The immunological monitoring of alloreactive responses in liver transplant recipients: A review

REVIEW

The Immunological Monitoring of AlloreactiveResponses in Liver Transplant Recipients:A ReviewRaymond Reding,1 Jeremie Gras,1,2 Dinh Quang Truong,1 Gregoire Wieers,1 and Dominique Latinne2

1Pediatric Liver Transplant Program and 2Laboratory of Transplant Immunology, Saint-Luc University Clinics,Universite Catholique de Louvain, Brussels, Belgium

The aim of this work is to review the current knowledge in the field of immunological monitoring of allogenic responsivenessin clinical liver transplantation. When compared to other solid-organ transplants, liver allografts are considered asimmunologically privileged, and, accordingly, constitute a favorable setting to develop experimental as well as clinicalstrategies for minimization of immunosuppression and even induction of operational tolerance. The validation of simple,reliable, noninvasive assays exploring antidonor alloreactivity will constitute a crucial step toward implementing suchapproaches in the clinic. In contrast to research in rodents claiming the development of donor-specific tolerance in case of graftsurvivals of over 100 days without immunosuppression, it is impractical to confirm tolerance induction in this way in humans.Promising candidate assays include the detection of post-transplant immune deviation, of circulating precursors of dendriticcells subtypes, and of regulatory T cells. A conceptual framework for the development of tolerance assays in clinical livertransplantation is also proposed. Liver Transpl 12:373-383, 2006. © 2006 AASLD.

Received July 5, 2005; accepted November 30, 2005.

Since the first human liver transplant (LT) some 52years ago, the vast majority of hepatic allograft recipi-ents have remained under life-long maintenance immu-nosuppression (IS). Despite the dramatic improvementof early graft survival, late graft losses due to chronicrejection as well as the morbidity and mortality second-ary to long-term IS remain major concerns in thisfield.1,2 Induction of tolerance to the transplant wouldprevent these complications, and clinical LT probablyconstitutes an ideal setting to develop such strategies,considering its immunologically privileged status whencompared to other types of allografts.3 However, despitethe observation that tolerance has been relatively“easy” to induce in rodent models, the translation ofthese researches in humans has not yet been shown toreproducibly induce IS-free tolerant state in transplantrecipients. In experimental models, tolerance can be

assessed by graft survival beyond 100 days without IS,and by absence of rejection of a donor strain skin trans-plant; such an approach is of course impractical inhumans, and the only currently available method toascertain tolerance in a given patient is the ability toprogressively withdraw all IS with preservation of graftfunction and histology. Unfortunately, although a smallnumber of grafts are not rejected after removal of allmedications used for the maintenance IS, trials involv-ing the deliberate withdrawal of immunosuppressivedrugs according to a protocol do not suggest that it issafe to do so for most patients: Such trials are associ-ated with the inherent risk to induce acute or evenchronic rejection with the need for higher levels of ISwhen compared to the stable situation before attempt-ing at IS withdrawal.1,4,5

In this context, the development of tolerance assays

Abbreviations: LT, Liver transplantation; IS, Immunosuppression; TCR, T-cell receptor; Treg, regulatory T cell(s); DC, dendritic cell(s);APC, antigen presenting cell(s); IL, interleukin; Th, T helper cell(s); IFN, interferon; pDC, precursor(s) of dendritic cell(s); mRNA,messenger RNA; ELISPOT, enzyme-linked immunosorbent spot assay; HLA, human leukocyte antigens; DTH, delayed-type hyper-sensitivity; sCD30, soluble CD30.Supported in part by a grant from the Fondation de la Recherche Scientifique Medicale (FRSM 3.4567.05), Brussels, Belgium, and by a grant fromthe Commission Universitaire pour le Developpement (Projet Interuniversitaire Cible), Brussels, Belgium (D.Q.T.).Address reprint requests to: Pr. R. Reding, MD, PhD, Department of Surgery (1401), Saint-Luc University Clinics, 10, Hippocrate Ave., B 1200Brussels, Belgium. Telephone: 32 2 764 1401; FAX: 32 2 764 9001; E-mail: [email protected]

DOI 10.1002/lt.20704Published online in Wiley InterScience (www.interscience.wiley.com).

LIVER TRANSPLANTATION 12:373-383, 2006

© 2006 American Association for the Study of Liver Diseases.

and the implementation of clinical tolerance trials areintimately dependent upon each other: The introduc-tion of tolerance induction protocols in solid-organtransplantation will require the identification of simple,robust, ideally noninvasive surrogate biomarkers re-flecting the immune alloreactivity of the recipient to-ward his/her donor. After a brief introduction summa-rizing the terminology and overall mechanisms ofallogenic tolerance, the candidate assays proposed forimmunological monitoring in allograft recipients will besystematically reviewed, with particular respect to hu-man LT. A tentative approach toward research and de-velopment in tolerance monitoring in the clinical settingwill also be outlined.

