Extracorporeal photopheresis: From solid organs to face transplantation

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Review Extracorporeal photopheresis: From solid organs to face transplantation Mikaël Hivelin a,b, , Maria Siemionow b , Philippe Grimbert c , Laurent Lantieri a a Plastic Surgery Department, Henri Mondor Hospital, University Paris 12, Creteil, France b Plastic Surgery Department, Cleveland Clinic, Ohio, USA c Department of Nephrology, Henri Mondor Hospital, University Paris 12, Creteil, France abstract article info Article history: Received 9 February 2009 Received in revised form 19 April 2009 Accepted 24 April 2009 Keywords: Early apoptotic Composite tissue allotransplantation Face transplantation Tolerance Photopheresis Photochemotherapy Composite tissue allotransplantations (CTA), were introduced with the rst successful hand transplantation and are now a part of reconstructive surgery armamentarium. These reconstructive procedures for non life- threatening indications remain rare due to adverse effects of the associated lifelong immunosuppressive therapy. Indeed, despite recent progress, immunosuppressive therapies remain non-specic to the type of donor and still bear signicant risks of serious side effects. Extracorporeal photopheresis (ECP), also called photochemotherapy, has been introduced in the composite tissue allotransplantation eld as a part of acute rejection treatment in face transplantations. ECP has been performed after solid organ transplantations as a supportive therapy for acute rejection episodes. It has also been used to treat graft versus host diseases, which can occur after bone marrow or stem cell transplantations. ECP is also used to treat dermatologic diseases, such as cutaneous T-cell lymphoma, or autoimmune diseases, such as scleroderma or pemphigus vulgaris. The principle of ECP is to induce leucocyte apoptosis with UVA radiation after their presentation by psoralens. These leucocytes are immediately re-infused into the patient, where they undergo early apoptosis. Following apoptosis, the leucocytes are engulfed by macrophage or other antigen-presenting cells, such as immature dendritic cells, in an anti-inammatory cytokine environment. The anti-inammatory cytokine secretion pattern, with a switch from TH1 to TH2 for CD4+ lymphocytes, and the engulfment by immature cells without co-stimulatory molecules induces anergy, by deleting effector T-cells that responded to the presented antigens. An increase in regulatory T-cells (T-regs) is also induced after ECP and may contribute to allograft acceptance by the recipient. ECP has already been used for the great majority of solid organ transplantations to cure acute rejection episodes or in an attempt to prevent or cure chronic rejections, such as bronchitis obliterans, which occurs after lung transplantation. Considering composite tissue allotransplantations, ECP was used in two face transplantations after the occurrence of second rejection episodes triggered by viral infections. ECP therapy, associated with maintenance immunosuppressive therapy and doses of methylprednisolone, and the control of viral infection, succeeded to reverse the rejection process without the development of other side effects. Despite the fact that the mechanism of action of ECP has not been fully elucidated, this therapy could be a useful supportive therapy during the treatment of acute rejection episodes in composite tissue allotransplantations. In this review, we introduce the interest of ECP implementation in CTA in face allotransplantations. © 2009 Elsevier B.V. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 2. UV-induced apoptosis, modication of cytokine secretion patterns, and T-regs induction after ECP . . . . . . . . . . . . . . . . . . . . . 118 2.1. Denition of extracorporeal photopheresis (ECP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 2.2. Donor apoptotic cells injections as experimental models to study ECP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 2.3. Different steps in cell apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 2.4. Apoptosis induced during ECP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 2.5. Different delays in apoptosis occurrence after UV radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 2.6. Characteristics of these apoptotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Transplant Immunology 21 (2009) 117128 Corresponding author. Department of Plastic Surgery, Henri Mondor Hospital, 51, av Mal de Lattre de Tassigny, 94010 Créteil Cedex, France. Tel.: +33149812533. E-mail address: [email protected] (M. Hivelin). 0966-3274/$ see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2009.04.005 Contents lists available at ScienceDirect Transplant Immunology journal homepage: www.elsevier.com/locate/trim

Transcript of Extracorporeal photopheresis: From solid organs to face transplantation

Transplant Immunology 21 (2009) 117–128

Contents lists available at ScienceDirect

Transplant Immunology

j ourna l homepage: www.e lsev ie r.com/ locate / t r im

Review

Extracorporeal photopheresis: From solid organs to face transplantation

Mikaël Hivelin a,b,⁎, Maria Siemionow b, Philippe Grimbert c, Laurent Lantieri a

a Plastic Surgery Department, Henri Mondor Hospital, University Paris 12, Creteil, Franceb Plastic Surgery Department, Cleveland Clinic, Ohio, USAc Department of Nephrology, Henri Mondor Hospital, University Paris 12, Creteil, France

⁎ Corresponding author. Department of Plastic SurgerE-mail address: [email protected] (M. Hiv

0966-3274/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.trim.2009.04.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 February 2009Received in revised form 19 April 2009Accepted 24 April 2009

Keywords:Early apoptoticComposite tissue allotransplantationFace transplantationTolerancePhotopheresisPhotochemotherapy

Composite tissue allotransplantations (CTA), were introduced with the first successful hand transplantationand are now a part of reconstructive surgery armamentarium. These reconstructive procedures for non life-threatening indications remain rare due to adverse effects of the associated lifelong immunosuppressivetherapy. Indeed, despite recent progress, immunosuppressive therapies remain non-specific to the type ofdonor and still bear significant risks of serious side effects.Extracorporeal photopheresis (ECP), also called photochemotherapy, has been introduced in the compositetissue allotransplantation field as a part of acute rejection treatment in face transplantations.ECP has been performed after solid organ transplantations as a supportive therapy for acute rejectionepisodes. It has also been used to treat graft versus host diseases, which can occur after bone marrow or stemcell transplantations. ECP is also used to treat dermatologic diseases, such as cutaneous T-cell lymphoma, orautoimmune diseases, such as scleroderma or pemphigus vulgaris.The principle of ECP is to induce leucocyte apoptosis with UVA radiation after their presentation bypsoralens. These leucocytes are immediately re-infused into the patient, where they undergo early apoptosis.Following apoptosis, the leucocytes are engulfed by macrophage or other antigen-presenting cells, such asimmature dendritic cells, in an anti-inflammatory cytokine environment. The anti-inflammatory cytokinesecretion pattern, with a switch from TH1 to TH2 for CD4+ lymphocytes, and the engulfment by immaturecells without co-stimulatory molecules induces anergy, by deleting effector T-cells that responded to thepresented antigens. An increase in regulatory T-cells (T-regs) is also induced after ECP and may contribute toallograft acceptance by the recipient.ECP has already beenused for the greatmajority of solid organ transplantations to cure acute rejection episodes orin an attempt to prevent or cure chronic rejections, such as bronchitis obliterans, which occurs after lungtransplantation.Considering composite tissue allotransplantations, ECPwasused in two face transplantations after the occurrenceof second rejection episodes triggered by viral infections. ECP therapy, associated with maintenanceimmunosuppressive therapy and doses of methylprednisolone, and the control of viral infection, succeeded toreverse the rejection process without the development of other side effects.Despite the fact that the mechanism of action of ECP has not been fully elucidated, this therapy could be a usefulsupportive therapy during the treatment of acute rejection episodes in composite tissue allotransplantations.In this review, we introduce the interest of ECP implementation in CTA in face allotransplantations.

© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182. UV-induced apoptosis, modification of cytokine secretion patterns, and T-regs induction after ECP . . . . . . . . . . . . . . . . . . . . . 118

2.1. Definition of extracorporeal photopheresis (ECP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182.2. Donor apoptotic cells injections as experimental models to study ECP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.3. Different steps in cell apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.4. Apoptosis induced during ECP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.5. Different delays in apoptosis occurrence after UV radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.6. Characteristics of these apoptotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

y, Henri Mondor Hospital, 51, av Mal de Lattre de Tassigny, 94010 Créteil Cedex, France. Tel.: +33 149812533.elin).

ll rights reserved.

118 M. Hivelin et al. / Transplant Immunology 21 (2009) 117–128

2.7. Differences between early or late apoptotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.8. The need for early apoptotic cells to induce anti-inflammatory effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.9. Engulfment by monocytes, macrophages, and immature dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1202.10. Interactions between the early apoptotic cells and immature dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

2.10.1. A total and rapid clearance of early apoptotic cells is required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212.10.2. Apoptotic lymphocytes induce changes in cytokine secretion patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

2.11. Replacement of the “suppressor” T-cell concept by the regulatory T-cell (T-Regs) idea . . . . . . . . . . . . . . . . . . . . . . . . 1222.12. Donor-specific or non-specific immunomodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

3. Indications for photopheresis in solid organ transplantations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223.1. ECP in experimental organ transplantations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223.2. Clinical implementation in transplantations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

3.2.1. Kidney transplantation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223.2.2. Heart transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233.2.3. Lung transplantation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233.2.4. Pancreas transplantations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233.2.5. Liver transplantations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233.2.6. Intestinal transplantations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233.2.7. Phototherapy and bone marrow allografts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

3.3. ECP sessions schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1243.4. Adverse effects of extracorporeal photopheresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

4. Introduction and relevance of ECP for composite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.1. Composite tissue allotransplantations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.2. Rejection and immunosuppressive therapy in CTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.3. Opportunistic infections management and immunosuppressive therapy modulation . . . . . . . . . . . . . . . . . . . . . . . . . 1244.4. GVHD in CTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254.5. ECP in CTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

tissue allotransplantations

1. Introduction

Composite tissue allotransplantations (CTA), such as hand, face, orabdominal wall transplantations are new promising options inreconstructive surgery. Despite encouraging functional results ofthese procedures, indications remain limited by adverse effectsrelated to a lifelong immunosuppression.

Mono or polyclonal antibodies, steroids, cyclosporine A, FK506,rapamycin, or mycophenolate mophetil are current immunosuppres-sive protocols used for induction and maintenance therapy. Co-stimulation blockade by monoclonal antibodies is a new promisingoption that seems to reduce the risks for diabetes and opportunisticinfections, but it can increase the incidence of hyperlipidemia [1].

Major adverse effects such as hypertension, diabetes, lipidmetabolismdisorders, opportunistic infections, andmalignancies result from thenon-specific modes of action of immunosuppressive protocols. More selectiveand safer therapies for induction, maintenance, or rejection treatmentcould be obtained through donor-specific tolerance inducing strategies.

Extracorporeal photopheresis (ECP) has already been used formore than 30 years for solid organ transplantations and can serve asan additional therapy during acute rejection episodes.

ECP has been recently implemented in the CTA field to treat acuterejection episodes in face allotransplantation [2,3].

In this review,wedescribe themechanismsof action for ECP, includingthe induction of peripheral lymphocyte apoptosis, the modification ofcytokine patterns, and the activation of regulatory T-cells. Then, wepresent the indications of ECP in transplantation, with emphasis on itsimplementation to composite tissue allotransplantations.

2. UV-induced apoptosis, modification of cytokine secretionpatterns, and T-regs induction after ECP

Ultraviolet (UV) light is the primary cause of non melanoma skincancer, the most prevalent form of human cancer [4,5]. Within

ultraviolet light, 3 subtypes are distinguished based on theirwavelengths: UVA (400 nm–315 nm) and a subset called UVA1(400–340 nm), UVB (315 nm–280 nm), or UVC (280 nm–100 nm).UVA radiation contributes to skin cancer via indirect DNA damagedue to highly reactive chemical intermediates, such as hydroxyl andoxygen radicals. UVA radiation penetrates deeply but does not causesunburn. UVA1 radiation has to be used in conjunctionwith psoralens(PUVA therapy) to induce direct DNA lesions.

UVB light can cause direct DNA damage. UVB radiation excites DNAmolecules in skin cells, causing aberrant covalent bonds to formbetween adjacent cytosine bases, producing a dimer. UVB radiationcan cause sunburn and induce skin tumors through direct effects onDNA, leading to gene mutations [6,7] and to indirect effects onthe immune system [4]. UVA and UVB are both used in clinicalapplications.

UVC radiation has a shorter wavelength and is not used clinically,except for its germicidal properties. Little attention is usually given toUVC rays since they are filtered out by the atmosphere.

The tolerogenic effect on tumor cells induced by UVB radiation hasbeen described since 1974 [8], when UVB-induced skin tumors inmicewere not rejected when the recipient was exposed to the sameradiation. These immunosuppressive properties were also transferredby adoptive therapy, thanks to T-cells called “suppressive” T-cellsfound in UVB exposed mice.

