Proc. Natl. Acad. Sci. USAVol. 89, pp. 4353-4357, May 1992Immunology

Evidence for a functional receptor for cyclosporin A on the surfaceof lymphocytes

(immunosuppression/cyclophilin/tolerance/membranes/transplantation)

NICHOLAS A. CACALANO*, B.-X. CHEN*, W. Louis CLEVELAND*t, AND BERNARD F. ERLANGER*t*Department of Microbiology, Columbia University, New York, NY 10032; and tDepartment of Medicine, St. Lukes/Roosevelt Hospital Center, New York,NY 10019

Communicated by Harold S. Ginsberg, February 7, 1992 (received for review March 12, 1991)

ABSTRACT Cyclosporin A (CsA) is an immunosuppressiveagent that inhibits the synthesis of lymphd by T lympho-cytes at the level of transcription. A cytoplasmic protein, cyclo-philin, is the most thoroughly studied CsA-binding protein, butits ubiquitous presence in cells of all types raises questions aboutits role in immunsuppression. In an attempt to ascertain thepresence of a cell surface receptor, we synthesized two polyva-lent macromolecular CsA derivatives, CsA-BBa-ovalbumin andCsA-BBa-aminodextran (CBD), from the product of the photo-chemical reaction ofCsA and 4-benzoylbenzoic acid (CsA-BBa).(t) They inhibited the peptidylprolyl cis-trans isomerase activityof cyclophilin and the synthesis of interleukin 2 by phorbolester-activated EL-4 cells. (fi) CBD also inhibited interleukin 2secretion by Con A-activated T-cell-enriched mouse splenocytes.4-Benzoylbenzoic acid (BBa)-aminodextran and aminextranwere inactive. (iii) Direct binding and competition studies with(3HJCsA indicated that CBD does not enter EL-4 cells (i.e., itacted at the surface). (iv) CBD caused agglutination of EL-4cells, murine B and T lymphocytes, human thymocytes, and twoT-cell hybridomas. Agglutination was inhibited by a monoclonalantibody to CsA and by CsA and CsA-BBa, but not by BBa. Noagglutination was seen with BBa-aminodextran or aminodex-tran. HeLa cells, Vero (monkey kidney) cells, a mouse plasma-cytoma, COS cells, and a poorly differentiated B-cell lymphomawere not agglutinated. (v) EL-4 cells failed to be agglutinatedafter treatment with trypsin or chymotrypsin. Specific aggluti-nation was again possible after incubation for 5 h at 3TC in theabsence of enzyme. (vo) CBD covalently linked to crosslinkedagarose beads inhibited interleukin 2 production by phorbolester-stimulated EL-4 cells. No activity was seen if cell-to-beadcontact was prevented by a 0.02-,gm microporous filter that didnot interfere with the passage of CBD. Our findings support thepresence of a functional receptor on the surface of selected cellsof the immune system.

Cyclosporin A (CsA) is a cyclic undecapeptide whose im-munosuppressive properties were documented by Borel andhis colleagues in a series of publications beginning in 1976(1-6). In 1977, Calne and White (7) demonstrated that CsAcould prolong the survival oforgan allografts in rats and dogs.CsA is now widely used in human recipients of kidney, liver,heart, combined heart-lung, and bone marrow transplantsand most recently in the treatment of such autoimmunediseases as uveitis and type I diabetes (8, 9).The nature of the functional receptor(s) for CsA has

become an issue of intense interest. Merker and Handschu-macher (10), in their early studies, reported that between 70%oand 80% of CsA concentrated by a T-cell lymphoma(BW5147) was located in the cytoplasm. The macromoleculeresponsible for binding had a molecular weight of 18,000 andwas called cyclophilin. It is now known that there are

isoforms of cyclophilins, which make up a conserved class ofproteins, all of which bind CsA (11, 12). Cyclophilin is nowconsidered by many to be the likely site of action of CsA, butits ubiquitous presence in cells of all types, including cells notinvolved in the immune response (13), raises questions aboutits role in immunosuppression. Interest in cyclophilins hasbeen intensified by the finding that they are enzymes-namely, peptidylprolyl cis-trans isomerases (14, 15)-theactivity of which is inhibited by CsA. The connection be-tween immunosuppression and enzymic activity, however,remains obscure, as does its link to transport of CsA andanalogues into the cell (16).The immune system is a network in which cells that

