Nel 1988, per iniziativa dell’ Università di Roma “La Sapienza” e delle Università di Perugia e Milano, prese corpo l’idea di formare un gruppo di lavoro capace di sviluppare ed estendere le ricerche sui trapianti d’organo, anche con l’intento di coinvolgere e formare giovani medici neolaureati nella difficile disciplina chirurgica; a questo scopo il Consorzio, sin dalla sua costituzione, ha assegnato oltre 160 borse di formazione favorendo l’inserimento nella ricerca e nelle strutture universitarie di una gran parte di neoformati. La bontà dell’idea iniziale delle tre Università promotrici è riscontrabile nel fatto che dalla sua costituzione il Consorzio si è progressivamente allargato ad altre 13 Università italiane e 3 straniere, una delle quali è la Columbia University di New York, sicché oggi il Consorzio Interuniversitario per i Trapianti d’Organo annovera un totale di 19 Università. La sua attività istituzionale è quella di promuovere e coordinare le ricerche ed altre attività scientifiche ed applicative nel campo dei trapianti d’Organo tra le Università consorziate favorendo, da un lato, le collaborazioni tra le Università italiane e quelle straniere ed altri Enti di ricerca, nonché le industrie e, dall’altro il loro accesso e la loro eventuale partecipazione alla gestione di laboratori esteri o internazionali operanti nel settore. L’ambizione del Consorzio è quella di estendere nei prossimi anni i lusinghieri risultati, sin qui raggiunti, ad un sempre maggior numero di pazienti attraverso il potenziamento della ricerca, il perfezionamento delle tecniche e la razionalizzazione delle risorse presenti nel paese, nonché una valorizzazione delle competenze delle giovani leve favorendone la formazione professionale. Attualmente il Consorzio è attivamente impegnato in diverse linee di ricerca ed ha conseguito importanti obiettivi, alcuni dei quali già trovano applicazioni cliniche in pazienti sottoposti a trapianto. Il Consorzio ha sede in Roma ed è sottoposto alla vigilanza del Ministero Istruzione Università e Ricerca. La personalità giuridica dell’Ente è riconosciuta con D.P.R. del 31/X/88.
Il Consorzio Interuniversitario per i Trapianti d’Organo racchiude in se i seguenti organi:
I nostri risultati sono conseguiti perseguendo obiettivi individuabili in:
Università di Roma "La Sapienza"Prof. Pasquale Berloco Università di Roma Tor VergataProf. Carlo Umberto Casciani Università Campus Biomedico di RomaProf. Roberto Coppola Università di PerugiaProf. Riccardo Calafiore Università di Milano Prof. Massimo Colombo Università di PalermoProf. Maurizio Romano Università di SienaProf. Mario Carmellini Università di Napoli "Federico II"Prof. Andrea RendaSeconda Università di NapoliProf. Biagio Lettieri Università di CagliariProf. Carlo Carcassi Università di CataniaProf. Pierfrancesco Veroux Università di MessinaProf. Maria Gioffrè Florio Università di SassariProf. Francesco Carta Università di TorinoProf. Giuseppe Segoloni Università di Musei Vatican. Antonio Famulari Università di BolognaProf. Eraldo Seren Università Cattolica del Sacro CuoreProf. Marco Castagneto
Università di Cluj - Napoca (Romania)Prof. Mihai Lucan Università di Birmingham (UK)Prof. Paul Mc MasterColumbia University di New York (Usa)Prof. Raffaello CortesiniUniversity St.Cyril and Methodius- Skopje (Macedonia)Prof. Zivko Popov
Università di Roma “La Sapienza”
Prof. Pasquale Berloco
Tra le tre Università che nel 1988 hanno dato vita al Consorzio. È attualmente rappresentata dal prof. Pasquale B. Berloco. Il prof. Berloco è attualmente direttore del Centro Trapianti del Policlinico Universitario Umberto I di Roma, nonché Professore Ordinario di Chirurgia presso l’ Università Sapienza di Roma. Nel 2001 ha diretto, insieme al prof. Massimo Rossi, il primo trapianto da vivente a Roma.
Università di Roma “Tor Vergata”
Prof. Carlo Umberto Casciani
L’ Università di Tor Vergata è attualmente rappresentata dal prof. Carlo Umberto Casciani. Il Professore ha creato e presiede l’Agenzia regionale del Lazio per i Trapianti e Patologie Connesse.
Università di Campus Bio-Medico di Roma
Prof. Roberto Coppola
L’ Università Campus Biomedico è attualmente rappresentata dal prof. Roberto Coppola. Il Professore è ordinario di chirurgia generale.
Università di Perugia
Prof. Riccardo Calafiore
Tra le tre Università che nel 1988 hanno dato vita al Consorzio. È attualmente rappresentata dal prof. Riccardo Calafiore. Il prof. Calafiore è Professore Associato di Endocrinologia e Responsabile del Laboratorio per lo Studio e il Trapianto delle Insule Pancreatiche, Dipartimento di Medicina Interna (Di.M.I e Adjunct Professor of Medicine, University of Miami School of Medicine, Miami, USA.