2.1. Definition of extracorporeal photopheresis (ECP) (Fig. 1)

ECP, or photochemotherapy [9], consists of re-infusion of 8-methylpsoralen-sensitized and UVA-radiated blood mononuclear cells.Plasma and leukocyte-rich fractions from the patient's peripheral bloodare separated by centrifugation. A thin buff layer is then wedgedbetween a clear layer (theplasma) anda red layer containingmostof thered blood cells. This 1% of the total blood volume contains most of thewhite blood cells and platelets.

119M. Hivelin et al. / Transplant Immunology 21 (2009) 117–128

The 8-methoxypsoralen (200–300 ng/mL) can be given systemi-cally or can be infused ex vivo, into the plasma, after its extraction byapheresis. This latter method allows increased drug levels during theradiation treatment and limits adverse effects, such as nausea.

The leukocyte-rich fraction is then passed through a 1mmdiameterplastic channel and is exposed to UVA1 light (wavelength: 340–400 nm,energy: 2 J/m2). The photoexposed white cells are then infused backinto the patient, and the total procedure lasts from 4 to 5 h.

During one ECP session, about 10 to 15% of all the peripherallymphocytes are treated and re-infused into the patient [10], with theexpectation that amajority of the cellswill undergo apoptosiswithin 48 h.

The number of ECP sessions depends on the indications, type oftransplant, type and intensity of rejection, and also on ECP's efficacy.

ECPproperties have beenused since the 1970s for all diseases that canbe mediated by unregulated T-cell populations, such as cutaneous T-celllymphoma; graft versus host disease (GVHD) [11]; autoimmune diseaseslike systemic lupus erythematosus, rheumatoid arthritis, scleroderma,and pemphigus vulgaris [12,13]; and also many types of allotransplanta-tions [2,14–21] so as to control acute or chronic rejection episodes.

2.2. Donor apoptotic cells injections as experimental models to study ECP

The apheresis process used for ECP is difficult to implement in smallanimalmodels, such as rodents.Most small animal experimental studiesthat can be directly compared with ECP used apoptotic cells created exvivo that were then injected into recipients. These studies described thesame effects on the rejection process.

2.3. Different steps in cell apoptosis (Fig. 2)

Apoptotis or Programmed Cell Death (PCD) allows the suppressionof all damaged or dangerous cells without initiation of theinflammatory cascade. This physiological process controls cell num-bers during growth or healing, with lower risks of collateral damage tothe surrounding tissues.

Fig. 2 illustrates different steps of cellular apoptosis with acomparison to the process of cellular necrosis.

2.4. Apoptosis induced during ECP

Exposure of cells treated with 8-MOP to UVA radiation inducesDNA monoadducts and covalent crosslink formation [10] (Fig. 1).Photo-activated 8-MOP covalently binds to DNA pyrimidine bases, cellsurface molecules, and cytoplasmic components of the exposed whitecells [22]. These covalent links result in cellular lethal effects, andleukocytes are re-infused when entering the apoptotic phase. Thegreat majority of blood lymphocytes undergo apoptosis within 48 hafter ECP [23].

2.5. Different delays in apoptosis occurrence after UV radiation

UV radiation can induce two types of cell apoptosis, immediateapoptosis or pre-programmed cell death (Pre-PCD) versus delayedapoptosis (or PCD) [24,25], depending on the wavelength (UVA orUVB), the radiation energy, and the presence or absence of aphotosensitizer [24,26,27].

Pre-PCD can be induced by UVA1 without psoralens through adirect membrane rupture by the oxygen-singlet oxidative process[27]. This Pre-PCD appears immediately after the radiation.

Immediate apoptosis induced by UVA1 is caused by membranealterations and requires a significantly higher energy level than thatused to activate psoralens [24].

On the contrary, both UVA1 radiation associated with psoralensand UVB radiation induce direct DNA damage of the lymphocyteswithout direct membrane alterations, leading to PCD. This PCD canalso be divided into intermediate and delayed apoptosis.

Intermediate apoptosis, imputed to direct DNA lesions, is due to adepolarization of the inner mitochondrial membrane [25], withcessation of ATP production, and inversion in the ratio of themitochondrial pro-apoptotic protein Bcl-2 to the anti-apoptoticprotein Bax [28]. The level of the pro-apoptotic protein p53 increases,shifting the p53 to Bcl-2 ratio, which activates the caspase apoptoticpathway. The scramblase protein is also activated and provokes thephosphatidyl-serine flip-flop described above.

Delayed apoptosis is observed 20 h after UV radiation and isimputable to the Fas (CD95)–Fas ligand interaction through thecaspase pathway [25,29,30].

2.6. Characteristics of these apoptotic cells [31]

During the first stages of apoptosis, phosphatidyl-serine residues(PS), which are present on the inner leaf of the cell membrane underbasal conditions, “flip-flop” to the outer leaf, allowing for theirphagocytosis by antigen-presenting cells (APCs) [32,33] (Fig. 1). Thesephosphatidyl-serine residues can be identified by annexin V staining[34,35]. From 0 to 1 h after ECP, the number of annexin V-positivelymphocytes rises to 15% and remains quite stable for the first 20 h.After 20 h, however, the number of annexin V-positive cells increases,as does the number of necrotic cells [29].

Indeed, annexin V can also detect necrotic cells, which alsoundergo a loss of membrane integrity [36]. Propidium iodine (PI)specifically stains necrotic cells, thanks to their membrane alterations,and it enables the differentiation of necrotic from apoptotic cells.

2.7. Differences between early or late apoptotic cells (Fig. 2)

Two apoptotic stages can be determined with the use of annexinand propidium iodine (PI) staining. During the first, or early, stage ofapoptosis, cells will stain positively for annexin V, thanks to the PSflip-flop that occurs early; but cells will stain negatively for PI, as thereis nomodification in theirmembrane permeability. During the second,or late, stage of apoptosis, cells will stain positively for annexin V andfor PI since this dye can penetrate the damaged membranes. The lateapoptotic cells progressively flake into apoptotic bodies (Fig. 2).

Thus, cells can be divided into three groups; intact (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), and late apoptotic ornecrotic (Annexin V+/PI+) cells [37] (Fig. 2).

In conclusion, we emphasize the different types of apoptosis,immediate, intermediate or delayed, as well as the early and late stagesin the apoptotic process that should be considered when analyzingapoptosis.

2.8. The need for early apoptotic cells to induce anti-inflammatory effects

As illustrated in Fig. 2, the pro- or anti-inflammatory reactionstriggered by antigen-presenting cells (APCs) are dependent on theapoptotic stage of the lymphocytes during their engulfment by APCs.

