Human Leukocyte Antigen-G ExpressionAfter Heart Transplantation Is Associated With a Reduced

Incidence of Rejection

Nermine Lila, DVM; Catherine Amrein, MD; Romain Guillemain, MD; Patrick Chevalier, MD;Christian Latremouille, MD; Jean-Noël Fabiani, MD; Jean Dausset, MD, PhD;

Edgardo D. Carosella, MD; Alain Carpentier, MD, PhD

Background—Human leukocyte antigen (HLA)-G, a nonclassic major histocompatibility complex class I moleculeexpressed in the extravillous cytotrophoblast at the feto-maternal interface, is known to protect the fetus from maternalcellular immunity. In a preliminary study, we showed that HLA-G is expressed in the hearts of some patients after hearttransplantation.

Methods and Results—In the present study, a larger number of patients was investigated to confirm this finding and to lookfor possible correlations between HLA-G expression and the number and types of rejection. Expression of HLA-G inendomyocardial biopsy specimens was investigated by immunohistochemical analysis, and detection of the solubleHLA-G in the serum was performed by immunoprecipitation followed by Western blot analysis. HLA-G was detectedin the biopsy specimens and serum of 9 of 51 patients (18%). The number of episodes of acute rejection wassignificantly lower in HLA-G–positive patients (1.261.1) as compared with HLA-G–negative patients (4.562.8)(P,0.001). No chronic rejection was observed in HLA-G–positive patients, whereas 15 HLA-G–negative patients hadchronic rejection (P,0.032). A longitudinal study of these patients reveals that the status of HLA-G expression wasmaintained after 6 months both in serum and in biopsy specimens. During this period, HLA-G–positive patients did nothave chronic rejection.

Conclusions—There is a significant correlation between rejection and HLA-G expression in the heart after transplantation.HLA-G expression and its effect in reducing the incidence and severity of rejection seem to be stable throughout theevolution.(Circulation. 2002;105:1949-1954.)

Key Words: transplantationn genesn rejection n grafting

I nduction of donor-specific tolerance is a major goal inorgan transplantation. It is well known that many patients

are free from significant graft rejection under medical treat-ment, whereas others have several episodes of acute rejectionor have signs of chronic rejection.1–3 The search for thereasons of these variations is important to understand themechanism of such tolerance.

The nonclassic human leukocyte antigen (HLA) class Imolecule HLA-G, expressed on extravillous cytotrophoblastsat the feto-maternal interface during pregnancy,4,5 has beenreported to play a role in mediating maternal tolerance of thefetal “semiallogeneic” graft.6 This HLA-G antigen exhibitslow polymorphism and can be expressed as both membrane-bound proteins (HLA-G1, HLA-G2, HLA-G3, HLA-G4) andsoluble isoforms (HLA-G5, HLA-G6, HLA-G7).7–9 The sol-

uble isoforms HLA-G5 and HLA-G6 have been detected inamniotic fluid and serum of pregnant women.10,11 HLA-Gexpression was initially found to be restricted to placenta.More recently, its expression has been detected in thymicepithelial cells,12 various malignant cells,13–15 and peripheralblood monocytes activated by IL-10.16 HLA-G is known toinhibit cytotoxic activity of T lymphocytes and natural killercells (NK),17,18 which are essential effector cells in graftrejection. We postulated that induction of HLA-G expressionby grafting might represent a means to disallow immunesurveillance and to favor tolerance of the transplanted organ.In a preliminary study, we showed that HLA-G was ex-pressed in the hearts of some patients after heart transplan-tation, but the number of patients was limited.19 The aim ofthe present retrospective study was to confirm this finding in

Received October 25, 2001; revision received February 7, 2002; accepted February 7, 2002.From the Laboratory for the Study of Cardiac Grafts and Prostheses, University of Pierre et Marie Curie, Broussais Hospital (N.L., A.C.); the

Department of Cardiovascular Surgery and Organ Transplantation, Georges Pompidou Hospital (C.A., R.G., P.C., C.L., J.-N.F., A.C.); and ResearchInstitute for Hemato-Immunology, CEA Laboratory, Saint-Louis Hospital (J.D., E.D.C.), Paris, France.

