Defining the Outcome of Immunosuppression WithdrawalAfter Liver Transplantation

JOHN DEVLIN, DEREK DOHERTY, LINDA THOMSON, TERENCE WONG, PETER DONALDSON,BERNARD PORTMANN, AND ROGER WILLIAMS

Successful immunosuppression withdrawal should ben-efit the natural history of organ transplantation patients. Toidentify the clinical hazards of removing drug treatment andpossible characteristics that predict a favorable outcome inlong-term liver recipients, immunosuppression was with-drawn completely and the clinicopathological outcomedocumented in 18 liver recipients. Indication for transplan-tation, HLA matching, early rejection history, and presenceof microchimerism were examined as predictors of out-come. Chimerism was determined by polymerase chainreaction–based examination for donor-specific HLA-DRB1alleles and Y-gene–specific nucleotide sequences. At 3years, 5 patients (28%) remained completely off immunosup-pression; 12 patients (67%) experienced histological graftchanges: acute rejection in 4, portal tract inflammation/hepatitis in 7, and necrosis in 1. Hepatitis B or C viralinfections did not account for the nonrejection patterns.Unmasking of systemic disorders occurred. Chimerism,demonstrated in 7 patients (39%), with skin the optimaltissue, was not associated with tolerance. Parameters associ-ated with successful drug withdrawal were transplantationfor non–immune-mediated liver disorders, fewer donor–recipient HLA A, B, and DR mismatches, and a lowincidence of early rejection. Immunosuppression with-drawal is a feasible option in a proportion of selected liverrecipients, but identification of tolerant patients remainsimprecise. (HEPATOLOGY 1998;27:926-933.)

Withdrawal of maintenance immunosuppressive agents inorgan recipients would represent a major advance in clinicaltransplantation, with benefits derived from the return innatural immunity and reduction in drug-related toxicity. Thepotential hazards of such an undertaking would be lessened if

clinicians were able to base it on the identification of either aclinical or immunologic marker of operational tolerance. Thetechnical limitations surrounding the in vitro assessment ofdonor–recipient immune responsiveness are such that as yetno parameter reliably predictive of tolerance has foundclinical application. Accordingly, considerable interest anddebate presently surrounds the thesis proposed by Starzl1 thatthe establishment and augmentation of systemic microchime-rism, after multilineage cell migration from a graft or viaadditional donor cell infusion, is an important factor inimmunomodulation of the allograft response with promotionof donor-specific unresponsiveness and hence long-term graftacceptance.2,3 The demonstration of either clinical toleranceor immune nonreactivity in apparent association with chime-rism in long-term liver and renal recipients respectively at theUniversity of Pittsburgh was the stimulus to the clinical trialreported here, in which immunosuppression drug with-drawal was attempted in a group of our long-term liverrecipients who were experiencing a number of side effects ordisorders related to continued immunosuppression. In addi-tion to a description of the clinical, biochemical, and histologi-cal outcome of such a patient cohort, we present correlativedata on the relationship between operational tolerance andthe presence of microchimerism and more readily identifiableclinical parameters such as donor–recipient HLA matching,early posttransplantation rejection history and the pretrans-plantation disorder.

PATIENTS AND METHODS

Patients

Eighteen liver recipients with a functioning primary allograft for.5 years (median, 7; range, 5-11) were entered into a program ofimmunosuppressive drug withdrawal (Table 1). All patients had aminimum of one side effect or disorder either initiated or aggravatedby maintenance immunosuppression, namely recurrence of hepato-cellular carcinoma (n 5 1) and hepatitis C virus (HCV)–related liverdisease (n 5 2), de novo malignancies (cervical n 5 1, breast n 5 1,and skin n 5 2), posttransplantation lymphoproliferative disorder(n 5 1), renal impairment (n 5 5), diabetes mellitus (n 5 2),hypertension (n 5 9), osteoporosis (n 5 3), severe weight gain (n 51), lipid metabolism abnormalities (n 5 7), severe hirsutism (n 51), neurotoxicity (n 5 4), azathioprine-related bone marrow toxic-ity (n 5 1), and various biochemical derangements (hyperkalemia,hypomagnesemia, hyperuricemia; n 5 7). The causes of the primarydisease comprised primary biliary cirrhosis (n 5 3), primarysclerosing cholangitis (n 5 3), Budd–Chiari syndrome (n 5 3), HCVand autoimmune hepatitis (n 5 1), HCV cirrhosis (n 5 1), cysticfibrosis (n 5 1), acute liver failure (n 5 3), alcohol-related cirrhosisand HCV (n 5 1), hepatocellular carcinoma and HCV cirrhosis (n 51), and hepatic metastases from treated testicular teratoma (n 5 1).