Early apoptotic cells are the only ones to provide immunomodu-latory effects. On the contrary, normal, late apoptotic, or necrotisedcells can stimulate the recipient's lymphocytes.

A recent study describes an absence of tolerogenic effects afterinfusion of donor apoptotic cells [38]. Donor lymphocyteswere renderedapoptotic by gamma radiation or UVA radiation after sensitization bypsoralens, and their infusion reduced the engraftment of donor bonemarrow when compared to living cells. Interestingly, this study wasperformed with the co-stimulation blockade using anti-CD 154antibodies, which can interfere with the tolerogenic process initiatedby apoptotic cells. Moreover, the assessment method of the apoptoticstagewas performedwith annexin V and actinomycinD staining, but thepercentage of early or late apoptotic cells infused was not described.Indeed, a high proportion of injected cells in a late apoptotic stage couldinduce an inflammation that could explain these results. The same result

120 M. Hivelin et al. / Transplant Immunology 21 (2009) 117–128

could be expected if an excessive amount of early apoptotic cells thatoverwhelmed the clearance processwas infused into the recipient. Someof these cells would undergo late apoptosis and, thus, would becomepro-inflammatory before their delayed engulfment.

The total clearance of early apoptotic cells before they undergo lateapoptosis is crucial to induce tolerance.

Fig. 1. Principles of extracorporeal photopheresis in composite tissue allotransplantationphotopheresis remain hypothetical). APC: Antigen-presenting cell. CM: Co-stimulatory molerequired to activate the effector T-cells, thus leading to anergy).

2.9. Engulfment by monocytes, macrophages, and immature dendriticcells (Fig. 1)

The fate of these early apoptotic cells is to be engulfed as fast aspossible by macrophage and immature dendritic cells, both immatureantigen-presenting cells. This engulfment mainly occurs in the spleen.

s (some of the mechanisms described to explain the immunomodulatory effect ofcule (the immature dendritic cells do not express the co-stimulatory molecules that are

121M. Hivelin et al. / Transplant Immunology 21 (2009) 117–128

As described above, the removal, or clearance, of apoptotic cells is amechanism used to eliminate all unnecessary cells without inducing animmune response, thus limiting the risk of causing collateral damage inhealthy tissue.

This rapid clearance has been demonstrated to have immunosup-pressive effects [39] due to inhibitory signals and the absence of co-stimulation of the effector T-cells.

Dendritic cells, as professional antigen-presenting cells (APCs)presentdonor allo- or auto-antigens to T-cells in the presence or absence of anassociationwith co-stimulatorymolecules, depending on their mature orimmature stage. This presentation is called direct whenperformed by thedonor's APCs or indirect when performed by the recipient's APCs.

Apoptotic cells produce anti-inflammatory cytokines, such as IL-10 orTGF-beta. This anti-inflammatory environment stimulates monocyte–machrophage cells or immature dendritic cells to engulf the apoptoticcells. These APCs then produce immunoregulatory cytokines (IL-10 orTGF-beta) and decrease their production of pro-inflammatory cytokines(e.g., TNF-alpha, IL-1, or IL-12) [40].

Recent studies demonstrated that there is a secretion of anti-inflammatory cytokines resulting from the phagocytosis of apoptoticcells. Before these studies, the lack of an anti-inflammatory responseafter apoptosis was only attributed to the rapid engulfment of theapoptotic cells before cell lysis occurs, thus avoiding the release of theirinflammatory contents into the local environment [41]. It has also beensuggested that the greater resistance to apoptosis induced by ECP ofperipheral blood monocytes, when compared to lymphocytes, couldexplain the anti-inflammatory response observed [42].

2.10. Interactions between the early apoptotic cells and immaturedendritic cells

2.10.1. A total and rapid clearance of early apoptotic cells is requiredThe concept of apoptotic cell clearance has been well studied for

autoimmune diseases such as systemic lupus erythematosus (SLE) or

Fig. 2. Differences in staining, phagocytes, and anti- or pro-inflammatory effects for the diffeerythematosus. Rheum Dis Clin North Am, 2004. 30(3): p. 505–27). Annexin V: Staining whmembrane, which occurs at a very early stage of apoptosis. Propidium iodine (PI), staining calate stages of apoptosis.

pemphigus. Indeed, a deficiency in apoptotic cell clearance issuspected to lead to auto-reactivity [43–45].

When the apoptotic load exceeds the phagocytic capacity toengulf the cells before they become late apoptotic or even necrotic,the cells secrete pro-inflammatory cytokines and are then phagocy-tosed by mature antigen-presenting cells, such as mature dendriticcells. This last pattern of phagocytosis leads to pro-inflammatoryeffects such as rejection of an allotransplant in a recipient or toautoimmune disease.

As this rapid clearance of the early apoptotic cells is mandatory toavoid an auto- or allo-immune reaction, any protocol based on theinjection of apoptotic cells should consider the precise amount of earlyapoptotic cells to inject, which could differ, depending on someone'sclearance capacity.

2.10.2. Apoptotic lymphocytes induce changes in cytokine secretionpatterns [31]

T-lymphocyte cytokine secretion patterns are described by a TH1pattern, with IL-2, IFNγ, or TNFα secretion, and by a TH2 pattern, withIL-4 or IL-5 secretion.

However, changes in cytokine levels and changes in the Th1/Th2ratio induced after the phagocytosis of apoptotic cells, from a pro- to ananti-inflammatory profile, remain controversial [31]. Some studiespresent a stimulation of effector T-cells [46], while others relate anti-inflammatory responses [47]. A pro-inflammatory response couldexplain the efficiency of ECP in CTCL, and ananti-inflammatory responsecould explain the effects on allotransplant rejection or autoimmunediseases.

Finally, the decrease in pro-inflammatory cytokines post ECP is notrelated to a simple decrease in the number of T-cells due to apoptosis[48]. Indeed, only 15% of the total number of T-cells is treated duringone ECP session. Moreover, UV-induced early apoptotic cells alsosecrete immune suppressive cytokines, such as IL-4 and IL-10 [49].

rent stages in apoptosis. (Modified from Sheriff, A. et al., Apoptosis and systemic lupusich binds phosphatidyl-serine residues once they are exposed on the outside of the celln penetrate cells after the alteration of their membranes during necrosis and during the

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2.11. Replacement of the “suppressor” T-cell concept by the regulatory T-cell(T-Regs) idea

The benefits of ECP on autoimmune diseases and transplantrejection as well as the T-cell phenotypes responsible for inhibition ofthe immune response remain unclear, and two main theories areconsidered from a historical perspective.