Correspondence to Nermine Lila, DVM, Laboratory for the Study of Cardiac Grafts and Prostheses, University of Pierre et Marie Curie, BroussaisHospital, 96 rue Didot 75014 Paris, France. E-mail nerminelila@yahoo.fr

© 2002 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/01.CIR.0000015075.89984.46

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a larger number of patients followed for a longer period oftime and to assess whether there was a correlation betweenthe rejection phenomena and HLA-G expression.

A longitudinal study of HLA-G expression was performed6 months after the first investigation to evaluate the persis-tence of HLA-G expression and the clinical evolution of thesepatients.

MethodsPatientsThis study comprises 51 patients (42 men and 9 women) whose agesranged from 20 to 73 years (mean, 55.5 years) and who received aheart transplant in our institution. Heart transplantation had beenperformed because of dilated cardiomyopathy in 28 patients (54.9%),coronary artery disease in 12 (23.5%), valvular disease in 6 (11.8%),and congenital heart disease and other reasons in 5 patients (9.8%).Donors included 42 men and 9 women with a mean age of 32.5years. All recipients were screened before transplantation for HLAantibodies, and no patient was found to have HLA-specific antibod-ies. All recipients and donors were typed for HLA-A, HLA-B, andHLA-DR by means of routine serological methods.

Length of follow-up was 1 to 5 years for 13 patients, 6 to 10 yearsfor 22 patients, and 11 to 15 years for 16 patients. All patientsreceived induction therapy with rabbit anti-thymocyte immunoglob-ulin (2.5 mg/kg body weight per day for 3 days). Immunosuppressivetreatment consisted of cyclosporine, prednisone, and azathioprine ormycophenolate. Prednisone was given on the first day after trans-plantation at 4 to 5 mg/kg body weight for 3 days. The dose ofprednisone then was decreased gradually in increments of 0.2 mg/kgbody weight per day. Thereafter, an attempt was made in all patientsto reduce or stop prednisone according to the results of endomyo-cardial biopsy specimens, the body weight, and possible side effects.

Azathioprine was given on the first day after transplantation atdoses of 3 mg/kg body weight per day. In 1997, azathioprine wasreplaced by mycophenolate at a rate of 2 to 3 g/d, according toleukocyte counts (target, 4000 to 6000/mm) and digestive tolerance.

Cyclosporine was given the third day after transplantation at 3 to4 mg/d. The dose of cyclosporine was adapted according to its bloodconcentration measured by radioimmunoassay (range, 100 to 150ng/mL the first year after transplantation), renal function, and thenumber of episodes of rejection. The episodes of acute rejection weretreated, according to the grade and type of rejection, either byreinforced doses of prednisone at 15 mg/kg body weight per day(orally) or by using the prednisone with rabbit anti-thymocyteimmunoglobulin.

The degree of rejection was classified according to the workingformulation of the International Society for Heart and Lung Trans-plantation (ISHLT)20: 0, no rejection; 1A, minimal graft rejection;1B, mild to moderate rejection; 3A, moderate to severe graftrejection; and 4, very severe graft rejection.

Five to six endomyocardial specimens were taken from the rightventricle of each patient after transplantation; at least 4 of the biopsyspecimens were used for the histopathological diagnosis of rejection,and 1 biopsy specimen was used for immunohistochemical analysisof HLA-G expression. The number of 1B and 3A acute rejectionsduring the first postoperative year was assessed.

Chronic rejection and its characteristic graft vascular disease weredetected by systematic angiography of the coronary arteries once peryear in every patient. Patient characteristics and associated diseaseswere recorded: age, sex, race, donors (age, sex, and race), tumor,diabetes, arterial hypertension, serum creatinine level, anddyslipidemia.