Università di Milano
Prof. Massimo Colombo
Tra le tre Università che nel 1988 hanno dato vita al Consorzio. È attualmente rappresentata dal prof. Massimo Colombo. Il prof. Colombo è Professore Ordinario di Gastroenterologia, Direttore Dipartimento di Malattie dell’Apparato Digerente ed Endocrino-Metabolico dell’Ospedale Maggiore IRCCS, Milano
Università di Palermo
Prof. Maurizio Romano
L’ Università di Palermo è attualmente rappresentata dal prof. Maurizio Romano. Il Professore è Direttore dell’Unità Operativa di Chirurgia Generale, dell’Uremico e dei Trapianti d’Organo.
Università di Siena
Prof. Mario Carmellini
L’ Università di Siena è attualmente rappresentata dal prof. Mario Carmellini. Il Professore è Responsabile Programma Trapianto Rene A.O.U. Senese.
Università di Napoli “Federico II”
Prof. Andrea Renda
L’ Università di Napoli “Federico II” è attualmente rappresentata dal prof. Andrea Renda. Il Professore è Ordinario di Chirurgia generale.
Seconda Università di Napoli
Prof. Biagio Lettieri
La Seconda Università di Napoli è attualmente rappresentata dal prof. Biagio Lettieri. Il Professore è Ordinario di Anestesia.
Università di Cagliari
Prof. Carlo Carcassi
L’ Università di Cagliari è attualmente rappresentata dal prof. Carlo Carcassi. Il Professore è Presidente, nel secondo mandato triennale, della A.I.B.T., Associazione Italiana di Immunogenetica e Biologia dei Trapianti.
Università di Catania
Prof. Pierfrancesco Veroux
L’ Università di Catania è attualmente rappresentata dal prof. Pierfrancesco Veroux. Il Professore è Direttore del centro trapianti reni del Policlinico di Catania.
Università di Messina
Prof. Maria Gioffrè Florio
L’ Università di Messina è attualmente rappresentata dalla prof.ssa Maria Gioffrè Florio. La Professoressa è direttore dell’ U.O.C. Medicina e Chirurgia d’Accettazione e d’Urgenza.
Università di Sassari
Prof. Francesco Carta
L’ Università di Sassari è attualmente rappresentata dal prof. Francesco Carta. Il Professore è direttore della Clinica Oculistica dell’ Università.
Università di Torino
Prof. Giuseppe Segoloni
L’ Università di Torino è attualmente rappresentata dal prof. Giuseppe Segoloni. Il Professore è Direttore SCDU Nefrologia Dialisi e Trapianto – ASO.
Università di l’Aquila
Prof. Antonio Famulari
L’ Università di Torino è attualmente rappresentata dal prof. Antonio Famulari. Il Professore è titolare della cattedra di Chirurgia Sostitutiva e dei Trapianti d’Organo.
Università di Bologna
Prof. Eraldo Seren
L’ Università di Bologna è attualmente rappresentata dal prof. Eraldo Seren. Il Professore è ordinario di Fisiologia Veterinaria.
Università Cattolica del Sacro Cuore
Prof. Marco Castagneto
L’ Università Cattolica è attualmente rappresentata dal prof. Marco Castagneto. Il Professore è direttore del Dipartimento di scienze chirurgiche.
Università Di Cluj - Napoca (Romania)
Prof. Mihai Lucan
L’ Università di Cluj è attualmente rappresentata dal prof. Mihai Lucan. Il Professore è direttore della Clinica di Urologia e trapianti renali.
Università Di Birmingham (UK)
Prof. Paul Mc Master
L’ Università di Torino è attualmente rappresentata dal prof. Giuseppe Segoloni. Il Professore è Direttore SCDU Nefrologia Dialisi e Trapianto – ASO.
Columbia University di New York (Usa)
Prof. Raffaello Cortesini
L’ Università di Columbia è attualmente rappresentata dal prof. Raffaello Cortesini. Il Professore è Professore di Trapiantologia e fondatore del Consorzio.
University St.Cyril and Methodius- Skopje(Macedonia)
Prof. Zivko Popov
L’ Università di St. Cyril and Methodius è attualmente rappresentata dal prof. Zivko Popov.
