Bone Marrow Transplantation (2002) 29, 719–725  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt Mini-review Extracorporeal photopheresis in chronic graft-versus-host disease

FM Foss, G Gorgun and KB Miller

Bone Marrow Transplantation and Experimental Therapeutics, Tufts New England Medical Center, Boston, MA, USA

Summary: Histopathologic changes which include sclerodermatous skin changes resulting from collagen deposition, pulmonary Despite significant advances in stem cell manipulation fibrosis, esophageal dysfunction, dry mouth or mucocutane- and post-transplant immunosuppression, chronic graft- ous ulcerations, cholestasis and myositis or fasciitis are versus-host disease (cGVHD) remains a cause of major thought to be initiated, in part, by autoantibodies to cell long-term morbidity in survivors of allogeneic stem cell surface and intracellular proteins.9 Conventional therapeutic transplantation. Extracorporeal photopheresis (ECP) is approaches for cGVHD, including corticosteroids and a novel therapeutic intervention which has demon- immunosuppressive agents have demonstrated limited effi- strated efficacy in patients with refractory acute and cacy in patients with extensive disease. Ultraviolet A ther- chronic GVHD. Clinical responses have been reported apy (PUVA), while effective in alleviating the symptoms in skin and visceral GVHD. While the long-term immu- of chronic skin GVHD, has had no impact on visceral nomodulatory effects of ECP in cGVHD are unknown, involvement.10–13 Novel strategies, including humanized recent studies of patients undergoing a 6- to 12-month anti-CD25 antibody (dacluzimab) and and anti-TNF-␣ anti- course of ECP treatment demonstrated an attenuation body (infliximab), have shown promise in limited pilot of Th1-mediated cytokine secretion by activated T- studies.14–17 helper cells, a shift in the DC1/DC2 ratio favoring plas- macytoid rather than monocytoid dendritic cell profiles, and a decrease in antigen responsiveness by dendritic Extracorporeal phototherapy cells. The implications of these immunomodulatory effects of ECP on pathogenesis and clinical outcome Extracorporeal phototherapy (ECP) is an immunothera- remains a fertile area for future research. peutic modality that has demonstrated clinical efficacy in Bone Marrow Transplantation (2002) 29, 719–725. DOI: cutaneous lymphoma/Sezary syndrome (CTCL), 10.1038/sj/bmt/1703529 scleroderma, and other autoimmune disorders. ECP Keywords: extracorporeal photopheresis; dendritic cells; involves extracorporeal exposure of peripheral mono- graft-versus-host disease; T cell subsets; allogeneic BMT nuclear cells to photoactivated 8-methoxypsoralen (8- MOP), followed by reinfusion of the treated cells. 8-MOP is a naturally occurring furocourarin that is biologically inert, unless exposed to ultraviolet A light, whereupon it Graft-versus-host disease (GVHD) remains a major cause becomes photoactivated and covalently binds and cross- of morbidity and mortality after allogeneic stem cell trans- links DNA, leading to initiation of . During a sin- plantation. While improvements in immunosuppressive gle treatment cycle of ECP, approximately 240 cc of buffy regimens have reduced the frequency and severity of acute coat and 300 ml of plasma are collected into a GVHD, the incidence of chronic GVHD remains bag from six collection cycles. The cells are exposed to unchanged at 27–50% after sibling matched related donor UVA at 2 Jcm2/cell beginning immediately after the first transplants and 42–72% after unrelated donor bone marrow cells are collected.18 Examination of the cells after UVA 1–6 or peripheral blood stem cell transplanted. Factors asso- exposure and prior to reinfusion demonstrates that about 2– ciated with cGVHD have been well-described and include 5% of the total circulating peripheral blood mononuclear increased donor and recipient age, HLA-disparate and unre- cells undergo apoptosis.18 An intravenous formulation of 8- lated donor transplants, prior acute GVHD, and use of allo- MOP, UVADEX, allows direct instillation of the photosen- 7,8 immune female donors. sitizing agent into the collected plasma and buffy coat ex The onset of cGVHD has arbitrarily been defined as vivo prior to UVA exposure. occurring 100 days after allogeneic stem cell infusion, and ECP was initially developed by Edelson et al19 as a ther- its clinical features are distinguished from acute GVHD in apy for patients with cutaneous T cell lymphoma with cir- 7,8 that they more closely resemble autoimmune diseases. culating Sezary leukemia cells. Response rates of up to 60% were reported in the initial study with a median time Correspondence: Dr FM Foss, Bone Marrow Transplantation and Experi- to response of 4–6 months, when the treatment was admin- 20–22 mental Therapeutics, Tufts New England Medical Center, Boston, MA istered for 2 consecutive days each month. In a retro- 02111, USA spective analysis of 450 patients treated in the United States Extracorporeal photopheresis in chronic GVHD FM Foss et al 720 and Europe, response rates of 56% and 66%, respectively, chronic stimulatory graft-versus-host reaction with clinical were reported. Response rates were higher in patients with features of systemic lupus erythematosis, injection of circulating clonal CD4+/CD7− Sezary cells and in those UVA/8-MOP-treated D2 splenocytes was capable of atten- with an intact immune system as determined by a normal uating the effects of GVHD that had been initiated by prior CD4/CD8 ratio.23 The anti-tumor effect correlated with the injection of D2 cells to initiate lupus-like