Laboratory InvestigationExpression of the HLA-G molecule was analyzed by immunohisto-chemical analysis of endomyocardial biopsy specimens, and detec-

tion of its soluble forms in the serum was achieved by immunopre-cipitation followed by Western blot analysis.

In addition to the biopsy specimens used for histopathologicalanalysis and immunohistochemistry, control specimens were ob-tained from 4 human trophoblasts as positive control and healthyskin from nonpregnant women (after mammoplasty) as negativecontrol of HLA-G expression.

Blood was collected from the patient at the time of the biopsy andcentrifuged to obtain the serum. Serum of 8 healthy volunteer donors(4 men and 4 nonpregnant women) was used as control.

Monoclonal and polyclonal antibodies were used for the detectionof HLA-G molecules in the biopsy specimens and serum of patients.

Monoclonal antibodies (mAbs) used were 87G IgG2a anti–HLA-G1 and -G5 (provided by D. Geraghty, Fred HutchinsonCancer Research, Seattle, Wash), 4H84 IgG1 antidenatured HLA-Gheavy chain (provided by M. McMaster, University of California,San Francisco), and W6/32 IgG2a anti-HLA class I heavy chainsassociated withb2m (Sigma, Milwaukee, Wis). An isotype-matchedantibody (Sigma) was used as control.

A rabbit polyclonal antibody PAG5-6 generated against theC-terminal peptide of the HLA-Ga-chain encoded by intron 4sequences was used to specifically recognize the soluble formsHLA-G5 and HLA-G6.9

Histology and ImmunohistochemistryFor histology, 4-mm–thick sections were obtained from each paraffinblock and stained with hematoxylin and eosin for diagnosis ofrejection.

For immunohistochemical studies, 6-mm–thick sections of frozentissues were fixed for 10 minutes in cold acetone, dehydrated, andpermeabilized with saponine in PBS. Staining procedures wereprocessed with the Dako Envision System (DAKO). Samples wereincubated for 30 minutes in 50% human normal serum in PBS toeliminate nonspecific bindings. Samples were incubated with thefollowing primary mAbs for 30 minutes: W6/32, 87G, 4H84 mAbsand control antibody and followed by incubation with a secondaryconjugated goat anti-mouse/antibody coupled with peroxidase(DAKO) for 30 minutes. After incubation for 10 minutes withsubstrate, sections were counterstained with hematoxylin dye andmounted with antimounting medium (DAKO).

Immunoprecipitation and Western Blot AnalysisThe class I positive M8 melanoma cell line kindly provided by F.Jotereau (INSERM U211, Nantes, France) was transfected with afull-length HLA-G5 cDNA subcloned in a vector pcDNA (Invitro-gen, San Diego, Calif), as previously described.9 The M8-HLA-G5transfectant and M8-pcDNA (transfected with the vector alone) wereused, respectively, as positive and negative controls for Western blotanalysis. One milliliter of both soluble HLA-G5 transfectant super-natant and serum samples were incubated overnight at 4°C withPAG5-6 polyclonal antibody.

Protein A–sepharose beads were added and incubated for 1 hour at4°C. The beads bearing the immune complexes were washed andincubated for 5 minutes at 95°C with 1% SDS sample buffer and150 mmol/L b-mercapto ethanol; 25-mL aliquots of total proteinfrom the indicated samples were separated on 12% SDS-PAGE andtransferred onto nitrocellulose membrane (Hybond-C, Amersham).After blocking in 5% nonfat milk in PBS–0.2% Tween-20 (Sigma),the membrane was incubated with the 4H84 mAb overnight at 4°C.After washing in PBS–0.2% Tween, the membrane was incubatedfor 30 minutes at room temperature with anti-mouse peroxidaseconjugate reagent (Amersham). After washing, the staining reactionwas carried out with the use of enhanced chemiluminescenceWestern blotting detection (Amersham), after which the membranewas exposed to Kodak film.

Statistical AnalysisData are presented as mean6SEM. The Student’st test was used,and a value ofP,0.05 was considered significant.