participate communicate with one another by means oflymphokines that act on cell surface receptors. It seemedlogical, therefore, to consider the possibility that CsA mightact on a cell surface receptor. There have been reports ofCsA-binding activity in membranes of some cells (17, 18).However, the question ofthe existence ofa membrane-boundreceptor is difficult to resolve because CsA is lipophilic. Anapproach to this problem has become possible as a result ofour recent synthesis of a CsA derivative [CsA-BBa; theproduct of the photochemical reaction of CsA and 4-ben-zoylbenzoic acid (BBa)] (19) that is biologically active andthat, because it contains a carboxyl group, can be convertedinto another family of CsA derivatives. In this paper, wereport findings that support the presence of a functional CsAreceptor on the surface of lymphocytes.

MATERIALS AND METHODSCsA was from Sandoz Research Institute (East Hanover,NJ). Cyclophilin was generously supplied by R. E. Hands-chumacher (Department of Pharmacology, Yale University).CsA-BBa Derivatives. Two high molecular weight, water-

soluble polyvalent derivatives ofCsA-BBa were prepared bycovalent linkage to ovalbumin (OVA) and to aminodextran.CsA-BBa-OVA was prepared in the same way as CsA-BBa-bovine serum albumin (19). The aminodextran derivative(CsA-BBa-aminodextran; CBD) was prepared as follows.The N-hydroxysuccinimide ester of CsA-BBa (19) preparedfrom 5.0 mg (-0.4 ,mol) of CsA-BBa in 0.3 ml of dimeth-ylformamide was added in 100-pLI aliquots to 5 mg of amino-dextran (Mr 70,000, 30 amino groups per molecule; MolecularProbes) in 0.5 ml of dimethyl sulfoxide. The solution wasallowed to stand overnight at room temperature; 3.2 ml ofdistilled water was then added. The reaction mixture wasthen dialyzed against phosphate-buffered saline (PBS) for 20h at 40C and passed through a Sephadex LH-20 column toremove low molecular weight components. Substitution was

Abbreviations: CsA, cyclosporin A; BBa, 4-benzoylbenzoic acid;CsA-BBa, the product of the photochemical reaction of CsA andBBa; CBD, CsA-BBa-aminodextran; OVA, ovalbumin; PMA, phor-bol 12-myristate 13-acetate; IL-2, interleukin 2.*To whom reprint requests should be addressed.

4353'

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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estimated to be 10 molecules of CsA-BBa per molecule ofaminodextran, based on a known amount of [3H]CsA-BBaN-hydroxysuccinimide ester in the reaction mixture. Theprocedure for the removal ofuncoupled CsA-BBa was testedby using mixtures of CsA-BBa (a portion of which wastritiated) and aminodextran, which were dialyzed and passedthrough Sephadex LH-20 to remove low molecular compo-nents. The high molecular weight fraction was free of radio-activity. This procedure served as a control in each experi-ment, and, in addition, the high molecular weight fractionwas tested for biological activity.CBD was covalently linked to Reacti-Gel (6x) agarose

beads (no. 20260; Pierce), as follows. CBD was concentratedto about 4 mg/ml and dialyzed against 0.1 M NaHCO3/Na2CO3, pH 9.0 overnight at 4TC. The solution (2.5 ml) wasthen added to 3 ml of Reacti-Gel (6x) beads and mixedovernight at 40C. The efficiency of coupling was determinedfrom the amount of radioactivity in the supernatant; about35-50%o ofthe CBD remained with the beads. The beads werewashed with PBS to remove unreacted CBD, with methanolto sterilize the beads, and then with sterile PBS to remove themethanol. This was followed by exhaustive washing with BM(Iscove's modified Dulbecco's medium containing F-12 nu-trient mixture and 5% fetal calf serum) until interleukin 2(IL-2) production by activated EL-4 cells was no longerinhibited by supernatant (see below for assay). The washingprocedure consisted of repeated incubation of the beads inBM for 1 h at 370C, discarding the supernatant, and addingnew medium. In the assay, a control was always run in whichsupernatant was taken from beads that were incubated withBM for 48 h (see assay for immunosuppression below). Thefinal suspension of the beads used in the assays was in 3 mlof BM; 1 ml of beads contained 200 ,ug of CBD (-20%ocoupling efficiency).

