738

Imaging the Rejecting HeartIn Vivo Detection of Major Histocompatibility

Complex Class II Antigen Induction

Mitsuaki Isobe, MD; Jagat Narula, MD; James F. Southern, MD, PhD;H. William Strauss, MD; Ban An Khaw, PhD; and Edgar Haber, MD

Background. Mice with abdominal heterotopic heart transplants were studied to determinewhether scintigraphic detection of an increase in major histocompatibility complex (MHC) classII antigen expression could be used as a noninvasive method for diagnosing early rejection.Methods and Results. Allografts from C3H/He (H2k) donors were transplanted into BALB/c

(112d) recipients (n=18). Two of the 18 allografted mice were treated with cyclosporine (15mg/kg/day), and two isografted mice served as controls. Each mouse was injected intravenouslywith 100 uCi of `1In-labeled anti-MHC class II monoclonal antibodies (10-2-16 and 14-4-4S) 24hours before scintigraphy. After imaging, the mice were killed for tissue counting and histopa-thology. Radiotracer uptake in the grafts reflected the severity of rejection as determined byhistopathological criteria. The percent injected dose per gram of tissue in excised grafts was4.8± 1.8 (mean± SD) for normal grafts (n=8), 11.1±9.7 for grafts with grade IA rejection (n=3,NS), 18.0±3.8 for grafts with grade IILA rejection (n =4, p <0.001 versus normal), 18.7±3.2 forgrafts with grade IIIB rejection (n=3, p<0.001 versus normal), and 22.6±5.4 for grafts withsevere rejection (grade IV) (n =3, p <0.001 versus normal). Rejecting allografts with lymphocyteinfiltration but without significant myocyte necrosis could be identified by this scintigraphicmethod. In the BALB/c donor-C57BL/6 (H2b, IE-) recipient combination, rejecting allograftswere visualized by 14-4-4S (anti-IE4P,r) antibody but not by 10-2-16 (anti-IAgrS.) antibody. Thisdifference shows that class II antigens induced on donor hearts are solely responsible for theantibody uptake in positive scintigrams of rejecting allografts.

Conclusions. We conclude that `1In-labeled anti-MHC class II antigen antibody imaging is asensitive and noninvasive method for detecting cardiac allograft rejection. (Circulation1992;85:738-746)

A lthough a variety of techniques for the detec-tion and quantification of cardiac allografttransplant rejection have been explored,

only histological inspection of myocardial specimensobtained through endomyocardial biopsy has gainedwidespread use.' Endomyocardial biopsy is invasive,costly, and prone to sampling error, and biopsyinterpretation is subjective and possibly arbitrary.We2'3 and other laboratories4-6 have shown that1"'In-antimyosin scintigraphy is useful for detectingmoderate and severe cardiac rejection in humanpatients and experimental animals. Myocyte necrosis

From the Cardiac Unit (M.I., E.H.), Department of Pathology(J.F.S.), and Nuclear Medicine Division, Department of Radiology(J.N., H.W.S., B.A.K.), Massachusetts General Hospital and Har-vard Medical School, Boston, Mass.

Supported in part by a grant from the Bristol-Myers SquibbPharmaceutical Research Institute.Address for correspondence: Mitsuaki Isobe, MD, Cardiac

Unit, Jackson 14, Massachusetts General Hospital, Boston, MA02114.

Received April 1, 1991; revision accepted September 24, 1991.

is essential to the detection of rejection by antimyosinscintigraphy in a mouse ectopic heart transplantationmodel.3 Because myocyte necrosis is an indicator ofrelatively advanced rejection, a more sensitive, non-invasive method of detecting cardiac transplant re-jection is desirable.The distribution of major histocompatibility com-

plex (MHC) antigens in various organs and tissues iswell documented in animals and humans.7'8 Normal,nucleated, nonlymphoid cells such as cardiac myo-cytes express low levels of MHC class I antigens(MHC K and D in mice) and do not express detect-able levels of MHC class II antigens (MHC IA andIE in mice). Recent immunohistological investiga-tions show that expression of class II antigens in-creases in rejecting organs,9-14 in tissues undergoingautoimmune injury,15-'7 in viral disease,18-20 and ininflammatory states.2' Enhanced class II antigenexpression correlates with clinical evidence of rejec-tion in transplanted human heart tissue obtained byendomyocardial biopsy.22,23 Sell et a122 found that