Abbreviations: HCV, hepatitis C virus; MHC, major histocompatibility complex; PCR,polymerase chain reaction; SSO, sequence-specific oligonucleotide; PCR-SSP, PCR withsequence-specific primers; RFLP, restriction fragment length polymorphism; AST,aspartate aminotransferase.

From the Institute of Liver Studies, King’s College School of Medicine and Dentistry,London.

Received December 23, 1996; accepted December 2, 1997.Roger Williams’s present address is Institute of Hepatology, University College

London Medical School, 69-75 Chenies Mews, London WC1E 6HX.Sections of this manuscript were presented at the Annual Meeting of the American

Association of the Study of the Liver Diseases, Chicago, IL, November 11-15, 1994.Address reprint requests to: John Devlin, M.D., Institute of Liver Studies, King’s

College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, England.Fax: 44-171-346-3167.

Copyright r 1998 by the American Association for the Study of Liver Diseases.0270-9139/98/2704-0006$3.00/0

926

Methods

After baseline determination of standard liver biochemical testsand a liver biopsy to give a complete assessment of graft status aswell as exclusion of allograft rejection, systematic immunosuppres-sion withdrawal was performed to a standard protocol. The baselinemaintenance regimens varied considerably (Table 1). The mediandaily dosage schedules were prednisone 5 mg (0.08 mg/kg),azathioprine 75 mg (1.1 mg/kg), and Cyclosporine A 250 mg (3.4mg/kg). In those patients still administered corticosteroids, predni-sone dosage was gradually withdrawn over a period of 4 to 6 weeks.The remaining immunosuppressive agents were then discontinuedand patient clinical status and biochemical graft function monitoredinitially in hospital (2-week period) and thereafter in the clinic at 1-to 4-week intervals. All patients experiencing an episode of signifi-cant biochemical graft dysfunction (level of any routine biochemicalliver parameter two or three times the upper limit of normal)underwent percutaneous biopsy of the graft and histopathologicalassessment, with liver allograft rejection defined by standard crite-ria.4 Immunosuppression was recommenced when episodes of liverbiochemical abnormalities were accompanied by any significant deterio-ration in histological appearance compared with baseline. After stabiliza-tion, a further slower weaning schedule of immunosuppressive drug

withdrawal was attempted. Further follow-up biopsies were performedbetween 8 and 24 months after attempted drug withdrawal.

The presence of donor-derived microchimerism was examined bymolecular analysis for class II major histocompatibility complex(MHC) donor-type genotype sequences in recipient peripheraltissue. Whole blood, bone marrow, and skin samples were collectedfrom the patients before immunosuppression withdrawal and ge-nomic DNA, from which the analyses were performed, was ex-tracted. Three separate methodologies were then evaluated for thedetermination of donor HLA-DRB1 alleles after polymerase chainreaction (PCR) amplification of the DNA. These techniques weresequence-specific oligonucleotide (SSO) analysis (PCR-SSO dotblot5; kits supplied by British Society for Histocompatibility andImmunogenetics, Molecular Special Interest Group, University ofBristol, England), one-step PCR with sequence-specific primers(PCR-SSP6; kit supplied by Dynal Inc., Wirral, England), andtwo-step nested sequence-specific PCR7 (kit supplied by BritishSociety for Histocompatibility and Immunogenetics). Maximal strin-gency and sensitivity conditions were employed (details on request).All recipients had previously been genotyped and assigned HLA-DRB1 specificities by molecular methods (restriction fragmentlength polymorphism [RFLP] and/or PCR-SSO). Further confirma-

TABLE 1. Summary Baseline Clinical and Immunosuppression-Related Data

Patient

AgePosttrans-plantation

(years)TransplantIndication

Major Immunosuppressant Complications Immunosuppression Dosages

Hypertension Nephrotoxicity Diabetes Malignancy Other CyA mg/kg Azathioprine mg/kg Prednisolone mg/kg

1 39 (5) sclerosing chol-angitis

Neurotoxicity 300 5.45 75 1.36 7.5 0.14

2 48 (6) Budd–Chiari syn-drome

1 breast 400 8 75 1.5 5 0.10

3 44 (8) Budd–Chiari syn-drome

1 skin 250 3.13 75 0.94 10 0.13

4 50 (6) alcoholic liverdisease/HCV

1 1 Avascularnecrosis of hipazathioprinetoxicity

100 1.64 50 0.82 5 0.08

5 49 (8) acute liver failure(NANB)

