Journal of Immunological Method¢, 128 (1990) 133-142
133
Elsevier JIM05537
Anti-idiotypic antibodies derived against C8, C9 and perforin bind homologous restriction factor John Ding-E Young, Shibo Jiang, Chau-Ching Liu and Cynthia S. Hasselkus-Light Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Ave., New York, N Y 10021, U.S.A.
(Received13 November1989,revisedreceived13 December1989,accepted21 December1989)
A pore-forming protein (PFP/perforin/cytolysin), stored in the cytoplasmic granules of cytolytic lymphocytes, lyses a variety of target cells but not the cytol~tic lymphocytes. In the complement (C) system, a CS-binding protein (CSbp) or homologous restriction factor (HRF) has been described that protect,, ~ells against lysis mediated by homologous C. C 8 b p / H R F is known to bind to C8 and C9 and has also been suggested to protect lymphocytes against perforin-mediated lysis. Here, using an anti-idiotypic antibody approach, several polyclonal antisera were raised against IgGs that are specific for mouse perforin, and human C8 and C9. These anti-idiotypic antisera were shown to react against an overlapping epitope(s) on C S b p / H R F as indicated by the following evidence: (i) all three types of antisera reacted against partially purified C 8 b p / H R F and against a 65 kDa protein banci in cell lysates; reactivity was only observed against disulfide-reduced antigens; (ii) the three antibodies react with a protein band in normal erythrocytes (E) but not with type III E of patients with paroxysmal nocturnal hemoglobinuria or with a mutant B lymphoblastoid cell line, both of which cell types are known to be deficient in CSbp/HRF; and (iii) the three antibodies compete with each other for binding to CSbp/HRF. Type III E and the C8bp/HRF-deficient mutant lymphoblastoid cell line, however, are as susceptible to perforinmediated lysis as type I E and wild-type lymphoblastoid cell line, respectively, indicating that C S b p / H R F does not play a role in protecting cells against perforin-mediated lysis. These paradoxical findings suggest that perforin may share with C8 and C9 the same domain(s) that bind to C S b p / H R F and yet, unlike C8 and C9, perforin is not inactivated by this type of putative interaction. Since C8 and C9 are now readily available, the anti-idiotypic approach described here provides a convenient protocol for production of antisera specific for CSbp/HRF. Key words: Anti-idiotypicantibody;Complement;Cytolysis;Protection;Membranerestriction;Perforin;Pore-fornfingprotein
Correspondence to: J. D.-E Young, Box 37, Laboratoryof CellularPhysiologyand Immunology,The RockefellerUniversity, 1230York Ave.,New York, NY 10021, U.S.A. Abbreviations: a, antibodyagainstthe specifiodantigen; C, complemenl; CSBP, C8-blndingprotein; CTL, cytotoxicT lymphocyte; E, erythrocytes; HRF, homologous restriction factor; NK, natural killer; PFP, pore-formingprotein; PNH, paroxysmalnocturnalhemoglobinuria
ln~oduction Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells produce a pore-forming protein (PFP, also termed perform or cytolysin) stored in their cytoplasmicgranules (Henkart, 1985; Young and Cohn, 1987; Mfiller-Eberhard, 1988; Podack,
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishersB.V.(BiomedicalDivision)
134 1988; Tschopp and Jongeneel, 1988). Though perforin lyses a variety of target cells, it does not kill CTLs or N K cells (Blakely et al., 1987; VeiTet et al., 1987; Jiang et aL, 1988; Shinkai et al., 1988a). The basis of this resistance phenomenon is presently unclear. In the C system, a protective mechanism also exists that ensures the protection of e,'Tthrocytes (E) from lysis by C of homologous species, a phenomenon known as homologous species restriction (Hansch et al., 1981; Hu and Shin, 1984). This type of resistance is thought to be mediated in part by an E membrane polypeptide of 65 kDa termed CS-binding protein (C8bp) (Schonermark et al., 1986; Shin et al., 1986) or homologous restriction factor (HRF) (Zalman et al., 1986). C 8 b p / H R F appears to bind to C8 and C9 in the target membrane and to thereby block lytic channel formation. CSbp/HRF has also been implicated in the protection of lymphocytes against perforin-mediated cytolysis (Zalman et al., 1987a, 1988; Miiller-Eberhard, 1988). These latter observations, if correct, would be consistent with an unifying concept for resistance, e.g., one that suggests that a unique membrane protein protects against both C- and perforin-mediated lysis. This model is particularly attractive in view of the structural homologies that exist between perfofin and the terminal C components (Lichtenheld et al., 1988; Shinkai et al., 1988b; Kwon et al., !989; Lowrey et al., 1989). We have used an anti-idiotypic antibody approach to identify putative perforin-binding proteins which presumptively protect lymphocytes against perforin-mediated lysis. This study was based on the premise that a perforin-inldbitor (hypothetically termed protectin) should bind to perforin, just as CSbp/HRF binds to C8 and C9. The underlying principle of the anti-idiotypic antibody approach is as follows. Antibodies are generated against perforin; among these are antibodies that recognize perforin in a way that mimics the physiological perforin-protectin interaction. Consequently, the idiotypes of some of the antibodies against perforin may have structures in common with protectin. A second set of antibodies raised against the 'protectin-like' idiotypic determinants of the anti-perforin antibodies contain anti-idiotypic antibodies that recognize the per-
forin-binding site of the putative protectin. We have also used the same strategy to obtain antibodies against C 8 b p / H R F by raising anti-idiotypic antibodies agah~st IgGs specific for C8 and C9. Our results shov, that anti-idiotypic antibodies derived against IgGs specific for perfollhl, C8, and C9 detect unique and overlapping epitopes present in CSbp/HRF. This anti-idiotypic approach provides a simple protocol through which antibodies specific for C 8 b p / H R F can be produced. Materials and methods Cells The mul~ine CTL lines CTLL-R8 and CTLL-2 were maintained in medium containing a source of interleukin-2, as described (Young et al., 1986a). Mouse mastocytoma P815, T leukemia EL-4, and T lymphoma YAC-1 and human erythroleukemia K562 cell lines were maintained in stationary suspension cultures as described elsewhere (Young et al., 1986a). Wild-type (JY-25) and mutant (JY-5) human B lymphoblastoid cell lines were kindly provided by Dr. T. A. Springer, Boston, MA (Hollander et al., 1989). The mutant JY cell line is deficient in phosphatidylinositol glycan anchors. Human erythrocytes (E) were obtained from venous blood of volunteers. Type III E from four patients with paroxysmal nocturnal hemoglobinuria (PNH) were kindly provided by Dr. W. F. Rosse, Durham, NC and were separated from other types of E as described (Chow et al., 1986). Mouse E were collected from BALB/c mice. To obtain E ghost membranes, freshly collected human E were washed three times with phosphatebuffered saline (PBS). 1 ml of packed E was incubated with 40 mi of 5 mM NaKPO4, pH 7.6,1 mM EDTA for 10 rain. After centrifugation at 39,000 × g for 20 rain, the superuatant was discarded and the pellet was washed six times in the same buffer. Membranes were kept frozen at - 70 °C. C components and PFP/perforin Human C8 and C9 (Hammer et al., 1981) and mouse PFP/perforin (Persechini and Young, 1988) were purified essentially as described. In some experiments, commercial preparations of C8 and
135 C9 (Calbiochem-Behring, San Diego, CA) were used. Perforin activity is given in hemolytic units (HU) (Jiang et al., 1988).
Antibodies and IgG fractions Rabbit polyclonal antibodies against the reduced and alkylated forms of human C8, C9 and mouse PFP/perforin were obtained as described (Young et al., 1986b,c). IgG fractions were purified from antisera by affinity chromatography using protein A-coupled agarose columns (young et al., 1986b). 15 antisera were prepared against anti-perforin IgG, nine antisera against anti-C9 IgG, and six antisera against anti-C8 IgG. The purity of the IgG fractions used for immunization was ascertained by gel electrophoresis and Coomassie blue staining. Affinity purified IgG were injected into rabbits following immunization protocols similar to those described earlier (young et al., 1986b,c; Persechini and Young, 1988), with antigens ranging between 0.2-1 mg of protein per injection. Blood from each animal was collected 4-5 days following each booster injection. Dinitrophenol-modified bovine serum albumin (DNP-BSA; 11 tool of DNP per mol of albumin) was obtained and separated from free DNP by elution through a DEAE-52 colur.m as described (Unkeless, 1977). Rabbit antibody against DNP. BSA was obtained as described above. Column chromatography 15 ml of packed human E ghost membranes, washed six times with PBS and twice with water, were resuspended to 200 ml in 20 mM Tris-HCl, pH 7.4, containing 1% Nonidet P-40 (NP-40), 1 mM DFP, and I mM EDTA. After centrifugation at 100,000 × g for 90 min, the supernatant was applied onto DEAE-Sepharose (fast flow resin, 2.5 × 30 cm, Pharmacia) column equilibrated with buffer A (20 mM Tris-HCl, pH 7.4, 0.1% NP-40) and eluted with a 0-2 M NaC1 linear gradient in 350 ml of buffer A at 1 ml/min. 4 ml fractions were collected and assayed for C 8 b p / H R F by immunodotblotting, as described below. Active fractions were pooled, concentrated under vacuum and applied to a Superose 12 column (Ph~rmacia), eluted with buffer A at 0.3 ml/min and collected as 0.5 ml fractions.