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Isobe et al MHC Class II Scintigraphy in Cardiac Rejection 739

histological evidence of moderate rejection in sequen-tial heart biopsies was always preceded by a markedincrease in the index of MHC antigens measured byradioimmunoassay. They also showed that this en-hancement of MHC class II antigen expression couldbe normalized by immunosuppressive therapy. How-ever, because diagnosis and follow-up of rejection bythis method would require endomyocardial biopsy, theusefulness of the approach is limited by the invasive-ness and sampling error inherent to the biopsy proce-dure. Therefore, the purpose of the present study wasto determine whether scintigraphic detection of anincrease in class II antigen expression could be used asa noninvasive method for diagnosing early cardiacallograft rejection.

MethodsAnimals

Male, inbred BALB/c (H2d) and C3H/He (H2k)mice (4-6 weeks old) were obtained from theCharles River Breeding Laboratory (Boston, Mass.).C57BL/6 (H2", IE-) male mice were purchased fromJackson Laboratory (Bar Harbor, Me.). All animalexperiments were approved by the Committee onResearch Animal Care Protocol Review Group andcarried out according to Massachusetts General Hos-pital guidelines and the National Institutes of HealthGuide for Care and Use of Laboratory Animals.

ScintigraphyHybridoma cell lines 10-2-16 (anti-mouse IAtrsf)24

and 14-4-4S (anti-mouse IEkdpr)25 were obtainedfrom ATCC (Rockville, Md.). The specificity andaffinity of the monoclonal antibodies have been de-scribed. Hybridoma cells were grown in Dulbecco'sminimum essential medium supplemented with 10%fetal calf serum and 0.1% gentamycin, and monoclo-nal immunoglobulins were purified by protein A affin-ity chromatography (Pharmacia, Piscataway, N.J.).Monoclonal antibodies 10-2-16 and 14-4-4S werelabeled with `11In by use of a bifunctional chelatingagent (diethylenetriamine pentaacetic acid[DTPA]).26-28 Approximately 100 gCi of `11In-DTPAantibody (10-2-16, 14-4-4S, or both) was injected intothe tail vein of the recipient mouse 24 hours beforescintigraphy. Scintigraphy was performed with agamma camera (Ohio Nuclear 100) equipped with a3-mm pinhole collimator as described.3 Both energypeaks (173 and 247 keV) of "'In were used for countacquisition. For each scintigram, the intensity of ra-dioactivity in the graft was compared with that of thenative heart after the cardiac area had been set bycomputer planimetry.

Tissue AnalysisMice were killed after scintigraphy. Venous blood

was withdrawn and the autologous heart, trans-planted heart, liver, spleen, kidneys, and lungs wereexcised. Both hearts were washed thoroughly withsaline. The biodistribution of radioactivity was deter-

mined as described.3 The ratio of percent injecteddose per gram of grafted heart (%ID) to that ofautologous heart was determined for each mouse.

Organ Grafting and Animal GroupsHeterotopic mouse heart transplantation was per-

formed by the microvascular technique as described.3In 18 mice, C3H/He hearts were transplanted intoBALB/c recipients. Two of this group were treatedwith cyclosporine (15 mg/kg/day s.c. injection). Twocontrol mice were isografted (BALB/c heart intoBALB/c recipient and C3H heart into C3H recipi-ent). Experimental and control mice were randomlychosen for scintigraphy 2-8 days after transplanta-tion and were injected with radiolabeled 10-2-16 and14-4-4S antibodies.

In our second set of experiments, we analyzedradiolabeled antibody uptake in rejecting allograftsin various donor-recipient combinations to show thespecificity of antibody uptake. `I1n-labeled 14-4-4Santibody was tested in the BALB/c recipient-C3H/He donor combination, `In-labeled 10-2-16antibody was tested in the C3H/He recipient-BALB/c donor combination, and each antibody wastested in two mice in the C57BL/6 recipient-BALB/cdonor combination. Cardiac graft contractility wasassessed daily by direct palpation,3 and scintigraphywas performed right after confirmation of a signifi-cant decline in the graft beat.

