Immunohistochemical phenotyping of theinflammatory infiltrate in de novoautoimmune hepatitis after livertransplantation in children

dn-AIH is a form of insidious late liver graftdysfunction, defined by the presence of serumautoantibodies, hypergammaglobulinemia andhistological features similar to those of classicalAIH (1). This condition, which develops inpatients transplanted for non-autoimmune dis-orders, responds well to the treatment schedulefor classical AIH, including intermediate-dosesteroids in addition to azathioprine or myco-phenolate mofetil (1, 2). In contrast, although ithas been suggested that it might be a form of

ACR (3), dn-AIH does not respond to conven-tional treatment of cellular rejection with shortcourses of high-dose steroids (1).Development of circulating autoantibodies is

relatively common after liver transplantation,affecting 13–42% of pediatric patients five yrafter grafting (4–6), but the prevalence of fullblown dn-AIH is only 2.4–5% (1, 7, 8). Thisprevalence is much lower than that of ACR (60–70%) (9), but considerably higher than that ofclassical AIH in the non-transplanted population(1.9 cases per 100 000 in Norway and 1 per200 000 in the USA) (10, 11).Although the term ‘‘autoimmune’’ to define

hepatitis affecting an allogeneic organ is oftenchallenged, the concept of recurrence of AIHafter grafting in patients transplanted because ofAIH has never been questioned, despite the fact

Had�zic N, Quaglia A, Cotoi C, Hussain MJ, Brown N, Vergani D,Mieli-Vergani G. Immunohistochemical phenotyping of the inflamma-tory infiltrate in de novo autoimmune hepatitis after liver transplanta-tion in children.

Abstract: We have investigated the inflammatory infiltrate in post-transplant dn-AIH, a form of late insidious graft rejection, focusing ontranscription factors defining effector and T-regs, using an antigenretrieval immunohistochemical method on archived liver tissue, andcompared it with ACR and classical AIH. Paraffin-embedded liverbiopsies from pediatric patients with dn-AIH (n = 10), ACR (n = 10),and AIH (n = 13) were selected randomly and stained using antibodiesdirected to CD4, CD8, T-bet (marker of Th1 polarization), GATA-3(marker of Th2 polarization), FOXP3 (marker for T regulatory cells),IL-17, CD56 (NK cells), and perforin. Portal and lobular lymphocyticinfiltrate was assessed semi-quantitatively. Prominent CD4, CD8, andT-bet positivity were present in both the lobular and portal infiltrate ofall three conditions. Overall T-bet score of lobular inflammation in thedn-AIH group was lower than in the ACR and AIH groups (p = 0.02).In contrast, most samples showed absent or minimal GATA-3 posi-tivity. FOXP3, CD56, IL-17, and perforin staining of mild to moderateseverity were present in all three groups in both the portal and lobularinfiltrate. A Th1 polarization of the inflammatory infiltrate character-izes dn-AIH, but also ACR and AIH.

Nedim Had�zic1,2, Alberto Quaglia2,Corina Cotoi2, Munther J. Hussain2,Nigel Brown2, Diego Vergani2 andGiorgina Mieli-Vergani1,2

1Department of Child Health, 2Institute for LiverStudies, King�s College Medical School at King�sCollege Hospital, Denmark Hill, London, UK

Key words: liver transplantation – de novoautoimmune hepatitis – cellular rejection –autoimmune hepatitis

Dr. Nedim Had�zic, Professor of Paediatric Hepatology,Department of Child Health, King�s College Hospital,Denmark Hill SE5 9RS, London, UKTel.: 44 20 32994120Fax: 44 20 32994228E-mail: nedim.hadzic@kcl.ac.uk

This manuscript was presented in abstract form atAnnual Meeting of British Association for Study ofthe Liver (BASL) in London, UK, 10–12 September2008.

Accepted for publication 2 April 2012

Abbreviations: ACR, acute cellular rejection; AIH, auto-immune hepatitis; DAB, diaminobenzidine; dn-AIH,de novo autoimmune hepatitis; GATA-3, GATA-bindingprotein 3; HLA, human leukocyte antigen; HRP, horse-radish peroxidase; IFNc, interferon-gamma; IL, interleukin;T-regs, regulatory T cells.