1 1 250 3.33 75 1 7.5 0.10

6 64 (6) hepatitis C (cir-rhosis)

skin recurrence ofHCV

200 2.7 100 1.35 7.5 0.10

7 40 (6) primary biliarycirrhosis

1 cushingoid 100 0.91 75 0.68 0 0.00

8 46 (6) sclerosing chol-angitis

1 1 lymphoma 300 4.29 75 1.07 5 0.07

9 48 (7) hepatic metas-tases

1 1 migraine, warts 125 1.54 100 1.23 0 0.00

10 69 (10) autoimmune-hepatitis/HCV

recurrence ofHCV

150 1.88 0.75 0.94 7.5 0.09

11 49 (8) acute liver failure(NANB)

1 1 cervical 200 3.64 75 1.36 7.5 0.14

12 63 (6) primary biliarycirrhosis

200 2.50 75 0.94 7.5 0.09

13 64 (6) primary biliarycirrhosis

1 bone marrow tox-icity

350 6.36 50 0.91 10 0.18

14 38 (7) sclerosing chol-angitis

1 osteoporosis 300 5.05 50 0.84 0 0.00

15 58 (6) hepatocellularcarci-noma/HCV

1 recurrence ofHCC

300 3.53 75 0.88 7.5 0.09

16 28 (7) cystic fibrosis genital warts 600 11.32 75 1.42 10 0.1917 23 (10) Budd–Chiari severely cush-

ingoid, gout100 0.91 75 0.68 7.5 0.07

18 39 (5) acute liver failure(drug toxicity)

oesophagitis 250 3.68 100 1.55 10 0.16

HEPATOLOGY Vol. 27, No. 4, 1998 DEVLIN ET AL. 927

tion of recipient HLA-DRB1 genotype was gained by PCR-SSPexamination of genomic DNA retrieved from a fresh protocol liverbiopsy. In male donor–to–female recipient pairings, the presence ofY-gene–specific nucleotide sequences in recipient DNA was alsodetermined by two separate primer pairs.8,9

The influence of HLA mismatching on the outcome of immuno-suppression withdrawal was evaluated. Donor HLA A, B, and DRtyping was performed by four U.K. donor centers and cross-checkedfor accuracy before assigning consensus specificities. Recipient class1 and DR types were performed by both serology and RFLP orPCR-SSO techniques. Common (public) antigen designations wereused in assigning mismatch scores.

Presence and/or reactivation of hepatitis B or C infection beforeand during immunosuppression withdrawal was examined for in theserum of all patients irrespective of the primary transplantationindication. HBV status was determined by standard commerciallyavailable kits. Radioimmunoassay or microparticle capture enzymeimmunoassay was used to measure levels of hepatitis B surfaceantigen, antibody to hepatitis B surface antigen, and total coreantibody (Ausria II, Abbott HBe [rDNA], Ausab; Abbott, NorthChicago, IL). HBV DNA was examined for by the hepatitis B viralDNA assay (Abbott). HCV antibody was determined by second-generation (United Biomedical Inc., Lake Success, NY) and/orthird-generation enzyme immunoassays (Murex, Dartford, Kent,England). HCV RNA was also sought using the sensitive and specificAmplicor PCR assay.10

Results are presented at a minimum follow-up of 3 years afterimmunosuppression withdrawal.

RESULTS

Clinical and Biochemical Outcome

Of the 18 patients, 5 have experienced no deterioration in graftfunction, continue to be without significant abnormalities inbiochemical tests, and remain clinically well (Table 2). Three ofthese patients developed minor fluctuating but spontaneouslyresolving increases in serum aspartate aminotransferase (AST)levels and biliary enzyme activities to just above the upper limit ofnormal at some point after immunosuppression withdrawal.

The remaining 13 patients experienced some derangement

in biochemical tests such that a degree of immunosuppres-sion was recommenced in 12 at a median of 3 weeks (range,2-24 weeks) after initial withdrawal of drugs. Several patternsof biochemical abnormalities in graft dysfunction were ob-served. Variable and often initially fluctuating increases inAST activity levels (median peak value, 288 IU/L; range,73-830 IU/L) occurred. These were first noted at a significantlevel (100 IU/L; i.e., two times the upper limit of normal) at amedian of 25 days (range, 10-176 days) postwithdrawal. Parallelincreases in levels of the biliary enzymes (alkaline phosphatase,g-glutamyltransferase) accompanied these changes in AST. Anincrease in serum total bilirubin occurred in 7 patients (medianpeak value, 53 mmol/L; range, 28-474 mmol/L) at a median of 32days after withdrawal. In one patient immunosuppression wasintentionally not recommenced because of the presence of aposttransplantation lymphoproliferative disorder, with retransplan-tation after an immunosuppression-free period being seen as thepreferred clinical option. This was the patient with the outlyinghigh bilirubin of 474 mmol/L.