FIGURE 1. Light microscopic sections of C3H/He ectopiccardiac allografts transplanted into BALBIc recipients. PanelA: Grade L4, focal interstitial infiltrate without necrosis. PanelB: Grade IIIA, multifocal infiltrates with myocyte necrosis.Panel C: Grade IIIB, diffuse cell infiltration and necrosis.Panel D: Grade ItV diffuse infiltrates with hemorrhage andnecrosis.

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740 Circulation Vol 85, No 2 February 1992

TABLE 1. Donor-Recipient Combination, Immunosuppression, Days After Transplantation, Anti-MHC Class II Antibody Uptake, andHistological Degree of Rejection

Graft/native heart HistologicalMouse Cyclosporine Days after Tissue degree ofNo. Donor Recipient (15 mg/kg/day) transplant %ID* counting Scintigram rejectiont1 C3H BALB/c - 3 4.00 2.0 1.8 O0:2 C3H BALB/c - 3 4.19 1.7 1.4 03 C3H BALB/c - 3 4.82 2.0 2.2 0§4 C3H BALB/c - 4 5.12 1.8 1.9 05 C3H BALB/c - 4 8.84 3.3 2 06 C3H BALB/c - 4 2.63 3.4 3.3 IA7 C3H BALB/c - 4 19.80 10.6 5.2 IA8 C3H BALB/c - 5 16.26 10.6 4.5 IIIA§9 C3H BALB/c - 5 22.32 20.1 7.1 IIIA10 C3H BALB/c - 6 19.14 10.4 4.1 IA11 C3H BALB/c - 6 22.40 11.3 7.1 IIIB12 C3H BALB/c - 6 26.46 20.0 8.6 IV13 C3H BALB/c + 6 3.83 1.4 2.2 014 C3H C3H - 6 5.29 2.6 1.4 015 BALB/c BALB/c - 7 2.91 1.9 2.5 IA16 C3H BALB/c + 7 2.95 2.5 2.0 0§17 C3H BALB/c - 7 15.33 9.7 4.5 IIIA18 C3H BALB/c - 7 18.79 14.3 6.5 IV19 C3H BALB/c - 7 16.50 12.2 5.7 IIIB20 C3H BALB/c - 9 17.25 14.6 5.3 IIIB

*Percent injected dose per gram organ. tSee Reference 1. tQuilty effect A. §Ischemia A. -, Not administered; +, administered.

Histological ExaminationGrafted and autologous hearts were embedded in

paraffin and stained with hematoxylin and eosin. Thesamples were submitted for blinded histopathologicalevaluation by a cardiac pathologist without experimen-tal history and were classified according to the Interna-tional Heart Transplantation Working Formulation.'

Statistical AnalysisA value ofp>0.05 was considered nonsignificant in

comparisons between multiple groups of data.29 Alldata were expressed as mean+ SD. Linear regressionwas computed by the least-squares method.

ResultsHistopathology and Radiotracer Uptake

Histological findings ranged from nearly normal tosevere rejection (Figure 1). Labeled antibody uptakeand histopathological data are listed in Table 1. Theratios of radioactivity by tissue counting of thegrafted versus native heart in the two isografted micewere 1.9 and 2.6. This slight increase in radioactivityin isografts was also observed after injection ofradiolabeled mouse immunoglobulin of irrelevantspecificity. We conclude that nonspecific inflamma-tion caused by operative manipulation was responsi-ble for the increase in radioactivity.