ALLOGENIC TOLERANCE: TERMINOLOGYAND GENERIC MECHANISMS

A clear, widely accepted definition of the immune sta-tuses underlying the term “tolerance” constitutes aminimal requirement for the study of immunologicalmonitoring in solid-organ transplant recipients. Thefollowing concepts should be distinguished. (1) Trans-plantation tolerance refers to an actively acquired, per-manent immunological unresponsiveness, as describedin mice that had been injected in utero or neonatallywith bone marrow–derived donor cells6; this “plain”tolerant state is characterized by an absence of donor-specific alloreactivity in vivo as well as in vitro, and it isbelieved to occur through central mechanisms in thethymus. (2) Operational tolerance is defined as the ab-sence of acute and chronic rejection, and indefinitegraft survival with normal function in an IS-free, fullyimmunocompetent host, usually as the end result of asuccessful attempt at IS withdrawal. In contrast withtransplantation tolerance, operational tolerance doesnot necessarily mean complete unresponsiveness of therecipient immune system toward the donor cell (splittolerance), but rather refers to the lack of a destructiveimmune response toward the graft despite the presenceof generalized immune competence.5 (3) Prope toler-ance, as proposed by Calne, describes a state of “almosttolerance” in patients who maintain normal allograftfunction and histology under minimal IS, usually amonotherapy calcineurin inhibition with infrathera-peutic blood levels.7-9 (4) Graft acceptance defines thecommon situation in which a transplant recipient has anormal immunosuppressive load, with absence of im-mune injury toward the graft; this condition is typicallyobserved in the subgroup of recipients without acuterejection episode in the early and middle-term post-transplant period, under routine immunoprophylaxis,usually with combination of drugs including cal-cineurin inhibition.

Several review papers are available in the literature,providing in-depth discussion of the putative mecha-nisms of operational and transplantation toler-ance.5,9-16 Such detailed description is beyond thescope of this work. In brief, central and peripheralmechanisms have been described to be involved in thedevelopment of allogenic tolerance, as studied in vari-

ous experimental models.17 (1) Central tolerance is op-erative through intrathymic clonal deletion of precursorT cells expressing T-cell receptor (TCR) with high affin-ity for self-antigens or alloantigens belonging to themajor histocompatibility complex; the generation ofnatural T regulatory cells (Treg) has also been recentlydescribed as another possible mechanism to maintaincentral tolerance.12 (2) Various types of tolerogenic den-dritic cells (DC) have been identified, including imma-ture DC that, as antigen presenting cells (APC), fail toprovide adequate costimulation for T cell activation andtend to promote tolerance induction.18 At the alloreac-tive T-cell level, the end result of an encounter with atolerogenic DC may be deletion, anergy, or induction ofTreg. (3) Peripheral clonal deletion of allogenic T cellsmay occur peripherally via passive death, or activation-induced cell death which constitutes an active, donorAPC-induced process leading to apoptosis of alloreac-tive T cells.9,14 (4) Anergy of allogenic T cells refers to astate of T-cell inactivation following antigenic stimula-tion, characterized by a functional inertia with inabilityto respond to subsequent antigenic stimuli that in-cludes an impaired capacity to produce interleukin(IL)-2, in the absence of T-cell deletion.14,19,20 (5) Im-mune regulation through Th1/Th2 cytokine deviation isbased on the subdivision of CD4� cells into T helper(Th)1 and Th2 cells on the basis of their pattern ofcytokine secretion: Th1 type cells produce essentiallythe proinflammatory, immunogenic cytokines IL-2,IL-3, interferon (IFN)-�, tumor necrosis factor-�, andgranulocyte-macrophage colony stimulating factor, andare involved in cellular immunity and allograft rejec-tion; in contrast, Th2 type cells produce essentially theregulatory, tolerogenic cytokines IL-3, IL-4, IL-5, IL-6,IL-10, and transforming growth factor-� possibly in-volved in graft acceptance; however, this simplisticmodel of immune deviation leading to tolerancethrough preferential Th2 cytokine production has beenseriously challenged in experimental models and is notyet fully validated in humans.19,21-25 (5) Cellular regu-lation of T cells is emerging as a key mechanism foractively inducing and maintaining unresponsiveness todonor alloantigens.10,12,26,27; several populations ofTreg have been identified in tolerance models as well asin clinical settings, including CD4�CD25�Foxp3�,CD4�CD8�, and CD8� cells.28-30

CANDIDATE ASSAYS OF ALLOGENICIMMUNE RESPONSE AND TOLERANCE

From a clinical perspective, the assessment of alloreac-tivity using immunological monitoring in transplant re-cipient may be directed at the following aims: (1) toallow early, noninvasive detection of acute or chronicallograft rejection, before effector mechanisms and or-gan destruction have been initiated; (2) to evaluate thelevel of IS required for a given patient (choice of immu-nosuppressor molecule, dosage adjustment, and targettherapeutic window according to blood monitoring),these adjustments being currently determined only onan empiric basis; and (3) to determine the immunolog-