Oltre ai finanziamenti pubblici previsti per le attività istituzionali erogati dal Ministero dell’ Università e della Ricerca, il Consorzio ha ricevuto e riceve contributi da enti pubblici e privati e fondazioni tra le quali annoveriamo:
Pubblica AmministrazioneFinanziamenti PubbliciOrganizzazioniContributi e DonazioniPrivati CittadiniContributi e Donazioni
Consorzio Interuniversitario Trapianti d’Organo
Sede Legale: II Clinica Chirurgica “Policlinico Umberto I” - 00161 - Roma
Sede Amministrativa: Via G. M. Lancisi, 31 - 00161 - Roma
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Anti-CD25 Treatment and FOXP3-Positive Regulatory T Cells in HeartTransplantationG. Vlada, E. K. Hoa, E.R.Vasilescua, J. Fana, Z. Liub, J.W. Caia, Z. Jinc, E. Burked,M. Dengd, M. Cadeirasd, R. Cortesinia, S. Itescud, C. Marboea, D. Mancinid, and N.Suciu-FocaaThe Departments of Pathologya, Surgeryb, Biostatisticsc, Medicined, and ofColumbia University, New York, NY 10032Running Title: Daclizumab and T Regulatory CellsWord count: 3655Key Words: Heart Transplantation, T regulatory cells, Rejection, FOXP3,DaclizumabCorresponding Author:Dr. Nicole Suciu-FocaDepartment of PathologyColumbia University630 West 168thStreet – P&S 14-401New York, NY 10032Tel: (212) 305-6941; Fax: (212) 305-3429E-mail: firstname.lastname@example.orgThe interleukin-2 receptor alpha chain (IL-2Ra, CD25) plays a major part inshaping the dynamics of T cell populations following immune activation, due to itsrole in T cell proliferation and survival. Strategies to blunt the effector responses intransplantation have been developed by devising pharmaceutical agents to blockthe IL-2 pathways. However, such strategies could adversely affect the CD25+FOXP3+Treg populations which also rely on intereukin-2 signaling for survival. Thepresent study shows that a cohort of heart allograft recipients treated withDaclizumab (a humanized anti-CD25 antibody) display FOXP3 expression patternsconsistent with functional T regulatory cell populations. High levels of FOXP3 wereobserved to correlate with lower incidence of and recovery from acute rejection, aswell as lower levels of anti-donor HLA antibody production. Therefore, T regpopulations appear fully functional in patients treated with Daclizumab, even when
5 doses were administered. By comparison, patients treated with fewer doses orno Daclizumab had a higher incidence of acute rejection, antibody production andgraft failure. Therefore, our data indicates that Daclizumab treatment does notinterfere with the generation of regulatory T cells and has a beneficial effect onheart allograft survival.IntroductionThe use of calcineurin inhibitors as part of standard immunosuppressivetherapy in organ transplantation has resulted in a remarkable increase in the rateof allograft survival. The additional use of various monoclonal (anti-CD3) andpolyclonal (antithymocytic/lymphocytic) antibodies (as depletional induction agents)has further contributed to prevention of early episodes of acute rejection (1).However, the risk of infection, malignancy and late rejection remains a loomingconcern. More recently, a new generation of immunosuppressive agents has beenintroduced to fine tune T cell responses by specifically targeting activated T cells.The humanized anti-interleukin-2 receptor alpha chain (IL-2R, CD25) monoclonalantibody (Daclizumab, Zenapax®, Hoffmann-La Roche) is such an example. Itsefficacy in conjunction with triple immunosuppression has been documented inseveral studies (1-4).The underlying hypothesis for the development of this drug has been thatblocking of the interleukin-2 receptor may prevent the proliferation anddifferentiation of effector T cells. However, studies in the field of regulatory T cellshave shown that CD25 represents a crucial marker of FOXP3+T cells withsuppressor function (5-12). In the light of this data, the obvious question iswhether anti-CD25 treatment results in the blockade or depletion of regulatory Tcells and whether quiescence can be attained in their absence. To answer thisquestion we have performed serial determinations of markers deemed to becharacteristic of regulatory/suppressor T cells (Treg/Ts), such as FOXP3, CTLA-4,and IL-10, in a cohort of 110 heart recipients transplanted between 2003 and 2006(Table 1). As putative markers for rejection we used IFN-γ and VEGF. In addition,given the fact that development of anti donor HLA antibodies is detrimental to thegraft we monitored such antibodies in patients’ sera. We now report on thepositive correlation between quiescence and expression of these Treg markers inperipheral blood CD4+and CD8+T cells, demonstrating that the development ofregulatory T cells is not impaired in patients treated with Daclizumab. Suchregulatory T cells were negatively associated with the occurrence of early acuterejection episodes and development of allo-antibodies. (13-15)Material and MethodsPatients110 heart allograft recipients (84 males and 26 females) with a mean age of49.4 years old (49.4±13.0 SD, range 18-72) were recruited for these studies at the
time of transplantation. The demographic of this study population is shown in Table1. All patients gave informed consent under the auspices of the appropriateInstitutional Review Board. Patients were treated with cyclosporine (CSA),mycophenolate mofetil (MMF), prednisone (MP) and with or without Daclizumab(administered at 1mg/kg body weight).Endomyocardial biopsies (EMB)Endomyocardial biopsies were performed by the standard transjugularapproach weekly for the first month and then at progressively longer intervals to abaseline schedule of every 6 months. The mean number of biopsies per patientwas 10 + 2. A minimum of four biopsy fragments were fixed in 4% bufferedformalin, paraffin embedded, and multiple hematoxylin and eosin stained sectionsfrom the three levels in the block were examined. An additional fragment wasfrozen for RT-PCR and Lymphocyte Growth Assays (LGA). Histological gradeswere assigned according to the criteria of the International Society for Heart andLung Transplantation (ISHLT): no rejection (grade 0), focal (grade 1A) or diffuseinterstitial mononuclear infiltrates without myocyte necrosis (grade 1B), multifocalaggressive infiltrates with myocyte damage (grade 3A), diffuse inflammatoryprocesswithnecrosis(grade3B)anddiffuseaggressivepolymorphous/mononuclear infiltrate with edema, hemorrhage, vasculitis andnecrosis (grade 4) (16).A baseline angiogram was performed