The first is based on an anticlonal immunity theory, which involvessuppressor T-cells that act by a “vaccine-like” process [50], as firstintroduced by Kripke's studies [51].

The second is based on tolerance induction by anergy or peripheraldeletion and by regulatory T-cell activation.

Early descriptions of antigen-specific suppressor T-cells generatedafter UV radiationwere observed after UVB radiation [8,51]. This T-cellsubset was supposed to inactivate alloreactive T-cells. In the samestudy, immunosuppressive properties were transferred by adoptivetherapy of suppressive T-cells found in previously UVB-radiated mice.

Other studies used adoptive therapy between UVB-radiated miceto search for lymphocyte subsets supporting the alloreactive T-celldeletion concept [52,53]. The existence of suppressor cells, to explainimmunomodulatory effects following UV radiation, was demonstratedin the 1970s [54] when donor-specific immunosuppression wasobtained in mice and when the host was UVB radiated beforesensitization with allogenic spleen cells [55]. Interestingly, the sameeffect was obtained by the direct radiation of the donor skin [56].

The second theory to explain the immunomodulativeproperties relieson the induction of regulatory T-cells [5,57–63]. CD4+CD25+Foxp3+cells are known todown-regulate the immune response. This last conceptwas first introduced by Rivas and Ulrich [64], with immunomodulatoryeffects explained by IL-4 and IL-10 regulatory cytokines, provided byregulatory T-cells CD4+CD8−.

This theory is compatible with the induction of effector T-cellanergy after ECP. Anergy is the peripheral inactivation of alloreactiveT-cells after antigen presentation by APCs without co-stimulationmolecules in an anti-inflammatory cytokine environment.

ECP seemed to enhance the immune tolerance instead of inducingan immunosuppression [65].

2.12. Donor-specific or non-specific immunomodulation

The first studies on suppressor T-lymphocytes induced by UVradiation promoting tolerance for tumor engraftment in micedescribed this suppression to be cell-mediated, adoptively transferred,and specific [53,54,66]. It is still unclear whether the effects are donorspecific or whether the immunomodulation is non-specific and onlylinked to the secretion of immunoregulatory interleukins, such as IL-4,IL-10, or TGF-beta.

If the immunomodulation through apoptotic cells injections is donorspecific, ECP could be performed on the donor lymphocytes after theircollection before organ harvesting, and these donor-origin leukocyteswould be injected during the immunosuppressive induction protocol.

This description of apoptosis stages after ECP is helpful tounderstand the molecular principles, but some of the mechanismsdescribed to explain the immunomodulatory effect of photopheresisremain hypothetical. It even remains controversial as to whether cellapoptosis is relevant for immunomodulation.

3. Indications for photopheresis in solid organ transplantations

All UV-mediated therapies such as UVA with or without psoralensensitization, and UVB radiation are efficient therapies in immunologicdisorders via their immunomodulating effects [67–69].

ECP has been used in dermatology for cutaneous T-cell lymphoma[9,70,71] and in autoimmune diseases, such as systemic sclerosis,systemic lupus, pemphigus, ulcerative colitis, or atopic dermatitis[5,72]. Other autoimmune diseases, such as inflammatory bowel

disease or rheumatoid arthritis, can also benefit from this therapy[72,73].

Indications in hematology include graft versus host diseasetreatment and after bone marrow or stem cell allotransplantation,particularly for steroid refractory transplantations [74–80].

ECP indications in solid organ transplantation can be divided intoacute [15,20,81–84] and chronic [85] rejection episodes treatments.

3.1. ECP in experimental organ transplantations

The use of ECP implementation to prevent or treat rejectionepisodes can be traced back to 1985, when the first transplantation ofa UVA-radiated rat kidney after administration of 8-methylpsoralenwas reported [86]. In 1989, the first ECP implementation in a primatemodel was performed for heterotrophic heart xenotransplantation(from cynomolgus monkey to baboon) and for allotransplantation(baboon to baboon) [87,88]. Animals were submitted to ECP twice aweek, starting 3 days after transplantation; controls only receivedcyclosporine and steroids. In this study, an absence of hyperacuterejection, an increased graft survival, and a suppression of response inmixed lymphocyte reaction (MLR) were observed in recipientspresensitized by donor blood. In two animals, biopsy-proven rejectionepisodes were reversed in the photochemotherapy groups, whereascontrols suffered from progressive rejection.

This study demonstrated how ECP could specifically suppressrecipient immune responses to donor xenoantigens, while maintainingthe capacity to respond to other unrelated antigens.

Thus, the suppression induced by photochemotherapy seemsspecific for the immunoreactive cell type [12].

For small bowel transplantation, ECP was only introduced inexperimental models [89]. In this study, small bowel transplantationswere performed from ACI rat donors to LEW rat recipients. These ratsreceived either donor splenocytes (radiated or not) or FK506, or boththerapies combined.

The non-radiated splenocytes did not delay the rejection, whichoccurred in one week. The donor splenocytes submitted to radiationslightly improved the survival, as did the FK506 cells alone. Whenradiated splenocytes and FK506 were combined, the small bowelsurvival was dramatically increased: 86 days on average (Pb0.005),and up to 100 days for 7 animals out of 24.

Using this last example, we want to insist that there are numerouscommon points between tolerance induction through apoptoticlymphocyte injections and extracorporeal phototherapy.

The main difference between these two types of treatments is theorigin of the apoptotic cells: from donor origin in the studies withapoptotic cell injections and from the recipient (collected byapheresis) in ECP. The second main difference is the timing withregard to the transplantation procedure: before or during thetransplantation for the tolerance inductions with apoptotic cells, andduring or after transplantation for ECP.

3.2. Clinical implementation in transplantations

Since thefirst clinical applications of ECP, its potential as an additionaltherapy to treat the rejection of all kinds of allografts or autoimmunediseases was sensed, leading to many experimental protocols [12] andclinical implementations in solid organ transplantation (Table 1).