Cells. The following cell lines were used in these studies:CTLL-2 (20), DO-11.10 (21), A20.2J (22, 23), P3X63-Ag8.65(24), 32-6.F12 (J. Vloka and W.L.C., unpublished), humanthymocytes, T-cell- and B-cell-enriched mouse splenocytes(25), HeLa cells, COS cells, Vero cells (monkey kidneyfibroblasts), and L-929 cells (mouse fibroblasts).

Assay for Immunosuppression by Soluble Agents by UsingEL-4 Cells and Nylon Wool-Nonadherent BALB/c Spleno-cytes. The conjugates were tested for their ability to inhibitthe production of IL-2 by EL-4 cells after stimulation byphorbol 12-myristate 13-acetate (PMA) (26). This process isinhibited by CsA (27). Prior to testing, both derivatives werepassed through Sephadex LH-20 to remove unreacted CsA-BBa. As a control, mixtures of CsA-BBa and aminodextranor OVA were passed through Sephadex LH-20, and the highmolecular weight fractions were tested for activity. A com-bination oftwo procedures was used (26,28). Before assayingCsA and the various derivatives, a standard curve wasdetermined for the effects of dilution of supernatants fromPMA-stimulated EL-4 cells on the viability of CTLL-2 cells.The dilution that gave 50o survival was selected for theassays. Results were consistent and reproducible. EL-4 cells(105 cells) in 250 ,l of BM were incubated in Corningflat-bottom 96-well plates with CsA-BBa-OVA, CBD, orcontrols in the presence ofPMA at 20 ng/ml for 40 h at 37°C.To quantitate IL-2 secretion, supernatant (8 ,ul) from eachwell was added to 200 ,ul of BM containing IL-2-dependentCTLL-2 (2 x 104) indicator cells. After 20 h at 37C, survivalof CTLL-2 cells was determined by the addition of 25 ,ul of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (5 mg/ml in sterile PBS) to each well, followed byincubation for 4 h at 37°C. Supernatant (150 ,l) from eachwell was removed, and 150 1A of isopropanol containing 0.04M HCO was added to each well and mixed with the cells todissolve crystals. Absorbance at 570 nm (vs. 630 nm refer-ence) was read on a Dynatech microplate reader. The back-

ground without cells was Zlot. As a control for the activityof the various derivatives, each assay included a standardcurve derived in the presence of CsA at concentrations from1 fM to 1 1LM. The dilution of supernatant obtained in thepresence of CsA that gave 50% viability of CTLL-2 cellsremained essentially unchanged from experiment to experi-ment. In one experiment, the assays were run over a range ofdilutions of CBD; the results were consistent with thoseobtained by the procedure described above.To assay for activity against stimulated murine T cells,

spleen cells from three BALB/c mice were enriched for Tcells by passage through nylon wool (25). The enriched T-cellpreparation was suspended in BM at a concentration of 2 x106 cells per ml. A portion of the cell suspension (100 IL) wasincubated with an additional 150 jld ofBM and Con A (Sigma;no. C2010) at 5 pug/ml, with and without inhibitors, for 48 hat 37rC. Supernatants were assayed for the presence of IL-2by using CTLL-2 cells as described above.An additional control was run to determine if CsA-BBa or

CBD affected the growth of CTLL-2 cells: neither did.Activity of CBD Reacti-Gel Beads. To show that cell-to-

bead contact was necessary for activity, the following ex-periments were run. BM (0.5 ml) orBM containing one ofthefollowing was introduced into Corning 24-well tissue cultureplates: CsA (0.1 AuM), CBD (1 ,uM), or CBD-Reacti-Gelagarose beads (a suspension of 0.5 ml). For each inhibitor,two wells were set up. EL-4 cells (0.5 ml of a suspension of106 cells per ml) were added directly to one of the pair; aninsert with a microporous filter (Nunc; no. 162332) wasinserted into the other well before the cells were added.Incubation and assay for IL-2 were as for the soluble com-pounds; samples for assay were taken from inside and outsideofthe cup with the microporous filter. There were two majorcontrols to eliminate the possibility that the cells, by someenzymic process, were releasing a soluble immunosuppres-sive agent. One was described above: supernatant from amixture of PMA-activated EL-4 cells and beads incubated at3TC for 48 h was assayed for immunosuppressive activity.None was found. A second control consisted of having cellsand beads (instead of beads only) in the outer compartment(in addition to the cells inside the insert). There was norelease of a soluble immunosuppressive component into theinsert containing EL-4 cells by the action of cell-associatedenzymes (i.e., the cells within the insert produced IL-2). Itshould be noted that the bond between CsA and BBa inCsA-BBa is a carbon-carbon bond. An amide bond linksCsA-BBa to aminodextran, but the low activity of CsA-BBarelative to that of CsA would require that almost all of it bereleased from the beads. Our controls also showed that themicroporous filter was permeable to CBD. (Unlike our expe-rience with the Reacti-Gel beads, we were unable to preventleakage from Affi-Gel 10 beads linked to CBD despite re-peated washing with medium.)