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ResultsExpression of HLA-G in EndomyocardialBiopsy SpecimensExpression of HLA-G antigens in sections of endomyocardialbiopsy specimens after heart transplantation was investigatedthrough immunohistochemical analysis with the use ofHLA-G mAbs: 87G IgG2a specific for HLA-G1 andHLA-G5 and 4H84 IgG1, which recognizes thea1 domain(pan–HLA-G).

We detected HLA-G proteins in 9 of 51 patients. Sevenpatients’ specimens were positively stained by both 87G and4H84 mAbs and two were positively stained by 4H84 mAbonly. Trophoblasts used as positive control exhibited strongstaining with 87G and 4H84 mAbs, whereas skin biopsyspecimens taken from healthy nonpregnant women (aftermammoplasty) were negative (Figure 1). Staining withW6/32 IgG2a was used to control the presence of intact HLAclass I molecules in different tissues and was positive in alltested tissue biopsy specimens (data not shown).

Detection of Soluble HLA-G Proteins in the Serumof Heart-Transplanted PatientsWe characterized the HLA-G–soluble antigens in the serumof 9 of 51 patients investigated by immunoprecipitation withPAG5-6, a rabbit polyclonal antibody specific for the solubleHLA-G5 and HLA-G6 proteins, followed by Western blotanalysis with 4H84 mAb.

Two isoforms of HLA-G antigens, with the sizes of 37 and27 kDa corresponding to the reported products of the alter-natively spliced transcripts sHLA-G5 and HLA-G6, could bedetected in the serum. In 7 patients, the soluble form HLA-G5was detected, and in 2 patients, a band corresponding to thesoluble form G6 was found (Figure 2).

In summary, HLA-G was detected both in the serum andmyocardial biopsy specimens in 9 patients (18%). Amongthese patients, 7 were positive for 87G and 4H84 mAbs, andthe soluble HLA-G5 was detected in their serum. Theremaining 2 patients were positive only for 4H84 mAb, andthe soluble HLA-G6 was detected in their serum. The

Figure 1. Immunohistochemical analysis ofHLA-G expression in endomyocardial biopsysamples (a, b), trophoblast as positive control (c),and healthy skin as negative control (d). Controlantibody 87G detects HLA-G1 and HLA-G5 iso-forms, and 4H84 detects the denatured form ofHLA-G. a, Endomyocardial biopsy specimen:positive staining with both 87G and 4H84; b, en-domyocardial biopsy specimen: negative stainingwith 87G and positive staining with 4H84; c, tro-phoblast biopsy specimen: positive staining withboth 87G and 4H84; d, healthy skin biopsy spec-imen: negative staining with both 87G and 4H84.

Figure 2. Detection of soluble forms of HLA-G5 and HLA-G6 in serum samples of 9 patients without chronic rejection (T1, T2, T3, T4,T5, T6, T7, T8, T9). M8 HLA-G5 transfectant cells used as positive control; M8 pc-DNA cells used as negative control. One control cor-responds to the serum of a healthy donor used as negative control.

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longitudinal study of these patients reveals that the status ofHLA-G expression was maintained after 6 months both in thebiopsy specimens and in the serum. HLA-G–positive patientsremained positive, and HLA-G–negative patients remainednegative.

Clinical AnalysisHLA-G–positive patients (18%) were 7 men and 2 women(all white), with a mean age of 58611.3 years (Table). Theexpression of HLA-G was detected during the followingposttransplantation period: (1) 1 to 5 years in 2 patients, (2) 6to 10 years in 5 patients, and (3) 11 to 15 years in 2 patients.Three HLA-G–positive patients (33.3%) had a pretransplan-tation diagnosis of coronary artery disease, and 6 had dilatedcardiomyopathy (66.7%). None of the HLA-G–positive pa-tients had a history of diabetes mellitus when compared with6 (14.3%) HLA-G–negative patients. Five HLA-G–positivepatients (55.6%) had a history of hypertension compared with38 HLA-G–negative patients (90.5%) (P,0.008). Renalfunction, as reflected by mean serum creatinine, was140645.12 in HLA-G–positive patients compared with187.56131.02 in HLA-G–negative patients. No tumor wasdetected in HLA-G–positive patients, whereas tumors devel-oped in 8 HLA-G–negative patients. No significant differencein age, sex, or race was observed between HLA-G–positiveand HLA-G–negative patients.

