3' granules contained in killer cells (9-12). Cytolytic granules end were sequenced by using synthetic 17-mer primers contain several related serine proteases (13-17), proteogly- spaced every 200 bp on either strand to prime synthesis cans (18), and cytotoxic proteins (19-21). One of the major instead of the universal 17-mer M13 primer. DNA sequences cytotoxic proteins found in granules is perforin 1 (P1). were analyzed with the Beckman Microgenie computer Incubation ofgranules or ofpurified P1(12) with erythrocytes software (28). or nucleated cells in the presence of calcium results in lysis Peptide Synthesis. On the basis of the deduced amino acid and the formation of membrane lesions of 160-A diameter. sequence of P1, peptides 15 and 18 amino acids long were These transmembrane lesions are indistinguishable from synthesized by using the fluoren-9-ylmethoxycarbonyl membrane lesions formed on target cells by intact NK and (Fmoc) methodology on a DuPont Coder 250. The peptides CTL (19, 20). By morphological criteria, P1 membrane were coupled with 0.025% glutaraldehyde to ovalbumin at a lesions resemble those formed by complement components 1:1 ratio (wt/wt) and used as immunogens to prepare specific C5b-9 during hemolysis. This observation, and immunolog- rabbit antisera. Hyperimmune sera were tested against pu- ical crossreactivity of P1 with C6, C7, C8, and C9, has led to the suggestion that P1 and terminal complement components Abbreviations: P1, perforin 1; NK, natural killer cells; CTL, cytotoxic T lymphocytes. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper is being deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. EMBL/GenBank data base (accession no. J04148).

247 Downloaded by guest on September 30, 2021 248 Immunology: Lowrey et al. Proc. Natl. Acad. Sci. USA 86 (1989)

A CLONED Pi cDNA ps 2413 described (29) and used to transform E. coli pop 2136. pEX1, Ps 1.Sc -2, and -3 are frame-shifted by one nucleotide each and thus ps 2.9 allow expression ofinserted DNA in all three reading frames. Three clones reactive with anti-P1 antisera were further studied by DNA sequence analysis and immunogenicity. E. coli inclusion bodies were prepared by sonication followed by B GEL 5,- - - =, READINGS centrifugation at 14,000 x g for 10 min. The pellet was -5' washed once with 0.15 M NaCl/10 mM Tris HC1, pH 7.5, and then used to immunize rabbits. Inclusion bodies analyzed by sodium dodecyl sulfate/PAGE were composed of approxi- 2000 mately 20-50% fusion protein. 1000 Northern Blot Analysis. Total RNA prepared by CsCl step BASE PAIR NUMBER gradients (30) or by the protocol of Chomczynski and Sacci (31) was analyzed by using standard Northern blot proce- FIG. 1. Strategy used for sequencing P1 cDNA. (A) Relative dures for P1 mRNA. positions of the three isolated and sequenced cDNA clones. (B) Individual sequences used to assemble complete P1 cDNA sequence. RESULTS rifled P1 by Western blot analysis. In the case of the Isolation of P1 cDNA Clones by Prokaryotic Expression. 15-residue peptide, the corresponding hyperimmune serum Approximately 100,000 recombinant clones from a CTL was also tested against ghosts of Pi-lysed erythrocytes. library (3F9) contained in the Agtll expression system were Pi-lysed membrane ghosts were prepared by lysing sheep screened with the P1 antiserum described previously (23). erythrocytes with P1 in 5-fold excess of the amount required One clone (ps2413) was isolated that directed the synthesis of for 100% lysis. The membranes were then pelleted in an a perforin-galactosidase fusion protein reactive with anti-P1 Eppendorf table-top centrifuge and washed twice in 1.0 mM in Western blots. In addition, the 600-bp insert cDNA of this EDTA. The final pellet was then analyzed by Western clone showed limited homology to C9 and was therefore used blotting subsequent to sodium dodecyl sulfate/PAGE. to obtain a 1.8-kb clone (psl.8c8) and full-length clones of P1 pEX Subcloning and Prokaryotic Expression of P1 cDNA. cDNA (ps2.9) by screening of additional CTL libraries. The Alu I fragments of P1 cDNA were subcloned in pEX1-3 as longest DNA insert obtained was 2.9 kb, which agreed with

aTTCCCCAGTCGTGAGAGGTCAGCATCCTTCATCCCTGTTCCCACAGCTTTCCAGAGTTTATGACTACTGTGCCTGCAGCATC 148

A8G ACG CTG C CCG T CTG CTG CTG CGA CCT GTC CpT CCC TAC ACT GCC AT CGG TCA G TC G CAG AAG CAC Cp GGGT GOC T8C TFC C GGC C TIC Cp AAGCCGfT TEC TGAGIG NA 271