Systemic complications were also observed after drugwithdrawal. Two of the three patients receiving liver trans-plants for primary biliary cirrhosis complained of arthralgia.There was no evidence of an erosive arthritis, and thissymptom settled spontaneously. Classical acute gout oc-curred in three patients and onset of previously undiagnosedpsoriasis in one patient. One further patient receiving atransplant for primary sclerosing cholangitis had a significantflare-up of previously quiescent ulcerative colitis.

Histological Outcome

Biopsies of the graft performed in the group of five patientsin whom immunosuppression did not have to be recom-menced between 8 and 24 months after withdrawal showedno major changes from findings present at baseline, and inparticular no evidence of either acute or chronic rejection.

Histopathological assessment of the graft during the epi-sodes of biochemical dysfunction in the other 13 cases

TABLE 2. Summary of Histological Outcome and Relationship Between Chimerism and Immunosuppression Requirements

Patient Baseline Biopsy

Histological Outcome Microchimerism Immunosuppression Recommencement

Biochemical Derangement Follow-up None Partial Complete

1 cholangiopathy not required see baseline 1 1 2 2

2 sinusoidal congestion and dilatation hepatitis see baseline 2 2 1 2

3 mild fatty change only not required see baseline 2 1 2 2

4 focal parenchymal hyperplasia not required hepatitis 2 1 2 2

5 mild portal tract inflammation hepatitis see baseline 1 2 2 1

6 chronic hepatitis hepatitis see baseline 1 2 1 2

7 chronic hepatitis rejection see baseline 2 2 2 1

8 cholangiography rejection not available* 2 2 1 2

9 minor non-specific changes hepatitis see baseline 1 2 1 2

10 chronic hepatitis hepatitis see baseline 1 2 1 2

11 minimal, focal portal tract inflammation rejection hepatitis 1 2 2 1

12 mild portal tract inflammation hepatitis see baseline 2 2 1 2

13 chronic hepatitis rejection see baseline 2 2 1 2

14 mild portal tract inflammation acute hepatic necrosis post-collapse 2 2 1 2

15 chronic hepatitis hepatitis not available† 2 2 1 2

16 fatty change not required see baseline 2 1 2 2

17 sinusoidal congestion and dilatation not required see baseline 2 1 2 2

18 chronic hepatitis hepatitis not available‡ 2 2 1 2

*Patient retransplanted.†Patient died.‡Patient refused.

928 DEVLIN ET AL. HEPATOLOGY April 1998

revealed characteristic histological features of acute cellularallograft rejection in 4 patients. One specimen was graded assevere rejection, the others as mild to moderate. A morecommon pattern of a mixed inflammatory portal tract infil-trate composed of predominantly lymphocytes with fewerplasma cells and occasional eosinophils was seen in 8 patients(Fig. 1). Two of this group also had severe neutrophilicinfiltration of the portal tract. In 3 patients of this group,minimal or mild limiting plate spillover and a lobularhepatitis in the form of occasional spotty necrosis and mixedinflammatory cells present within sinusoids was found. In 2patients, these features were accompanied by minimal focalendotheliitis. Infiltration within bile ducts was rare. Thisoverall picture of a hepatitislike disorder appeared to repre-sent exacerbation of similar but less severe baseline findingsin 5 of the patients and a de novo appearance in 2 cases. In 2patients this histological picture was observed in the contextof a coexisting viral illness (Coxsackie in one, unidentified inthe other [atypical lymphocytosis present on blood filmexamination]). One further case showed features of acutehepatic necrosis with evidence of recent parenchymal col-lapse along with a moderate mixed inflammatory cell infil-trate in both postnecrotic areas and portal tracts (Fig. 2).Extensive hepatocyte ballooning and regeneration and severecholestasis were also present. This patient had a precedinghistory of a virallike illness and nonsteroidal anti-inflamma-tory agent usage. In the final patient in this group, nosignificant abnormalities were detected in the liver biopsy.Overall, in these 13 patients, and perhaps not surprisingly, nocorrelation between biochemical abnormalities and accompa-nying histological features was detected. In particular, norelationship between hepatitis and/or rejection and the heightof the serum transaminase activity was found. None of thepatients had serological evidence of HBV infection (hepatitis

B surface antigen, total core antibody, and HBV DNA undetect-able) before or after immunosuppression withdrawal. In 4patients, HCV antibody and RNA were present in the serumat the time of withdrawal. Two of these 4 patients had anepisode of hepatitis during withdrawal, with 1 patient show-ing significant histological progression of disease (on 50% ofbaseline immunosuppression 18 months later). In the remain-ing 5 patients who experienced hepatitis, there was noevidence of HCV infection.