Regardless of the time after transplantation andthe presence or absence of cyclosporine therapy, thelevel of radiotracer uptake reflected the histologicalseverity of rejection. The %ID in excised grafts was

4.8±1.8 (mean±SD) for normal grafts (n=8),11.1±9.7 for grafts with grade IA rejection (n=3,NS), 18.0±3.8 for grafts with grade IILA rejection(n=4, p<0.001 versus normal), 18.7±3.2 for graftswith grade IIIB rejection (n=3,p<0.001 versus nor-mal), and 22.6±5.4 for grafts with severe rejection(grade IV) (n=3,p<0.001 versus normal) (Figure 2).The ratio of radioactivity in the graft versus nativeheart was similar. The ratio was 2.2±0.6 for normalgrafts, 6.6±4.6 for grade IA grafts (p<0.05 versusnormal), 13.5±5.8 for grade IIIA grafts (p<0.001versus normal), 12.7±1.7 for grade IIIB grafts(p<0.001 versus normal), and 17.2±4.0 for grade IVgrafts (p<0.001 versus normal) (Figure 3). The %IDin excised grafts increased progressively with time inallografts (%ID=2.9xdays-1.0, r=0.65, p<0.01,n=16) (Figure 4). The ratio of radioactivity in thegraft versus native heart also increased progressivelywith time (G/N=2.3xdays-4.4, r=0.65, p<0.05,n=16). Radiolabeled antibody uptake in cyclospor-ine-treated allografts, for which there was no histo-logical evidence of rejection, did not increase relativeto that in isografts even 7 days after transplantation.

ScintigraphyA good linear correlation was observed between the

ratio of graft to native heart radioactivity measured bytissue counting and that measured by computer planim-etry from the scintigrams (P=0.33 xG/N+ 1.4, r=0.95,n=20, p<0.001, where P=ratio of radioactivity in thegraft versus autologous heart measured by computer

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Isobe et al MHC Class II Scintigraphy in Cardiac Rejection 741

p<0.00 1

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FIGURE 2. Scatterplot showing percent injected dose ofanti-MHC class II antibody per gram (%ID) ofgrafted heartcompared with histological degree of rejection. The %ID inmice with grade IILA, IIIB, or IV rejection is significantlygreater than the %oID in mice with normal grafts. *, Non-treated allograft; A, isograft; o, cyclosporine-treated allograft.

planimetry). Correlation between the intensity of ra-diotracer signal from the scintigram and the %ID in thegraft was also good (P=0.25 x %ID+0.96, r=0.93,n=20,p<0.001). Strong and unequivocal accumulationof labeled antibodies was apparent in scintigrams ofrejecting allografts from day 4 of transplantation (Fig-

ure 5). In contrast with mice with rejecting allografts,isografted mice and cyclosporine-treated mice showedno specific accumulation of radiolabeled antibodies 6and 7 days after transplantation.

In an allografted mouse imaged on day 4 oftransplantation (No. 7, Table 1) and another imagedon day 6 (No. 10), the tissue counting and scintigramrevealed significant uptake of radiotracer, whereasthe histological studies showed cell infiltrates but nosignificant myocyte necrosis. In two mice (No. 3 andNo. 16) with significant myocyte necrosis probablycaused by perioperative ischemia but without evi-dence of active rejection, there was no specific accu-mulation of radiotracer.

Dependence ofAntibody Binding on the Class IIAntigen Expressed

In the C3H/He (H2k) donor-BALB/c (H2d) recip-ient combination, the rejecting graft was clearlyvisualized by radiolabeled 14-4-4S antibody (anti-IE dPr). In contrast, in the BALB/c donor-C3H/Herecipient combination, there was no increase in ra-diolabeled 10-2-16 (anti-IA)rJsf) antibody uptake inthe rejecting allograft relative to nonrejecting al-lografts, even though the graft showed histologicalevidence of severe rejection 10 days after transplan-tation. In the BALB/c donor-C57BL/6 (H2b, IE-)recipient combination, in which one mouse was in-jected with radiolabeled 10-2-16 at 8 days aftertransplantation and another with radiolabeled 14-4-4S at 7 days after transplantation, rejecting al-lografts with H2d antigens in mice with H2b antigens

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FIGURE 3. Scatterplot showing the ratio of radioactivitydetermined by tissue counting in the graft versus autologousheart compared with histological degree of rejection. The ratioin mice with grade L4, IIL4, IIIB, or IV rejection is signifi-cantly greater than that in mice with normal grafts. 0,

Allografted mouse without cyclosporine; A, isografted mouse;o, allografted mouse treated with cyclosporine.