within one month of transplantation todetect unsuspected vascular disease. Angiograms were repeated annually. Thediagnosis of CAV was made when contrast angiography demonstrated diffusedsmall vessel involvement, defined as concentric and symmetrical narrowing ofterminal branches.Molecular HLA typing and testing of anti-HLA antibodiesHLA genotypes of transplant recipient/donor pairs were determined by PCRwith sequence specific primers (SSP) using commercially available kits (OneLambda, Los Angeles, CA). Sequential samples of sera obtained from eachpatient prior to and following transplantation were tested for anti-HLA alloantibodiesby lymphocytotoxicity and solid-phase assays to determine the frequency of panelreactive antibodies (PRA). The specificity of the antibodies for the donor’s HLAantigens was assessed by screening and direct cross-matching procedures aspreviously described (17).Lymphocyte Growth AssayA biopsy fragment (1mm3piece) was placed in medium supplemented withrecombinant IL-2 (5 units/ml) and examined microscopically at 48h. Growth was
scored on a semi quantitative scale from 0 to 3 on the basis of circumferential Tcell aggregation. A score of 1 or greater was deemed positive (18).Quantitative Real Time (qRT)-PCRA total of 508 peripheral blood samples from 110 transplant patients were tested byqRT-PCR for expression of various genes at the transcriptional level. CD4 andCD8 cells were isolated from each of these samples by use of commerciallyavailable magnetic isolation kits (Miltenyi Biotec, Gladbach, Germany).Additionally, qRT-PCR studies of were performed on 104 EMB samples. Biopsieswere stored overnight at 4oC in 5 volumes of RNAlater tissue collection/RNAstabilization solution (Ambion, Foster City, California). After removal of the RNAstabilization solution biopsies were stored at -80oC for subsequent RNA extraction.Total RNA was isolated from biopsies with the RNAqueous-4PCR kit(Ambion) following the manufacturer’s recommendations. Complementary cDNAwas synthesized using the 1stStrand cDNA Synthesis Kit for RT-PCR (RocheDiagnostics, Basel, Switzerland). Quantitative real-time PCR was performed usingTaqman gene expression assays (Applied Biosystems, Foster City, CA). The geneexpression assays used in these studies are identified below, by gene name,manufacturer’s abbreviation and part number: Cytotoxic T Lymphocyte Antigen 4(CTLA-4, Hs00175480_m1), Forkhead box P3 (FOXP3, Hs00203958_m1),Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 4326317E), Interleukin-10(IL-10, Hs00174086_m1), Interferon-gamma (IFN-γ, Hs00174143_m1), andvascular endothelial growth factor (VEGF, Hs00173626_m1) all from AppliedBiosystems. Forward and reverse primers are contained in different exons, toprevent amplification of genomic DNA. We used the 7300 RT-PCR instrument(Applied Biosystems), applying the manufacturer’s protocols and recommendedamplification conditions, as follows: one cycle at 50°C (2 min), then 95°C (10 min),followed by 50 cycles at 95°C (15 s) and 60°C (1 min). Data were collected andanalyzed using the 7300 SDS 1.3.1 software (Applied Biosystems). Each assayplate included “no template” negative controls and a control cDNA. The relativeamount of gene expression was calculated according to the formula: 2−∆Ct, where∆Ct = [Ct(gene) − Ct(GAPDH)] and Ct is the “crossing threshold” value returned bythe PCR instrument for every gene amplification.Statistical AnalysisThe generalized estimating equation (GEE) approach with workingindependence was used to study the association between two variables. Two GEEmodeling approaches were used, one with constant intercept and the other withdifferent intercept term for each subject. The identity link was used for continuousvariables, while the logit link was used for rejection, which is binary.Time to death was used as the end point in overall graft survival analysis,
and patients who were alive were censored at the date of last clinical contact. TheCox proportional hazards regression model was used for multivariate analysis ofDaclizumab treatment. Overall graft survival was computed using Kaplan-Meierproduct-limit estimators and compared by log rank tests. The influence ofDaclizumab treatment on the development of anti-HLA antibodies was evaluatedby Kaplan-Meier estimators, with P values obtained using log rank statistics. Forcategorical variables, a 2x2 table was constructed and compared by either chi-square analysis or Fisher’s exact (2-tail) test. A non-parametric Wilcoxon exacttest was used for analysis of changes in the level of FOXP3 expression in CD4+and CD8+T cells from patients with resolving acute rejection episodes. P valuesless than 0.05 were considered significant. All statistical tests were 2-sided.Statistical analysis of data was performed using the SAS version 9.1 softwarepackage (SAS Institute, Cary, NC) (19, 20).ResultsExpression of FOXP3 in CD4 and CD8 T cells from the peripheral blood ofheart allograft recipientsRegulatory T cells are characterized by their capacity to suppress thereactivity of other effector populations of antigen-stimulated T cells. Several distinctpopulations of regulatory T cells, such as thymus-derived CD4+CD25+FOXP3+natural T reg (5, 6), antigen-induced CD4+CD25+FOXP3+peripheral T reg (7-9),IL-10-producing CD4+CD25+FOXP3-Tr1 (10, 11), CD8+FOXP3+Ts (8, 9, 21-23)and CD8+IL-10-producing Ts (12) have been shown to play a role in humantransplantation and experimental models (8). To assess the presence of T reg inhuman allotransplantation, we analyzed the expression of FOXP3 in peripheralCD4 and CD8 T cell populations as well as in heart allograft biopsies. EMB grade3A or higher was considered indicative of acute cellular rejection (16). Analysis of508 peripheral blood samples showed a negative correlation between the level ofFOXP3 expression in CD4+T cells and EMB (N=1557) grade 3A (p=0.033, Figure1).Although within the CD8 compartment the level of FOXP3 expression was5-fold higher during quiescence compared to high-grade rejection, the differencedid not reach significance (p>0.296).Similarly, analysis of FOXP3 expression in biopsy tissue (N=103) showed anegative association with the grade of rejection which did not reach statisticalsignificance (P=0.416). However, the consistent trend for high expression ofFOXP3 in peripheral blood and biopsies during quiescence and low expressionduring episodes of acute rejection suggests that regulatory T cells play a role inallotransplantation.