AG GGGG GG ATG G6T GG AC C C CpA CAG C GJT ATO~ AtT CkC TEC TC T;C GTG MC AfCA AGG T;C TG AGG CpCT CGC AfC TC TGT MC TCC CTA ATG GAC 394 GATGC YC GG' GQRIG%GCACAICCe TL AM N S IOG DEXPI/9 ACA CAG COC CTA CCT GTG OCGA ATC ACC CAC T5G CCT CAC AGC TCA CAC TGC CAG COT MT TG bICC GCA 0CC AMG GTC C"AC T5CCPAfCAGGOT 0CC COG GCA JCT GCT AAT 517 GJC CfG p S R N A A A K A R A L0 QC Ve H iod' A N

AC MT AMC GAC T5G CjT GTG G8G CTG GAT GTG MC CCT AGG CCA GAG GCA MAC ATG GCC TCC GTG GCT G8C TCC CAC TCCMG TVTTA 0CC MT OCT SAG TAT CAG SAC 640 N DV N PR P N N CRGC A S V OCA IC I0110 E 1 S S K A N A E 14Ib Y 0D

AG A T a F C CTG K CCTPPCCA CTC DKTTC AAA K ACC CTCL R A CTCL CCC CCCR N TTTF N S 763 TN~ TITA5Aj DtC TJA VAAv eG TOT ~ pF 0C s lTd LC VGGTA CAA AMA 1711. SAC AAMG AGA 0CC 1Id AMC CA

CAT GCT TAC CAC AGG CTC TC TCC C TAT 8 A CHAC TT ATC ACG GCT GTG GAC CTC GGT GGC CGC ATC TCG GTC CTT ACA GCC CTG CT CC TT CAG CTG ACC CTG MT 886 Ti TENH190 Y NH RL s 20 l G 218 R I S V 01L TIN oEXP1/8 GOG CLTC AJCA GJT GAT CeAC GTA GGA GAC TOC CLTG A"AC VTG G8AG GCC CaAG GTC A5CC AIC GOT GCC CaAA GCC A$CC GVTC T5CC A5CT CeAA TYAC AtAA GCfT TGCT GeAG G8AG AA AKAG AKAA %GA CHAC AKAA ATG 1009 A~ H LGAE V C H L LTOTA P E 0 CCCTCcT TIC CAC CAG TOC CGT G#G CGT CAC GTC GAA GTA CTT GOT GGC CCT CTG GAC TC G CAT T F G 30 E T AMC GCT

H C Q E a C¶Ci 6;Q EXP1KHA/57 S S3 9 AJ Tr8 LT PC SGC NA PF 8G 7T vT 6A 320° SG L GEG CpCC CTG CHAC ATCA TLTA CTG Grk GEM CQAG A"AC CpCG XG CGG GEAG G^CT CLTG ARGA CAG GCT ATC AGC CAT TAT ATA ATG AGC 1255 340 Peptide I AGAGJC CRG T&G CAG AyAC T8T AGC: AGG CpCC TgCC.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ARCG T5A GOC CaAG CHAT AKAG A5CT A5CC C"AT GDAT T5CA TGC: CaAG TeCT GAG TFC CaAG rf T5CA AAG GVTC AfCC A"AC CaAG CQC TGC: TGCC CpCA CGCA CtAG 1378 3L 0I H E0H lL 1

D V w N N NN T P R 44A T 6 K M F E N V 45b L S T G G P R V 46AV W AAG W0L Peptide 2 470 CCT GOTTC TTCCTCATCGTC TFACGGT GGT GAG GT ACT GOT GGC CT CTG SAC TCCCAC ACTGC CTT C GGGCCM GCTA CCASAGCATACTC ACTGGCC 1747