3.2.1. Kidney transplantationThe first clinical application of UV radiation in transplantationwas

the treatment of kidney allograft acute rejection [90]. This firstclinical attempt was performed using PUVA therapy (UVA radiationand 8-methylpsoralen), on the donor-kidney before its transplanta-tion. Thirty-three PUVA-pretreated kidneys were compared totwenty-six non-pretreated kidney allografts. The results wereencouraging, as significantly fewer rejection episodes were reported

Table 1Implementation of extracorporeal photopheresis (ECP) in solid organ transplantationsfor the treatment of acute rejection episodes.

Type of solid organ allotransplant Year of introduction (andreference)

Kidney 1987 [88]Heart 1992 [12]Lung 1995 [99]Pancreas 1995 [90]a

Liver 2000 [103]Composite tissue allotransplantation (partial facetransplantation)

2006 [2]

Small bowel –

a The third patient presented among the case reports had received a combinedkidney and pancreas transplantation and is cited as the first implementation of ECP forpancreas transplantation, even if the function of the pancreas is not described after theECP therapy.

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at 3 months, even if the difference considering the allograft survivalitself was not significant. The number of patients in the studies onkidney rejection treated by extracorporeal photopheresis is relativelylow, when compared to studies involving other solid organs. Twoarticles presenting the same protocols and number of patients forkidney allotransplant rejection, presented contradictory results[91,92].

The extracorporeal photopheresis, also called photochemotherapy,was then used to treat recurrent acute rejection episodes [16] andsteroid refractory acute rejection episodes in kidney transplantation[82,84,92–94].

3.2.2. Heart transplantationCostanzo-Nordin et al. first described the efficiency of ECP as a

complementary treatment for acute rejection in a heart allotrans-plantation [14,95,96] and even described a single cycle of ECP able toreverse moderate acute rejection. After 25 days, the inflammatoryinfiltrate disappeared from the transplants. The recurrent acuterejection of heart allotransplant [15] also became another indicationfor this therapy. A study on the effect of ECP on recurrent or refractorycardiac allograft rejection was performed on 11 patients (with goodresponses for 7), with sessions scheduled as described for heartallograft rejection therapy and a follow up from two to five years [18].The B lymphocytes and the macrophages were also lowered moresignificantly by ECP compared to steroids alone [14]. Because ECP wasworking on the levels of B lymphocytes and cytotoxic antibodies, itwas also indicated for hypersensitized patients [97]. Finally, thebenefits of ECP have even clearly been demonstrated on patientsurvival and allograft function during multiple and or refractoryrejection [98,99].

3.2.3. Lung transplantationLung transplantation is the standard therapy for end-stage lung

deficiency resulting from chronic obstructive pulmonary disease andan increasing number of pulmonary fibrosis cases. Despite potentimmunosuppressive maintenance treatments incorporating tacroli-mus, mycophenolate mofetil, and corticosteroids [100], the develop-ment of chronic allograft rejection, with bronchiolitis obliteranssyndrome (BOS), continues to decrease the long-term survival oflung transplant recipients.

The first introduction of ECP in human lung transplantation wasperformed for an acute rejection episode occurring in a severelyinfected patient [101], who improved clinically after 3 weeks andhistologically after 4 weeks. The same year, ECP was used for lungchronic rejection refractory to steroid doses in 3 patients, allowingstabilization of the degradation of their pulmonary function [102]. ECPfor lung transplantation was then implemented for refractory BOS[103], and the stabilization of pulmonary function and the improve-ment of survival was related tomonthly ECP treatments performed on

2 consecutive days [104]. Patients were receiving a range of 3 to 13ECP, with a median number of 6 [17].

It is interesting to notice that after ECP for lung transplantation,levels of T-Regs did not correlate with the number of photopheresis,but rather, with the lung function itself [58].

There are no studies addressing the prophylactic effect of ECP forlung transplantation.

3.2.4. Pancreas transplantationsAmong the three cases reported in 1995 by Sunder-Plassman about

the use of ECP for acute kidney rejection [92], the third patient hadreceived both a kidney and a pancreas transplant, and was treated byECP for acute rejection. The function of the pancreas allotransplant isunfortunately not reported before or after the ECP therapy. There is noother study on ECP for pancreas transplantation or for Langerhansislets in humans. This case can be considered to be the first onereported about ECP after pancreas transplantation (Table 1).

3.2.5. Liver transplantationsThe implementation of ECP among treatments for liver allograft

rejection was introduced later [105], which could be explained by itssuspected tolerogenic properties, with fewer rejection episodescompared to other solid organ transplantations [106–108].

The studies on ECP for liver transplantation suggested that ECPwould be an alternative to treat recalcitrant graft rejection in somepatient subsets. Rejections occurring with HCV recurrence, steroid-resistant acute rejection, or acute rejection in a major ABO-mismatched liver graft were particularly considered as electiveindications for ECP [20]. ECP was then studied for rejectionprophylaxis. First, ECP was used to delay the calcineurin inhibitorintroduction in high-risk liver allograft recipients (18 patients), so asto decrease the calcineurin inhibitor toxicity without an increase inthe rejection risk in the immediate post-operative time [21].

Then, ECP was applied to ABO-incompatible liver transplantationto prevent antibody-mediated rejection (AMR) and cell-mediatedrejection. The study consisted of 19 patients receiving ABO-incompa-tible allografts, 11, treated by therapeutic plasma exchange (TPE), and8 treated by TPE but also by ECP and receiving immunoglobulin IV. Allpatients also received an immunosuppression regimen, includingsteroids, mycophenolate mofetil, and tacrolimus. The graft survival at6, 12, and 18 months was significantly higher in the second group,treated by ECP and anti-CD25monoclonal antibodies (basiliximab) IV,and no case of acute rejection was noticed after up to 568 days offollow up. The main drawback that can be outlined in the design ofthis study is the introduction of IV immunoglobulin in addition to ECP,which makes it difficult to draw any conclusion about ECP efficiencyon the control of rejection in these ABO-incompatible patients.

Finally, the last implementation of ECP for liver transplantation hasbeen performed in HCV RNA-positive patients receiving livers fromdonors older than 40 years (median age: 66 years). The objective of thisstudy was to reduce the immunosuppressive regimen so as to begin(69.2% of 78 patients could receive the antiviral prophylaxis) to improvethe efficacy of antiviral treatment (interferon and ribavirin) [109].

3.2.6. Intestinal transplantationsTo our knowledge, there is still no description of the use of ECP for

small bowel transplant rejection except for an experimental protocolof an induction therapy with apoptotic splenocytes in a rat smallbowel transplantation model [89].