The dynamic of FOXP3 expression in peripheral blood T cells duringresolving rejectionResolving rejections were defined as lymphocytic infiltrates grade 3A EMBsubsiding to grade 1 or 0 within 1 week. We examined the expression of FOXP3in peripheral blood T cells from 9 such cases. In one of these 9 cases, the CD4+Tcells were not testable for technical reasons. Of particular note was the finding thatthe level of FOXP3 expression went from low levels to higher values duringresolving rejection. Thus, in 7/8 rejection episodes, the level of FOXP3 inperipheral blood CD4 T cells increased 7 fold (P=0.036) during the resolvingrejection. Similarly, peripheral CD8 T cells showed a mean fold increase of 13(P=0.028), in 9/9 cases of resolving rejection (Figure 2).An exception seemed to occur in a patient who showed low FOXP3 (in both CD4and CD8 peripheral cells) in conjunction with EMB grade 3A, followed one weeklater by a 10-fold decrease in the level of FOXP3 as rejection appeared to resolveto grade 1 EMB. This patient, however, showed a second episode of rejectiongrade 3, two weeks after the first grade 3 episode (Figure 3) consistent with the lowlevel of FOXP3 seen at the time when EMB showed only grade 1B infiltrates. Thefinding that during recurrent rejection the level of FOXP3 expression in peripheralblood T cells stays at a low value reinforces the concept that FOXP3 is associatedwith T regulatory cells that may play a role in protecting the graft.FOXP3 associations with the expression of other markers of T cell functionCytotoxic lymphocyte associated antigen 4 (CTLA-4) negatively regulatesthe classical costimulation pathways required for efficient T cell activation. Thismolecule is highly expressed by both CD4 and CD8 regulatory T cells (6, 17, 18).There is also evidence that interleukin-10 mediates the suppressor function ofseveral types of regulatory T cells (8, 10, 11, 12). Because different populations ofregulatory T cells may be simultaneously present in the circulation (24, 25), weanalyzed the relationship between the level of expression of FOXP3 and that ofthese suppressor markers in CD4 and CD8 cells from sequential samples of blood.We found a significant correlation between the level of expression of FOXP3 andCTLA-4 as well as FOXP3 and IL-10, irrespective of rejection status (Table 2).No association between the level of expression of FOXP3 and that of IFN-γin CD4 and CD8 T cells from the circulation of transplant patients was found duringquiescence or rejection (GEE, P-values >0.05, Table 2).Studies of EMB tissue showed no significant relationships between the levelof expression of FOXP3, IL-10, CTLA-4 or IFN-γ (P values >0.05). This most likelyreflects the fact that within the EMB tissue, infiltrating T cells represent only a smallfraction of the cell sample. Unexpectedly however, there was a significantcorrelation between the levels of expression of FOXP3 and VEGF (P=0.007, Table2). This observation was unexpected because VEGF, a growth factor active inangiogenesis, vasculogenesis and endothelial cell growth, was previously shown tobe increased in allograft rejection (26).