Pediatr Transplantation 2012 � 2012 John Wiley & Sons A/S.

Pediatric TransplantationDOI: 10.1111/j.1399-3046.2012.01723.x

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that also in this condition the target organ isallogeneic. As the antigenic targets for liver-specific autoimmunity are species specific andshared by both recipient and donor livers, andthe transplanted organ is repopulated by recipi-ent�s immune cells with antigen-presenting abil-ity, an autoimmune attack to the liver can alsoarise in the context of allotransplantation (12).The pathogenesis of dn-AIH remains uncer-

tain (12). In classical AIH, where autoaggressivecellular immunity is thought to be the mechanismleading to liver damage, most of the liver-infiltrating T cells are positive for the CD4helper/inducer phenotype (13). Autoimmuneliver damage is believed to be orchestrated byCD4-positive T lymphocytes recognizing a self-antigenic peptide embraced by an HLA class IImolecule and presented to uncommitted T helper(Th0) cells by professional antigen-presentingcells. The Th0 cells become activated and differ-entiated into functional phenotypes according tothe nature of the antigen and the cytokinesprevailing in the microenvironment. Arising inthe presence of the macrophage-produced IL-12,Th1 cells secrete mainly IL-2 and IFNc, whichactivate macrophages, enhance expression ofHLA class I, and induce expression of HLAclass II molecules on hepatocytes, rendering themable to present the autoantigenic peptide to Th0cells, thus perpetuating the immune recognitioncycle. Th2 cells, which differentiate from Th0 ifthe microenvironment is rich in IL-4, producemainly IL-4, IL-10, and IL-13, all of whichinduce autoantibody production by B lympho-cytes. It has recently emerged that key players inthe commitment of CD4+ T cells toward theTh1 or the Th2 pathways are the transcriptionfactor T-bet (T-box expressed in T cells) (14) andGATA-3 (15), respectively. Production of T-betis upregulated by IFNc, a signature cytokine forTh1 response, which prevails in most autoim-mune disorders (14).The process of autoantigen recognition is

controlled by regulatory mechanisms, the failureof which perpetuates the autoimmune attack. Inclassical AIH, CD4+CD25+ T-regs, which con-trol the innate and adaptive immune responses bypreventing the proliferation and effector functionof autoreactive T cells, are defective in numberand function (16). Moreover, the percentage ofT-regs inversely correlates with biomarkers ofdisease severity (17). Expression of forkhead/winged-helix transcription factor (FOXP3) inCD4+CD25+ T cells is associated with theability to exert regulatory functions (18, 19).To investigate whether the immune mecha-

nisms leading to dn-AIH are similar to those

leading to classical AIH or ACR, we havecompared the inflammatory infiltrate in threeconditions, focusing on transcription factorsdefining effector and T-regs.

Material and methods

Ten archived liver biopsies from pediatric patients withdn-AIH were selected randomly from the database. Noneof these patients had received a blood group–mismatchedliver graft. All had abnormal liver function tests, normalultrasound Doppler studies, negative viral screening,hypergammaglobulinemia, and/or elevated titers of auto-antibodies (Table 1a). Their liver biopsies were compatiblewith the diagnosis of chronic hepatitis, showing a portalinfiltrate of moderate degree composed predominantly oflymphocytes with a variable proportion of plasma cells, withmild to moderate interface activity. A lobular inflammatorycomponent was invariably present with sinusoidal lympho-cytosis, necro-inflammatory foci, and in most cases,perivenular liver cell necrosis with associated inflammation,including plasma cells. Archived paraffin-embedded liverbiopsies from 10 children with ACR and 13 with classicalAIH, diagnosed according to international criteria (20, 21),were also randomly selected from the database and used ascontrols (Table 1b,c). Histological forms of atypical cellularrejection such as idiopathic chronic hepatitis or isolatedcentral perivenulitis were not included in this analysis. All 10children with ACR responded to high-dose steroids and all13 children with AIH achieved initial biochemical remissionwith immunosuppressive treatment. Of the 13 children withclassical AIH, eight were biopsied before treatment, whilefive were on immunosuppression at the time of biopsy: twohad already been treated with prednisolone for four andseven wk, respectively, because of severe coagulopathy atpresentation; one was treated with cyclophosphamide andchloroquine for associated systemic lupus erythematosus;and two were biopsied during biochemical relapse while onlow-dose immunosuppression with prednisolone combinedwith azathioprine (1) and mycophenolate mofetil (1). Allbiopsies were performed for clinical reasons, after obtainingwritten informed consent from older patients, or parents orguardians for the procedure and tissue storage.