Immunosuppression Requirements

The reintroduction of immunosuppression, in 12 of the 18patients, and the schedule followed were determined predomi-nantly and arbitrarily on the basis of biochemical dysfunctionrather than pathological features. In 4 patients with modestincreases of AST levels (,150-300 IU/L), short courses ofadditional oral prednisolone (30 mg/day for 2 weeks, 20mg/day for 1 week, then gradually reduced to 5 mg/day orwithdrawn) in addition to reintroduction of ,50% to 75% oftheir maintenance baseline immunosuppression. With higherAST activities, as seen in 8 patients, a conventional high-dosecorticosteroid antirejection regimen (methylprednisolone 1 gdaily for 3 days) was administered in addition to a reintroduc-tion of the complete baseline immunosuppression.

After either of these regimens, AST activities returned tothe normal range rapidly in 9 of 12 patients. In the remaining3 patients, in whom graft function did not normalize quickly,an additional course of high-dose corticosteroids and, in 2 ofthese 3 patients, temporary conversion to tacrolimus wasrequired. OKT3 was not used in any patient. Increases in theserum bilirubin level returned to within the normal rangeconsiderably more slowly (median 3 weeks after commence-ment of treatment) than the increases in levels of liverenzymes. In 2 patients in whom the serum bilirubin level was

FIG. 1. Liver histology 87 days after therapy withdrawal. Portal tracts (LEFT and BOTTOM of the field) are infiltrated by predominantly lymphocytes, whichare largely spilling over the parenchymal limiting plate. A lobular hepatitis is seen with a mild cell dropout around the hepatic venule (HV).

HEPATOLOGY Vol. 27, No. 4, 1998 DEVLIN ET AL. 929

slow to fall and in whom histological features of only mildhepatitis or rejection had been present on biopsy, investiga-tions of biliary drainage were performed. In both patients,cholangiograms revealed dilated biliary tracts and stasiswithin the gallbladder conduit that had been used in the originalanastomosis. Biliary reconstruction was subsequently performed,with resolution of the biochemical changes.

In 11 of the 12 patients in whom immunosuppression wasreintroduced and after an interval to allow stabilization ofgraft function, a further attempt at drug withdrawal wasinitiated (this was not attempted in the single case who hadexperienced a severe episode of acute cellular rejection). Aweaning regimen, which comprised a stepwise progressivereduction of immunosuppressive drugs, was now employed.In 9 of the 12 patients, further minor fluctuating increases inliver enzyme activity levels occurred. Despite this commonpattern of increases in enzyme levels, 9 patients were success-fully weaned to a significant degree and are presentlyreceiving lower dosages than at baseline, with normal graftfunction. Of these, 7 patients are presently receiving ,50% ofthe baseline corticosteroid and azathioprine dosage accompa-nied by either whole blood Cyclosporin A levels ,60 ng/mL(our lower limit for maintenance immunosuppression; n 55) or complete withdrawal of this agent (n 5 2).

Prednisolone has been stopped in 4 patients and azathio-prine in 6. The median daily dosage schedules and percentagechanges for the immunosuppressive agents at latest follow-upare prednisolone, 0.06 mg/kg (225%; range, 100%-0%);azathioprine, 0.39 mg/kg (264%; range 100%-0%), andCyclosporin A, 1.77 mg/kg (237%; range, 218%-100%).

Predicting a Favorable Outcomefor Immunosuppression Withdrawal

Tolerance and Chimerism. Two-step nested sequence-specificPCR was the most sensitive methodology for the detection of

donor-type genotypic sequences. This method detected HLA-DRB1 allele specificities of donor type in 7 of the 18 patients(39%). PCR-SSO and single-step PCR-SSP methodologieswere less sensitive (22% with PCR-SSO and 11% withsingle-step PCR-SSP) and were abandoned after 12 and 9patient samples respectively. Demonstration of donor ge-nomic elements by Y-gene primers concurred entirely withthe positive and negative results achieved with all of theabove techniques and confirmed chimerism in 3 femalerecipients. No sample resulted in a false-positive result withrespect to presence of Y-gene sequences and not the appropri-ate donor HLA-DRB1 alleles. Of the tissues examined, thepresence of donor genomic sequences was found most oftenin skin. Employing the nested PCR-SSP methodology, donorHLA-DRB1 was found in 5 patient samples from peripheralblood, 4 from bone marrow, and 7 from the skin sample. Ofthe 7 patients with evidence of microchimerism, 2 (29%)have been successfully withdrawn and maintained off immu-nosuppressive drugs. A further 3 (43%) have been weanedsignificantly after partial reintroduction of immunosuppres-sion. The final 2 chimeric patients (29%) experienced severegraft dysfunction and remain on standard dose regimens.Both cases experienced an episode of acute allograft rejectionconfirmed histologically. Of the 11 patients who had noevidence of microchimerism, 3 have successfully been re-moved from immunosuppression (27%) and a further 6 havebeen weaned significantly (55%). In the remaining 2 nonchi-meric patients (18%), a return to the baseline immunosuppres-sion level was required. Comparing the frequencies of com-plete or partial drug withdrawal, there was no statisticaldifference between the number of patients who were chimericand the number who were nonchimeric.