An alternative protocol was used in some ex periments to partially enrich for C8bp/HRF. E ghost membranes, solubilized in PBS containing 1% deoxycholate and 1 mM DFP to a final protein concentration of 10 mg/ml, were centrifuged at 100,000 x g for 90 min. 2 nil of the clear supernatant were applied to a TSK-G4000 SWG column (2.15 × 60 cm, LKB, Sweden), equilibrated and eluted with PBS containing 0.1% deoxycholate at a flow rate of 0.25 ml/min. 2 lnl fractions were collected and tested by immunodothlotting as before. The peak HRF-containing fractions were lyophilized, resuspended in PBS to 5 mg of protein/m] and used in competition radioimmunoassays. Protein determinations were performed by using the BCA-protein assay kit (Pierce, Rockford, IL).
Gel electrophoresis and immunoblotting For immunoblotting experiments, cells were washed three times in PBS. Nucleus-free lysates of cells were then obtained by rupturing cells in PBS containing 1% Nonidet P-40 (NP--40; CalbiochemBehring), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO), followed by sedimentation of nuclei and unLroken cells by centrifugation (3000 × g, 15 rain). Protein samples in 1% SDS were reduced and alkylated with 20 mM dithiothreitol (DTr) and 50 mM iodoacetamide, respectively (Young et al., 1986b). After boiling for 5 rain, the samples were applied to mini-gels (Model 360, Bio-Rad Laboratories, Richmond, CA) ,~th 10% polyacrylamide, followed by elec~i transfer of proteins to Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) at a constant 400 mA for 2.5 h (Young et al., 1986b). Membranes were fixed in water containing 10% acetic acid and 15% isopropanol for 10 rain and blocked with 1% milk/2% glycine in Tris-buffered saline for 16 h. Antisera were used at a dilution of 1/50, and goat ~ I F(ab')2 anti-rabbit IgG (5.6 #Ci/#g; New England Nuclear, Boston, MA) was used as the secondary label at 0.12 #Ci/m]. Membranes were washed with 20 mM Tris-buffer, pH 7.4, containing 0.4 M NaC1, 0.25% Tween 20 (Bio-Rad Laboratories), 0.25% NP-40, and 2 mM EDTA. Autoradiography was performed for 16-48 h with intensifying screens. The Mr standards used con-
136 sisted of prestained protein markers obtained from Amersham (Arlington Heights, IL). To assay for C8bp/HRF, immunodothlotting was performed on PVDF membranes using the Bio-Dot apparatus from Bio-Rad. 50/tl of protein samples were added to each well and allowed to sit for 4-8 h. After blocking with gelatin-containing buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaC1, 2% glycine, 0.05% scdinm azide, 1% gelatin), the membrane was incubated with Tris-buffered saline, pH 7.4, containing 0.1% gelatin and 50 ~tg/ml of purified C8 for 4 h at room temperature. After three washes in detergent-containingbuffers, as above, the membrane was further incubated with rabbit anti-C8 antiserum at 1/50 dilution. The blots were developed with mI-labeled goat F(ab')2 anti-rabbit IgG as before.
was centrifuged and 70/~1 of the supernatant was collected from each well for determination of radioactivity and the percentage of cytotoxi~ty (Jiang et al., 1988).
Competition radioimmunoassay Protein A-purified IgGs specific for antibodies derived against mouse perforin, and human C8 and C9 were labeled with 125I to specific activities of 3000-5000 cpm per ng of protein, using the Iodogen (Pierce) procedure (Fraker and Speck, 1978). 50 /tl of a CSbp/HRF suspension (at 25 /tg/ml in PBS), partially purified on a TSK-G4000 column, were allowed to sit in each well in flexible plastic dishes for 4 h. Plates were then washed with PBS and blocked with 1% gelatin in PBS for 4 h. A mixture of 125I-labeled IgGs at 10,000 cpm and various cold IgGs of given specificities and at indicated concentrations were added to each well to a final volume of 50 ml and incubated for I h at 4°C. After washing (as in immunoblotting), the individual wells were counted in a gamma counter. The percentage of inhibition of binding produced by the indicated cold IgGs was determined as {1 - (cpm of binding observed in the presence of the cold IgG)/(cpm of binding observed in the presence of cold IgG specific for DNP-BSA)}.
Anti-idiotypic antibodies bind to a 65 kDa membrane protein Polyclonal antisera of three different specificities were derived against IgGs specific for mouse perforin and for human C8 and C9. These antisera were screened by immunoblottingfor their reactivities against proteins of various cell types (Fig. 1; data shown here only for human and mouse E). Anti-idiotypic antisera of all three specificities reacted with unique protein bands of 65 kDa. reactive band of this molecular mass was observed in all cell types tested (in addition to E shown in Fig. 1, two CTLL and four tumor cell lines were tested). The following antisera were shown to react against the 65 kDa protein band: nine out of 15 anti-idiotypic antisera against anti-perforin IgG, six out of nine antisera against anti-C9 IgG, and four out of six antisera against anti-C8 IgG. Reactivity was observed only when cell proteins were disulfide-reduced as non-reduced blots did not react with any of the antisera used (Fig. 1). Preimmune sera used as controls did not give any specific reaction with the cell lysates tested (Fig. 1). As all three a~,~ti-idiotypic antibodies reacted with E ghost membranes, it is likely that the antigen is localized on the cell surface. However, attempts to localize the antigen by immunofluorescence or FACS analysis have failed to give any specific signal (data not shown), a result consistent with the fact that the antibodies described
Cytotoxicity assay Perforin-mediated lysis of tumor cells was detected by 51Cr release, as previously described (Jiang et ai., 1988). Briefly, 50 HU of purified murine perforin were mixed with 5 x 104 51Crlabeled JY-5 or JY-25 cells in 140 #1 serum-free RPMI 1640 m a round-bottom well of a microtiter plate. After incubation at 37°C for 3 h, the plate
Immunofluorescence and fluorescence-activated cell sorter (FA C$) analysis Indirect immunofluorescence was carried out exactly as described (Liu et al., 1987) and FACS analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA) using anti-idiotypic antisera at 1/50 dilution as primary antibodies, followed by fluorescence-conjugated goat anti-rabbit IgG F(ab')2 (Tagn, Burlingame, CA).
Results
137
g
I
2
I
2
I
2
I
2
1
2
1
2
1
2
93~ 69~
46~ +SH -SH --SH -SH +SH +SH Fig. 1. lmmunoblots of cell iysate prc:,ins re~,;ted with anfi-idiotypic antisera. 5 x 104 cell equivalents of either human (1) or mouse (2) E were electrophoresed per lane, and reacted with anti-idiotypic antise::a derived against IgG specific for mouse perfofin, and human C~ and (::9. Th,~ ~ three ~ti-idiotypic anfisera are indicated by aaPFP, aaC8 and aaC9, respectively. Cell proteins were eleetrophoresod in either reducing (+SH) or non-reducing ( - S H ) conditions. For the last two lanes, the pre-inanunv ~ were obtained from the three rabbits that yielded the anti-idiotypic sera, each of which was used at a dilution of 1/50. Autoradiography was performed for 16 h.
h e r e only detect a n t i g e n s w h e n t h e y h a v e b e e n disulfide-reduced.
Demonstration that the antibody-reactive 65 kDa protein is H R F Previous studies h a v e s h o w n t h a t C S b p / H R F h a s a m o l e c u l a r m a s s o f 65 k D a , s u g g e s t i n g to u s
t h a t o u r a n t i b o d i e s m a y react with this molecule. H R F is a b s e n t f r o m t y p e lII E b u t p r e s e n t in t y p e I E o f P N H p a t i e n t s ( H a n s c h et al., 1987; Z a l m a n et ~ . , 198To). T y p e III E d o n o t c o n U d n p h o s p h a t i d y l i n o s i t o l glycan a n c h o r s (Low a n d S~tiel, 1988), s u g g e s t i n g t h a t H R F is a n c h o r e d to E m e m b r a n e s via a p h o s p h o i n o s i t o l linkage. T h e
B A
~
g
g
g
~
o
~
~
~,
=
~
93--
93-69-
69--
46-
46--
g I
I
Type IE
I
J
Type ITTE
I
I I
JY-25
I
JY-5
Fig. 2. Immunoblots of cell lysate proteins reacted with anti-idiotypic antisera. 5 × 10" cell equivalents from E (A) or J'Y cells (B) were electrophoresed per lane and reacted under reducing conditions with the indicated anti-idiotypie anfisera (same notation as that shown in the legend to Fig. 1). In A, type I and type Ill E from a PNH patient were obtained as described in the materials and methods section. In B, either ~ - 2 5 or YY-5 cell proteins were reacted with the indicated antisera. Autoradiography was performed for 48 h.