CTG GtG TIT GCC Cf.C CAG G2O CTT CTG GAG ATC C1TC CAG GAA AfC GACA T T GGG GCT GTG TaG TAI 1888

AGACAGGTGGrATACTATGAGTTCGAGGCCAGCCTGGGTCTACAGGGTCTCAAAAAAAAATTTTTAATrCMTTCAAACC AAGCCAGAGCGCTCTCCCACGCAC AGCTCAGTCGTCACTATCAGACAGTGTCTCATACTGCGCTGAGAGGAGCGG 205 1 GGCAGGCTCCTCTCCAGATGCGGTTCTGTTGATGTGGAGCTGCTTGGGTGATGGCAGAGGGGCGCATGCGATCTGTGTAGGTGGTGTACTGCTGAGCATCCGTATGGTCAGAGGGTTGTAAGGGAACTGAGTAGACCTGTTGAAGAGGGCCGATCTTGCCATG 2214 CCCTACCTTGGTGTCTGCCTGTGGTAATCACCTTCTGAATGCACAGAGGAGACAGAAGAGGCCrTTCAGTTCCTAAAAAGCTTTGCCCAGGAATAGGAGArTTAGACTTCCTGCTGrTTGArTTGTTGATCAGCrTTGGrTTGCATACTCCTGCATGAGGT 2377 GATGGCTCCCCTCATCTCTCCGTGCCTCGCTGCTCGTCACCACTGAGGTGCTGGCACTACCAGGCATAATGCCCACAGGATGGGACATGACAACACCATGATCTTCAACTTrATCATCAAACTTCCCATGAGAGCCrTTCTGATGATGTCTTCTAGACCAAG 2540 TTACTGGCGGGATCAGCACAAGAATGGCTTGCTrrGA~CACTGTCGATGTGGTACTGCT r~rGGCAATGAGATCTCGCTGTACAGrCCATTGAGArGTGCaA T~CCTCCCAGGA aGTCAAC ACGAATTCCTGrTTCrTTTATTACCAT 2703 GGGTGATGTGCTTTCAAGCTTGGTGAATGAAGAGTAGCTTATACTTCCTGGTGATCGAGTCACTCCTTTGCTCAGCAGGGTCaCACCCCCTCTGT>GACAAGACTGCTGTCCTGCTTCTCATGCACGCAGGGCCTTGGGGTGGGAGAG 286 GGCTCATAGGGCTCTGATGGGATATGACTCCTCTCTGAGATTTrCCAGGCCTG 2920 FIG. 2. Nucleotide and derived amino acid sequence of murine P1. Amino acids (standard one-letter symbols) are enumerated below each line and nucleotides are numbered above each line. The two N-linked glycosylation sites are underlined and marked "N." Also shown are the sequences ofboth pEXPI clones and synthetic peptides. pEXP1 cDNA clones have been shown, including the Alu I sites. pEXP1/95 constituted the 5' end of psl.8c8. Downloaded by guest on September 30, 2021 Immunology: Lowrey et A Proc. Nati. Acad. Sci. USA 86 (1989) 249

* * * * * * * * * *

PERFORIN 1 (359-387)O- - - R P G R S W -H K SS H D|S C O C E C Q D S[ V T N Q[ - -m

C9 (336-366) CO HT C G N G T -V L MD Ga C A C - - P FH F E G I JE I S

FACTOR X (80-113) C S - - L D N G G C D - F C R E E R E V C S C- A H G Y V L G D D S K S[C

EGF-P (445-482) C S S P D N G G - C Sj I C L P L R P G S W E C D C- F P G Y D L S D R L S C UROKINASE (13-42) CL- - - N G G T C V S - -N L Y F S N H W C N - P K j]F G G Q HF E I D LDL-R (342-374) C - - -O L C V N L E - - - -G Y L QC E G F Q L D PH -T K AWK A TPA (56-90) 0 F --- F N[E T C Q Q - 0QP E G F A GiZIC C E E]T RA