Immunohistochemical studies

Immunohistochemistry on paraffin-embedded biopsies wasperformed using two techniques: the HRP-based two-stepNovolink system (Novocastra, Newcastle upon Tyne, UK)– which has a sensitivity several-fold greater than conven-tional HRP techniques – when using primary murinemonoclonal antibodies (22), and the avidin–biotin complextechnique when using anti-IL-17 primary goat anti-humanantibody (23).Briefly, the biopsies were deparaffinized in xylene and

rehydrated through descending concentrations of ethanol.Heat-induced epitope retrieval in Tris–citrate–EDTA buffer(pH 9.0) was performed before application of the antibody.For the Novolink technique (22), peroxidase and protein

block reagents were used before and after the primaryantibody. The primary mouse anti-human antibodies wereused at the following dilutions: CD4 1/50 (Vector Lab,Peterborough, UK), CD8 1/10 (gift from MH Brown, JohnRadcliffe Hospital, Oxford, UK), T-bet 1/100 (Santa CruzBiotechnology Inc, Santa Cruz, CA, USA), GATA-3 1/200(Santa Cruz Biotechnology Inc,), FOXP3 1/100 (Abcam plc,

Had�zic et al.

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Cambridge, UK), CD56 1/20 (Invitrogen, Camarillo, CA,USA), and perforin 1/200 (gift from Dr. GM Griffiths,MRC Laboratory for Molecular Cell Biology, UniversityCollege London, London, UK). The sections were incu-bated with antibodies for 60 min in a humid chamber atroom temperature, followed by the addition of Novolinkrevealing polymer anti-mouse peroxidase-conjugated anti-body. The peroxidase reaction was developed using theDAB-chromogenic substrate for 10 min.For IL-17 detection (23), an avidin–biotin block reagent

was used before the primary antibody. The sections wereincubated with a polyclonal goat anti-human IL-17antibody (R&D Systems Europe, Abingdon, UK) at a

dilution of 1/30 for 60 min at room temperature.After washing, the sections were incubated with biotin-conjugated secondary anti-goat antibody (horse anti-goatIgG; Vector Laboratories, Burlingame, CA, USA) at adilution of 1/50 for 30 min. Immune reactivity wasvisualized by avidin–biotin–peroxidase complex (ABCcomplex; Vector Laboratories). Peroxidase activity wasdeveloped by DAB for eight min.All sections were counterstained in hematoxylin and

water-mounted in glycerol. Negative controls were the sameslides without the primary antibody. Nuclear staining inkeratinocytes was used as positive control for GATA-3immunostaining.

Table 1. Clinical features of the patient groups at the time of liver biopsy

Age(months) Sex

AST (IU/L,nr < 50)

GGT (IU/L,nr < 55)

Bilirubin(lM, nr < 20) Autoantibodies

IgG (mM,nr 5–15)

Intervalpost-LT(months)

Original diagnosisbefore LT

Treatment beforebiopsy

(a) dn-AIH1 52 F 139 22 7 SMA 1/40 16.9 9 Bile salt export pump

deficiencyCyA, Aza, Pred

2 69 F 266 31 9 ANA 1/2560 15.2 54 Biliary atresia CyA, Aza, Pred3 155 F 1304 146 251 SMA 1/160 13.4 116 Biliary atresia CyA, Aza, Pred4 105 F 238 81 8 Anti-LC1 19.4 7 Glycogen storage disease

type IbTac, Pred

5 167 M 133 65 25 ANA 1/640 15.2 77 Biliary atresia CyA, Aza, Pred6 139 F 69 7 5 SMA 1/40 22.0 36 Biliary atresia Tac, Pred7 73 M 255 53 15 ANA 1/160 15.3 65 Biliary atresia CyA, Aza, Pred8 204 M 102 69 18 ANA 1/640 14.3 50 Familial hypercholesterolemia Tac, MMF, Pred9 160 M 100 87 19 ANA 1/160 15.2 118 Biliary atresia CyA, Aza, Pred