138 1
2
3
4
5
93 m 69--
46-30-Fig. 3. Anti-kh'otypic andsera react against CSbp/HRF. Lane
l: SDS-PAGEprofde of CSbp/HRF of human E, eiuted from a Superose 12 cohmm, as described in the materials and methods section. 20 nag protein was applied per lane. Eleetrophoresis was performed under reducing conditions. Lanes 2-5: C8bp/HRF, eleetrophoresedas in lane 1, was transferred to blots and reacted with aaPFP 0ane 2), aaC8 (lane 3), aaC9 (lane 4), and pre-immune serum (lane 5). The notations used for the anti-idiotypicantisera are the same as those given in the legend to Fig. 1.
immunoblot analysis in Fig. 2A (data shown here for only one patient; similar data were obtained for all four PNH patients) indicates that all three types of antisera reacted against a 65 kDa protein |00
........ J ........ '
.........
band of type I E and of normal, untreated E but not against proteins derived from type III E of P N H patients. H R F is also absent from the mutant B lymphoblastoid cell line JY-5 but not from the parental cell line JY-25 (Hollander et ai., 1989). As shown in Fig. 2B, a protein band of 65 kDa from JY-25 cells reacted with all three types of antisera. This same band was absent from JY-5 cells (Fig. 2B). C S b p / H R F was partially purified by sequential appfication of human E membrane proteins through DEAE-Sepharose and Superose 12 columns and by using a sandwich immunobloldng assay for screening active fractions (see materials and methods section). This assay relied first on binding of C S b p / H R F to C8, followed by reaction with anti-C8 antibodies (Schonermark et al., 1986). The activity eluted from the Superose 12 column contained a major band of 65 kDa (see gel profile in Fig. 3, lane 1). This band was shown to react with all three types of anti-idiotypic antisera (Fig. 3, lanes 2-4) but not with pre-immune sera (lane 5). Taken together, these data indicate that the anti-idiotypic antisera described here react against C 8 b p / H R F .
'
:'C
'
...... '
........ "
50
!
2
5
I,I
0.1
,/ k
~
. . . . . . . .
,
! .0
. . . . . . . .
I
10
.
. . . . . . . .
i,i
100
0.1
! .0
10
100
0.1
........
i
1.0
........
i
10
........
i
100
Concentration o f I g G (gg]ml) Fig. 4. Competition solid-phase radioimmunoassay showing that the anti-idiotypicantisera react against overlappingepitopes on CSbp/HRF. Plates were coated with partially purified C8bp/HRF and reacted with 125I-labaledIgO of one of three speeificities (aaPFP in A; aaC8 in B; and aaC9 in C) and the followingcold IgGs at the given concentrations: aaPFP (o), aaC8 (it), and aaC9 (,,). The ~ inhibitionof binding was determined as describedin the materials and methods section,
139 100 F A "'"""~
'
!75Iff ~
....... ' ........ ' ........ '
]
1 ~~ o.1
~ l.o
-~ 1o
loo
t~ . . . . . . . ÷ .......~ . , o.l
1.o
1o
1oo
o.!
Lo
1o
loo
Concentration of IgG ( g g / m l ) Fig. 5. Assessmentof the idiotypic-anti-idiotypicreaction.The experimentalprotocolwas the sameas that in Fig.4, exceptthat the followingcold IgGswere used for competition:anti-PFPlgG (or aPFP; o), aC8 (®), aC9 (A),and aDNP-BSA(A).123blabeled lgGswereof the followingspeciflcities:(A) aaPFP, (B) aaCS,and (C) aaC9.
Anti-idiotypic antibodies react against overlapping or same epitopes on C8bp / HRF The possibility that the three types of antisera react against a unique epitope on C 8 b p / H R F was assessed by a competition radioimmunoassay. l~I-labeled IgGs of one of the three given specificities were reacted against partially purified human E C S b p / H R F eluted from a TSK G-4000 column. The extent of inhibition in binding produced by cold IgGs of the same Or different specificities was quantified (Fig. 4). The three unlabeled, anti-idiotypicIgGs competed avidly for binding to C S b p / H R F mediated by themselves and by each other (Fig. 4). We also tested the inhibitory effect of cold IgGs specific for perforin, C8, C9, and DNP,BSA on the binding reaction between ~25I-labeled antiidiotypic IgG and CSbp/HRF-coated plates. As shown in Fig. 5, IgGs specific for perforin, C8 and C9, but not anti-DNP-BSA IgG, inhibited this binding reaction in a dose-dependent manner, confirming that the labeled antibodies described here are anti-idiotypic since they must have bound to epitopes of the anti-perforin, anti-C8 or anti-C9 IgGs close to or in their antigen-combining sites. "[he lack of inhibitory effect associated with the presence of IgG specific for DNP-BSA (Fig. 5) or
IgG from preimmune serum (not shown) rules out the possibility that the inhibitory effect observed here could be attributed to nonspecific binding between the l~I-labeled anti-idiotypic IgGs and non-idiotypic determinants on the cold IgGs.
C8bp / H R F is ineffective in protecting cells against perforin-mediated &sis The results presented above would be consistent with the view that C 8 b p / H R F confers
80[ 6O
40
2O o
dY-5
dY-25
Fig. 6. C8bp/HRF-deficientmutant cellsare as susceptibleto perforin-mediatedlysisas wild-typecells.JY-25 and JY-5 cells were exposedto purifiedperforin and SICr releasewas measured as describedin the materialsand methodssection.Data representquadriplicates+ standarddeviation.
140
resistance to ceils against both perforin- and homologous C-mediated lysis, as suggested (Zalman et al., 1987a, 1988; Miiller-Eberhard, 1988). Since HRF is absent from type III E of PNH patients, it would follow then that these E should be more sensitive to perforin-mediated lysis than normal or type I E. We and others have recently shown, however, that type III E are indeed more susceptible to lysis mediated by homologous C than control or type I E, but they are as susceptible to perforin-mediated lysis as type I E (Hollander et al., 1989; Jiang et ai., 1989; Krahenbuhl et al., 1989). These results are further co, roborated by experiments done here with JY-5 and JY-25 ceils. As shown in Fig. 6, the HRF-deficient JY-5 cells are just as susceptible to perforin-mediated lysis as JY-25 cells, further supporting the notion that HRF does not play a role in protecting against perforin-mediated lysis.
Discussion The idiotypic network theory of Jeme (1974) proposes that idiotypes can act as immunogens. Anti-idiotypic antibodies elicited this way have been used in the past to identify receptors and other binding proteins (Pain et aL, 1988). We reasoned that, for a candidate surface protein to function as a 'protectin', it should bind first to perforin and that this interaction could presumably be mimicked by an idiotypic-anti-idiotypic reaction. Anti-idiotypic antibodies elicited against anti-perforin antibodies were shown here to bind to C8bp/HRF. Anti-idiotypic antibodies raised similarly against anti-C8 and anti-C9 antibodies also detected C 8 b p / H R F and in fact, the three antibodies were shown to compete with each other, apparently for the same or overlapping binding site(s) on C8bp/HRF. It should be noted that, by using the same strategy, several antisera with the same reactivities were obtained, indicating that these results cannot be explained by non-specific or spurious reactivities conferred by in&vidual batches of antisera. Since C8 and C9 are readily available now in large quantities, the procedure described here provides a convenient way of deriving CSbp/HRF-specific antibodies that could be used for further analysis of this polypeptide.
The main evidenc~ used to argue in favor of a role for C 8 b p / ! i R F in protecting lymphocytes against perforin-mediated lysis is that isolated HRF, reconstituted into sheep E membranes by a detergent-dilution protocol, protects these cells against lysis mediated by both homologous C and perforin (Zahnan et al., 1987a). The results obtained here with anti-idiotypic antibodies would appear to be consistent with this interpretation. It was therefore surprising to find out that type III E from PNH patients, known to lack C8bp/HRF, are highly susceptible to lysis mediated by homologous C but are equally susceptible to perforin-mediated lysis, when compared to normal human E (Hollander et al., 1989; Jiang et al., 1989; Krahenbuhl et al., 1989). These latter results, and the cytotoxicity data presented here on JY-5 and JY-25 cells, argue clearly against a role for I-IRF in restricting perforin-mediated lysis. It is paradoxical that perforin and C 8 b p / H R F should share antigenic determinants that are complementary and yet the latter should not play any role in restricting perforin-mediated lysis. Since antibodies raised against perforin, C8, and C9 share the same idiotypes or antigenic determinants, it is likely that perforin, C8, and C9 are structurally homologous in the region where C8 and C9 bind to C8bp/HRF. This inference is consistent with recent cloning studies which have revealed the existence of partial amino acid identity between perforin, C8, and C9 (Lichtenheld et al., 1988; Shinkai et al., 1988b; Kwon et ai., 1989; Lowrey et al., 1989). It is possible that additional domains, present only on C8 and C9, are involved in rendering these molecules susceptible to CSbp/HRF-mediated block on pore-formation. We speculate that C S b p / H R F is also structurally homologous to perforin, C8, and C9, as indicated by preliminary findings that antibodies raised against the disulfide-reduced forms of C8 and C9 react against C S b p / H R F (unpublished). The anti-idiotypic antibodies described here only reacted with C 8 b p / H R F in its disulfide-reduced form, suggesting that a major structural rearrangement is necessary before the idiotypic determinant is exposed. It is not clear whether a similar conformational change could occur when C 8 b p / H R F is extracted and reincorporated into E membranes, in which condition it may conceivably have some
affinity for perforin, and, at sufficiently high c o n centrations, it m a y exert s o m e protective function against perforin-mediated lysis, as a d v o c a t e d b y Z a l m a n et al. (Zalman et al., 1987a). F u r t h e r structural studies are w a r r a n t e d to substantiate these a n d any o t h e r speculations. T h e molecular basis o f the perforin-resistance p h e n o m e n o n remains to b e determined. Since H R F , the only putative protective factor studied to date, does n o t s e e m to explain this type o f reaction, other protective molecules (protectins) n e e d to b e sought. It should b e n o t e d that p e r forin-resistance is a n inducible p h e n o t y p e a n d appears to correlate with the level o f cytotoxicity o f t h e effector cells (Nagler-Anderson et al., 1988; Liu et al., 1989). T h e s a m e p h e n o m e n o n has b e e n described for yeast killer toxin, against which toxin-producing yeasts are resistant (Boone et al., 1986). I n general, it would m a k e sense for any cytolytic cell type to acquire s o m e f o r m o f defense against d a m a g e t h a t might b e inflicted b y its o w n lyric mediators.