374 REDING ET AL.

LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

ical phenotype related to operational tolerance and toallow proper identification of the allograft recipients inwhom IS withdrawal could be safely conducted. Thisreview essentially concentrates on the latter issue, witha deliberate emphasis on the tolerance assays evalu-ated (or to be evaluated) in the context of clinical LT(Table 1).31

Detection of Microchimerism

Chimerism is defined as the existence of replicatingcells from different genetic backgrounds in a single or-ganism.11 Macrochimerism or mixed chimerism is de-fined by the persistence of more than 5% of circulatingdonor-derived cells, as observed following hematopoi-etic stem cell grafting, a condition classically associatedwith intrathymic deletion of donor-reactive T cells andcentral tolerance.32-35 In contrast, in microchimerism,donor-specific cells, usually DC, are present at low fre-quencies (�1/104 to 105 cells) in the transplant recip-ient. This latter finding has been related to the migra-tion of donor passenger leukocytes from the graft-to-recipient lymphoid organs early after transplantation,

as shown in the rat LT allograft model with spontane-ous tolerance induction.36 Despite much controversy, acorrelation between the degree of donor microchimer-ism in transplant patients and alloreactivity or allograftacceptance/tolerance has not been clearly estab-lished22,37-41; particularly in LT patients, high levels ofearly microchimerism did not abrogate the persistenceof an alloreactive response at 1 year posttransplant,such microchimerism being now considered as a con-sequence of graft acceptance under maintenance ISrather than a marker of allogenic unresponsive-ness.42,43 Moreover, it was suggested recently that chi-meric cells identified in transplantation studies maynot necessarily be donor-derived, and could have beenpresent in recipient tissues before transplantation.44

Consequently, microchimerism detection is no longerregarded as a promising tool for immunological moni-toring.

Detection of Dendritic Cells Precursors

These critically important APC circulate as immatureDC precursors (pDC), which exit the blood to reside asimmature DC in tissues throughout the body, and traf-fic via lymph or blood to secondary lymphoid organsfollowing antigen uptake. In humans, 2 major sub-populations of blood pDC have been described, mono-cytoid pDC and plasmacytoid pDC.45 Monocytoid DC(CD11c�) can be derived from circulating monocytes inresponse to granulocyte-macrophage colony-stimulat-ing factor and IL-4, whereas plasmacytoid DC (CD123�)develop after stimulation with IL-3 and CD40L. Mono-cytoid DC, which induce Th1 cell differentiation in vitro,and plasmacytoid DC, which promote Th2 cell re-sponses, have been designated DC1 and DC2, whichmay be specialized for the induction of immunity andtolerance, respectively.46 Mazariegos and colleagues,from the Pittsburgh group, hypothesized that the circu-lating levels of these pDC subsets, relative to one an-other, might reflect the status of liver graft recipientswith respect to clinical tolerance and safety of IS with-drawal vs. dependence on antirejection therapy with apersisting risk of rejection.47 Accordingly, these au-thors found that circulating pDC2 were more prevalent,relative to pDC1, in stable LT patients off IS and inthose undergoing successful IS withdrawal, comparedto recipients on maintenance therapy.18 According totheir results, the authors speculated that a pDC2/pDC1 subset ratio of 0.1 could serve as the thresholdabove which a patient might be considered for ISweaning. Interestingly, since pDC2 could be mobi-lized selectively in living donors in response to gran-ulocyte-macrophage colony-stimulating factor, suchimmunoregulatory approach may be of potentialtherapeutic value for the induction of tolerance tosolid-organ grafts.48

Measurement of T cell Alloreactivity

Beside classical in vitro analyses of T-cell alloreactivityusing mixed lymphocyte cultures,3 several approaches

TABLE 1. Candidate Tolerance Assays in Human

Allograft

Detection of tolerogenic dendritic cell precursorsRatio between pDC1 and pDC2*

Measuring T cell alloreactivityMixed lymphocyte reaction*Cytokine production by PBMC stimulated by donor-

specific alloantigen*Frequencies of cytokine-producing donor-reactive

cells (ELISPOT)Trans vivo delayed-type hypersensitivity assay*

Detection of Th1/Th2 immune deviationCytokine gene polymorphism (pretransplant analysis

in the recipient)*Profiles of circulating cytokines*Profiles of intragraft mRNA precursors of cytokines*

sCD30 determinationProfiling of alloreactive T cells (detection of

regulation/deletion)Detection of circulating regulatory T cells

(CD4�CD25�, CD4�CD8�, CD8�)*Detection of alloreactive T cell apoptosis (TUNEL

assay)Patterns of V� usage (indirect detection of clonal

deletion)Graft immunohistochemistry

Other assaysAlloantibody and autoantibody titers (humoral

immune response)Defining “tolerance genes” by gene chip microarray

technologyDefining “tolerance proteins” by proteomics

technology

Abbreviations: PBMC, peripheral blood mononuclear cells;TUNEL, terminal deoxynucleotide transferase-mediateddUTP nick end labeling.*Assays with data available in liver transplant recipients.