10 151 F 112 65 12 AMA 1/160 14.4 33 Biliary atresia Tac, MMF, Pred

(b) ACR1 13 F 73 94 40 neg 8.85 0.5 Acute liver failure Tac, Pred2 159 M 129 210 11 neg 25.5 1 Autoimmune liver disease Tac MMF, Pred3 16 M 74 779 9 neg 15.4 1 Glycogen storage disease

type IVTac, Pred

4 10 M 135 67 16 neg 6.53 1 Biliary atresia Tac, Pred5 152 F 270 160 249 neg 7.85 1 Acute liver failure

(drug-related)Tac, Pred

6 22 F 226 302 27 neg 6.94 1 Acute liver failure Tac, Pred7 21 F 100 711 147 neg 6.94 0.5 Acute liver failure Tac, Pred8 23 F 58 153 335 neg 5.93 0.5 Biliary atresia Tac, Pred9 181 F 526 64 59 neg 4.44 0.25 Acute liver failure Tac, Pred

10 18 F 296 87 19 neg 4.82 1 Biliary atresia Tac, Pred

Age(months) Sex

AST (IU/L,nr < 50)

GGT (IU/L,nr < 55)

Bilirubin (lM,nr < 20) Autoantibodies

IgG (mM,nr 5–15)

Treatment beforebiopsy

(c) Autoimmune hepatitis1 165 M 225 259 23 ANA 1/640; SMA 1/640 24.6 Nil2 155 F 426 516 26 ANA 1/40; SMA 1/640 24.8 CP, chloroquine3 123 F 1624 94 58 ANA 1/20; SMA 1/160 28.9 Nil4 186 F 378 63 40 SMA 1/40 31.9 Nil5 33 M 5922 165 63 ANA 1/80; SMA 1/40 13.3 Nil6 158 F 637 250 38 SMA 1/2560 54.3 Nil7 113 F 260 75 20 ANA 1/40; LKM 1/1280 21.7 Nil8 183 M 531 65 73 SMA 1/640 53.1 Nil9 189 M 157 60 27 ANA 1/80 18.0 Pred, MMF

10 160 M 227 48 386 ANA 1/40; SMA 1/80 19.0 Pred, MMF11 188 M 886 164 85 ANA 1/40 12.4 Pred12 130 F 585 86 63 ANA 1/2560; SMA 1/2560 50.4 Nil13 209 F 44 99 16 ANA 1/160 20.3 Pred, Aza

ANA, anti-nuclear antibody; SMA, anti-smooth-muscle antibody; LKM, anti-liver-kidney-microsomal antibody; anti-LC1, anti-liver cytosol-1 antibody; AMA, anti-mitochondrial antibody; Tac, tacrolimus; CyA, cyclosporine A; Pred, prednisolone; MMF, mycophenolate mofetil; Aza, azathioprine; CP, cyclophosphamide.

Immunohistochemical phenotyping of the inflammatory infiltrate

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The immunostained slides were then examined by a liverhistopathologist (AQ), blinded to clinical details. CD4,CD8, T-bet, GATA-3, FOXP3, perforin, and IL-17-positiveportal and lobular lymphocytes were assessed semi-quanti-tatively using the following scoring system:

Grade 0 = no positive cells identified;Grade 1 = occasional positive cells identified after a search

at high magnification;Grade 2 = sporadic positive cells identified easily, even at

low magnification;Grade 3 = dense infiltrate of positive lymphocytes repre-

senting approximately one-third of the inflammatory cellinfiltrate;

Grade 4 = dense infiltrate of positive lymphocytes repre-senting more than one-third of the inflammatory cellinfiltrate.

This scoring system was based on a previously publishedstudy (22).

Statistical methods

Statistical analysis was carried out using SPSS version 14.0(SPSS Inc, Chicago, IL, USA). Differences in portal andlobular histological grading between the different patientgroups were assessed for each antibody by chi-square test. Ap value of <0.05 was considered significant.

Results

The immunostaining results in dn-AIH, ACR,and AIH are shown in Table 2 and Figs. 1–3.