Acknowledgements W e t h a n k R a g h u r a m G o r t i a n d Leila Posaw for excellent technical assistance, Drs. D a v i d M. Ojcius a n d Sanjay 30ag for critical reading o f the manuscript, a n d Dr. Zanvil A. C o h n for continuo n s s u p p o r t a n d advice. This w o r k was s u p p o r t e d in p a r t b y grants f r o m the Cancer Research I n s t i t u t e / F r a n c e s L. a n d E d w i n L. C u m m i n g s Memorial F u n d Investigator A w a r d a n d the N.I.H. (CA-47307) a n d b y a Scholarship f r o m the Lucille P. M a r k e y Charitable Trust. Post-doctoral support was f r o m the C a n c e r Research Institute (S.J.) a n d the Irvington H o u s e Institute for Medical Research (C.-C.L.).
References Blakely, A., Gorman, K., Ostergaard, H., Svoboda, K., Liu, C.-C., Young, J.D.-E and Clark, W.R. (1987) Resistance of cloned cytotoxic T lymphocytes to cell-mediated cytotoxicity. J. Exp. Med. 166,1070. Boone, C., Bussey, H., Greene, D., Th6mas, D.Y. and Vernet, T. ¢1986) Yest killer toxin: site-directed mutations ira-
pficate the precursor protein as the immunity component. Cell 46,105. Chow, F.-L., Hall, S.E., Rosse, W.F. and Telen, M.J. (1986) Separation of the acetylcholineslerase-deflcient red cells in paroxysmal nocturnal hemoglobinuria. Blood 67, 893. Fraker, P.J. and Speck, J.C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6*tetrachloro-3a,6a-diphenylglycoluril. Binchem. Biophys. Res. Commun. 80, 849. Hammer, C.H., Wirtz, G.H., Renfer, L., Gresham, H.D. and Tack, B.F. (1981) Large scale isolation of functionally active components of the human complement system. J. Biol. Chem. 256, 3995. Hansch, G.M., Hammer, C.H., Vanguri, P. and Shin, M.L. (1981) Homo!J~ous species restriction in lysis of erythrocytes by terminal complement proteins. Proc. Natl. Acad. Sci. U.S.A. 78, 5118. Hansch, G.M., Schonermark, S. and Roelcks, D. (1987) Paroxysmal nocturnal hemoglobinuria type III: lack of an erythrocyte membrane protein restricting the lysis by C5b-9. J. Clin. Invest. 80, 7. Henkart, P.A. (1985) Mechanism of lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 3, 31. Hollander, N., Shin, M.L., Rosso, W.F. and Springer, T.A. (1989) Distinct restriction of complement- and cell-mediao ted lysis. J. ImmunoL 142, 3913. Hu, V.W. and Shin, M.L. (1984) Species-restricted target cell lysis by human complement: complement-lysed erythrocytes from heterologous and homologous species differ in their ratio of bound to inserted C9. J. ImmunoL 133, 2133. Jerne, N.K. (7974) Towards a network theory of the immune system. Ann. Inst. Pasteur 125C, 373. Jiang, S., Persechini, P.M., Zychlinsky, A., Lin, C.-C., Perassia, B. and Young, J.D.-E (1988) Resistance of cytolytic lymphocytes to perforin-mediated kilting: Lack of correlation with complement-associated homologous species restriction. J. Exp. Med. 168, 2207. Jiang, S., Persechini, P.M., Rosse, W.F., Perussia, B. and Young, J.D.-E (1989) Differential susceptibility of type III erythrocytes of paroxysmal nocturnal hemoglobinuria to lysis mediated by complement end perforin. Biochem. Biophys. Res. Commun. 162, 316. Krahenbuhl, O.P., Peter, H.H. and Tschopp, J. (1989) Absence of homologous restriction factor does not affect CTL-mediated ¢ytolysis. Eur. J. lmmunol. 19, 217. Kwon, B.S., Wakulchik, M., Lin, C.-C., Persechini, P.M., Trapani, J., Haq, A.K., Kim, Y. and Young, J.D.-E (1989) The structure of the lymphocyte pore-forming protein perforin. Biochem. Biophys. Res. Commun. 158, I. Lichtenheld, M.G., Olsen, K., Lu, P., Lowrey, D.M., Hameed, A., Hengarmer, H. and Podack, E.R. (1988) Structure and function of human perforin. Nature 335, 448. Liu, C.-C., Steffen, M., King, F. and Young, J.D.-E (1987) Identification, isolation, and characterization of a novel cytotoxin in routine cytolytic lymphocytes. Cell 51, 393. Lin, C.-C., Jiang, S., Persechini, P.M., Zychlinsky, A., Kaufmann, Y. and Young, J.D.-E (1989) Resistance of cytolytic lymphocytes to perforin-mediated killing, Induction of re-
142 sistance correlates with increase in cytotoxicity.J. Exp. Med. 169, 2211. Low, M.G. and Salfiel, A.R. (1988) Structural and functional roles of glyco~l-phosphatldylinositolin memhrunes. Science 239, 268. Lowrey, D.M., Aebischer, T., Olsen, K., Lichtenheld, M., Rupp, F., Hengarmer, H. and Podack, E.R. (1989) Cloning, analysis, and expression of routine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9. Proc. Natl. Acad. Sci. U.S.A. 86, 247. Mllller-Eberhard, HJ. (1988) The molecular basis of target cell killing by human lymphocytes and of killer cell self-protection. Immonol. Rev. 103, 87. Nagler-Anderson, C., Verret, C.R., Firmenich, A.A., Berne, M. and Eisen, H.N. (1988) Resistance of primary CD8 + cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines. 3. Immanul. 141, 3299. Pain, D., Kanwar, Y.S. and Blobel, G. (1988) Identification of a receptor for protein import into chloroplasts and its locafization to envelope contact zones. Nature 331, 232. Persechini, P.M. and Young, J.D.-E (1988) The primary structure of the lymphocyte pore-forming protein perforin: partial amino acid sequencing and determination of isoelectric point. Biochem. Biophys. Res. Commun. 156, 740. Podack, E.R. (1988) Granule-mediated cytolysis of target cells. Curt. Top. Microbial. ImmunoL 140,1. Schonermark, S., Rauterberg, E.W., Shin, M.L., Loke, S., Roelcke, D. and Hunsch, G.M. (1986) Homologous species resW.'ctiun in lysis of human erythrocytes: a membrane-derived protein with CS-binding capacity functions as an inhibitor. J. Immunol. 136,1772. Shin, M.L., Hansch, G., Hu, V.W. and Nicholson-Weller, A. (1986) Membrane factors responsible for homologous species restriction of complement-tnediated lysis: evidence for a factor othe~ than DAF operating at the stage of C8 and C9. J. ImmunoL 136,1777. Shinkai. Y., Ishikawa, H., Hattori, M. and Okumura, K. (1988a) Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis. Eur. J. Immanol. 18, 29. Shinkal, Y., Takio, K. and Okumura, K. (1988b) Homology of perforin to the ninth component of complement (C9). Nature 334, 525.
Tschopp, J. and Jongencel, C.V. (1918) Cytotoxic T lymphocyte mediated cytolysis. Biochemistry 27, 2641. Unkeless, J.C. (1977) The presence of two Fc receptors on mouse macrophages: Evidence from a variant cell line and differential trypsin sensitivity. J. Exp. Med. 145, 931. Verret, C.R., Firmeulch, A.A., Kr~nz, D.M. and ~sen, H.N. (1987) Resistance of cytotoxic T lymphocytes to the lyric effects of their toxic granules. J. Exp. Med. 166,1536. Young, LD.-E and Cohn, Z.A. (1987) Cellular and humoral mechanisms of cyV0toxicity: structural and functional analogies. Adv. Immunol. 41, 269. Young, J.D.-E, Hengartner, H., Podack, E.R. and Cohn, Z.A. (1986a) Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell 44, 849. Young, J.D.-E, Cohn, Z.A. and Podack, E.R. (1986b) The ninth component of complement and the pore-forming protein (Perforin 1) from cytotoxic T cells: structural, immunologicul, andfunctionul similarities. Science 233,184. Young, J.D.-E, Liu, C.-C., Leong, L.G. and Colin, Z.A. (1986c) The pore-forming protein (Perforin) of cytolytic T lymphocytes is immonologlcally related to the components of membrane attack complex of complement through cysteine~-rich domains. J. Exp. Med. 164, 2077. Zvlmav. L.S., Wood, L.M. and Mtiller-Eberhurd, H.J. (1986) Isolation of a human erythzocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels. Proc. Natl. Acad. Sci. U.S.A. 83, 6975. Zalman, L.S., Wood, L.M. and Miiller-Eberhar~ H.J. (1987a) Inhibition of antibody-dependent lymphocyte 6yt_otoxicity by homologous restriction factor incorporated into target cell membranes. J. Exp. Med. 166, 947. Zalman, L.S., Wood, L.M., Frank, M.M. and Mtiller-Eberhard, H.J. (1987b) Deficiency of the homologous restriction factor in paroxysnTalnocturnal hemoglobinuria. J. EXp. Med. 165, 572. Zulman, L.S., Brokers, M.A. and Mllller-Eberhard, H.J. (1988) Self-protection of cytotoxic lymphocyte.s:A soluble foml of homologous restriction Tactor in cytoplasmic granules. Prco. Natl. Acad. Sci. U.S.A. 85, 4827.