IMMUNE MONITORING IN LIVER TRANSPLANTATION 375


have been proposed to more accurately assess the re-activity of recipient T cell toward donor antigens.

In Vitro Cytokine Production by Recipient Cells

Levels of IL-2, IL-4, IFN-�, IL-10 and transforminggrowth factor-� produced in vitro by peripheral bloodmononuclear cells from 15 LT patients at 5-7 monthsposttransplant were analyzed by the Vancouver group,after stimulation by donor-specific alloantigens (liquidnitrogen-stored donor spleen cells), by third-party an-tigens (from other donors spleen cells), or by nonspe-cific pokeweed mitogen.49 Mononuclear cell response tostimulation, cytokine levels and cytokine messengerRNA (mRNA) from the cell cultures were assayed, andcytokine production was correlated with the clinicalcondition of the patient, including biochemical dataand graft histology. When compared to third-partystimulation, results showed a highly significant corre-lation between donor-specific-stimulated IL-4 andIL-10 production from circulating mononuclear cells ofrecipients with stable liver graft function when com-pared to acute rejection, this correlation being indepen-dent of the level of IS.49 Using a similar approach, Zhouand colleagues in Brussels group recently described areal-time polymerase chain reaction method based onIL-2 and IFN-� mRNA quantification upon in vitro stim-ulation of recipient whole blood with allogenic T-cell-depleted peripheral blood mononuclear cells.50 Asstudied in a tolerant recipient of a liver and hematopoi-etic stem cell transplant from the same living donor,this technique may constitute a promising whole bloodmixed lymphocyte reaction assay to monitor allogenicimmune responsiveness, with the particular advan-tages to only require small volumes of patient’s blood, toprovide results within 48 hours, and to allow storage ofmRNA samples for later analysis of other genes of in-terest.50 Flow cytometric measurement of the capacityof recipient T cells for intracellular cytokine productionwas also tested by several groups as a tool for immunemonitoring; it was essentially assessed in renal trans-plant recipients, where the technique could not, however,differentiate long-term outcomes of the allograft.51-53

Enzyme-Linked Immunosorbent Spot (ELISPOT)Assay

The frequencies of IFN-�- or IL-10-producing donor-reactive cells in recipient blood can be determined bymeans of the ELISPOT assay, and this technique hasbeen proposed as a surrogate marker of allogenic re-sponsiveness, essentially in renal transplantation.54-59

Pretransplant frequencies of donor-specific IFN-� ELI-SPOT were recently significantly correlated with post-transplant outcomes in 2 series of kidney recipients,high frequencies of alloreactive cytokine-producingcells in the recipient being associated with an increasedrisk of severe acute rejection episodes as well as withlower long-term glomerular filtration rate;54,55 accord-ingly, these authors suggested that such an approachmay serve as a pretransplant “cellular cross-match,” inaddition to the classical detection of preformed anti-

donor antibodies. Similarly, in the posttransplant pe-riod, ELISPOT measurements of IFN-�-producing recip-ients cells could be correlated with acute rejection andrenal function at 6 and 12 months.56-58 Interestingly,using the ELISPOT technique to monitor the indirectalloreactivity, the Harvard group studied the frequen-cies of Th1 (IFN-�-producing) and Th2 (IL-5- and IL-10-producing) peripheral blood lymphocytes reactive witha panel of synthetic peptides corresponding to se-quences from donor (HLA)-DR molecules (instead ofdonor cells)59: Such approach allowed the separation ofpatients with stable or impaired renal function accord-ing to a cutoff value below or higher than 60 IFN-�spots/106 cells, and constitutes a promising tool forlong-term immunological monitoring. In LT recipients,the ELISPOT technique has been used to assess hepa-titis C virus eradication and Epstein-Barr virus cyto-toxic T cells, but not yet, to our knowledge, to exploreallogenic responsiveness.60,61 Finally, the limitations ofthe ELISPOT approach should be mentioned, includingthe relatively reduced number of human cytokines as-sessable so far through this technique, as well as itsquestionable reliability particularly for stored cells.