3.2.7. Phototherapy and bone marrow allograftsThe main indication of ECP after bone marrow transplantation is

for the treatment of graft versus host disease (GVHD), which can alsooccur after stem cell allotransplantation or even after solid organtransplantations. We will not describe the principle and interests ofECP for the treatment of GVHD.

124 M. Hivelin et al. / Transplant Immunology 21 (2009) 117–128

Nevertheless, a better donor bone marrow engraftment has beendescribed inmanyexperimentalmodels after injections of donor-derivedearly apoptotic cells.

In experimental mouse models, injecting UVB-induced apoptoticleukocytes intravenously can facilitate allogenic bone marrow engraft-ment [110].

3.3. ECP sessions schedule

There is no exact number of ECP sessions required to treat acuterejection after organ transplantation, as it is used as an additionaltherapy. ECP used as a treatment for CTCL involved treatment on twosuccessive days every four weeks. This number of session implied thatless than 5% of total body lymphocytes were irradiated each month [9].

Considering the average number of sessions of ECP per patient,after a transplantation, themedian number of ECP sessions per patientwas 20 in a liver transplantation study reported in 2004 [20].

For transplanted patients, the therapy can begin with one to twosessions a week for four weeks and are then tapered, depending onthe effect on the rejection episode.

3.4. Adverse effects of extracorporeal photopheresis

No significant or major side effects are generally observed duringand after the ECP therapy for GVHD treatment, as for the treatment ofrejection episodes in transplantations, in adults or children[17,20,111,112]. However, some adverse effects are reported, such asmalaise, low-grade fever, and gastrointestinal troubles [113].

Some side effects are not specific but linked to repeated and long-term vascular access with large catheters used for apheresis [114]. Theuse of large catheters and the risk of hemostasis troubles withformation of massive thrombosis [115] need to be carefully weighedagainst the benefits.

The number of opportunistic infections is not increased by ECPtreatment, confirming that the immunoregulation induced is specific[16,116]. The reported episodes of sepsis [103] seem to be more likelyrelated to the increased vascular access of the procedure inconjunction with the generally poor health status of the patient.

Minor side effects observed for patients receiving photopheresisinclude hypotension and syncope resulting from volume shifts duringthe leukapheresis phase of treatment.

Although many studies describe ECP as helpful to control allograftrejection [85,95,96,117], there is a lack of prospective randomized trialsand no sufficient evidence to demonstrate an advantage of photopher-esis over othermethods used to treat rejection exists. Thus, ECP remainsan additional therapy in transplantation, rather than a standard therapy.

4. Introduction and relevance of ECP for compositetissue allotransplantations

Composite tissue allotransplantation indications will remain excep-tional until the lifelong immunosuppressive therapy and its adverseeffects are reduced. The immunosuppression currently used in CTA iscomparable with that used for solid organ transplantation. Althoughsome attempts at tolerance induction protocols have been performed inthe clinic, for kidney [118], liver [119], pancreatic islets [120], andcomposite tissue allotransplantations [2,121], tolerance has not beenroutinely achieved, and themost encouraging results obtained in kidneytransplantation were obtained with family-related living donors [118].

4.1. Composite tissue allotransplantations

The first successful hand transplantation was performed in 1999,thanks to amore efficient immunosuppressive treatment than the oneused for the first attempt in 1964 [122,123].

The donor and recipient had the same blood group (O+), but 6human leukocyte antigen (HLA) group mismatches [124]. Therecipient did not observe a continuous immunosuppressive therapy,and signs of cutaneous rejection appeared at 15 months post-transplantation and were partially reversed after reintroduction ofanti-CD25 antibodies and steroids. However, the patient did notpursue the immunosuppressive protocol for a second time, and thus,the transplanted hand was re-amputated after 29 months post-transplantation. No rejections have been reported to lead to a handloss when the immunosuppressive therapy has been well observed.

Despite 19 unilateral and 11 bilateral hand transplantations, 3 facetransplantations, and 38 other subtypes of CTA performed throughoutthe world [125,126] with mainly fair or good functional results and norejection justifying the removal of these allografts when theimmunosuppressive treatment was not interrupted [127], the justi-fication for such procedures is still debated.

For face transplantation, themajority (78.4%) of a panel of 162NorthAmerican plastic surgeons agreed about the limitations of autologousreconstructions for severe facial injuries, but only 27% were in favor ofsuch a procedure under the current immunosuppressive protocols. Theothers would prefer to wait for 10 years of hindsight (16%), for animprovement in immunosuppression (23.8%), or for the clinicalapplication of tolerance induction protocols (28%) [128].

Considering these last two positions, one canwonder what preciselevel of improvement in the immunosuppressive protocols wouldmake the CTA as acceptable as another type of transplantation.

4.2. Rejection and immunosuppressive therapy in CTA

The clinical and histological assessments of the allograft arenecessary and sufficient to detect and prove the rejection in compositetissue allotransplantation because the skin is visible and is the firstcomponent to suffer from rejection.

It is interesting that, contrary to primate models, acute rejectionepisodes in human hand allotransplantation have been completelyreversedwith intravenousmethylprednisolone, provided that thepatientshad a good observance of the immunosuppressive therapy [129]. Somecases also benefited from topical applications of steroid cream.

Considering the face transplantation rejections, this one can bemonitored clinically as a hand allograft. Mild or moderate rejectionepisodes led to methylprednisolone doses but also to antibodyinjections, when the steroid doses were inefficient [2].

In contrast with rejection episodes occurring in hand transplanta-tion which could be controlled by an increase in the dose ofprednisone and local application of tacrolimus and corticosteroidcream, intravenous doses of corticosteroids were necessary to reverseacute rejection in face transplantation.

The numbers of hand and face transplantations remain too small toperform prospective and randomized studies, but different teams arealready using the same patterns for induction and maintenancetherapies. Nevertheless, considering the indication of the varioustreatments for acute rejection, and even maintenance therapy, there isno consensus on the grade of rejection and time to wait for its responseafter methylprednisolone doses before beginning the antibody treat-ment. The same statement can be made for the topical therapy.

4.3. Opportunistic infections management and immunosuppressivetherapy modulation

Risk of opportunistic infection is related to the deepness ofimmunosuppression after the transplantation. For viral infections, thelevel of serology matching between the donor and the recipient alsohas to be considered.

CMV infection is one of the most frequent opportunistic infectionsand is the most frequent systemic viral infection complicating solidorgan transplantation [130–133].