Tolerance and HLA/Clinical Parameters. Patients in whomimmunosuppression could not be withdrawn had moredonor HLA mismatches than did those in whom it could be

FIG. 2. Liver histology some 9 months after stopping immunosuppression. The architecture is distorted because of severe parenchymal damage withbridging collapse (upper part of the field), early nodular hyperplasia, hepatocyte ballooning, and comparatively mild portal and lobular inflammation.

930 DEVLIN ET AL. HEPATOLOGY April 1998

significantly reduced or withdrawn completely. The presenceof two mismatches at the DR locus (n 5 3) was accompaniedby failure of withdrawal in all patients. Additionally, all of the4 patients who failed withdrawal had two mismatches at the Blocus (100%), compared with 6 of 9 who were partially with-drawn (67%) and 2 of 5 in whom all drugs were removed (40%;Table 3). The median number of mismatches for all three loci washigher in the unsuccessfully withdrawn patients. With respect tothe pretransplantation indication, the 5 patients who were success-fully completely withdrawn from immunosuppression had allreceived transplants for nonimmunologic or nonviral disorders(Budd–Chiari syndrome [2], alcoholic liver disease [HCV present],cystic fibrosis, and primary sclerosing cholangitis). In compari-son, the 4 patients in whom immunosuppression had to bereturned to baseline had received transplants for either immuno-logic or viral-related disorders (autoimmune hepatitis [2, HCVpresent in one], HCV cirrhosis, and late-onset hepatic failure).The transplantation indications of the intermediate group aresummarized in Table 4. Finally, we examined the influence ofearly rejection posttransplantation and the subsequent ability tobe withdrawn from immunosuppression. A pattern toward lowerrejection frequency in the successfully withdrawn patients ispresent. Clinical rejection (i.e., where a supplemental course ofcorticosteroids was administered) occurred in 1 of the 4 patientscompletely withdrawn, whereas this was required in 3 of the 4failed cases (Table 4).

DISCUSSION

This report provides further evidence for the uniqueimmunologically privileged position of liver allografts com-pared with other solid organ transplants. From the initialobservations that genetically disparate porcine and canineliver allografts were spontaneously accepted in the absence ofimmunosuppression, considerable experimental and clinicalobservations of the reduced immunogenicity and tolerance-inducing properties of a liver graft have been reported.11,12

The clinical tolerance that we have demonstrated adds to theobservations of a low incidence of immunologic graft failuressecondary to a reduced susceptibility to hyperacute rejection,resistance to positive cross-match, less frequent and severeacute and chronic rejection, and an ability to induce donor-specific tolerance.13-15 In the present series, it was possible toeither completely withdraw (5 of 18 patients) or significantlyreduce (9 of 18) maintenance immunosuppression to levelspreviously considered subtherapeutic. Such results differfrom those seen in other solid organ recipients, for whom thehazards of even partial immunosuppressive drug withdrawalfrom maintenance regimens are well recognized.16 The resultsin the present study are very similar to the earlier reports fromthe University of Pittsburgh with respect to the proportionsbeing weaned completely (e.g., complete withdrawal, 27%

and 28% of cases respectively).17 The larger experience fromPittsburgh is also comparable to our results, with 19% of the95 patients drug-free at follow-up.18

The optimal regimen for drug withdrawal is unknown. Inthe absence of any experimental data, we chose to discon-tinue the immunosuppressive drugs abruptly (corticosteroidswere weaned to offset any adrenal suppression). This high-risk strategy requires very close monitoring and may beviewed as being unnecessarily hazardous. It could be hypoth-esized that a weaning regime would allow donor–recipientimmunologic engagement and promote the establishment oftolerance if a mechanism such as suppression is at play.Regardless, a slower weaning protocol may promote bothclinician and patient acceptability. Our criteria for resumingimmunosuppression were based for the most part on thepresence of significant biochemical graft dysfunction. Hence,some patients were restarted on immunosuppression eventhough the accompanying histological features may havebeen a mild exacerbation of the baseline findings. Withmaintenance of liver function as our primary aim, we placedlittle store on the histological findings because, as we discussbelow, the significance of common lobular hepatitis is unknown.