Trans Vivo Analysis of Delayed-TypeHypersensitivity

It may be of interest to evaluate in vivo cell-mediatedallogenic immunity without exposing patients directlyto the challenge antigens, in order to avoid the risk ofsensitization.62 An alternative method for human de-layed-type hypersensitivity (DTH) was described; it in-volves the transfer of peripheral blood mononuclearcells plus donor antigen in the footpads of naıve, severecombined immunodeficiency mice.63 Antigen-drivenswelling is then measured after 24 hours, postinjectionmeasurements being compared to preinjection mea-surements to obtain specific swelling and quantify DTHreactivity. Such trans vivo DTH assay not only allowsthe determination of nonresponsiveness or of the levelof sensitization of the recipient toward donor-specificantigen(s), but also permits identification of the exis-tence of regulatory mechanisms operative in the trans-plant recipient, particularly of tolerizing antigens64: Inbrief, the colocalization of donor and recall tetanus tox-oid or Epstein-Barr virus antigens can induce donor-antigen-linked suppression of the response to theserecall antigens, an active downregulation mechanismpartially dependent upon the local activity of trans-forming growth factor -� and IL-10.63 In LT patients,the trans vivo DTH assay was shown to be a valuablemethod for identifying operationally-tolerant recipi-ents, in vitro mixed lymphocyte cultures tests failing todo so63; furthermore, co-localized soluble HLA-A and -B(and not HLA-DR) antigens were shown to allow thedetection of such DTH regulation phenomenon.65 How-ever, the logistical limitations of this assay should bementioned, including the need to maintain immunode-ficient mice in costly facilities.

376 REDING ET AL.


Cytokine Immune Deviation

Despite the limitations of the Th1/Th2 paradigm inhuman immunology and particularly of the interpreta-tion of preferential Th2-type cytokine environment as asurrogate marker of operational tolerance, 3 differentapproaches have been developed to assess the cytokinegenotype and phenotype in transplant recipients in thecontext of immunological monitoring.

Cytokine Gene Polymorphisms

The production of cytokines varies among individualsand these variations are determined by genetic poly-morphisms, most commonly within the regulatory re-gion of the relevant gene. Such polymorphisms in thecytokine genes may be associated with the magnitudeof cytokine production, usually categorized as “low,”“intermediate,” or “high,” and, accordingly, with theintensity of alloimmune responses following allogenicstimulation. As performed in the recipient before trans-plantation, the rationale of this type of genetic analysiswould consist in the ability to evaluate the productioncapacity of Th1/proinflammatory vs. Th2/tolerogeniccytokines in a given patient, in order to predict his/heroverall alloreactivity.66 Consequently, the aims of suchstudies would be to determine if an individual patient’spropensity to develop acute or chronic rejection is re-lated to the presence of these genetic polymorphisms(either alone or in combination). Unfortunately, suchan approach has several theoretical limitations, includ-ing the overly simplistic view of the Th1/Th2 immunedeviation paradigm, as well as the lack of considerationfor the allogenic disparity between the allogenic recipi-ent and his/her organ donor. Nevertheless, data fromthe Pittsburgh group suggested that genetic predispo-sition toward low tumor necrosis factor-� and high/intermediate IL-10 production may facilitate IS with-drawal in pediatric LT.67 Considering the lack ofstatistical power to detect small or moderate gene ef-fects in limited clinical series, a meta-analysis studyingthe impact of cytokine gene polymorphism on graft ac-ceptance in clinical LT was recently published, combin-ing the data available from 7 centers.68 In the overallanalysis, the only genetic risk factor associated withacute liver graft rejection was IL-10 polymorphism atposition 1082.A, an allele corresponding to low in vitroproduction of IL-10.69

Circulating Cytokines Levels

The analysis of published clinical studies correlatingcirculating cytokines levels to the immunological statusafter human LT (rejection vs. graft acceptance vs. oper-ational tolerance) only provides confusing and contra-dictory results.70-76 The immunological and clinical rel-evance of circulating cytokine levels in LT isconsiderably limited by several drawbacks, includingthe presence of confounding factors (surgical stress,blood transfusions, ischemic-reperfusion injury, he-patic regeneration, infectious complications), and thelack of serial cytokine profiles in most published se-ries.70 Moreover, circulating cytokines levels should

only be regarded as an indirect evaluation which maynot necessarily reflect the exposure to cytokine locallywithin the allograft or the lymphoid organs of the recip-ient. And indeed, these works, most of them publishedin the mid 1990s, did not result in standard guidelinesfor the immunological monitoring in clinical LT.

Detection of In Vivo mRNA Precursors ofCytokines

The analysis of cytokine mRNA precursors using thereverse transcriptase-polymerase chain reaction tech-nology was proposed to palliate the limitations of ana-lyzing the circulating cytokine levels as surrogatemarker of tissue exposure. In the clinic, such analysishas been essentially limited to the allograft, with therequirement to obtain control biopsy samples before aswell as after graft reperfusion. Unfortunately, the pub-lished studies providing data concerning cytokine pre-cursors with respect to graft acceptance were againrather conflicting, particularly concerning IL-2, IL-4,IL-15, and IFN-�.71-73,77,78 As already suggested forcirculating cytokines, these contradictory results makevery hazardous the use of isolated determinations ofintragraft cytokines precursors to predict the level ofallogenic responsiveness of a given patient in order topropose IS withdrawal.