Portal inflammation

CD4, CD8In all patients the portal infiltrate was composedof a mixture of CD4+ and CD8+ lymphocyteswithout obvious predominance of either popula-tion and with no significant difference betweenthe three groups, grade 3 and 4 being the mostfrequent scores for all groups.

T-betAll 33 samples showed prominent T-bet-positivelymphocytes in theportal tracts, thescoringbeing2andabove in all but onepatientwith classicalAIH.There was no significant difference in portal T-betexpression among the three groups of patients.

GATA-3In contrast to the Th1 T-bet immunostaining,most samples (24, 72.7%) did not show GATA-3positivity, with eight samples (24.2%; six AIH,one dn-AIH and one ACR) showing occasional(grade 1) GATA-3-positive portal lymphocytes,and one sample only (from the ACR group)showing a conspicuous (grade 3) GATA-3-positive portal infiltrate. There was no signif-icant difference in portal GATA-3 expressionbetween the three groups of patients.

FOXP3Portal FOXP3-positive lymphocytes were presentin all samples; grade 2 positivity was detected in12 samples (36.4%; four dn-AIH, six AIH, twoACR) and grade 3-4 positivity in 16 samples(48.5%; four dn-AIH, five AIH, seven ACR),with no significant difference in FOXP3 expres-sion between the three patient groups.

IL-17Grade 1–2 positivity was detected in 23 samples(74%; seven dn-AIH, six AIH, 10 ACR) andgrade 3–4 positivity in none of the samples, withno significant difference in IL-17 expressionbetween the three patient groups.

Table 2. Results of the immunohistochemistry grading expressed as number ofpatients with a given score

Group

Portal tracts

CD4 CD8 T-BET GATA-3 FOXP3 NK IL-17 Perforin

dn-AIHGrade 0 – – – 9 – 1 3 –Grade 1 – – – 1 2 3 7 –Grade 2 1 – 4 – 4 2 – 8Grade 3 6 4 6 – 4 – – 2Grade 4 3 6 – – – – – –

AIHGrade 0 – – – 7 – 4 5 –Grade 1 – – 1 6 2 3 5 1Grade 2 1 1 4 – 6 5 1 6Grade 3 9 7 8 – 4 – – 6Grade 4 3 5 – – 1 – – –

ACRGrade 0 – – – 8 – 3 – –Grade 1 – – – 1 1 5 10 –Grade 2 – 1 2 – 2 2 – 2Grade 3 6 2 7 1 6 – – 5Grade 4 4 7 1 – 1 – – 3

Group

Lobule

CD4 CD8 T-BET GATA-3 FOXP3 NK IL-17 Perforin

dn-AIHGrade 0 – – – 10 2 3 4 –Grade 1 5 – 1 – 3 3 5 –Grade 2 2 2 7 – 2 – 1 9Grade 3 2 5 1 – 3 – – 1Grade 4 1 3 1 – – – – –

AIHGrade 0 – – – 12 1 5 4 –Grade 1 1 – 1 1 1 3 7 3Grade 2 5 – 1 – 5 4 – 8Grade 3 6 4 8 – 5 – – 1Grade 4 1 9 3 – 1 – – –

ACRGrade 0 – – – 9 1 3 – –Grade 1 2 1 – – 2 4 6 –Grade 2 6 – 2 1 4 3 4 1Grade 3 2 2 7 – 3 – – 7Grade 4 – 7 1 – – – – 2

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PerforinGrade 1–2 positivity was detected in 17 samples(51%; eight dn-AIH, seven AIH, two ACR) andgrade 3–4 positivity in 16 samples (48%; two dn-AIH, six AIH, eight ACR), with no significantdifference in perforin expression between thethree patient groups.

CD56Grade 1–2 positivity was detected in all samples(six dn-AIH, 12 AIH, 10 ACR) and grade 3–4positivity in none of the samples, with nosignificant difference in CD56 expression betweenthe three patient groups.

Fig. 1. dn-AIH. A portal tractshows a mild inflammatory cellinfiltrate with plasma cells(hematoxylin and eosin [400·magnification]). Serial sectionsfrom the same portal tracts areimmunostained for CD4, CD8,T-bet, GATA-3, FOXP3, andperforin. Additional immuno-stains for NK cells (CD56) andIL-17 were performed on differ-ent sections from the samehistological block.