133
Elsevier JIM05537
Anti-idiotypic antibodies derived against C8, C9 and perforin bind homologous restriction factor John Ding-E Young, Shibo Jiang, Chau-Ching Liu and Cynthia S. Hasselkus-Light Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Ave., New York, N Y 10021, U.S.A.
(Received13 November1989,revisedreceived13 December1989,accepted21 December1989)
A pore-forming protein (PFP/perforin/cytolysin), stored in the cytoplasmic granules of cytolytic lymphocytes, lyses a variety of target cells but not the cytol~tic lymphocytes. In the complement (C) system, a CS-binding protein (CSbp) or homologous restriction factor (HRF) has been described that protect,, ~ells against lysis mediated by homologous C. C 8 b p / H R F is known to bind to C8 and C9 and has also been suggested to protect lymphocytes against perforin-mediated lysis. Here, using an anti-idiotypic antibody approach, several polyclonal antisera were raised against IgGs that are specific for mouse perforin, and human C8 and C9. These anti-idiotypic antisera were shown to react against an overlapping epitope(s) on C S b p / H R F as indicated by the following evidence: (i) all three types of antisera reacted against partially purified C 8 b p / H R F and against a 65 kDa protein banci in cell lysates; reactivity was only observed against disulfide-reduced antigens; (ii) the three antibodies react with a protein band in normal erythrocytes (E) but not with type III E of patients with paroxysmal nocturnal hemoglobinuria or with a mutant B lymphoblastoid cell line, both of which cell types are known to be deficient in CSbp/HRF; and (iii) the three antibodies compete with each other for binding to CSbp/HRF. Type III E and the C8bp/HRF-deficient mutant lymphoblastoid cell line, however, are as susceptible to perforinmediated lysis as type I E and wild-type lymphoblastoid cell line, respectively, indicating that C S b p / H R F does not play a role in protecting cells against perforin-mediated lysis. These paradoxical findings suggest that perforin may share with C8 and C9 the same domain(s) that bind to C S b p / H R F and yet, unlike C8 and C9, perforin is not inactivated by this type of putative interaction. Since C8 and C9 are now readily available, the anti-idiotypic approach described here provides a convenient protocol for production of antisera specific for CSbp/HRF. Key words: Anti-idiotypicantibody;Complement;Cytolysis;Protection;Membranerestriction;Perforin;Pore-fornfingprotein
Correspondence to: J. D.-E Young, Box 37, Laboratoryof CellularPhysiologyand Immunology,The RockefellerUniversity, 1230York Ave.,New York, NY 10021, U.S.A. Abbreviations: a, antibodyagainstthe specifiodantigen; C, complemenl; CSBP, C8-blndingprotein; CTL, cytotoxicT lymphocyte; E, erythrocytes; HRF, homologous restriction factor; NK, natural killer; PFP, pore-formingprotein; PNH, paroxysmalnocturnalhemoglobinuria
ln~oduction Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells produce a pore-forming protein (PFP, also termed perform or cytolysin) stored in their cytoplasmicgranules (Henkart, 1985; Young and Cohn, 1987; Mfiller-Eberhard, 1988; Podack,
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishersB.V.(BiomedicalDivision)
134 1988; Tschopp and Jongeneel, 1988). Though perforin lyses a variety of target cells, it does not kill CTLs or N K cells (Blakely et al., 1987; VeiTet et al., 1987; Jiang et aL, 1988; Shinkai et al., 1988a). The basis of this resistance phenomenon is presently unclear. In the C system, a protective mechanism also exists that ensures the protection of e,'Tthrocytes (E) from lysis by C of homologous species, a phenomenon known as homologous species restriction (Hansch et al., 1981; Hu and Shin, 1984). This type of resistance is thought to be mediated in part by an E membrane polypeptide of 65 kDa termed CS-binding protein (C8bp) (Schonermark et al., 1986; Shin et al., 1986) or homologous restriction factor (HRF) (Zalman et al., 1986). C 8 b p / H R F appears to bind to C8 and C9 in the target membrane and to thereby block lytic channel formation. CSbp/HRF has also been implicated in the protection of lymphocytes against perforin-mediated cytolysis (Zalman et al., 1987a, 1988; Miiller-Eberhard, 1988). These latter observations, if correct, would be consistent with an unifying concept for resistance, e.g., one that suggests that a unique membrane protein protects against both C- and perforin-mediated lysis. This model is particularly attractive in view of the structural homologies that exist between perfofin and the terminal C components (Lichtenheld et al., 1988; Shinkai et al., 1988b; Kwon et al., !989; Lowrey et al., 1989). We have used an anti-idiotypic antibody approach to identify putative perforin-binding proteins which presumptively protect lymphocytes against perforin-mediated lysis. This study was based on the premise that a perforin-inldbitor (hypothetically termed protectin) should bind to perforin, just as CSbp/HRF binds to C8 and C9. The underlying principle of the anti-idiotypic antibody approach is as follows. Antibodies are generated against perforin; among these are antibodies that recognize perforin in a way that mimics the physiological perforin-protectin interaction. Consequently, the idiotypes of some of the antibodies against perforin may have structures in common with protectin. A second set of antibodies raised against the 'protectin-like' idiotypic determinants of the anti-perforin antibodies contain anti-idiotypic antibodies that recognize the per-
forin-binding site of the putative protectin. We have also used the same strategy to obtain antibodies against C 8 b p / H R F by raising anti-idiotypic antibodies agah~st IgGs specific for C8 and C9. Our results shov, that anti-idiotypic antibodies derived against IgGs specific for perfollhl, C8, and C9 detect unique and overlapping epitopes present in CSbp/HRF. This anti-idiotypic approach provides a simple protocol through which antibodies specific for C 8 b p / H R F can be produced. Materials and methods Cells The mul~ine CTL lines CTLL-R8 and CTLL-2 were maintained in medium containing a source of interleukin-2, as described (Young et al., 1986a). Mouse mastocytoma P815, T leukemia EL-4, and T lymphoma YAC-1 and human erythroleukemia K562 cell lines were maintained in stationary suspension cultures as described elsewhere (Young et al., 1986a). Wild-type (JY-25) and mutant (JY-5) human B lymphoblastoid cell lines were kindly provided by Dr. T. A. Springer, Boston, MA (Hollander et al., 1989). The mutant JY cell line is deficient in phosphatidylinositol glycan anchors. Human erythrocytes (E) were obtained from venous blood of volunteers. Type III E from four patients with paroxysmal nocturnal hemoglobinuria (PNH) were kindly provided by Dr. W. F. Rosse, Durham, NC and were separated from other types of E as described (Chow et al., 1986). Mouse E were collected from BALB/c mice. To obtain E ghost membranes, freshly collected human E were washed three times with phosphatebuffered saline (PBS). 1 ml of packed E was incubated with 40 mi of 5 mM NaKPO4, pH 7.6,1 mM EDTA for 10 rain. After centrifugation at 39,000 × g for 20 rain, the superuatant was discarded and the pellet was washed six times in the same buffer. Membranes were kept frozen at - 70 °C. C components and PFP/perforin Human C8 and C9 (Hammer et al., 1981) and mouse PFP/perforin (Persechini and Young, 1988) were purified essentially as described. In some experiments, commercial preparations of C8 and
135 C9 (Calbiochem-Behring, San Diego, CA) were used. Perforin activity is given in hemolytic units (HU) (Jiang et al., 1988).
Antibodies and IgG fractions Rabbit polyclonal antibodies against the reduced and alkylated forms of human C8, C9 and mouse PFP/perforin were obtained as described (Young et al., 1986b,c). IgG fractions were purified from antisera by affinity chromatography using protein A-coupled agarose columns (young et al., 1986b). 15 antisera were prepared against anti-perforin IgG, nine antisera against anti-C9 IgG, and six antisera against anti-C8 IgG. The purity of the IgG fractions used for immunization was ascertained by gel electrophoresis and Coomassie blue staining. Affinity purified IgG were injected into rabbits following immunization protocols similar to those described earlier (young et al., 1986b,c; Persechini and Young, 1988), with antigens ranging between 0.2-1 mg of protein per injection. Blood from each animal was collected 4-5 days following each booster injection. Dinitrophenol-modified bovine serum albumin (DNP-BSA; 11 tool of DNP per mol of albumin) was obtained and separated from free DNP by elution through a DEAE-52 colur.m as described (Unkeless, 1977). Rabbit antibody against DNP. BSA was obtained as described above. Column chromatography 15 ml of packed human E ghost membranes, washed six times with PBS and twice with water, were resuspended to 200 ml in 20 mM Tris-HCl, pH 7.4, containing 1% Nonidet P-40 (NP-40), 1 mM DFP, and I mM EDTA. After centrifugation at 100,000 × g for 90 min, the supernatant was applied onto DEAE-Sepharose (fast flow resin, 2.5 × 30 cm, Pharmacia) column equilibrated with buffer A (20 mM Tris-HCl, pH 7.4, 0.1% NP-40) and eluted with a 0-2 M NaC1 linear gradient in 350 ml of buffer A at 1 ml/min. 4 ml fractions were collected and assayed for C 8 b p / H R F by immunodotblotting, as described below. Active fractions were pooled, concentrated under vacuum and applied to a Superose 12 column (Ph~rmacia), eluted with buffer A at 0.3 ml/min and collected as 0.5 ml fractions.