Cell Agglutination Assay. Cells (8-12 x 104) were added to200 ,lj of BM without fetal calf serum containing 50 "I ofeither PBS, aminodextran, BBa-aminodextran, or CBD at afinal concentration of 0.5 ,uM (35 pg/ml), in the presence orabsence of various inhibitors or solvent controls. The cellswere gently agitated on a platform shaker for 15 min at 250C,incubated without agitation for 2-3 h at 250C, placed in arefrigerator, and examined for agglutination periodically. Theend points usually became apparent after about 2-3 h.Treatment of Cels with Trypsin and Chyypin. Cells

were treated with trypsin or chymotrypsin at concentrationsof 1% and 5%, respectively, as described (29). Incubationwas at 370C for 5 min. Digestion was terminated by theaddition ofBM, centrifugation, and resuspension ofthe pelletin fresh medium for the agglutination assay. Treated cellssuspended in fresh medium were also kept at 370C and testedhourly for their ability to be agglutinated.

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Assay for Peptidylprolyl cis-trans Isomerase Activity. Pep-tidylprolyl cis-trans isomerase activity was measured by amodification of the previously described assay (14). Cyclo-philin (332 nM) in 35 mM Hepes (pH 7.8) was incubatedwithout inhibitor or in the presence of CsA, CsA-BBa, orCBD at increasing concentrations for 1 min on ice in apolycarbonate disposable cuvette. The test peptide (Suc-Ala-Ala-Pro-Phe-4-nitroanilide), dissolved in dimethyl sulfox-ide/35 mM Hepes, pH 7.8 (60:40, vol/vol) was added to afinal concentration of 45 uM, and the cuvette was removedfrom the ice bath and incubated for 2 min in a spectropho-tometer at room temperature. Any condensed moisture onthe window of the cuvette was removed, and the reaction wasstarted by adding chymotrypsin to a final concentration of 15kLM; absorbance was read at 390 nM at different time points.

Assay for the Entrance of CBD into EL-4 Cells. Twoprotocols were used. The first was modeled after that of Sigalet al. (16) in which displacement of [3H]CsA from thecytoplasm of cells was measured. EL-4 cells (106) wereincubated at 370C for 1 h with 4150,000 cpm of [3H]CsA(Amersham; 8 Ci/mmol; 1 Ci = 37 GBq) to allow entranceinto the cells. This was followed by incubation with theinhibitor (CsA or CBD) for periods from 3to 24 h. Assay forintracellular [3H]CsA was as described (16).The second assay was a direct determination ofthe entrance

of CBD into the cells by using radiolabeled CBD, which wasprepared as follows. CBD (1 mg in 500 1,l ofPBS, pH 7.5) wasincubated overnight in the cold with 250 Al (=0.5 ,Amol) of[3H]acetic anhydride (Amersham; no. TRK.2; 6 Ci/mmol) intetrahydrofuran. After exhaustive dialysis, the product wasassayed and estimated to have about two of its amino groupsacetylated, for a specific activity of 10 Ci/mmol.

Entrance into EL-4 cells was determined by the protocoldescribed above for [3H]CsA.

RESULTSInhibition of Peptidylprolyl cis-trans Isomerase Activity.

CBD inhibited prolylpeptidyl cis-trans isomerase activity;50% inhibition occurred at a concentration of about 100 nM(Fig. 1). For comparison, the data for CsA (50% inhibition at30 nM) and CsA-BBa (50% inhibition at 250 nM) are alsoshown. No inhibition by BBa-aminodextran occurred at aconcentration of 1 ,uM.Immunosuppression by CsA-BBa Conjugates. CsA-BBa-

OVA and CBD were tested for their ability to inhibit IL-2production by EL-4 cells stimulated by PMA. Both showeddose-dependent activity; 50% inhibition with the OVA con-jugate occurred at about 100 nM and with the aminodextranconjugate at about 60 nM (Fig. 2). Under the same conditions,50% inhibition by CsA was at about 2.5 nM.CBD was equally as active in suppressing IL-2 secretion by

Con A-stimulated murine T splenocytes (data not shown).As described in Materials and Methods, low molecular

weight material was removed from the macromolecular con-jugates by passage through Sephadex LH-20.