Several novel patterns of liver blood test abnormalities andhistological graft appearances were observed after withdrawalof immunosuppressive drugs. The expected biochemicalpattern of acute liver allograft rejection in the early posttrans-plantation period, with increases in biliary and hepatocellularenzyme levels and an associated increase in bilirubin levels,was uncommon. Biochemically, the variable but occasionallyvery severe increases in serum transaminase activity levels(up to 40-fold) indicate a hepatocellular-directed reaction, apattern that is consistent with the predominant damage tothis cell found in pharmacologically unmodified liver recipi-ents undergoing rejection.19,20 As was also noted in thePittsburgh patients, a biochemical flare-up of enzymes canresolve spontaneously or with minor reintroduction of immu-nosuppression. This pattern has also been observed inexperimental animals either when immunosuppression wasnot introduced or after discontinuation.21,22 However, classi-cal features of liver allograft rejection, as characterized by thetriad of endotheliitis, portal tract inflammation, and bile ductdamage, developed in a minority of patients only (4 of 18).More commonly when there was an increase in liver enzymelevels, the graft histology demonstrated moderate or severeportal tract inflammation, occasionally with features of alobular hepatitis. This pattern, the significance of which isunknown, is a common histological feature in liver graftbiopsies from immunosuppressed long-term recipients andcannot be attributed to known hepatitic viruses.23 Whetherthese graft-infiltrating cells represent an immune response toa latent serologically undiagnosed virus or a previouslyunrecognized forme fruste of allograft rejection is specula-

TABLE 3. Relationship Between Outcome of Immunosuppression Withdrawal and HLA Matching. A Lower Frequency of Class 1 and 2 Mismatches IsAssociated with a Favorable Outcome

ImmunosuppressionWithdrawal

HLA Loci Mismatch Scores

A Locus B Locus DR Locus Total

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Complete (n 5 5) 1 1 2 median 1 2 3 median 1 2 3 median 1 2 3 medianPartial (n 5 9) 2 1 2 median 2 3 4 median 2 3 4 median 2 3 4 medianAbsent (n 5 4) 0 1 2 median 3 4 5 median 3 4 3 median 3 4 5 median

HEPATOLOGY Vol. 27, No. 4, 1998 DEVLIN ET AL. 931

tive. Perhaps the response to steroids is more suggestive of arejection process. Further, the Pittsburgh group have sug-gested that late rejection does not show the features ofsubendothelial venular inflammation but is characterized by moreprominent periportal and lobular necroinflammation and paren-chymal regeneration than is classical early rejection.24

The apparent influence of systemic viral infections on graftfunction, possibly seen in 3 patients, is of interest and is inkeeping with our previous observation that such infectionscan be associated with late acute liver allograft rejection inconventionally immunosuppressed patients.25 In patientswho did not receive transplants, withdrawal of both chemo-therapy and immunosuppression can result in a biochemicaland histological flare-up of an underlying hepatitis, althoughin this study no evidence of the hepatitis viruses (hepatitis Bvirus or HCV) that cause chronic liver inflammation waspresent in the majority of cases.26 Close clinical surveillancewill be required to monitor other effects that may follow fromthe return of immunocompetence, such as recurrence ordeterioration of primary diseases that have an immunologicpathogenesis, which we have previously reported can occurin autoimmune hepatitis.27

Before clinicians embark on withdrawing immunosuppres-sion on a routine basis, criteria that can be used to selectappropriate patients are required. There has been consider-able interest in the observation from Starzl’s group that thepresence of microchimerism, derived from the donor, was auniversal feature of long-term liver and renal recipients, andthe thesis that enhancing this phenomenon may be associatedwith graft acceptance.1,2 This phenomenon was present inapproximately 40% of the long-term liver transplant recipi-ents studied in this series. The methodology used in ourstudy does allow donor and recipient differentiation at thegenomic level, and we can confirm that the foreign DNA thatwas detected has donor rather than third-party characteristics(such as could have been derived from a blood producttransfusion). We also noted a variation in the detection rate ofchimerism in relation to the recipient tissue, with skin havingthe highest incidence. Our results with respect to tissuedistribution and the overall proportion of patients withchimerism, however, are virtually identical to a recent studyfrom Suberbielle performed in long-term renal recipients inwhich chimerism was found in 33% of studied patients, againpredominantly in the skin.28 Our preferred method fordetection of donor class II MHC, based on the nested PCRmethodology for donor HLA-DRB1 alleles, is readily available

in molecular transplant HLA laboratories and appears to behighly sensitive.29 However, this technique does not yield anyinformation on the localization or viability of the detecteddonor genotype or the cell type from which it is derived. Thisis a general limitation to PCR-based methodology. Whether,for example, the donor DNA we are detecting is shed from thegraft and could be derived from the soluble class I antigen,which is released from liver parenchymal cells, or representslymphoid cells that have migrated and become establishedfrom the substantial passenger cell pool present in humanliver grafts cannot be determined.30,31