An alternative protocol was used in some ex periments to partially enrich for C8bp/HRF. E ghost membranes, solubilized in PBS containing 1% deoxycholate and 1 mM DFP to a final protein concentration of 10 mg/ml, were centrifuged at 100,000 x g for 90 min. 2 nil of the clear supernatant were applied to a TSK-G4000 SWG column (2.15 × 60 cm, LKB, Sweden), equilibrated and eluted with PBS containing 0.1% deoxycholate at a flow rate of 0.25 ml/min. 2 lnl fractions were collected and tested by immunodothlotting as before. The peak HRF-containing fractions were lyophilized, resuspended in PBS to 5 mg of protein/m] and used in competition radioimmunoassays. Protein determinations were performed by using the BCA-protein assay kit (Pierce, Rockford, IL).
Gel electrophoresis and immunoblotting For immunoblotting experiments, cells were washed three times in PBS. Nucleus-free lysates of cells were then obtained by rupturing cells in PBS containing 1% Nonidet P-40 (NP--40; CalbiochemBehring), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO), followed by sedimentation of nuclei and unLroken cells by centrifugation (3000 × g, 15 rain). Protein samples in 1% SDS were reduced and alkylated with 20 mM dithiothreitol (DTr) and 50 mM iodoacetamide, respectively (Young et al., 1986b). After boiling for 5 rain, the samples were applied to mini-gels (Model 360, Bio-Rad Laboratories, Richmond, CA) ,~th 10% polyacrylamide, followed by elec~i transfer of proteins to Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) at a constant 400 mA for 2.5 h (Young et al., 1986b). Membranes were fixed in water containing 10% acetic acid and 15% isopropanol for 10 rain and blocked with 1% milk/2% glycine in Tris-buffered saline for 16 h. Antisera were used at a dilution of 1/50, and goat ~ I F(ab')2 anti-rabbit IgG (5.6 #Ci/#g; New England Nuclear, Boston, MA) was used as the secondary label at 0.12 #Ci/m]. Membranes were washed with 20 mM Tris-buffer, pH 7.4, containing 0.4 M NaC1, 0.25% Tween 20 (Bio-Rad Laboratories), 0.25% NP-40, and 2 mM EDTA. Autoradiography was performed for 16-48 h with intensifying screens. The Mr standards used con-
136 sisted of prestained protein markers obtained from Amersham (Arlington Heights, IL). To assay for C8bp/HRF, immunodothlotting was performed on PVDF membranes using the Bio-Dot apparatus from Bio-Rad. 50/tl of protein samples were added to each well and allowed to sit for 4-8 h. After blocking with gelatin-containing buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaC1, 2% glycine, 0.05% scdinm azide, 1% gelatin), the membrane was incubated with Tris-buffered saline, pH 7.4, containing 0.1% gelatin and 50 ~tg/ml of purified C8 for 4 h at room temperature. After three washes in detergent-containingbuffers, as above, the membrane was further incubated with rabbit anti-C8 antiserum at 1/50 dilution. The blots were developed with mI-labeled goat F(ab')2 anti-rabbit IgG as before.
was centrifuged and 70/~1 of the supernatant was collected from each well for determination of radioactivity and the percentage of cytotoxi~ty (Jiang et al., 1988).
Competition radioimmunoassay Protein A-purified IgGs specific for antibodies derived against mouse perforin, and human C8 and C9 were labeled with 125I to specific activities of 3000-5000 cpm per ng of protein, using the Iodogen (Pierce) procedure (Fraker and Speck, 1978). 50 /tl of a CSbp/HRF suspension (at 25 /tg/ml in PBS), partially purified on a TSK-G4000 column, were allowed to sit in each well in flexible plastic dishes for 4 h. Plates were then washed with PBS and blocked with 1% gelatin in PBS for 4 h. A mixture of 125I-labeled IgGs at 10,000 cpm and various cold IgGs of given specificities and at indicated concentrations were added to each well to a final volume of 50 ml and incubated for I h at 4°C. After washing (as in immunoblotting), the individual wells were counted in a gamma counter. The percentage of inhibition of binding produced by the indicated cold IgGs was determined as {1 - (cpm of binding observed in the presence of the cold IgG)/(cpm of binding observed in the presence of cold IgG specific for DNP-BSA)}.
Anti-idiotypic antibodies bind to a 65 kDa membrane protein Polyclonal antisera of three different specificities were derived against IgGs specific for mouse perforin and for human C8 and C9. These antisera were screened by immunoblottingfor their reactivities against proteins of various cell types (Fig. 1; data shown here only for human and mouse E). Anti-idiotypic antisera of all three specificities reacted with unique protein bands of 65 kDa. reactive band of this molecular mass was observed in all cell types tested (in addition to E shown in Fig. 1, two CTLL and four tumor cell lines were tested). The following antisera were shown to react against the 65 kDa protein band: nine out of 15 anti-idiotypic antisera against anti-perforin IgG, six out of nine antisera against anti-C9 IgG, and four out of six antisera against anti-C8 IgG. Reactivity was observed only when cell proteins were disulfide-reduced as non-reduced blots did not react with any of the antisera used (Fig. 1). Preimmune sera used as controls did not give any specific reaction with the cell lysates tested (Fig. 1). As all three a~,~ti-idiotypic antibodies reacted with E ghost membranes, it is likely that the antigen is localized on the cell surface. However, attempts to localize the antigen by immunofluorescence or FACS analysis have failed to give any specific signal (data not shown), a result consistent with the fact that the antibodies described
Cytotoxicity assay Perforin-mediated lysis of tumor cells was detected by 51Cr release, as previously described (Jiang et ai., 1988). Briefly, 50 HU of purified murine perforin were mixed with 5 x 104 51Crlabeled JY-5 or JY-25 cells in 140 #1 serum-free RPMI 1640 m a round-bottom well of a microtiter plate. After incubation at 37°C for 3 h, the plate
Immunofluorescence and fluorescence-activated cell sorter (FA C$) analysis Indirect immunofluorescence was carried out exactly as described (Liu et al., 1987) and FACS analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA) using anti-idiotypic antisera at 1/50 dilution as primary antibodies, followed by fluorescence-conjugated goat anti-rabbit IgG F(ab')2 (Tagn, Burlingame, CA).
Results
137
g
I
2
I
2
I
2
I
2
1
2
1
2
1
2
93~ 69~
46~ +SH -SH --SH -SH +SH +SH Fig. 1. lmmunoblots of cell iysate prc:,ins re~,;ted with anfi-idiotypic antisera. 5 x 104 cell equivalents of either human (1) or mouse (2) E were electrophoresed per lane, and reacted with anti-idiotypic antise::a derived against IgG specific for mouse perfofin, and human C~ and (::9. Th,~ ~ three ~ti-idiotypic anfisera are indicated by aaPFP, aaC8 and aaC9, respectively. Cell proteins were eleetrophoresod in either reducing (+SH) or non-reducing ( - S H ) conditions. For the last two lanes, the pre-inanunv ~ were obtained from the three rabbits that yielded the anti-idiotypic sera, each of which was used at a dilution of 1/50. Autoradiography was performed for 16 h.
h e r e only detect a n t i g e n s w h e n t h e y h a v e b e e n disulfide-reduced.
Demonstration that the antibody-reactive 65 kDa protein is H R F Previous studies h a v e s h o w n t h a t C S b p / H R F h a s a m o l e c u l a r m a s s o f 65 k D a , s u g g e s t i n g to u s
t h a t o u r a n t i b o d i e s m a y react with this molecule. H R F is a b s e n t f r o m t y p e lII E b u t p r e s e n t in t y p e I E o f P N H p a t i e n t s ( H a n s c h et al., 1987; Z a l m a n et ~ . , 198To). T y p e III E d o n o t c o n U d n p h o s p h a t i d y l i n o s i t o l glycan a n c h o r s (Low a n d S~tiel, 1988), s u g g e s t i n g t h a t H R F is a n c h o r e d to E m e m b r a n e s via a p h o s p h o i n o s i t o l linkage. T h e
B A
~
g
g
g
~
o
~
~
~,
=
~
93--
93-69-
69--
46-
46--
g I
I
Type IE
I
J
Type ITTE
I
I I
JY-25
I
JY-5
Fig. 2. Immunoblots of cell lysate proteins reacted with anti-idiotypic antisera. 5 × 10" cell equivalents from E (A) or J'Y cells (B) were electrophoresed per lane and reacted under reducing conditions with the indicated anti-idiotypie anfisera (same notation as that shown in the legend to Fig. 1). In A, type I and type Ill E from a PNH patient were obtained as described in the materials and methods section. In B, either ~ - 2 5 or YY-5 cell proteins were reacted with the indicated antisera. Autoradiography was performed for 48 h.