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FIGURE 4. Scatterplot showing time course of "1In-labeledanti-MHC class II antigen antibody uptake in grafted hearts.Localization is expressed as the percent injected dose pergram(%ID) of wet tissue. The %ID in nontreated allografts (-,C3HIHe heart in BALBIc recipient) increased progressivelywith time, whereas that in isografts (A, BALBIc heart inBALBIc recipient or C3H/He heart in C3H/He recipient) or

in cyclosporine-treated allografts (o, C3HIHe heart inBALBIc recipient) remained low.

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742 Circulation Vol 85, No 2 February 1992

FIGURE 5. Scintigrams showing temporal progression of `'In-labeled anti-MHC class II antigen antibody uptake in C3H/Hehearts grafted into BALBIc recipients. Labeled antibodies (10-2-16 and 14-4-4S) were injected into recipients 24 hours beforescintigraphy. Panel A: An allografted mouse (No. 2, Table 1) 3 days after transplantation. The graft with no histopathologicalevidence of rejection is not visualized. S, spleen. Panel B: An allografted mouse (No. 6) 4 days after transplantation. Uptake in themildly rejected graft is obscure. Panel C: Five days after transplantation (No. 9). Histopathological examination showed moderaterejection in this graft. Unequivocal, strong uptake of radiotracer is apparent in the graft. L, liver; K, kidney; B, bladder; G, graft.Panels D and E show grafts 6 days (No. 12) and 7 days (No. 19), respectively, after transplantation. Both grafts showed advancedrejection by histological examination and were identified by the scintigrams. Panel F: An allografted mouse treated with cyclosporine(No. 16), 7 days after transplantation. The graft, which showed no rejection by histopathological examination, is not visualized.

were visualized by an anti-IEkdYPr antibody but not byan anti-IAkrsf antibody (Table 2 and Figure 6).

DiscussionThe results of this study demonstrate for the first

time that anti-MHC class II antibody scintigraphycan be used to detect cardiac rejection. Scintigraphywas sufficiently sensitive to detect early rejectionbefore development of significant myocyte necrosis.The results also show that MHC class II antigensinduced on donor cardiac myocytes are the uniquesource of antigen accounting for the positive scan inthe rejecting heart.Although a variety of noninvasive techniques for

detection and quantification of cardiac rejection havebeen explored, none has gained widespread use.Antimyosin scintigraphy is a potentially usefulmethod for diagnosing cardiac rejection in the clini-cal setting because myocyte necrosis is a direct resultof rejection and is usually regarded as an indication

for additional rejection-directed therapy. Myocytenecrosis is not an early manifestation of rejection,however; it occurs at a relatively advanced stage.Also, antimyosin scanning cannot differentiate peri-operative necrosis from necrosis caused by rejection.A more sensitive and specific scintigraphic methodfor detecting cardiac rejection would be useful.MHC class I and class II molecules, known as

transplantation antigens, play an essential role in thepathogenesis of rejection.8,30 Although low levels ofclass I antigens are expressed in normal cardiacmyocytes731 class II antigens normally are not detect-ed.7'9 However, induction of both class I and class IIantigens has been reported in rejecting kidney,8'10"12heart 11,13,14,22,23 liver,32 and pancreas33 allografts.Milton et ali134 observed a massive induction ofdonor-type class II antigens in rejecting rat cardiacallografts. In the report by Sell et a122 of a series of sixpatients who underwent cardiac transplantation, anincrease in the level of MHC class II antigen expres-

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FIGURE 6. Scintigrams after injection of`11n-labeled anti-MHC class II antigenantibodies in mice with rejecting allograftsin various donor-recipient combinations.See Table 2for explanation. Panels A, B,C, and D conespond to mice No. 21, 22,23, and 24, respectively.

sion always preceded histological evidence of moder-ate rejection. In all six patients, the expression ofclass II antigens decreased when rejection abated.These results led us to conclude that donor-type classII antigens induced on allografted hearts should beuseful imaging targets for detecting early cardiacrejection.