Acute and Chronic RejectionClinical analysis of the patients showed that the number ofepisodes of acute rejection in the first year after transplanta-tion in HLA-G–positive patients was significantly decreased,1.261.1 when compared with 4.562.8 in HLA-G–negativepatients (P,0.001). In addition, no chronic rejection could bedetected in HLA-G–positive patients, whereas 15 HLA-G–negative patients had chronic rejection (P,0.032).

HLA Match in HLA-G–Positive andHLA-G–Negative PatientsIn HLA-G–positive patients (n59), 2 patients had 1 HLA-Aand 1 HLA-B match, 2 patients had 1 HLA-A and 1 HLA-Drmatch, 2 patients had 1 HLA-B match, 2 patients had 1HLA-DR match, and 1 patient had no HLA-A, HLA-B, orHLA-DR match.

In HLA-G–negative patients without chronic rejection(n527), 6 patients had 1 HLA-A match, 2 patients had 1HLA-B match, 2 patients had 1 HLA-DR match, 2 patientshad 1 HLA-A, 1 HLA-B, and 1 HLA-DR match, and 15patients had no HLA-A, HLA-B, or HLA-DR match.

In HLA-G–negative patients with chronic rejection(n515), 5 patients had 1 HLA-A match, 1 patient had 1HLA-A and 1 HLA-B match, 1 patient had 1 HLA-A and 1HLA-DR match, 1 patient had 1 HLA-B match, and 7patients had no HLA-A, HLA-B, or HLA-DR match.

DiscussionThe development of tolerance requires that the immunesystem may utilize several strategies for neutralizing self-

Baseline Characteristics of HLA-G–Positive and HLA-G–Negative Patients

HLA-G–PositivePatients(n59)

HLA-G–NegativePatients(n542) P

Age, years 58611.3 53.3613.3 0.347

Sex

Men 7 (77.8) 35 (83.3)

Women 2 (22.2) 7 (16.7) 0.691

Race

White 9 (100) 40 (95.2)

Black 0 2 (4.8) 0.504

Posttransplantation follow-up

1–5 years 2 (22.2) 11 (26.2)

6–10 years 5 (55.6) 17 (40.5)

11–15 years 2 (22.2) 14 (33.3)

Pretransplantation heart disease

Dilated cardiomyopathy 6 (66.7) 22 (52.4)

Coronary artery disease 3 (33.3) 9 (21.4)

Valvulopathy disease 0 6 (14.3)

Other 0 5 (11.9)

Hypertension

Yes 5 (55.6) 38 (90.5)

No 4 (44.4) 4 (9.5) ,0.008*

Diabetes mellitus

Yes 0 6 (14.3)

No 9 (100) 36 (85.7) 0.227

Dyslipidemia

Yes 6 (66.7) 29 (69)

No 3 (33.3) 13 (31) 0.888

Baseline serum creatinine,mg/dL

140.4645.12 187.56131.02 0.295

Cancer

Yes 0 8 (19)

No 9 (100) 34 (81) 0.153

Donor

Age, years 29.5610.9 35.5611.9 0.164

Sex

Men 6 (66.7) 36 (85.7)

Women 3 (33.3) 6 (14.3) 0.173

Race

White 9 (100) 41 (98)

Black 0 1 (2) 0.640

Mean rejection score in firstyear after transplantation

1.261.1 4.562.8 ,0.001*

Chronic rejection

Yes 0 15 (35.7)

No 9 (100) 27 (64.3) ,0.032*

Values are mean6SD or n (%).*Significant correlation.