Pretransplant and PosttransplantDetermination of Soluble CD30 (sCD30)

Originally described on Reed-Sternberg cells ofHodgkin’s disease, CD30 is a membrane glycoproteinthat belongs to the tumor necrosis factor superfamily. Itis expressed on activated T cells, preferentially thosesecreting Th2-type cytokines, although the CD30 mol-ecule is not considered as a physiological marker of Th2cells but rather as a costimulatory molecule regulatingthe balance between Th1/Th2 responses.79,80 After ac-tivation of CD30� cells, a soluble form of CD30 (sCD30)is released and can be measured in the serum. In kid-ney recipients, the pretransplant detection of highsCD30 level was shown to constitute a more accuratepredictor of acute rejection, when compared to panelreactive antibodies.81,82 Similarly, in the early post-transplant period, a significant decrement of sCD30levels was observed, with lower levels measured in thepatients without rejection.83,84 To our knowledge, nopublished data on sCD30 is available in LT recipients; apreliminary study in a limited number of patients inBrussels did not find any statistically significant corre-lation between pre-LT and early post-LT sCD30 valuesand the occurrence of acute rejection (D.Q.T., unpub-lished data).

Regulatory T Cells

Downregulation of immune responses by Treg is emerg-ing as a key mechanism for inducing and maintainingunresponsiveness to donor alloantigens, as well as inthe context of self-tolerance and autoimmun-ity.12,27,85,86 Studies of Treg in transplantation have



identified multiple populations of cells with differentcell-surface phenotypes and different mechanisms ofaction, as recently reviewed by Walsh and colleagues10

(1) Naturally occurring CD4�CD25� Treg, which areselected centrally in the thymus, are also characterizedby other cell-surface markers, such as CD45RB,CTLA-4 and glucocorticoid-induced tumor necrosis fac-tor receptor family–related receptor, as well as by theexpression of the forkhead/winged helix transcriptionfactor Foxp3; CD4�CD25� Treg inhibit effector T-cellproliferation apparently mainly through cell-contact-dependent mechanisms. (2) Inducible CD4� Treg,formed in the periphery following antigenic stimulation,included Th3 cells, known to promote tolerance follow-ing the oral ingestion of antigens through secretion oftransforming growth factor -�, and Tr1 cells, similar toTh3 cells but secreting large amounts of IL-10. (3) OtherTreg types have also been described outside the CD4�

compartment, including CD3�CD4�CD8� (double neg-ative) cells and CD8�CTLA4�Foxp3� cells.27-30,85,87,88

In vivo and in vitro approaches have been proposed todetermine the potential contribution of Treg detectionin the monitoring of allogenic responsiveness in trans-plant recipients.

In Vivo Detection of Treg

The Kyoto LT program has developed a clinical strategythat enables a substantial proportion (approximately40%) of LT patients to be weaned from IS completely,either in a physician-controlled, elective IS withdrawalprotocol or for the management of posttransplant com-plications including Epstein-Barr virus–related malig-nancy.4 However, a subgroup of recipients submitted tosuch therapy withdrawal (approximately 25%) encoun-tered rejection during the process of weaning from ta-crolimus and required the reintroduction of steroids.4

The peripheral blood mononuclear cells of these LTrecipients with successful or unsuccessful IS with-drawal were systematically phenotyped with the aim tocharacterize the immunological differences betweenthese 2 distinct populations of patients.89 Interestingly,an increase was observed in the frequency of circulatingCD4�CD25high� cells in operationally tolerant patients,when compared to age-matched volunteers or patientsstill on IS. Furthermore, an increase of the percentageof B cells and of V�1/V�2 �� T-cell ratio as well as adecrease of the percentage of natural killer cells werealso found in operationally tolerant cases. This studysuggests a role for cellular regulation through a circu-lating CD4�CD25high� subset acting as Treg in themaintenance of operational tolerance in LT recipients.Beside these measurements of Treg in the peripheralblood, it is of interest to mention the possibility of de-tecting CD8� and CD4�CD8� Treg among the graft in-filtrating T cells, as observed in animal models of trans-plantation tolerance30,88: Such an approach maypromise to better characterize the phenotype and sig-nificance of chronic cellular infiltrates within long-standing allografts in humans.

In Vitro Detection of Treg

The frequency of cytokine-producing indirectly primedalloreactive T cells (see Regulatory T Cells) determinedby ELISPOT analysis was further refined by the Har-vard group using in vitro depletion of putative CD25�

Treg.27 In a series of renal transplant recipients, incu-bation of peripheral blood mononuclear cells with anti-CD25 monoclonal antibody resulted in the change inHLA-allopeptide-specific IFN-� and IL-10 frequencies in8/17 (47%) of the patients with low allopeptide respon-siveness, whereas 0 of 8 with higher responses demon-strated such regulation (P � 0.05). The regulatory cellswere present in the circulation as early as 3 monthsposttransplant and persisted for a number of years,despite conventional IS.27 To our knowledge, such ap-proach has not been published for LT recipients, but itmay be valuable for assessing the existence of cellularregulation in the context of indirect alloimmunity in thelong-term.