Induced MHC class II expression on graft cells ismost likely a result of y-interferon, a lymphokinegenerated by activated T lymphocytes and naturalkiller cells.35 The donor MHC class II antigens canaugment both cellular sensitization in the recipientand susceptibility to MHC class I- and class II-

specific effectors in the donor targets. Because thelarge-scale induction of class II antigens could greatly

stimulate activation of T cell-derived y-interferon,induction of class II antigen would be maintained inmyocardial and endothelial cells. In this way, anexpanding and self-regenerating cycle would be ini-tiated. Induced class II antigens could also serve asnew targets for class Il-specific cytotoxic T cells. Thisenhancement in MHC antigen expression could sig-nificantly drive the rejection process.Two MHC molecules (IA and IE) have been

identified as class II antigens in mice. Therefore, weselected these antigens as targets for radioimmu-noimaging. In our experiments in the C3H/He do-nor-BALB/c recipient combination, we used anti-bodies against IA and IE antigens that react withboth donor and recipient antigens. In contrast to

TABLE 2. Uptake of `'In-Labeled Anti-MHC Class II Antibodies 10-2-16 (Anti-IAkrsf) and 14-4-4S (Anti-IEkdPr) inRejecting Mouse Heart Allografts in Various Donor-Recipient Combinations

Histological ScintigramMouse Days after degree of Graft/nativeNo. Donor Recipient transplant rejection Antibody heart (%ID)* Graft Spleen21 C3H (H2b) BALB/c (H2b) 6 IIIA 14-4-4S 12.5 + +

22 BALB/c C3H 10 IV 1O-2-16 1.6 +

23 BALB/c C57BL/6 (H2b) 8 IIIA 10-2-16 3.124 BALB/c C57BL/6 7 IIIA 14-4-4S 10.4 + -

*Percent injected dose per gram organ; +, positive; -, negative.

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nonrejecting allografts and isografts, which were notvisualized by the radiolabeled antibodies, allograftswith histological evidence of rejection were clearlyidentified. Radiolabeled antibody uptake in rejectingallografts increased progressively with time and inproportion to the severity of rejection. Although allallografts were beating at the various times at whichthe mice were killed, beating was obviously impairedin allografts in mice killed 7 and 9 days after trans-plantation. Significant loss of myocytes in these micemay account for the relative reduction in radiotraceruptake observed in the late stage of rejection. How-ever, the reason for the nonlinear correlation be-tween histological grade of rejection and class IIantibody uptake remains to be determined.

It is of interest that anti-class II antibody uptakewas dissociated from myocyte necrosis. In an al-lografted mouse imaged on day 4 after transplanta-tion and another imaged on day 5, the scintigramsrevealed strong uptake of radiotracer, whereas histo-logical studies showed evidence of cell infiltrates butno significant myocyte necrosis. There was a signifi-cant increase in %ID in these grafts relative to that inisografts and nonrejecting allografts. One allograftedmouse killed 3 days after transplantation and anothercyclosporine-treated mouse killed after 7 daysshowed significant ischemic myocyte necrosis (prob-ably caused by perioperative ischemia) and no evi-dence of rejection. These mice also showed no in-crease in radiotracer activity in comparison withhistologically normal grafts. Therefore, we concludethat myocyte necrosis does not affect anti-class IIantibody uptake in allografted hearts.

In our series of 20 mice, eight allografts weredeemed normal on histological examination and fourgrafts were given a rejection grade of IA (cell infil-trate without myocyte necrosis). The ratio of %ID forgraft versus native hearts in the eight mice whosegrafts were normal was always less than that in themice whose grafts showed histological evidence ofrejection. In two of the four allografts with grade IArejection (in which there was no evidence of myocytenecrosis), radiotracer uptake increased significantlyand was clearly visible in the scintigraphic image, andin one isograft with focal monocytic infiltrates con-sistent with grade IA rejection, there was no increasein tracer uptake in comparison with histologicallynormal grafts. All grafts with advanced rejection(grade III or IV) revealed statistically significantincreases in tracer uptake and were clearly visible byscintigraphy. Because the transition from mild tomoderate cardiac rejection is subtle in humans anddifficult to detect by histological examination, the useof anti-class II antibody scintigraphy to detect al-lograft rejection before the development of myocytenecrosis may have clinical relevance. It should benoted, however, that in a study by Sell et al,22 no strictcorrelation was observed between extent of leukocyteinfiltration and level of MHC antigen expressiondetermined by radioimmunoassay.