100OC 80-

60-

40-Ne 20- ,'

010-9 10-8 10-7 10-6 10-5

Inhibitor Concentration (M)

FIG. 1. Inhibition of peptidylprolyl cis-trans isomerase activityby CsA (o), CsA-BBa (o), and CBD (o).

CBD Does Not Enter the Cytoplasm ofEL-4 Cells. From theabove results, it appeared possible that the macromolecularconjugates were acting at a receptor on the surface of EL-4cells. On the other hand, the macromolecules might haveentered the cells by pinocytosis, either as intact molecules or,in the case of the OVA conjugate, as peptides after proteo-lytic digestion by a secreted cell component or by a compo-nent of the serum in the medium. With respect to the dextranconjugate, there are no known mammalian dextranases.

Entrance ofCBD into the cytoplasm ofEL-4 cells was firstinvestigated by a competition assay in which displacement of[3H]CsA from the cytoplasm by unlabeled CsA and by CBDwas determined. Shown in Table 1 are the results of a typicalexperiment. Competition by CsA was essentially complete;the remaining radioactivity was nonspecific. On the otherhand, no displacement of cytoplasmic [3H]CsA occurred inthe presence of CBD, indicating that CBD does not enter thecell. This was confirmed by a direct assay. In a typicalexperiment, 106 EL4 cells were exposed to tritiated CBD(78,700 cpm) at 37°C for 1, 4, and 24 h. Only backgroundcounts (=40 cpm) were found in the cells, which were morethan 80% viable after 24 h.

Agglutination of EL-4 Cells. If, in fact, there is a receptoron the cell surface, it should be possible for the polyvalent,macromolecular CsA-BBa derivatives to agglutinate EL-4cells. This possibility was tested, and the results of one ofmany replicate experiments are shown in Fig. 3. They can besummarized as follows:

(i) CBD (0.5 ,uM) agglutinated EL-4 cells.(ii) Agglutination was inhibited by CsA and CsA-BBa at

concentrations of 10 uM. BBa did not inhibit; neither BBa-aminodextran nor aminodextran agglutinated EL-4 cells.

(iii) A monoclonal antibody specific for CsA (30) inhibitedagglutination; PB001, a monoclonal IgG of unrelated speci-ficity, did not inhibit agglutination.

(iv) Treatment of the cells with trypsin or chymotrypsinabolished their ability to be agglutinated. It was restored byincubating the treated cells at 37°C in fresh medium for 5 h(data not shown).

Selectivity of Agglutination. The preponderance of evi-dence suggests that the in vivo immunosuppressive activity ofCsA is targeted at T cells, causing inhibition of the synthesisof lymphokines at the level of transcription (31-33), althougheffects on B cells have been observed in in vitro cell systems(34-36). We, therefore, examined the agglutination reactionfor its cell specificity (Table 2). We found agglutination oftwoother T-cell lines, DO-11.10 and 32-6.F12, both of which areT-cell hybridomas specific for OVA; in both cases, aggluti-nation was inhibited by CsA and CsA-BBa but not by BBa.32-6.F12 cells, after recognition of OVA, secrete IL-2, aprocess that is inhibited by CsA. We have no data on theeffect of CsA on DO-11.10. Specific agglutination was also

100-

c 80-0

n 60-'c 40-

0 20-

0-10-9nhbt C0-7ncntaton(MInhibitor Concentration (M)

FIG. 2. Inhibition of IL-2 secretion by CBD (e) and CsA-BBa-OVA (o). CsA caused 50%o inhibition at about 2.5 nM; CsA-BBacaused 50%o inhibition at about 175 nM. Supernatants from stimulatedEL-4 cells gave OD readings of 0.272 + 0.012; negative experimentsand controls gave OD readings of 0.030 + 0.008.