Long-Term Alterations of T-Cell-ReceptorRepertoires

T cells recognize antigens through their TCR that is aheterodimer of either �� or �� chains.90 During T-cellontogeny, the � chain undergoes somatic rearrange-ment of 4 noncontiguous gene clusters, V, D, J, and C,resulting in a large repertoire of TCR molecules. TheTCR antigen-binding site is formed by 3 complementarydetermining regions, regions 1 and 2 being encoded bysequences of C genes alone; region 3 consists of rear-ranged sequences of V, D, and J genes and representsthe most variable TCR region that contacts the centralresidues of the bound peptide.91 Therefore, the analysisof TCR-V� usage, as measured by the polymorphism ofcomplementary determining region 3, may providevaluable information on the composition of T-cell rep-ertoire selected during an immune response.90,92,93 Ac-cordingly, such analyses could also reflect the occur-rence of peripheral deletion of alloreactive T cells to becorrelated with a status of putative operational toler-ance. Using a global method of T-cell repertoire analy-sis, particularly polymerase chain reaction technique toanalyze mRNA precursors of TCR repertoires and anal-ysis of complementary-determining region 3 length dis-tribution, 2 groups almost simultaneously publishedcomparative studies of TCR V� usage in long-term kid-ney transplant recipients with variable levels of graftacceptance from chronic rejection to operational toler-ance, as well as in healthy individuals.90,92 Soulillou’steam in Nantes showed a unique blood TCR patterncharacterized by a restricted complementary determin-ing region 3 length distribution92; similarly, Opelz’sgroup in Heidelberg evidenced strongly altered V� us-age, including an increased frequency of oligoclonalityand a decreased frequency of polyclonality.90 Whethersuch studies will help to identify a potential surrogatepattern of operational tolerance in liver recipients un-der life-long IS remains to be determined.

378 REDING ET AL.


Alloantibodies

For many years, attention was focused on cellularmechanisms of allograft rejection, humoral mecha-nisms being considered mainly as inductors of hyper-acute rejection in presence of antidonor HLA antibodiesbefore grafting.94 Increasing evidence now suggeststhat humoral responses to alloantigens could play animportant role in both acute and chronic alloimmunity,particularly following activation of the indirect path-way; consequently, the detection of alloantibodiesshould be mentioned in the list of candidate assays forthe immunological monitoring of transplant recipi-ents.95,96 A positive correlation between anti-HLA anti-bodies and poor graft outcome was established in kid-ney, heart, lung and liver recipients, whether thoseantibodies were present before grafting or appear aftertransplantation.94,97 Similarly, posttransplant appear-ance of non-HLA antiendothelial antibodies could becorrelated with coronary artery disease and chronicrejection in heart and kidney allograft recipients, re-spectively.94 Moreover, particularly in LT recipients, denovo autoimmune hepatitis has been described as apathogenic mechanism in the long-term, a conditionassociated with the presence of autoantibodies.98 Thehypothesis was formulated that damage of the grafttissues through preservation or immunological injurymay lead to the exposure or release of putative autoan-tigens and secondary induction of autoimmune re-sponses.99 So far, nevertheless, the role of detection ofalloantibodies and autoantibodies in allograft monitor-ing is not yet fully defined, particularly in LT.

Microarray Analysis of Gene Expression

The term DNA microarray refers to a high-density arrayof oligonucleotides or polymerase chain reaction -prod-ucts immobilized onto a solid support such as glassslides; the immobilized DNA selectively retrieves genesor sequences of interest when the array is hybridized toa mixture of complementary sequences obtained fromtissue or blood samples of clinical relevance.100 Sinceminiaturization currently allows more than 50,000genes to be spotted on a single slide, such technology iscapable of generating genome-wide profiles of mRNAexpression, allowing the exploration of gene expressionpatterns and regulation, as well as the characterizationof the genetic diversity underlying pathological condi-tions. Accordingly, global microarray profiling does notrequire prior knowledge of the gene pathways involvedin a disease and brings the potential to unravel thestructured and coordinated physiological processes,within hypotheses-generation studies that might pro-vide unexpected pathogenic pathways, possibly far re-moved from the standard hypotheses-based gene anal-yses.100,101 In the context of transplant recipients, genechips hold great promise for discovering noninvasivebiomarkers for monitoring of intragraft events, and forstratifying patients toward more individualized treat-ment regimes, particularly using comparative analysesof the peripheral blood, of the graft and its local envi-

ronment (bile in LT).101 To date, only a few publishedstudies in organ transplantation use microarrays, thelimitations being the cost of the technique as well assome procedural restrictions, including the heterogene-ity of the sampling source, the need for sample RNAamplification, the variability in expression measure-ments, and the complexity of data management.102-105

Despite the current lack of published studies using themicroarray technique for immunological monitoring inclinical LT, this approach has the potential to createinnovative knowledge by better defining the complexinteractions of various genes orchestrating the immunesystem, which may result in a large spectrum of re-sponses in LT recipients, varying from rejection to tol-erance.