Fig. 3. a. Chronology of the acute rejection episodes and of the introduction of extracorporeal photopheresis after the face transplantations performed in Lyon (legend in b).b. Chronology of the acute rejection episodes and of the introduction of extracorporeal photopheresis after the face transplantations performed in Paris.

125M. Hivelin et al. / Transplant Immunology 21 (2009) 117–128

CMV infection can result in direct effects or CMV disease, related tothe presence of high rates of viral replication, and to indirect effectscaused by virus interactions with the host immune response which canincrease the risk of rejection and graft dysfunction, acceleratedatherosclerosis, opportunistic infections, malignancies, post-transplantdiabetes mellitus, and Guillain–Barré syndrome.

The prophylaxis of CMV infection is based on gancyclovir orvalgancyclovir and on the control of the immunosuppressive protocol.

CMV-related complications in composite tissue allotransplantationshave been described for hand [134], and face transplantations. Amongthe 18 patients who received uni or bilateral hand transplantationsbefore 2005,17 were tested for CMV infection. Three CMV-seronegativerecipients (Lyon-France, Louisville, USA and Insbruck, Austria) receivedCMV-positive hands andwere, thus, exposed to an important viral load.

Considering the increased risk of rejection caused by a CMVinfection, which is also suspected in CTA [134], transplantationsbetween CMV-matched donors and recipients appear mandatory.

ECP is already used to treat acute rejection episodes occurring afterliver transplantation in HCV-positive patients [20]. As CMV alsoincreases the risk of rejection [130–133], ECP would be a valuableadditional treatment for the acute rejection episode, without increas-ing in the level of immunosuppression.

Barr et al. performed a prospective randomized study on the effects ofECPoncardiac allograft acute rejection that showed that cytomegalovirusDNA was detected significantly less frequently in patients receivingphotopheresis than in those receiving the standard therapy [116].

4.4. GVHD in CTA

Another indication of ECP in composite tissue allotransplantationwould be the graft versus host therapy. Nevertheless, as inductionprotocols in CTA or in solid organ transplantation are non myeloa-blative, there is a reduced risk for GVHD, and it has not been reportedfor hand or face transplantation. The experimental models, includingvascularized and unvascularized bone marrow, highlighted this needfor myeloablation to obtain a GVHD in CTA [135,136].

4.5. ECP in CTA (Figs. 1, 3a and 3b)

Two human face transplantations were the first reported clinicalimplementations of extracorporeal photopheresis in composite tissueallotransplantations [2,3].

126 M. Hivelin et al. / Transplant Immunology 21 (2009) 117–128

In both cases ECP was used after the second acute rejectionepisode, to prevent both acute and chronic rejection withoutintensifying the immunosuppressive regimen.

In thefirst case, theauthors suspected aherpes virus infection tohavetriggered the second episode of rejection. On day 185, a Herpes SimplexVirus Type 1 (HSV1) infection of the patient's lip was documented andwas treated by valacyclovir systematically and acyclovir topically.

TheCMVstatuses of both thedonorand the recipient are not indicatedin the early report [137], nor in the 18 month follow up report [2].

ECP was started 2 months after the second rejection episode, thus10 months post-transplantation, in order to reduce the risk of furtheracute rejectionwithout intensifying the immunosuppressive regimen.ECP was performed twice a week for 4 weeks or once a week for8 weeks and was described as well tolerated (Fig. 3a). It was notdetermined whether the HSV1 infection, suspected to have triggeredthe second rejection, was already cured when this rejection began, orwhen the ECP was started.

The second patient who received face transplantation alsobenefited from ECP during the second rejection episode. This secondepisode of rejection was associated with a CMV viremia [3] (Fig. 3b)and required steroid boluses and anti-thymocyte globulin for 7 days.ECP was also started to control the rejection process withoutintensifying the immunosuppressive regimen, as the patient sufferedfrom valgancyclovir-resistant CMV viremia. ECP was performed twiceweekly for 2 weeks, thenwas reduced to one course every 2 weeks for3 months to control subclinical rejection, which persisted on mucosalbiopsies.

ECP has proven to be valuable to treat rejection in liver transplanta-tions challenged by HCV recurrences, and it has been used in facetransplantation for a rejection thatmay have been triggered by a herpesvirus infection.

As ECP has been demonstrated to be a powerful additional therapy toprevent and treat acute, recurrent, and refractory rejection in trans-plantation, the next step was to use it in the anti-rejection therapy forcomposite tissue allotransplantations (CTA), as most of them includeskin, which is considered a strong immunogenic component [138].

It is not clear why ECP was first implemented in face transplanta-tion rather than in hand or abdominal wall transplantations. ECPseems to be an interesting option as an additional therapy for acuterejection episodes, as it does not have the adverse effects ofimmunosuppressive treatment, such as steroid doses, which stillseem to be required for grade 2 rejection episodes.

Themechanisms bywhich ECPworks to prevent allograft rejections,like pathways for apoptosis induction or regulatory T-cells inductionbegin to be understood. Nevertheless, many unknowns remain such asthe precise modification of the cytokines secretion patterns, andwhether the apparent tolerance induction that occurs with ECPtreatment is donor specific or not.

5. Conclusions

Tolerance induction protocols that include apoptotic cell injectionsor extracorporeal photopheresis for the treatment of acute or chronicrejections have effects on apoptotic cells/monocyte–macrophages thepro/anti-inflammatory cytokine profile, and the induction of regulatoryT-cells.

There is still no evidence that these processes are donor specificsince the donor antigens presented after donor apoptotic cellinjections and the recipient antigens presented during photopheresisinduce the same results.

The efficiencyand safety of extracorporeal photopheresis has alreadyproven in solid organ transplantation and in bone marrow transplanta-tionwhen it is used post-operatively during acute rejection episodes oreven in chronic rejection states. Additionally, ECP appears to be atreatment of choice for treating rejections triggered by infections.

Face transplantation was the first implementation of extracorporealphotopheresis for composite tissue allotransplantations.

ECP on the donor and/or recipient blood or tolerance inductionprotocols based on donor early apoptotic cell injections can also be usedbefore the transplantation or during or after the immunosuppressiveinduction, even when there are not any signs of rejection.

Conflict of interest

None of the authors has a financial interest in any of the products,devices, or drugs mentioned in this article.

Acknowledgement

Mikael Hivelin was supported by an additional grant from thePhilippe Foundation, Inc.

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