FOXP3 is inversely related to the production of anti-donor HLA antibodiesThe generation, differentiation, and maturation of antibody producing B cellsare highly dependent on T cell help. Such help can be provided via the productionof Th2-type cytokines, or by direct T-B interaction (27). By inhibiting Th (28, 29,and reviewed in ref. 8), T reg can interfere with B cell proliferation anddifferentiation into Ig secreting plasma cells. To determine whether the presence ofT reg may affect the antibody status of the patients, we analyzed the relationshipbetween the level of FOXP3 expression in CD4 and CD8 T cells and the presenceof anti-HLA (IgG) antibodies in the patients’ circulation. This analysis revealed anegative correlation between high levels of FOXP3 in the CD4 compartment andthe development of anti-HLA IgG antibodies (p=0.014, Figure 4A). Patientsdeveloping high PRA following transplantation also showed low expression ofFOXP3 in CD8 T cells. However, this correlation was not statistically significant(p= 0.976, Figure 4). These findings are consistent with the hypothesis thatregulatory T cells suppress the activation and differentiation of alloantibodyproducing B cells, either directly or acting on allospecific T helper cells.Relationship between Daclizumab treatment and immune responses againstthe graftWithin this study cohort of 110 patients, 7 were not treated with Daclizumab.All 7 patients showed an early, post-transplantation “spike” of anti-HLA Ab. In threerecipients this phenomenon was transient, although the patients remained prone tosubsequent sporadic, short-term bouts of anti-HLA antibodies as late as one yearafter transplantation (Figure 5A, B, C). In the remaining four patients not receivingtreatment with Daclizumab anti-HLA antibodies persisted in the circulation over theentire period of observation as illustrated in Figure 5D. The anti-donor-HLAspecificity of the antibodies was demonstrated by a direct cross match of thepatients’ sera with T cells and B cells from the specific donor spleens.Study of patients treated with Daclizumab showed that only 42% of thepatients that have received 4 or more doses developed anti-HLA antibodies within24 months post transplantation, compared to 69% and 100% of those whoreceived 1-3 doses or no Daclizumab, respectively (P<0.0001) (Figure 5E, F).Thus, it appears that the activation of the humoral arm of the immune responseagainst the graft was inhibited by Daclizumab in a dose related manner.We analyzed the frequency of EMB grade 3A or higher in our cohort of patients,after completion of the Daclizumab treatment course. Patients receiving more than3 doses had a significantly lower incidence of EMB grade 3A rejection compared topatients receiving 0-3 doses (P<0.04, Figure 6). Acute rejection (EMB grade 3+)was strongly associated with positive LGA results which reflect the growth capacityof graft infiltrating cells and their implicitly aggressive nature (P<0.0001, Figure 7).This data indicates that Daclizumab inhibits both the cellular and humoral immuneresponse against the graft.
Effect of Daclizumab treatment on patient survivalAmong patients treated with Daclizumab (N= 103), 73.6% received 5courses of the drug while the remaining 25.4% received lesser amounts. The Coxproportionate hazards model shows that Daclizumab had a protective effectagainst graft loss, and that this effect was dosage dependent. Thus, receivingthree Daclizumab doses or less exposes a patient to 4.3 fold higher risk of graftloss compared to patients receiving four or more treatments (Figure 8A, B).Patients receiving five or more doses of Daclizumab showed the same (about 4-fold) protection from graft loss as those receiving 4 doses, suggesting no furtherbenefit from additional doses. The 12-month actuarial heart allograft survival was82% among patients receiving 0-3 doses of Daclizumab and 96% among thosereceiving more (Figure 8C, D).DiscussionOver the last decade, the literature has been dominated by the concept thatregulatory T cells which have the CD4+CD25+FOXP3+phenotype are cruciallyimportant to the maintenance of tolerance to self and non-self antigens (5-8).Studies in rodents have demonstrated unambiguously that this population ofregulatory T cells is primarily responsible for protecting the animal fromautoimmune diseases (5-8, 30, 31). Evidence that both CD4+and CD8+FOXP3+Tcells mediate allogeneic tolerance has also been provided by numerous studies inrodents (30, 32, 33).In spite of the large body on information about the capacity of T reg to inhibit T cellresponses in malignancy (34, 35) and certain autoimmune diseases, the dataregarding their mechanism of action and clinical significance is still controversial.The controversy emerges primarily from the finding that in human CD25, FOXP3and CTLA-4 are markers which are shared by Treg with a stable phenotype and by‘transient’ regulatory T cells which represent only a stage in the differentiation ofactivated T cells (36, 37). The promiscuity of FOXP3 expression in activatedhuman T cells may explain our finding that FOXP3 and IFN-γ showed no negativeassociation either in EMB or circulating T cells. Similarly, the positive correlationbetween the expression of FOXP3 and a putative rejection marker VEGF (26) maybe caused by the presence of activated rather than regulatory T cells within thegraft. On the other hand, it is possible that, by analogy to the situationencountered in cancer, VEGF and IL-10 create an immunosuppressiveenvironment within the graft, inhibiting T cell reactivity (38).Similarly, the finding of FOXP3 and CTLA-4 expression in peripheral blood CD4and CD8 T cells even during EMB grade 3 rejection episodes, may also indicateongoing T cell activation rather than presence of Treg. However, the significant risein the level of expression of these markers after successful treatment of a rejectionepisode, together with the negative association between high level of FOXP3 anddevelopment of alloantibodies indicates that FOXP3+CD4 and CD8 T cell subsets