CONCEPTUAL APPROACH TOWARDMONITORING OF ALLOGENIC RESPONSES

Considering the growing number of candidate assaysavailable to monitor allogenic responses, a strategy ofinvestigation in this field is clearly required to avoidtesting of proposed tolerance assays at random, as al-ready pointed out by the Pittsburgh group.106 Accord-ingly, we wish to propose hereafter an approach thatmight contribute to rationalize the investigation of pu-tative tolerance assays in clinical transplantation(Fig. 1).

Early allogenic activation of T cells and early post-transplant acute rejection have been shown to be es-sentially mediated through the direct recognition path-

Figure 1. Conceptual framework for research and develop-ment of immunological monitoring and tolerance assays, ac-cording to the putative chronology of tolerance induction andmaintenance mechanisms in the early and long-term post-transplant periods. Early immunological monitoring wouldrely on the study of early posttransplant profiles (Th1/Th2immune deviation, direct allogenic pathway assays), whereastolerance assays would rather rely on a “snapshot” evaluationof parameters related to T-cell regulation, to the indirect path-way of alloimmunity, alloantibodies, and T-cell receptor rep-ertoire.



way (presentation of allogenic stimulus by donor APC),whereas it is believed that the indirect recognition path-way (presentation of allogenic peptides in the context ofself major histocompatibility complex by recipient APC)predominates with increasing duration of engraft-ment.11 Immunoregulation of direct-pathway T cell isthought to be realized through a combination of anergicand deletional mechanisms, in contrast to the indirectpathway, which is considered to be primarily regulatedby Treg.27,107 The combination of these observationsled us to hypothesize that immunological monitoringshould be approached in 2 different ways, with respectto the interval post-transplantation. (1) Early monitor-ing requires the analysis of posttransplant profiles ofimmunological parameters, mainly centered on cyto-kine immune deviation and concurrent cell anergy/deletion (in vivo/in vitro detection of cytokines, directpathway ELISPOT assays); such profile studies shouldhelp to predict the immunological fate of the transplant,early rejection vs. graft acceptance vs. possibility offuture operational tolerance. (2) Tolerance assay(s)should be used in the longer-term in stable transplantpatients under maintenance IS as an immunologicalsnapshot of the residual reactivity of the recipientagainst his/her allograft; according to this interpreta-tion and the current knowledge, such tolerance assaysshould mainly focus on the detection of Treg (in vivo inthe peripheral blood and within the allograft, as well asin vitro using the indirect ELISPOT assay after CD25�

depletion), of major histocompatibility complex andnon–major histocompatibility complex alloantibodies(as markers of indirect alloimmunity), and on the alter-ations of TCR repertoires. In parallel, microarray tech-nology would allow the development of hypothesis-gen-erating studies to explore the mechanisms of graftacceptance, prope tolerance, and operational tolerancein long-standing allograft recipients. Such tolerancemonitoring should probably be done regularly to assurethe robustness of the tolerant state in patients con-stantly exposed to numerous environmental stimulithat could alter this putatively metastable condition.

CONCLUSION

The identification of a state of tolerance to an allograftrequires that clinicians develop appropriate interac-tions with immunologists and other basic scientists tostart the immunological evaluation of the patients ofinterest. This multidisciplinary approach should ideallybe organized in a prospective fashion from the time oftransplantation (evaluation of dynamic profiles), as wellas in longer-term recipients having putatively devel-oped operational tolerance (“snapshot” evaluation).Such clinical research will benefit from multicentricstudies as well as from concerted projects as developedby the Immune Tolerance Network at the National In-stitutes of Health.108 Defining the clinical and labora-tory phenotype of the tolerant patient should consider-ably facilitate the confident implementation of clinicaltrial of tolerance induction and IS withdrawal. A majorimpediment to the design and evaluation of new tools

for the immunological monitoring frequently resides,however, in the complete lack or limited supply of donorcells available to be used in assays evaluating donor-specific alloreactivity in the recipients. Accordingly,prospective measures should be developed by trans-plant centers to appropriately retrieve, process, andstore donor cells for later use in the immunologicalassays.109

Finally, the ethical aspects of immunological moni-toring should not be underestimated. Which patientsshould be selected for immunological monitoring? Whatabout the use of living donor blood in this assessment?On which basis could a patient be proposed for IS with-drawal? Accordingly, a new type of risk/benefit balancewill have to be developed, taking into account the ben-efit of successful IS withdrawal in a subgroup of pa-tients vs. the risk of acute/chronic rejection in case offailure in another subgroup. Such immunological mon-itoring protocols will require thorough discussionwithin the clinical teams, with the Institutional ReviewBoard, as well as with the patients themselves.

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The immunological monitoring of alloreactive responses in liver transplant recipients: A review

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