* * ** * * * * * * * * * PERFORIN 1 ( 218-234) L I S S Y G T H F I T T|AVID L G G

HUMAN CO (339-354) L E T Y G T H Y S S S G S - L G G MURINE CO (313-328) L E T -Y G T H Y S TS G S L G G

CS -ALPHA (303-319) F ND Y G T H Y I T S G S M G G

CS-BETA (282-298) JrF R D F G TH Y I T EA L G G C7 (273-289) LLMT D F G T H Y L OS G S L G G FIG. 3. (Upper) Homology of P1 with other proteins containing the cysteine-rich domain of epidermal growth factor receptor precursor (EGF-P). Asterisks were placed in the P1 sequence wherever the same amino acid was found to occur in at least two other proteins. LDL-R, low density lipoprotein receptor; TPA, tissue plasminogen activator. (Lower) Homology of P1 to the putative membrane-binding domain of C9, C8a, C8,3, and C7 (32-35). the size ofmRNA expressed by CTL and hybridizing with the Homology to C9. Computer alignment shows homology of original clone. the C-terminal part of C9 with the N-terminal part of P1. Sequence of Perforin cDNA and Derived Protein Sequence. These regions include a putative membrane-spanning am- The DNA sequence was obtained by using the M13-shotgun phipathic helix and the cysteine-rich epidermal growth factor strategy, combining the universal M13 primer with synthetic receptor precursor domain (Fig. 3). The spacing of the two primers complementary to sequences already obtained (Fig. domains also is retained. P1 does not contain the cysteine- 1). Ambiguous G+C-rich sequences in addition were se- rich domain homologous to the low density lipoprotein quenced with reverse transcriptase as DNA polymerase. receptor which is found in all terminal complement proteins The 2.9-kb perforin cDNA contained one open reading frame beginning with an ATG start codon and continuing for (32-35). 554 codons to a TAA termination site (Fig. 2). The derived Verification of Perforin cDNA as Coding for P1. An immu- amino acid sequence predicts a leader peptide with a prob- nological approach was taken to prove the identity of the P1 able cleavage site at position 1 (Fig. 2). Thus the mature clones. Two synthetic peptides (see Fig. 2 for sequence) and protein contains 534 amino acids. The sequence contains two three in-frame P1-B-galactosidase fusion proteins (Fig. 4) potential glycosylation sites and, as in the case ofthe terminal produced in E. coli were used to immunize rabbits. The complement proteins, no typical transmembrane domain. antisera were tested in Western blots for reactivity with purified P1. Fig. 5 shows the results of this analysis, indi- LO t- cating that the antisera produced from E. coli extracts oo CD cr. (anti-fusion protein) detect a protein isolated from cytolytic x X X(Xx LLw wL L ULN.^ granules with all the characteristics of P1. Similarly, the

CL~CLCL ~CL oo't'a;.'e' C anti-peptide antisera reacted with P1 and stained erythrocyte membranes previously lysed with P1 (Fig. 6). As these 200_.

116.,,- 0X X 92,,v J 0 E W 66,,, E~~. C _.( 45.,. Coomassie Anti-,3GAL Anti-PI Blue FIG. 5. Western blots with P1- FIG. 4. Reactivity ofpEXP1 plasmid fusion proteins with anti-P1 specific antisera generated by im- and anti-,B-galactosidase antisera. Each clone was induced to express 68KD--O' munization with P1 fusion proteins. its fusion proteins as described (29) and the proteins were separated pEXP1/8 and pEXP1/97 were used by electrophoresis on a sodium dodecyl sulfate/5-12.5% polyacryl- to immunize rabbits after prepara- amide gradient gel. Coomassie blue staining is shown on the left, tion ofinclusion bodies from E. coli. where positions of molecular mass standards (kDa) are indicated. pEXP1/57 antiserum was weakly After transfer to nitrocellulose, the proteins were stained with 1:200 reactive and its immunoblot is not dilutions of antisera. reproduced here. Downloaded by guest on September 30, 2021 i~~~~i1_; s~~~~~~~~T 250 Immunology: Lowrey et al. Proc. Natl. Acad. Sci. USA 86 (1989)

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FIG. 7. Northern blot analysis ofvarious tissues, CTL clone 28S- HY3-AG3, and control T-cell lymplioma clone EL4. The insert of P1 cDNA clone ps1.8c8 68KD- was radiolabeled by using a ran- l8s- dom priming kit (Boehringer Mannheim) and used to probe 10 ,ug of total RNA from each sam- ple. Total RNA was quantitated by both absorbance at 260 nm and ethidium bromide staining.