have a protective role against rejection in humans, similar to animal models (9, 22,23, 39, 40). Therefore, our data is consistent with a recently proposed model (36,37), which suggests that in humans, transient expression of FOXP3 ischaracteristic of all activated T cells. However, stable expression of FOXP3differentiates T regulatory cells from activated T cells committed to other effectorfunctions (28, 41). The clinical consequences of this observation cannot beunderestimated as they warn against the use of these markers for identifying Tsuppressors for either diagnostic or prognostic purposes.Although it is difficult to discriminate between the possibility that the cellsexpressing CD25, FOXP3 and CTLA-4 are precursors of Th or Treg, thequantitative changes which we observed during resolving rejection argue in favorof continuous monitoring of these markers to predict the outcome of a rejectionepisode. This view is further supported by the finding that declining levels ofFOXP3 occurring following treatment of rejection are indicative of recurrence ratherthan remission of acute rejection.The fact that CD25 is expressed by all T cells upon activation explains theapparent inconsistency between the clinical efficacy of Daclizumab as animmunosuppressive agent and its potential to block or deplete CD25+T reg. Ourdata contribute evidence that Daclizumab, particularly after repeatedadministration, inhibits the production of allo-antibodies and improves patientsurvival, consistent with other authors finding that it reduces the rate of acuterejection. (1-4, 42) Furthermore, the data indicate that CD4+and CD8+T regulatorycells develop in patients treated with Daclizumab together with standard calcineurin– inhibitory therapy arguing against the view that this type of immunosuppressionprevents the differentiation of regulatory T cells.AcknowledgementsThis work was supported by RO1 AI55234-04 and the Interuniversitary OrganTransplantation Consortium, Rome, Italy.FiguresFigure 1. The level of FOXP3 expression (relative to GAPDH) in peripheral CD4 Tcells (A), CD8 T cells (B) and EMB © is inversely related to acute rejection grade3A.Figure 2. The dynamic of FOXP3 expression in peripheral T cells during resolvingrejection.Figure 3. The dynamic of FOXP3 expression in CD8+ T cells from a patient withmultiple rejection episodes.Figure 4. FOXP3 expression in peripheral T cells is inversely related to thedevelopment of anti-HLA antibodies in heart transplant recipients.
Figure 5.Anti-HLA antibody production. Daclizumab non-treated patientsrecurrent (A, B, C) or prolonged (D) anti-HLA antibody production. Log rankstatistics (E) and cumulative incidence of anti-donor antibody production (F) inpatients receiving various Daclizumab treatments.Figure 6.Incidence of acute rejection after completion of Daclizumabtreatment(s).Figure 7. Relationship between development of acute rejection (EMB grade ≥3A)and LGA results.Figure 8. Protective effect of Daclizumab on heart graft survival. Daclizumabtreatment(s) and hazard ratios of graft loss (A, B) and actuarial survival (C, D).References1. Kirk AD. Induction immunosuppression. Transplantation 2006;82(5):593-602.2. Sandrini S. Use of IL-2 receptor antagonists to reduce delayed graft functionfollowing renal transplantation: a review. Clinical transplantation 2005;19(6):705-710.3. Figueras J, Prieto M, Bernardos A, Rimola A, Suarez F, de Urbina JO et al.Daclizumab induction and maintenance steroid-free immunosuppression withmycophenolate mofetil and tacrolimus to prevent acute rejection of hepaticallografts. Transpl Int 2006;19(8):641-648.4. Vo AA, Toyoda M, Peng A, Bunnapradist S, Lukovsky M, Jordan SC. Effect ofinduction therapy protocols on transplant outcomes in crossmatch positive renalallograft recipients desensitized with IVIG. Am J Transplant 2006;6(10):2384-2390.5. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annual review ofimmunology 2004;22:531-562.6. Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers.Nature reviews 2002;2(6):389-400.7. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Naturereviews 2003;3(3):253-257.8. Vlad G, Cortesini R, Suciu-Foca N. License to heal: bidirectional interaction ofantigen-specific regulatory T cells and tolerogenic APC. J Immunol2005;174(10):5907-5914.9. Manavalan JS, Rossi PC, Vlad G, Piazza F, Yarilina A, Cortesini R et al. Highexpression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells.Transplant immunology 2003;11(3-4):245-258.10. Roncarolo MG, Gregori S, Levings M. Type 1 T regulatory cells and theirrelationship with CD4+CD25+ T regulatory cells. Novartis Foundation symposium2003;252:115-127; discussion 127-131, 203-110.11. Levings MK, Gregori S, Tresoldi E, Cazzaniga S, Bonini C, Roncarolo MG.Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but notCD25+CD4+ Tr cells. Blood 2005;105(3):1162-1169.12. Endharti AT, Rifa IMs, Shi Z, Fukuoka Y, Nakahara Y, Kawamoto Y et al.Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells. J Immunol2005;175(11):7093-7097.