Immunohistochemical phenotyping of the inflammatory infiltrate

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Lobular inflammation

CD4, CD8In all three groups, the lobular infiltrate wascomposed of a mixture of CD4+ and CD8+lymphocytes without obvious predominance of

either population and with no significant differ-ence between the three groups.

T-betAll samples showed T-bet-positive lymphocytesin the lobule, mostly with an intrasinusoidal

Fig. 2. ACR. A portal tractshows a moderate mixedinflammatory cell infiltrate(hematoxylin and eosin [400·magnification]). Serial sectionsfrom the same portal tracts areimmunostained for CD4, CD8,T-bet, GATA-3, FOXP3, andperforin. Additional immuno-stains for NK cells (CD56) andIL-17 were performed on differ-ent sections from the same his-tological block.

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location. In 21 samples (63.6%; eight ACR, 11AIH and two dn-AIH), lobular T-bet-positivelymphocytes were prominent (grade 3 in 16samples; grade 4 in five samples). In the other 12samples (36.4%; two ACR, two AIH, and eight

dn-AIH), the lobular infiltrate of T-bet-positivelymphocytes was milder, either grade 1 or grade 2.There was a significantly lower T-bet score in thedn-AIH group, compared with the ACR and AIHgroups (p = 0.02).

Fig. 3. AIH. A portal tractshows a mild inflammatory cellinfiltrate with plasma cells(hematoxylin and eosin [400·magnification]). Serial sectionsfrom the same portal tracts areimmunostained for CD4, CD8,T-bet, GATA-3, FOXP3, andperforin. Additional immuno-stains for NK cells (CD56) andIL-17 were performed on differ-ent sections from the samehistological block.

Immunohistochemical phenotyping of the inflammatory infiltrate

7

GATA-3Most samples did not show lobular GATA-3positivity, with one sample (AIH; 2.9%) showinggrade 1 and another one (ACR; 2.9%) grade 2scoring.

FOXP3Most samples (29; 87.9%) showed FOXP3-positive lymphocytes in the lobule, predomi-nantly with an intrasinusoidal location. In 12samples (36.4%; three ACR, six AIH, threedn-AIH), lobular FOXP3-positive lymphocyteswere prominent: grade 3 in 11, and grade 4 in oneAIH sample. In the other 21 samples (63.6%;seven ACR, seven AIH, seven dn-AIH), thelobular infiltrate of FOXP3-positive lymphocyteswas either not present or was mild (grade 1–2).There was no significant difference between thethree groups.

IL-17Grade 1–2 positivity was detected in 23 samples(70%; six dn-AIH, seven AIH, 10 ACR) andgrade 3–4 positivity in none, with no significantdifference in IL-17 expression between the threepatient groups.

PerforinGrade 1–2 positivity was detected in 21 samples(66%; nine dn-AIH, 11 AIH, one ACR) andgrade 3–4 positivity in 11 samples (34%; onedn-AIH, one AIH, nine ACR), but there was nosignificant difference in perforin expressionbetween the three patient groups.

CD56Grade 1–2 positivity was detected in 17 samples(61%, three dn-AIH, seven AIH, seven ACR)while grade 3–4 positivity was not present in anyof the samples. No significant difference in CD56expression was found between the three patientgroups.No difference for any of the parameters

studied was observed between treated oruntreated patients with classical AIH.

Discussion

Using an immunohistochemical approach focusedon transcription factors defining specific T cellpopulations, we show that a Th1 lymphocytepolarization is present in dn-AIH after livertransplantation, but also in classical AIH and inACR.The finding that T-bet positive cells predom-

inate in the portal tract in all three disordersinvestigated is not surprising as in experimental

and clinical models of organ-specific autoim-mune disease and of cellular rejection, Th1 cellshave been consistently observed in the targettissue and have been implicated as mediators ofdamage. Th1 polarization of the portal infiltratehas been described in liver autoimmune condi-tions including AIH (24, 25), primary sclerosingcholangitis (26) and primary biliary cirrhosis(27), but also in other inflammatory conditionsof the liver, which are not necessarily perceivedas autoimmune, such as biliary atresia (28) ornon-alcoholic steatohepatitis (29).The reason why lobular T-bet infiltration is