stimulation of lymphocytes with mitogens or in mixed lym- A B C D E phocyte culture also results in perforin mRNA expression concomitant with the appearance of cytotoxicity (Table 1). FIG. 6. Western blots of purified P1 and Pi-lysed erythrocyte Importantly, perforin mRNA was also expressed in CTL ghosts with antisera prepared against peptide 1. P1-lysed ghosts were a choriomen- prepared as described in the text. Purified P1 was used as the antigen generated in vivo by hepatotropic lymphocytic in lanes A, B, and D, while Pi-lysed ghosts were run in lanes C and ingitis virus strain (36). These CTL were directly isolated E. Lane A was immunostained with preimmune sera, lanes D and E from the liver and analyzed without further culture. were immunostained with anti-Pi, and lanes B and C were immu- Genomic Structure ofPerforin. Fig. 8 shows a Southern blot nostained with anti-peptide 1 antisera. The same reactivity against of restriction endonuclease-digested genomic DNA hybrid- purified P1 was obtained with antisera made against peptide 2. ized with a full-length P1 cDNA clone. Perforin appears to be encoded in a single-copy gene of moderate size (<6 kb). So were to remove the membranes washed unbound protein, far no evidence for polymorphism has been detected in positive reaction indicated that P1 inserted and lysed the different mouse strains. erythrocytes and that this inserted P1 reacts with anti-peptide antisera. Of all known granule components, only P1 inserts into erythrocyte membranes. DISCUSSION Cell-Specific Expression ofPerforin. Northern blot analysis of RNA extracted from various tissues and cells (Table 1) We have determined the nucleic acid sequence of cloned P1 indicates that perforin is expressed specifically in cytolytic cDNA and deduced the amino acid sequence. The original lymphocytes and not in other cells or tissues. A heavily clone of perforin cDNA was obtained by antibody screening loaded Northern blot of HY3-AG3 shows a 2.9-kb band and of a prokaryotic expression library contained in Agtll. That some degradation products (Fig. 7). All CTL clones analyzed this cDNA in fact codes for perforin was verified by the to date contain perforin mRNA (Table 1). In addition, production of antisera to synthetic peptides and to perforin- Table 1. Expression of perforin mRNA in normal murine lymphocytes Cytotoxic activity, Perfoinn Clone Cell type and specificities* LUt per 106 cells mRNAt % HY3-AG3 NK-like CTL 50 100 3F9 H-2Db allospecific CTL 35 75 5/10-20 AED-specific CTL 25 25 8/10-20 H2-Kb-restricted CTL 30 25 50.1 j Three LCMV-specific, 30 75 532 H-2-restricted CTL 35 25 527 (various H-2 specificities) 35 25 Ova-7 Ovalbumin-specific, H2-IAb-restricted TH 0 None EL4 T-cell lymphoma 0 None P815 Mastocytoma 0 None MC57G Methylcholanthrene-induced fibrosarcoma 0 None L929 Fibrosarcoma 0 None Primary MLC (BALB/c anti-B6) ND 15 Con A-stimulated splenocytes ND 15 LPS-stimulated splenocytes ND None Secondary MLC (anti-VSV splenocytes) 6.5 15 Secondary MLC (anti-vaccinia splenocytes) ND 15 Primary in vivo (anti-LCMV splenocytes) 3 15 Primary in vivo (anti-LCMV liver lymphocytes)t 25 15 Normal splenocytes ND <5 *AED, N-iodoacetyl-N'-(5'-sulfonic-l-napthyl)ethylenediamine; LCMV, lymphocytic choriomeningitis virus; TH, helper T cells; MLC, mixed lymphocyte culture; Con A, concanavalin A; LPS, lipopolysaccharide; VSV, vesicular stomatitis virus. tOne lytic unit (LU) = no. of effectors to cause 50%o lysis of appropriate targets in a 4-hr 51Cr release assay. ND, not determined. tmRNA expression was compared to the expression of P1 mRNA in HY3-AG3 (100%o) cells from Northern analysis. §Anti-LCMV liver lymphocytes were isolated as described (36). Downloaded by guest on September 30, 2021 Immunology: Lowrey et A Proc. NatL. Acad. Sci. USA 86 (1989) 251