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reactive T cells. These strategies include deletion,21 anergy,22

and immunoregulatory pathways.23

This study suggests that the expression of HLA-G afterheart transplantation may allow an alternative strategy todownregulate the host immunoresponse and to limit graftrejection. In vitro, soluble HLA-G has been demonstrated toinduce apoptosis of activated CD81 T cells24 and to modu-late NK25 and allo-CTL response,26 whereas membrane-bound HLA-G proteins have been shown to inhibit both NKand T cell–mediated cytolysis,6,18 to suppress the prolifera-tion of allospecific CD41T lymphocytes,27 and to induceTh2 cytokine profile.28 Interestingly, we recently demon-strated that soluble HLA-G protein secreted by allo-specificCD41 T cells suppresses the allo-proliferative response.29

In the present study, HLA-G expression was found in 18%of the 51 patients who received a heart transplant 1 to 15years before this study was undertaken.

In HLA-G–positive patients, the incidence of acute rejec-tion was significantly decreased (P,0.001), and there was nocase of chronic rejection (P,0.032) when compared withHLA-G–negative patients. Expression of HLA-G after a6-month interval persisted both in the biopsy specimens andin the serum of HLA-G–positive patients, whereas the HLA-G–negative patients remained so. No significant differencebetween age, race, or sex could be detected between HLA-G–positive and HLA-G–negative patients. It is of note thatthe two women expressing HLA-G in this series were notpregnant after transplantation and that none of the HLA-G–positive patients had a tumor. Therefore, the expression ofHLA-G was likely activated during the process of transplan-tation, not by pregnancy or tumor development.

In heart-transplanted patients, HLA-G expression mayallow escape from recognition and destruction of the graft byalloreactive T cells. Furthermore, the soluble HLA-G formsHLA-G5 and HLA-G6 detected in the serum of some patientsmay play an additional role in inhibiting the cytotoxic activityof NK cells.25 The expression of HLA-G in this series wasrecognized at different times after transplantation from 1 to15 years. The fact that a repeated investigation after 6 monthsshowed a persistent expression of HLA-G may indicate thatthis expression can be activated during the first year aftertransplantation and maintained by factors yet to be identified.Experimentally, numerous factors have been shown to up-regulate HLA-G expression, such as IL-10.16 An IL-10–HLA-G autocrine effect may contribute to graft tolerance.However, we cannot exclude that HLA-G–positive patientsmay carry out specific HLA-G alleles associated with highHLA-G production.30

In addition, the degree of histoincompatibility betweendonor and recipient may play a role in stimulating HLA-Gproduction. Today, heart transplantation has been establishedas a valuable therapeutic approach to end-stage heart failure.The search for HLA-G expression in transplant recipientscould provide a new understanding of the factors favoringgraft tolerance and new modulation of immunosuppressivetherapy. For example, the use of soluble forms of HLA-Gcould contribute to immunosuppression and make it possibleto reduce the amount of other immunosuppressive agents.

The present study supports the hypothesis that HLA-Gexpression in heart-transplanted patients is associated with abetter graft tolerance. HLA-G expression seems to minimizeacute rejection and to protect the graft from chronic rejection.It remains to be understood why some heart-transplantedpatients express HLA-G, whereas others do not.

AcknowledgmentsThis study was supported by grants from the Société Française deTransplantation, the Vancaire la Mucoviscidose, the EtablissementFrançaise de Greffes, and the French Commissariat à l’EnergieAtomique. We thank Drs Daniel Geraghty and Micheal McMasterfor generously providing HLA-G antibodies, Pascal Paul and ImanKhalil-Daher for technical advice and helpful discussions, and SelimAractingi for providing skin biopsy specimens.

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Latremouille, Jean-Noël Fabiani, Jean Dausset, Edgardo D. Carosella and Alain CarpentierNermine Lila, Catherine Amrein, Romain Guillemain, Patrick Chevalier, Christian

a Reduced Incidence of RejectionHuman Leukocyte Antigen-G Expression After Heart Transplantation Is Associated With

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