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Table 1Study populationtransplanted between 06/21/2003 and 05/12/2006Total number of patients studied110Age (mean +S.D.)49+13Age range:18-72Gender: Male8476%Female2624%Endomyocardial Biopsy GradeTestsN(%)Grade 087957%Grade 1A56035%Grade 1B735%Grade 291%Grade 3A362%Total:1557100%Anti-HLA antibodies screeningTestsN(%)HLA-class I Positive764%HLA-class II Positive1599%Total1744100%Lymphocyte Growth Assay (LGA)TestsN(%)LGA+16111%LGA -136589%Total1526100%Table 1. Demographics and immunopathological findings
Figure 1A.B.FoxP3 expressioninperipheral CD4cells01234RejectionNo Rejection0246810RejectionNo rejectionFoxP3 expressioninperipheral CD8cellsmeanSErejectionn=510.88510.8402no-rejection n=1536.69454.8673meanSErejectionn=510.08830.0200no-rejectionn=1552.86571.3061C.D.0.00.100.20RejectionNo rejectionFoxP3 expressioninHeart BiopsiesResponseand CovariatenEstim.95% CIP-value(A)CD4 / FOXP3206-1.323(-2.476,-0.169)0.025(B)CD8 / FOXP3204-0.030(-0.085,0.026)0.296(N.S.)©BX / FOXP3103-0.115(-0.509,0.280)0.416(N.S.)meanSErejectionn=440.08830.0499no-rejectionn=570.14370.0855* GEE test
Figure 2CD4 T cells3A0 or 1Biopsy Grade0.0010.010.101.0010.00FoxP3ExpressionCD8 T cells3A0 or 1Biopsy Grade0.0010.010.101.0010.00100.00Expression of FoxP3 in CD4+ and CD8+ Peripheral Blood T Cellsafter Successful Treatment of High Grade (>3A) RejectionsCD4+ FoxP3N= 8CD8+ FoxP3N= 9Incidence of increasedexpression7/89/9Mean Fold Increase +SD7.54 + 8.8813.52 + 30.23Range4.41 – 27.880.47 – 93.83P-value 0.0360.028 Wilcoxon Exact Test
Figure 3CD8 T cells0.010.101.003A1B(1wk)3A(2wks)Biopsy GradeFoxP3Expression
Table 2CellsAnalyzedResponseCovariatenEstimate95% CIp-valueCD4FOXP3FOXP3FOXP3CD8FOXP3IL-102422.079(0.474, 3.684)0.011FOXP3FOXP3CTLA42510.765(0.213, 1.317)0.007CD8CTLA42460.594(0.109, 1.078)0.016CD4IL-102510.351(0.249, 0.452)<0.0001CD4IFN-G2472.004(-0.261,4.270)N.S.CD8IFN-G252-0.070(-2.257,2.118)N.S.Table 2. FoxP3 as a marker of suppression in transplant patients:. Strong associations with thesuppressive markers CTLA4 and IL-10 in T cells (irrespective of rejection status)CellsAnalyzedResponseCovariatenEstimate95% CIp-valueFOXP3FOXP3FOXP3EMBVEGF1021.235(0.853, 1.616)0.007EMBIFN-G1000.238(-0.415, 1.892)N.S.EMBIL-10992.547(-3.969, 9.064)N.S.* GEE tests
Figure 4Association between anti-HLA Ab and FoxP3 expression (GEE test)ResponseCovariatenEstimate95% CIP-value-0.0680.014N.S. (0.976)-0.0003Anti-HLA IgGCD4 / FOXP3255(-0.123, -0.014)Anti-HLA IgGCD8 / FOXP3252(-0.021,-0.020)FOXP3 expression in peripheral CD4 cells0.00.20.40.60.81.0020406080Anti HLA (IgG) AntibodiesFOXP3 expression in peripheral CD8 cells0.00.20.40.60.81.0020406080
Figure 5A.0204060801001 yr post-TxTransplantation020406080100Transplantation6 mo post-TxC.D.020406080100Transplantation1 yr post-TxAnti-HLA (IgM) abAnti-HLA (IgG) abLegendB.0204060801001 yr post-TxTransplantationF.E.Number of Daclizumab treatmentsNone1-3 Doses>3 DosesMo.N=7N=16N=87000061003823121003831181006939241006942Log rank statistics: P-value<0.00010204060801000612182401-3>3Number ofDaclizumabTreatmentsMonths (post transplantation)Cumulative Incidence(%) ofanti-donor Ab production
Figure 6AcuteRejection±0-3> 361622978871594109DaclizumabTreatment (doses)Chi square test, P <0.04
Figure 7LGA±±2014341381311144915813251483AcuteRej.Chi square test, P<0.0001
Figure 8C.A.808590951000122436Months post transplantationSurvival (%)Group A (0-3 Daclizumab Treatments)Group B (4+ Daclizumab Treatments)00.10.20.30.40.50.6> 0> 1> 2> 3> 4Number of Daclizumab treatment coursesGraft LossHazard RatioP=0.028D.B.Comparisonof # ofDaclizumabcoursesHazardRatio95% Hazard RatioConfidence limitsP-value*( 0 ) vs (>0)0.4200.0513.4340.419(≤1) vs (>1)0.4810.1002.3210.362(≤2) vs (>2)0.3420.0851.3790.132(≤3) vs (>3)0.2340.0620.8830.032(≤4) vs (>4)0.2690.0711.0160.053Patient Group APatient Group B(0-3 doses ofDaclizumab) N=23Actuarial Survival(%)(4+ doses ofDaclizumab) N=87Actuarial Survival(%)0100100686100128296188293248293308289368289Mo. Post-Tx* Cox proportional hazard modelLog rank statistics: P-value =0.028
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