Dr. Pat Grey and Sandra Soccaras, who provided their help in I - = > various parts of this work. This research was supported by National kb Institute ofArthritis and Infectious Disease Grant Al 21999, National 23.1 Cancer Institute Grant Ca39201, and American Cancer Society Grant IM-369A to E.R.P. D.M.L. is a Research Fellow of the Cancer 6.E Research Institute (New York). 4.4 1. Zinkernagel, R. M. & Doherty, P. C. (1975) J. Exp. Med. 141, 1427-1436. 2. McChesney, M. B. & Oldstone, M. B. A. (1987) Annu. Rev. Im- munol. 5, 279-304. 3. Herberman, R. B. & Ortaldo, J. R. (1981) Science 214, 24-30. 4. Mueller, C., Gershenfeld, H. K., Cobe, C. G., Okada, C. Y., Bleackley, R. C. & Weissman, I. L. (1988) J. Exp. Med. 167, 1124- 0.6 1136. 5. Dennert, G., Anderson, C. G. & Warner, J. (1985) J. Immunol. 135, 3729-3734. FIG. 8. Genomic Southern blot analysis ofthe P1 gene. Enzymes 6. Duke, R. C., Chervenak, R. & Cohen, J. J. (1983) Proc. Natl. Acad. used to cut DNA isolated from mouse strain BDF1 kidneys are shown Sci. USA 80, 6361-6365. above their lanes. The left lane contained markers of the indicated 7. Russel, J. H., Masakowski, V., Rucinsky, T. & Phillips, 0. (1982) length. J. Immunol. 128, 2087-2094. 8. Schmid, D. S., Tite, J. P. & Ruddle, N. H. (1986) Proc. Natl. Acad. galactosidase fusion proteins which reacted with authentic Sci. USA 83, 1881-1885. perforin isolated from CTL cytolytic granules. 9. Geiger, B., Rosen, D. & Berke, G. (1982) J. Cell Biol. 95, 137-143. 10. Kupfer, A., Dennert, G. & Singer, S. J. (1983) Proc. Nat!. Acad. The predicted perforin sequence shows considerable ho- Sci. USA 80, 7224-7228. mology with the terminal complement proteins C7, C8, and 11. Masson, D. & Tschopp, J. (1985) J. Biol. Chem. 260, 9069-9072. C9, as expected from previously reported immunological 12. Podack, E. R., Young, J. D. E. & Cohn, Z. A. (1985) Proc. Natl. cross-reactivity between those proteins (22, 23). The align- Acad. Sci. USA 82, 8629-8633. ment of P1 with C9 shows a homology region extending from 13. Pasternak, M. S. & Eisen, H. N. (1985) Nature (London) 314, 743- 745. C9's C terminus to approximately amino acid 120 of C9. This 14. Masson, D. & Tschopp, J. (1987) Cell 49, 679-685. region contains the putative membrane-binding site (37) and 15. Lobe, C. G., Finlay, B. B., Paranchych, W., Paetkau, V. H. & a cysteine-rich domain found in a number of proteins whose Bleackley, R. C. (1986) Science 232, 858-861. prototype is the epidermal growth factor receptor precursor. 16. Gershenfeld, H. K. & Weissman, I. L. (1986) Science 232,854-858. The first 120 amino acids of C9, which contain a thrombo- 17. Pasternak, M. S., Verret, C. R., Liu, M. A. & Eisen, H. N. (1986) spondin-like domain, and the cysteine-rich domain ofthe low Nature (London) 322, 740-743. 18. MacDermott, R. P., Schmidt, R. E., Caulfield, J. P., Herm, A., density lipoprotein (LDL)-receptor type are not represented Bartley, G. T., Ritz, J., Schlossman, S. F., Austen, R. F. & in perforin, even though they are found in C7 and the subunits Stevens, R. L. (1985) J. Exp. Med. 162, 1771-1787. of C8. The previous finding that an antibody to an acidic 19. Dennert, G. & Podack, E. R. (1983) J. Exp. Med. 157, 1483-1495. synthetic peptide corresponding to the LDL-receptor-like 20. Podack, E. R. & Dennert, G. (1983) Nature (London) 302,E442-445. domain of C9 (22) cross-reacted with perforin can be ex- 21. Liu, C. C., Steffen, M., King, F. & Young, J. D. E. (1987) Cell 51, plained by an acidic region in the C-terminal part of perforin 393-403. 