In this study, detection of systemic donor-type microchime-rism did not allow identification of tolerance and accordinglycannot be utilized as a tool in the patient selection forprospective drug withdrawal. A major role for microchime-rism as either a mechanism or a marker for graft acceptancewas not identified in the present series, with similar propor-tions of chimeric patients experiencing rejection and nonchi-meric patients exhibiting tolerance. Furthermore, a casereport of intractable acute allograft rejection developing inthe presence of chimerism 8 years after transplantation in aliver recipient in whom immunosuppression was reduced hasalso recently been reported.32 Our results also do not agreewith the observation that the presence of chimerism wasassociated with donor-specific unresponsiveness, as assessedby mixed lymphocyte reaction methodology in long-termkidney recipients.2 The sensitivity of in vitro observations ofantidonor reactivity using the mixed lymphocyte reactionmethodology, which employs recipient peripheral circulatingrather than graft-infiltrating cells retrieved under the um-brella of immunosuppression, is questionable. Despite theseresults, a central role for mixed cell chimerism in interpretingseveral immunologic observations and in the promotion oftolerance cannot be dismissed.

Because the presence of chimerism cannot be equated withtolerance, further investigations into identification of thisstate are required, taking into account the several controver-sial but presently unsubstantiated hypotheses relating to theunique position of liver grafts (for a review, see Wood andSachs33). The potential benefit of a degree of HLA matching insuccessful immunosuppression withdrawal should not beoveremphasized at present, given the comparatively smallstudy population presented. Nevertheless, the early patternsreported of lower mismatches in all loci being associated withtolerance are encouraging. Although the influence of HLAmatching in liver graft survival and rejection is complex,recent studies of large clinical populations, who are conven-tionally immunosuppressed, consistently demonstrate a lowerfrequency of rejection in the presence of fewer HLA A and Bloci mismatches.34,35 Such data alongside a possibly similarbut less important effect with DR matching provide evidenceto support the importance of our early observations.36 Remov-ing immunosuppression may amplify and hence unmask theeffects of HLA matching.

On the basis of our results, early rejection history, whichmay be related to the degree of HLA matching, potentiallyoffers a more readily accessible tool for assigning risk beforewithdrawal. Finally, and perhaps most surprisingly, an influ-ence of the original transplantation indication and subse-quent ability of a patient to be withdrawn from immunosup-pression has emerged. Disorders with a well-characterizedimmunologic or viral etiopathological basis appear morelikely to experience graft dysfunction after withdrawal. Such

TABLE 4. Relationship Between the Outcome of ImmunosuppressionWithdrawal, the Original Liver Disorder and History

of Early Acute Rejection

ImmunosuppressionWithdrawal Transplant Indication

Early Rejection n (%)

Clinical Histological

Complete (n 5 5) BCS 3 2, PSC, ALD (with HCV),CF

5 (56) 3 (33)

Partial (n 5 9) PSC, HCV, NANB, tumor, PBC 3 2,BCS, FHF

Absent (n 5 4) AIH, AIH and HCV, HCV, NANB 3 (75) 2 (50)

Abbreviations: AIH, autoimmune hepatitis; ALD, alcoholic liver disease;BCS, Budd-Chiari syndrome; CF, cystic fibrosis; FHF, fulminant hepaticfailure; NANB, non-A non-B hepatitis; PBC, primary biliary cirrhosis; PSC,primary sclerosing cholangitis.

932 DEVLIN ET AL. HEPATOLOGY April 1998

a pattern if confirmed raises several hypotheses. First, we areattributing graft dysfunction to rejection when we are in factobserving recurrence of the primary disease unmasked bydrug withdrawal or that such disorders in some mannerpromote the host versus allograft response. The latter possibil-ity is supported by reports, for example, demonstratingincreased liver rejection in the presence of active HCVinfection.37 The influence on rejection frequency of a disordersuch as autoimmune hepatitis, which is characterized byincreased immunologic responsiveness, is not known.

Regardless of the associations with or mechanisms that under-lie the presence of clinical tolerance in long-term liver recipients,this phenomenon, even in varying degree, has the potential forimproving their natural history. Longer follow-up is requiredbefore the beneficial effect of immunosuppression withdrawal ondrug-related toxicity or occurrence of de novo and recurrentinfectious and malignant complications can be determined. Re-ports regarding the efficacy of partial immunosuppressive drugwithdrawal in relation to reversal of drug toxicity complicationsare mixed, particularly when these are long-standing, as forinstance with chronic Cyclosporin A-related nephrotoxicity.38

Nevertheless, data from a recent study in renal patients of steroidwithdrawal and our own preliminary observations suggest thatsignificant reductions in the prevalence of hypertension andglucose and lipid abnormalities can be anticipated.39 It is ourpreliminary conclusion that immunosuppression withdrawal isfeasible in a proportion of patients without major hazard ifperformed with controlled clinical monitoring.

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