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Table 1. Competition assay for entrance of CBD into EL-4 cells

Component(s) cpm in cells

[3H]CsA 4808 ± 460[3H]CsA + CsA (2.5 ,uM) 745 ± 8[3H]CsA + CBD (48 AiM)* 4891 ± 232

Cells were incubated for 1 h at 370C in the presence of [3H]CsA(188,280 cpm). CsA or CBD was then added, and incubation wascontinued for 4 h. Radioactivity within the cells was then determined(see Materials and Methods).*Concentration as moles of CsA content.

seen with human thymocytes, T-cell-enriched mouse spleen-ocytes, and B-cell-enriched mouse splenocytes.On the other hand, no agglutination was seen with two

B-cell lines, P3x63-AG8.653, a murine plasmacytoma, and A20.2J, a murine B lymphoma line; agglutination was not seenwith HeLa, Vero, COS, or L-929 cells.Immunosuppression by CBD Linked to agarose beads. All of

the above findings are consistent with a functional CsAreceptor on the surface of lymphocytes. To test this possi-bility further, PMA-treated EL-4 cells were incubated withCBD that had been covalently linked to Reacti-Gel agarosebeads so that there was continuous contact between the cellsand the beads during the 48-h incubation period. The super-natant was assayed for IL-2 as described for the solubleagents. Immunosuppression occurred, which was not seen ifthe cells were separated from the beads by a microporousfilter that did not interfere with the passage of CBD or IL-2(Fig. 4). Various controls were run to ensure that no immu-nosuppressive component passed into solution by "leakage"or as a result of the action of EL-4 cells on the beads. Theseare described in detail in Materials and Methods.

DISCUSSIONOur findings can be summarized as follows:

(i) Two macromolecular, multivalent CsA derivatives,CsA-BBa-OVA and CBD, inhibit the cis-trans enzymic ac-tivity of cyclophilin and show immunosuppressive activity inthe EL-4 cell system. Although it is possible that the OVAderivative is fragmented by proteases and enters the cell asshort peptides, there are no known mammalian dextranases.

Table 2. Differential agglutination of cells by CBD

Positive agglutination* Negative agglutination

EL-4 (murine thymoma) P3 x63-AG8.63 (plasmacytoma)DO-11.10 (T-cell hybridoma) A20-2J (B-cell lymphoma)32-6.F12 (T-cell hybridoma) HeLa cellsHuman thymocytes Vero (monkey kidney fibroblasts)Mouse B splenocytes L-929 (mouse fibroblasts)

(nylon adherent)Mouse T splenocytes COS cells

(nylon nonadherent)*Agglutination was inhibited by CsA and by CsA-BBa but not byBBa. No agglutination occurred in the presence of BBa-amino-dextran or aminodextran.

(ii) Competition and direct binding assays confirm thatCBD does not enter EL-4 cells. Moreover, others havereported that Mr 70,000 aminodextran fluorescent conjugatesare membrane impermeant (37).

(iii) CBD inhibited IL-2 secretion by Con A-stimulatedmurine T splenocytes.

(iv) CBD agglutinated selected cells of the immune systemand had no effect on a number of nonimmune cells or on apoorly differentiated B-cell lymphoma. Agglutination wasinhibited by CsA, CsA-BBa, and an anti-CsA monoclonalantibody.

(v) Agglutination of EL-4 cells was no longer possible aftertreatment with trypsin or chymotrypsin. After 5 h at 37°C inthe absence of enzyme, agglutination was again possible.

(vi) CBD covalently linked to beads of cross-linked agaroseinhibited IL-2 production by activated EL-4 cells. Bead-to-cell contact was necessary.These findings strongly support the existence ofa functional

cell surface receptor on selected cells of the immune system.Consistent with the possibility that it could be a membrane-bound form of cyclophilin is the ability of CBD to inhibit thepeptidylprolyl cis-trans isomerase activity of cyclophilin.Some "cyclophilin activity" has been reported to be asso-

ciated with unspecified cell membranes (38). There is also areport of a CsA-binding peptidylprolyl cis-trans isomerase

300-

200

NI Nlo Nli CD CDo CDi Bd Bdo BdiInhibitor

FIG. 3. Agglutination of EL-4 cells by CBD. All experimentswere done in duplicate. Rows B and C: column 2, PBS; column 3,aminodextran; column 4, BBa-aminodextran; column 5, CBD; col-umn 6, CBD in 0.5% ethanol; column 7, CBD plus BBa in 0.5%ethanol; column 8, CBD plus CsA-BBa in 0.5% ethanol. Rows E andF: column 2, CBD in 0.5% methanol; column 3, CBD plus CsA in0.5% methanol; column 4, CBD in 0.5% dimethyl sulfoxide; column5, CBD plus CsA in 0.5% dimethyl sulfoxide; column 6, CBD plusPBOO1 (50 ,g/ml); column 7, CBD plus mouse IgG (50 ,ug/ml);column 8, CBD plus monoclonal anti-CsA antibody H4 1.3.