Cyclosporine inhibits lymphokine production byhelper T cells in vitro and is widely used to alleviatetissue allograft rejection in vivo.36 Cyclosporine alsosuppresses induction of MHC class II antigens, de-spite evidence of substantial leukocyte infiltrationshown in cyclosporine-treated grafts.37 This suppres-sion of MHC antigen induction is almost certainly aconsequence of suppression of y-interferon releasefrom the infiltrating leukocytes.38,39 Not surprisingly,radiolabeled antibody uptake in cyclosporine-treatedallografts did not increase relative to that in isografts,reflecting the absence of active rejection.As shown in Table 2, rejecting allografts were

imaged only by anti-class II antibody against donorantigen. Normal splenocytes, especially B lympho-cytes, express class II antigens.8 The strong uptake ofantibody in the recipient's spleen is consistent withthe reactivity of the antibody with the recipient'sclass II antigens. Regardless of the reactivity of theantibody with the recipient's antigens, the rejectingBALB/c (H2d) grafts were visualized by 14-4-4S(anti-IE;dPr) but not by 10-2-16 (anti-IAkrsf) anti-body. These results also rule out the possibility ofnonspecific accumulation of antibody (immunoglob-ulin) in sites of rejection.One difference between rejection in mice com-

pared with humans and other large mammals such aspigs requires consideration. Acute rejection in miceis provoked primarily by class I antigens. In themouse vascularized graft model with class II mis-matching, spontaneous long-term tolerance has beenobserved.40 Class II mismatched grafts are invariablyrejected in the pig kidney transplant model.41 Thisdiscrepancy may relate to the expression of class IIantigens on the vascular endothelium of large ani-mals (including humans) but not that of mice.42 Thedistribution of MHC antigens, as well as its effect oninduction of class II antigens associated with acuterejection, must be investigated because of its poten-tial to affect the sensitivity of anti-class II antibodyscintigraphy.

This study demonstrates that cardiac rejection canbe detected by anti-class II antibody scintigraphy atthe stage at which there is leukocyte infiltration butno significant myocyte necrosis. This method may beparticularly useful for distinguishing the active cellu-lar infiltrates that are the first sign of a rejectionepisode from the innocuous and self-limiting infil-trates frequently seen with cyclosporine and appar-ently seen in one of our isografted mice. Since thelevel of HLA-DR antigen on rejecting cardiac cellsdecreases after intensive immunosuppressive thera-py,22 MHC class II antigen scintigraphy may simplifythe follow-up of patients with episodes of rejection.Enhancement of class II antigen expression in

rejecting allografts occurs not only in heart but alsoin other organ transplants. Because of the lowexpression of class II antigens in normal kidneys8,10and pancreas,33 MHC class II antigen scintigraphymay also be useful for detecting rejection of theseorgans. In the detection of kidney rejection, this

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method would be particularly useful for distinguish-ing renal failure caused by rejection from that causedby cyclosporine toxicity.

AcknowledgmentThe authors thank Thomas McVarish for prepar-

ing the manuscript.

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4. Addonizio LJ, Michler RE, Marboe C, Esser PE, Johnson LL,Seldin DW, Gersony WM, Alderson PO, Rose EA, CannonPJ: Imaging of cardiac allograft rejection in dogs using indium-111 monoclonal antimyosin Fab. J Am Coll Cardiol 1987;9:555-564

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KEY WORDS * transplantation * scintigraphy * cyclosporine* MHC rejection

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M Isobe, J Narula, J F Southern, H W Strauss, B A Khaw and E HaberII antigen induction.

Imaging the rejecting heart. In vivo detection of major histocompatibility complex class

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