138 1
2
3
4
5
93 m 69--
46-30-Fig. 3. Anti-kh'otypic andsera react against CSbp/HRF. Lane
l: SDS-PAGEprofde of CSbp/HRF of human E, eiuted from a Superose 12 cohmm, as described in the materials and methods section. 20 nag protein was applied per lane. Eleetrophoresis was performed under reducing conditions. Lanes 2-5: C8bp/HRF, eleetrophoresedas in lane 1, was transferred to blots and reacted with aaPFP 0ane 2), aaC8 (lane 3), aaC9 (lane 4), and pre-immune serum (lane 5). The notations used for the anti-idiotypicantisera are the same as those given in the legend to Fig. 1.
immunoblot analysis in Fig. 2A (data shown here for only one patient; similar data were obtained for all four PNH patients) indicates that all three types of antisera reacted against a 65 kDa protein |00
........ J ........ '
.........
band of type I E and of normal, untreated E but not against proteins derived from type III E of P N H patients. H R F is also absent from the mutant B lymphoblastoid cell line JY-5 but not from the parental cell line JY-25 (Hollander et ai., 1989). As shown in Fig. 2B, a protein band of 65 kDa from JY-25 cells reacted with all three types of antisera. This same band was absent from JY-5 cells (Fig. 2B). C S b p / H R F was partially purified by sequential appfication of human E membrane proteins through DEAE-Sepharose and Superose 12 columns and by using a sandwich immunobloldng assay for screening active fractions (see materials and methods section). This assay relied first on binding of C S b p / H R F to C8, followed by reaction with anti-C8 antibodies (Schonermark et al., 1986). The activity eluted from the Superose 12 column contained a major band of 65 kDa (see gel profile in Fig. 3, lane 1). This band was shown to react with all three types of anti-idiotypic antisera (Fig. 3, lanes 2-4) but not with pre-immune sera (lane 5). Taken together, these data indicate that the anti-idiotypic antisera described here react against C 8 b p / H R F .
'
:'C
'
...... '
........ "
50
!
2
5
I,I
0.1
,/ k
~
. . . . . . . .
,
! .0
. . . . . . . .
I
10
.
. . . . . . . .
i,i
100
0.1
! .0
10
100
0.1
........
i
1.0
........
i
10
........
i
100
Concentration o f I g G (gg]ml) Fig. 4. Competition solid-phase radioimmunoassay showing that the anti-idiotypicantisera react against overlappingepitopes on CSbp/HRF. Plates were coated with partially purified C8bp/HRF and reacted with 125I-labaledIgO of one of three speeificities (aaPFP in A; aaC8 in B; and aaC9 in C) and the followingcold IgGs at the given concentrations: aaPFP (o), aaC8 (it), and aaC9 (,,). The ~ inhibitionof binding was determined as describedin the materials and methods section,
139 100 F A "'"""~
'
!75Iff ~
....... ' ........ ' ........ '
]
1 ~~ o.1
~ l.o
-~ 1o
loo
t~ . . . . . . . ÷ .......~ . , o.l
1.o
1o
1oo
o.!
Lo
1o
loo
Concentration of IgG ( g g / m l ) Fig. 5. Assessmentof the idiotypic-anti-idiotypicreaction.The experimentalprotocolwas the sameas that in Fig.4, exceptthat the followingcold IgGswere used for competition:anti-PFPlgG (or aPFP; o), aC8 (®), aC9 (A),and aDNP-BSA(A).123blabeled lgGswereof the followingspeciflcities:(A) aaPFP, (B) aaCS,and (C) aaC9.
Anti-idiotypic antibodies react against overlapping or same epitopes on C8bp / HRF The possibility that the three types of antisera react against a unique epitope on C 8 b p / H R F was assessed by a competition radioimmunoassay. l~I-labeled IgGs of one of the three given specificities were reacted against partially purified human E C S b p / H R F eluted from a TSK G-4000 column. The extent of inhibition in binding produced by cold IgGs of the same Or different specificities was quantified (Fig. 4). The three unlabeled, anti-idiotypicIgGs competed avidly for binding to C S b p / H R F mediated by themselves and by each other (Fig. 4). We also tested the inhibitory effect of cold IgGs specific for perforin, C8, C9, and DNP,BSA on the binding reaction between ~25I-labeled antiidiotypic IgG and CSbp/HRF-coated plates. As shown in Fig. 5, IgGs specific for perforin, C8 and C9, but not anti-DNP-BSA IgG, inhibited this binding reaction in a dose-dependent manner, confirming that the labeled antibodies described here are anti-idiotypic since they must have bound to epitopes of the anti-perforin, anti-C8 or anti-C9 IgGs close to or in their antigen-combining sites. "[he lack of inhibitory effect associated with the presence of IgG specific for DNP-BSA (Fig. 5) or
IgG from preimmune serum (not shown) rules out the possibility that the inhibitory effect observed here could be attributed to nonspecific binding between the l~I-labeled anti-idiotypic IgGs and non-idiotypic determinants on the cold IgGs.
C8bp / H R F is ineffective in protecting cells against perforin-mediated &sis The results presented above would be consistent with the view that C 8 b p / H R F confers
80[ 6O
40
2O o
dY-5
dY-25
Fig. 6. C8bp/HRF-deficientmutant cellsare as susceptibleto perforin-mediatedlysisas wild-typecells.JY-25 and JY-5 cells were exposedto purifiedperforin and SICr releasewas measured as describedin the materialsand methodssection.Data representquadriplicates+ standarddeviation.
140
resistance to ceils against both perforin- and homologous C-mediated lysis, as suggested (Zalman et al., 1987a, 1988; Miiller-Eberhard, 1988). Since HRF is absent from type III E of PNH patients, it would follow then that these E should be more sensitive to perforin-mediated lysis than normal or type I E. We and others have recently shown, however, that type III E are indeed more susceptible to lysis mediated by homologous C than control or type I E, but they are as susceptible to perforin-mediated lysis as type I E (Hollander et al., 1989; Jiang et ai., 1989; Krahenbuhl et al., 1989). These results are further co, roborated by experiments done here with JY-5 and JY-25 ceils. As shown in Fig. 6, the HRF-deficient JY-5 cells are just as susceptible to perforin-mediated lysis as JY-25 cells, further supporting the notion that HRF does not play a role in protecting against perforin-mediated lysis.
Discussion The idiotypic network theory of Jeme (1974) proposes that idiotypes can act as immunogens. Anti-idiotypic antibodies elicited this way have been used in the past to identify receptors and other binding proteins (Pain et aL, 1988). We reasoned that, for a candidate surface protein to function as a 'protectin', it should bind first to perforin and that this interaction could presumably be mimicked by an idiotypic-anti-idiotypic reaction. Anti-idiotypic antibodies elicited against anti-perforin antibodies were shown here to bind to C8bp/HRF. Anti-idiotypic antibodies raised similarly against anti-C8 and anti-C9 antibodies also detected C 8 b p / H R F and in fact, the three antibodies were shown to compete with each other, apparently for the same or overlapping binding site(s) on C8bp/HRF. It should be noted that, by using the same strategy, several antisera with the same reactivities were obtained, indicating that these results cannot be explained by non-specific or spurious reactivities conferred by in&vidual batches of antisera. Since C8 and C9 are readily available now in large quantities, the procedure described here provides a convenient way of deriving CSbp/HRF-specific antibodies that could be used for further analysis of this polypeptide.
The main evidenc~ used to argue in favor of a role for C 8 b p / ! i R F in protecting lymphocytes against perforin-mediated lysis is that isolated HRF, reconstituted into sheep E membranes by a detergent-dilution protocol, protects these cells against lysis mediated by both homologous C and perforin (Zahnan et al., 1987a). The results obtained here with anti-idiotypic antibodies would appear to be consistent with this interpretation. It was therefore surprising to find out that type III E from PNH patients, known to lack C8bp/HRF, are highly susceptible to lysis mediated by homologous C but are equally susceptible to perforin-mediated lysis, when compared to normal human E (Hollander et al., 1989; Jiang et al., 1989; Krahenbuhl et al., 1989). These latter results, and the cytotoxicity data presented here on JY-5 and JY-25 cells, argue clearly against a role for I-IRF in restricting perforin-mediated lysis. It is paradoxical that perforin and C 8 b p / H R F should share antigenic determinants that are complementary and yet the latter should not play any role in restricting perforin-mediated lysis. Since antibodies raised against perforin, C8, and C9 share the same idiotypes or antigenic determinants, it is likely that perforin, C8, and C9 are structurally homologous in the region where C8 and C9 bind to C8bp/HRF. This inference is consistent with recent cloning studies which have revealed the existence of partial amino acid identity between perforin, C8, and C9 (Lichtenheld et al., 1988; Shinkai et al., 1988b; Kwon et ai., 1989; Lowrey et al., 1989). It is possible that additional domains, present only on C8 and C9, are involved in rendering these molecules susceptible to CSbp/HRF-mediated block on pore-formation. We speculate that C S b p / H R F is also structurally homologous to perforin, C8, and C9, as indicated by preliminary findings that antibodies raised against the disulfide-reduced forms of C8 and C9 react against C S b p / H R F (unpublished). The anti-idiotypic antibodies described here only reacted with C 8 b p / H R F in its disulfide-reduced form, suggesting that a major structural rearrangement is necessary before the idiotypic determinant is exposed. It is not clear whether a similar conformational change could occur when C 8 b p / H R F is extracted and reincorporated into E membranes, in which condition it may conceivably have some
affinity for perforin, and, at sufficiently high c o n centrations, it m a y exert s o m e protective function against perforin-mediated lysis, as a d v o c a t e d b y Z a l m a n et al. (Zalman et al., 1987a). F u r t h e r structural studies are w a r r a n t e d to substantiate these a n d any o t h e r speculations. T h e molecular basis o f the perforin-resistance p h e n o m e n o n remains to b e determined. Since H R F , the only putative protective factor studied to date, does n o t s e e m to explain this type o f reaction, other protective molecules (protectins) n e e d to b e sought. It should b e n o t e d that p e r forin-resistance is a n inducible p h e n o t y p e a n d appears to correlate with the level o f cytotoxicity o f t h e effector cells (Nagler-Anderson et al., 1988; Liu et al., 1989). T h e s a m e p h e n o m e n o n has b e e n described for yeast killer toxin, against which toxin-producing yeasts are resistant (Boone et al., 1986). I n general, it would m a k e sense for any cytolytic cell type to acquire s o m e f o r m o f defense against d a m a g e t h a t might b e inflicted b y its o w n lyric mediators.