less prominent in dn-AIH when compared toACR and classical AIH remains to be elucidated.As the degree of cellular infiltrate was similar inthe three conditions, we investigated whether aproportion of the T-bet-negative cells infiltratingthe parenchyma were Th17 cells, a lymphocytesubset recently directly implicated in tissue dam-age (30). Although IL-17-positive cells werepresent in dn-AIH, their frequency was nothigher than in AIH and ACR.NK cells, belonging to the innate immunity

defence mechanisms, were sparsely but equallyrepresented in the three groups of pathologies,suggesting that by the time the liver damage isestablished, cells of the adaptive immunity pre-vail. Perforin, a cytolytic protein present in thegranules of NK cells and CD8 T cells, was alsosimilarly represented in the three conditions. Therole of innate immunity during the initial stagesof autoimmune liver disease or cellular rejectionshould be further investigated.The fact that our results do not demonstrate a

clear difference between the effector cells of tissuedamage in dn-AIH post-liver transplant, ACRand classical AIH suggests that possibledifferences should be sought at the level ofautoantigen/alloantigen specificity. This could beachieved, as reported inAIH type 2 (31), by the useof labeled tetramers loaded with auto/alloanti-genic peptides. A careful selection of antigenicpeptides and the use ofHLA class I andHLAclassII tetramers should enable immunohistochemicalcharacterization of the three disorders.We have also found no difference in the

presence and distribution of FOXP3-positiveT-regs among dn-AIH, ACR, and AIH. Again,possible differences could be demonstrated by theinvestigation of tissue antigen-specific T-regs, anapproach currently being developed in AIH type2. In addition, the study of immune regulatorycells should be extended to include more recentlydescribed populations with suppressor functions,such as CD8-positive/CD28-negative T cells andcd T cells (22).

Had�zic et al.

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An important aspect of this study is that it wasperformed on archived paraffin-embedded mate-rial. Until recently, the functional definition of Tcell subsets relied on the use of frozen tissue andintracellular identification of signature cytokines(IFNc or IL-2 for Th1 cells, IL-4 or IL-13 forTh2 cells). Such technique is marred by thedifficulty in recognizing definite T cell subtypes,because of cytokine leakage in frozen tissue.Moreover, in frozen sections, identification ofT-regs has been based on the simultaneousdetection of membrane CD4 and CD25 mole-cules in a double-staining technique, the readingof which is difficult and undependable. The use ofantigen retrieval in paraffin-embedded sectionshas two major advantages: first, to overcome theproblems in frozen tissue described above, andsecond, to allow the retrospective study ofclinically well-characterized patient cohorts.In conclusion, using immunohistochemical

techniques on paraffin-embedded material, wehave defined tissue T cell subsets – mostly throughthe detection of their lineage-specific transcriptionfactors – in dn-AIH post-liver transplant, classicalAIH and ACR. Although this study should beextended to a larger number of patients, possiblyrecruited through multicenter collaboration, ourresults suggest that a Th1-driven liver dysfunctionrepresents the final phase of the immune-mediatedevents leading to all three conditions. An expla-nation for their presumed distinct immunopath-ogeneses should be sought at an earlier diseasestage, for example, at the first appearance ofhypergammaglobulinemia and/or autoantibodyseropositivity, or when non-adherence to anti-rejection medications is suspected.

Acknowledgments

Drs. Had�zic and Quaglia have contributed equally to thismanuscript and share the first authorship. The authors whohave taken part in this study declare that they do not haveanything to disclose regarding funding or conflict of interestwith respect to this manuscript. They also wish to thank Ms.Chiara Serafini for help with the histology assessment.

Author contributions

The authors have participated in the following stages duringmanuscript preparation: concept/design (Drs. Had�zic andMieli-Vergani), data analysis/interpretation (Drs. Quaglia,Hussain, and Cotoi), drafting article (Dr. Had�zic), criticalrevisionof article (Drs.Mieli-Vergani, Vergani, andQuaglia),approval of article (all authors), and statistics (Dr. Brown).

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