22. Tschopp, J., Masson, D. & Stanley, K. K. (1986) Nature (London) (contained in peptide 2, Fig. 2). 322, 831-834. Perforin mRNA has been detected to date in all CTL, 23. Young, J. D. E., Cohn, Z. A. & Podack, E. R. (1986) Science 233, regardless of whether they were generated in vivo or cultured 184-190. in vitro. Moreover, the expression of perforin mRNA as 24. Rupp, F., Acha-Orbea, H., Hengartner, H., Zinkernagel, R. & detectable in Northern blots preceded the onset of cytotoxic- Joho, R. (1985) Nature (London) 315, 425-427. 25. Iwamoto, A., Ohashi, P. S., Pinder, H. P., Walker, C., Micha- ity by 12-24 hr (data not shown). Initial experiments indicate lopoulos, E. E., Rupp, F., Hengartner, H. & Tak, T. W. (1987) J. that both class I and class II major histocompatibility com- Exp. Med. 165, 591-600. plex-restricted CTL express perforin. These findings suggest 26. Young, R. A. & Davis, R. W. (1983) Science 222, 778-782. that perforin is intimately related to the expression of 27. Bankier, A. T. & Barrel, B. J. (1983) in Techniques in the Life cytotoxicity by lymphocytic killer cells. This is further Sciences, NucleicAcidBiochemistry, ed. Flavell, R.A. (Cambridge, supported by the finding that cells devoid of cytotoxic MA), pp. B508/1-B508/35. 28. Queen, C. & Korn, L. J. (1984) Nucleic Acids Res. 12, 581-599. activity and numerous tissues did not express perforin. The 29. Stanley, K. K. & Luzio, J. P. (1984) EMBO J. 3, 1429-1434. question whether peritoneal exudate lymphocytes (38) ex- 30. Maniatis, T., Fritsch, E. R. & Sambrook, J. (1983) Molecular press perforin needs to be reexamined with these more Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold sensitive techniques now available. Spring Harbor, NY), pp. 316-328. Although the perforin mRNA expression experiments 31. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. show a strong correlation between perforin transcription and 32. DiScipio, R. G., Gehring, M. R., Podack, E. R., Kaa, C. C., Hugh, T. E. & Fey, G. H. (1984) Proc. Natl. Acad. Sci. USA 81, 7298- cytotoxicity, they do not constitute proof that perforin is 7302. essential for target lysis by lymphocytes. Indeed, the obser- 33. DiScipio, R. G., Chakravati, D. N., Muller-Eberhard, H. J. & Fey, vations of CTL-mediated DNA degradation (6, 7), of soluble G. H. (1988) J. Biol. Chem. 263, 549-555. cytotoxic factors (8), of perforin-independent cytotoxic gran- 34. Haefliger, J. A., Tschopp, J., Nardelli, D., Wahli, W., Kocher, ule components (21), and of Ca-independent lysis of target H. P., Tosi, M. & Stanley, K. K. (1987) Biochemistry 26, 3551- 3554. cells (39) suggest that several pathways exist by which 35. Rao, A. G., Howard, 0. M. Z., Ng, S. C., Whitehead, A. S., lymphocytes exert cytotoxic activity. The availability of Colten, H. R. & Sodetz, J. M. (1987) Biochemistry 26, 3556-3564. cloned perforin cDNA will enable us to critically assess the 36. Zinkernagel, R. M., Haenseler, E., Leist, T. P., Cerny, A., Hen- role of perforin in lymphocyte-mediated cytotoxicity in vitro gartner, H. & Althage, A. (1986) J. Exp. Med. 164, 1075-1092. and in vivo. 37. Stanley, K. K., Page, M., Campbell, A. K. & Luzio, J. P. (1986) Mol. Immunol. 23, 451-458. 38. Berke, G. (1987) Transplant. Proc. 19, 412-416. We are grateful to Mi Kyung Lee and Laurie Fulton, who provided 39. Oostergaard, H. L., Kane, K. P., Mescher, M. F. & Clark, W. R. excellent technical support during this study. We are also grateful to (1987) Nature (London) 330, 71-72. Downloaded by guest on September 30, 2021