Acknowledgements W e t h a n k R a g h u r a m G o r t i a n d Leila Posaw for excellent technical assistance, Drs. D a v i d M. Ojcius a n d Sanjay 30ag for critical reading o f the manuscript, a n d Dr. Zanvil A. C o h n for continuo n s s u p p o r t a n d advice. This w o r k was s u p p o r t e d in p a r t b y grants f r o m the Cancer Research I n s t i t u t e / F r a n c e s L. a n d E d w i n L. C u m m i n g s Memorial F u n d Investigator A w a r d a n d the N.I.H. (CA-47307) a n d b y a Scholarship f r o m the Lucille P. M a r k e y Charitable Trust. Post-doctoral support was f r o m the C a n c e r Research Institute (S.J.) a n d the Irvington H o u s e Institute for Medical Research (C.-C.L.).
References Blakely, A., Gorman, K., Ostergaard, H., Svoboda, K., Liu, C.-C., Young, J.D.-E and Clark, W.R. (1987) Resistance of cloned cytotoxic T lymphocytes to cell-mediated cytotoxicity. J. Exp. Med. 166,1070. Boone, C., Bussey, H., Greene, D., Th6mas, D.Y. and Vernet, T. ¢1986) Yest killer toxin: site-directed mutations ira-
pficate the precursor protein as the immunity component. Cell 46,105. Chow, F.-L., Hall, S.E., Rosse, W.F. and Telen, M.J. (1986) Separation of the acetylcholineslerase-deflcient red cells in paroxysmal nocturnal hemoglobinuria. Blood 67, 893. Fraker, P.J. and Speck, J.C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6*tetrachloro-3a,6a-diphenylglycoluril. Binchem. Biophys. Res. Commun. 80, 849. Hammer, C.H., Wirtz, G.H., Renfer, L., Gresham, H.D. and Tack, B.F. (1981) Large scale isolation of functionally active components of the human complement system. J. Biol. Chem. 256, 3995. Hansch, G.M., Hammer, C.H., Vanguri, P. and Shin, M.L. (1981) Homo!J~ous species restriction in lysis of erythrocytes by terminal complement proteins. Proc. Natl. Acad. Sci. U.S.A. 78, 5118. Hansch, G.M., Schonermark, S. and Roelcks, D. (1987) Paroxysmal nocturnal hemoglobinuria type III: lack of an erythrocyte membrane protein restricting the lysis by C5b-9. J. Clin. Invest. 80, 7. Henkart, P.A. (1985) Mechanism of lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 3, 31. Hollander, N., Shin, M.L., Rosso, W.F. and Springer, T.A. (1989) Distinct restriction of complement- and cell-mediao ted lysis. J. ImmunoL 142, 3913. Hu, V.W. and Shin, M.L. (1984) Species-restricted target cell lysis by human complement: complement-lysed erythrocytes from heterologous and homologous species differ in their ratio of bound to inserted C9. J. ImmunoL 133, 2133. Jerne, N.K. (7974) Towards a network theory of the immune system. Ann. Inst. Pasteur 125C, 373. Jiang, S., Persechini, P.M., Zychlinsky, A., Lin, C.-C., Perassia, B. and Young, J.D.-E (1988) Resistance of cytolytic lymphocytes to perforin-mediated kilting: Lack of correlation with complement-associated homologous species restriction. J. Exp. Med. 168, 2207. Jiang, S., Persechini, P.M., Rosse, W.F., Perussia, B. and Young, J.D.-E (1989) Differential susceptibility of type III erythrocytes of paroxysmal nocturnal hemoglobinuria to lysis mediated by complement end perforin. Biochem. Biophys. Res. Commun. 162, 316. Krahenbuhl, O.P., Peter, H.H. and Tschopp, J. (1989) Absence of homologous restriction factor does not affect CTL-mediated ¢ytolysis. Eur. J. lmmunol. 19, 217. Kwon, B.S., Wakulchik, M., Lin, C.-C., Persechini, P.M., Trapani, J., Haq, A.K., Kim, Y. and Young, J.D.-E (1989) The structure of the lymphocyte pore-forming protein perforin. Biochem. Biophys. Res. Commun. 158, I. Lichtenheld, M.G., Olsen, K., Lu, P., Lowrey, D.M., Hameed, A., Hengarmer, H. and Podack, E.R. (1988) Structure and function of human perforin. Nature 335, 448. Liu, C.-C., Steffen, M., King, F. and Young, J.D.-E (1987) Identification, isolation, and characterization of a novel cytotoxin in routine cytolytic lymphocytes. Cell 51, 393. Lin, C.-C., Jiang, S., Persechini, P.M., Zychlinsky, A., Kaufmann, Y. and Young, J.D.-E (1989) Resistance of cytolytic lymphocytes to perforin-mediated killing, Induction of re-
142 sistance correlates with increase in cytotoxicity.J. Exp. Med. 169, 2211. Low, M.G. and Salfiel, A.R. (1988) Structural and functional roles of glyco~l-phosphatldylinositolin memhrunes. Science 239, 268. Lowrey, D.M., Aebischer, T., Olsen, K., Lichtenheld, M., Rupp, F., Hengarmer, H. and Podack, E.R. (1989) Cloning, analysis, and expression of routine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9. Proc. Natl. Acad. Sci. U.S.A. 86, 247. Mllller-Eberhard, HJ. (1988) The molecular basis of target cell killing by human lymphocytes and of killer cell self-protection. Immonol. Rev. 103, 87. Nagler-Anderson, C., Verret, C.R., Firmenich, A.A., Berne, M. and Eisen, H.N. (1988) Resistance of primary CD8 + cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines. 3. Immanul. 141, 3299. Pain, D., Kanwar, Y.S. and Blobel, G. (1988) Identification of a receptor for protein import into chloroplasts and its locafization to envelope contact zones. Nature 331, 232. Persechini, P.M. and Young, J.D.-E (1988) The primary structure of the lymphocyte pore-forming protein perforin: partial amino acid sequencing and determination of isoelectric point. Biochem. Biophys. Res. Commun. 156, 740. Podack, E.R. (1988) Granule-mediated cytolysis of target cells. Curt. Top. Microbial. ImmunoL 140,1. Schonermark, S., Rauterberg, E.W., Shin, M.L., Loke, S., Roelcke, D. and Hunsch, G.M. (1986) Homologous species resW.'ctiun in lysis of human erythrocytes: a membrane-derived protein with CS-binding capacity functions as an inhibitor. J. Immunol. 136,1772. Shin, M.L., Hansch, G., Hu, V.W. and Nicholson-Weller, A. (1986) Membrane factors responsible for homologous species restriction of complement-tnediated lysis: evidence for a factor othe~ than DAF operating at the stage of C8 and C9. J. ImmunoL 136,1777. Shinkai. Y., Ishikawa, H., Hattori, M. and Okumura, K. (1988a) Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis. Eur. J. Immanol. 18, 29. Shinkal, Y., Takio, K. and Okumura, K. (1988b) Homology of perforin to the ninth component of complement (C9). Nature 334, 525.
Tschopp, J. and Jongencel, C.V. (1918) Cytotoxic T lymphocyte mediated cytolysis. Biochemistry 27, 2641. Unkeless, J.C. (1977) The presence of two Fc receptors on mouse macrophages: Evidence from a variant cell line and differential trypsin sensitivity. J. Exp. Med. 145, 931. Verret, C.R., Firmeulch, A.A., Kr~nz, D.M. and ~sen, H.N. (1987) Resistance of cytotoxic T lymphocytes to the lyric effects of their toxic granules. J. Exp. Med. 166,1536. Young, LD.-E and Cohn, Z.A. (1987) Cellular and humoral mechanisms of cyV0toxicity: structural and functional analogies. Adv. Immunol. 41, 269. Young, J.D.-E, Hengartner, H., Podack, E.R. and Cohn, Z.A. (1986a) Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell 44, 849. Young, J.D.-E, Cohn, Z.A. and Podack, E.R. (1986b) The ninth component of complement and the pore-forming protein (Perforin 1) from cytotoxic T cells: structural, immunologicul, andfunctionul similarities. Science 233,184. Young, J.D.-E, Liu, C.-C., Leong, L.G. and Colin, Z.A. (1986c) The pore-forming protein (Perforin) of cytolytic T lymphocytes is immonologlcally related to the components of membrane attack complex of complement through cysteine~-rich domains. J. Exp. Med. 164, 2077. Zvlmav. L.S., Wood, L.M. and Mtiller-Eberhurd, H.J. (1986) Isolation of a human erythzocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels. Proc. Natl. Acad. Sci. U.S.A. 83, 6975. Zalman, L.S., Wood, L.M. and Miiller-Eberhar~ H.J. (1987a) Inhibition of antibody-dependent lymphocyte 6yt_otoxicity by homologous restriction factor incorporated into target cell membranes. J. Exp. Med. 166, 947. Zalman, L.S., Wood, L.M., Frank, M.M. and Mtiller-Eberhard, H.J. (1987b) Deficiency of the homologous restriction factor in paroxysnTalnocturnal hemoglobinuria. J. EXp. Med. 165, 572. Zulman, L.S., Brokers, M.A. and Mllller-Eberhard, H.J. (1988) Self-protection of cytotoxic lymphocyte.s:A soluble foml of homologous restriction Tactor in cytoplasmic granules. Prco. Natl. Acad. Sci. U.S.A. 85, 4827.