FIG. 4. Immunosuppressive activity of CBD linked to agarosebeads. NI, no inhibitor present during a 48-h incubation of PMA-stimulated EL-4 cells; CD, CBD present during incubation; Bd, CBDagarose beads present during incubation. The "i" and "o" endingssignify aliquots taken from inside or outside, respectively, of themicroporous filter cup when it was present during the incubation ofthe EL-4 cells. OD 570/630 is related to viability of CTLL-2 cells,which require IL-2 for growth (see Materials and Methods). There-fore, low OD values indicate inhibition of IL-2 secretion by activatedEL-4 cells.

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with a putative membrane-spanning domain, a product of theninA gene ofDrosophila (11, 39). It has 45% sequence homol-ogy with human cyclophilin. It is postulated to participate inthe folding of rhodopsin, presumably by interactions withinthe membrane of the outer segment of rod cells.

Cyclophilin has also been reported in Neurospora crassa,in the cytoplasm and in mitochondria (40). In the latter it ispresumed to be involved in the proper folding of proteins thatpass through the mitochondrial membrane. Hence, it mightbe membrane associated.A peptidylprolyl isomerase, cloned from a human cDNA

library, has been found to have a hydrophobic leader se-quence that could presumably anchor it to a cell membrane(41, 42).Most recently, CD3- large granular lymphocytes have been

reported to have a cell surface molecule involved in bothrecognition and triggering natural killer cell-mediated lyticfunction (43), which, when cloned and sequenced, has 50%6homology to cyclophilin (S. K. Anderson, S. Gallinger, J.Roder, J. Frey, H. Young, and J. Ortaldo, personal commu-nication).

It is conceivable, therefore, that there is a lymphocyte-specific membrane-bound cyclophilin homologue that is im-portant in membrane signal transduction or in the folding ofmembrane-associated proteins and that its inhibition is as-sociated with the immunosuppressive action of CsA.On the other hand, the membrane-associated target may not

be a cyclophilin. In this regard, Tropschung et al. (40) reportedsignificant residual CsA-binding activity in mitochondria ofmutants of N. crassa lacking immunodetectable cyclophilin.There has been a recent report in which evidence was

presented for the binding ofcytoplasmic complexes ofFK506and its binding protein and of CsA and cyclophilin to acommon target, calcineurin, a calmodulin-dependent phos-phatase (44). No evidence is presented for the relevance ofthis biochemical finding to the process of immunosuppres-sion; and it is not claimed. Nevertheless, calcineurin has beenfound to be associated with the plasma membrane of T cells(45, 46), which could make it available for interaction with amembrane-associated CsA-cyclophilin complex. This couldbe followed by dephosphorylation and inhibition of a factorthat translocates into the nucleus and regulates transcriptionof IL-2 (47). The NFcB-IKcB system, which is controlled byphosphorylation of IKB, can serve as a model for this kind ofa mechanism (48).

If, as our findings indicate, a membrane-bound cyclophilin-related receptor functions in the immune response, what isthe role of cytoplasmic cyclophilin? Our data provide noanswer to this question. On the other hand, CsA produces anumber of toxic effects in treated patients. The action ofCsAon cytoplasmic cyclophilin might be involved in these effects.If so, compounds that target only the surface receptor couldprove to be selective immunosuppressive agents.

This work was supported by National Institutes of Health (NIH)Grants R01-NS-15581, R01-DA-06333, and P01-HL-36581 and NIHTraining Grants 2-T32-AI-07161 and T32-CA-09503. This paper ispart of a dissertation to be submitted by N.A.C. in partial fulfillmentof the requirements for the Ph.D. degree in Microbiology.1. Borel, J. F. (1976) Immunology 31, 631-641.2. Borel, J. F., Feuer, C., Magnee, C. & Stahelin, H. (1977) Immu-

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