disease.30 These appearance of CD8+ cytotoxic T cells in the peripheral results supported the concept that photoactivated T cells blood as well as the cells infiltrating into cutaneous tumor might be responsible for induction of a vaccine-like effect, and plaque lesions. While the absolute number of Sezary although the putative anti-idiotype T cell populations have leukemia cells decreased in most patients during ECP, there not been fully characterized. was no significant long-term modulation of normal CD4+ T lymphocyte populations. Although the mechanisms are less clear, ECP has also demonstrated similar efficacy in a number of autoimmune disorders, including scleroderma, Effects of ECP on lymphocytes, monocytes and pemphigus vulgaris, rheumatoid arthritis, systemic lupus dendritic cells erythematosis, and solid organ allograft rejection (Table 1).22,24–28 The anti-idiotype response to clonal T cell populations induced by ECP is likely to be induced by UVA-mediated cellular damage.31 T lymphocytes appear to be most sensi- Mechanism of action of ECP: preclinical models tive to this effect, as demonstrated by Yoo et al,32 who showed that normal T cell and Sezary T leukemia cells, but The mechanism of action of UVA-mediated immunomod- not other mononuclear populations, demonstrated morpho- ulation was first described in mice exposed to ultraviolet A logic evidence of apoptosis within 24 h after ECP treat- light in the presence of 8-MOP (PUVA). Immunosuppres- ment. In this study, 8-MOP alone failed to induce sion was accompanied by a decrease in the number and apoptosis, but exposure to UVA alone was associated with function of epidermal Langerhans cells and alteration in induction of early markers of apoptosis, including Annexin cytokine production by keratinocytes.13 The administration V.18 Failure to detect apoptotic lymphocytes from 1–24 h of 8-MOP and UVA irradiated spleen and bone marrow after ECP may be related to uptake and clearance of these cells to mice receiving allografts resulted in a significant cells by the reticuloendothelial system.32 attenuation of GVHD compared to littermates who received In addition to initiating DNA strand breaks, upregulation non-UVA irradiated cells, demonstrating an effect of UVA of FAS expression on T lymphocytes may be another mech- exposure on effectors of alloreactivity.29 In another study, anism by which ECP induces apoptosis. Since T lympho- using C57BL/6 × DBA/2)F1 (B6D2F1) mice which, when cytes also express FAS ligand, self-induction of cell death inoculated with parental DBA/2 (D2) splenocytes, develop may occur in the treated lymphocytes in the absence of cytokine secretion from monocytes or other accessory 33 Table 1 Extracorporeal phototherapy: advantages, toxicities, uses cells. This may explain the observed selective effect of and results ECP on T lymphocytes, but not on B lymphocytes or mono- nuclear cells. Diseases in which ECP has been used Selective effects of ECP on subpopulations of activated Cutaneous T cell lymphoma T cells have been observed in treated patients. Normaliz- Scleroderma ation of CD4/CD8 ratios has been described in CTCL Pemphigus 34 Systemic lupus erythermatosis patients, as well as a shift from predominantly Th2 to Th1 Inflammatory bowel disease cytokine secretion. In the context of CTCL, the restoration Solid organ allograft rejection of Th1 cytokine secretion increases IL-12 production by Acute and chronic GVHD following allogeneic stem cell transplant monocytes, which might subsequently inhibit Th2 cytokine Outcomes from therapy secreting cells that are believed to be responsible for the Decrease in circulating Sezary leukemia cells in CTCL clinical manifestations of CTCL.35 Improvement in skin and visceral GVHD While monocytes appear to be resistant to the apoptotic Decreased lymphocytic infiltration in cardiac allografts ␣ Attenuation of rejection in liver and renal allografts effects of ECP, the induction of TNF- secretion by pho- 36 Improvement in skin thickening and motor dysfunction in scleroderma toactivated monocytes facilitates lymphocyte apoptosis. Tapering of immunosuppressive therapies in autoimmune disorders Further, photoactivation of monocytes by ECP has also been shown to enhance their differentiation into CD83+ Advantages + Not generally immunosuppressive CD36 dendritic cells capable of phagocytosing the apop- May allow tapering of immunosuppressive agents totic T cells.37 In the presence of the costimulatory mol- Little or no end organ damage ecules, B7.1 and B7.2, these dendritic cells are capable of No cumulative toxicity No increased incidence of infection presenting tumor antigens from phagocytosed tumor cells in the context of MHC molecules and thus initiating cellular Disadvantages immune responses.38 Upregulation of antigen presentation Inconvenience: must go to a center with ECP, long course of therapy Cost in concert with presence of exposure and processing of Requires adequate i.v. access; increased frequency of infections if tumor antigens from apoptotic cells may explain why only central access used those CTCL patients with circulating leukemia cells exhib- ited a clinical response to ECP.

Bone Marrow Transplantation Extracorporeal photopheresis in chronic GVHD FM Foss et al

ECP 721

TCR Class 1 MHC + CDB DC1 DC2 T cell CD80 CD123 CD83 CD86 IL-12 TCR

CD4+ T cell Th1 cell

TNF IFN Inflammation Macrophage activation Th2 cell

IL-4, IL-10, IL-13

Inhibits macrophage activation and inflammation

Figure 1 Proposed mechanism of action of ECP in cGVHD. ECP modulates dendritic cell populations, resulting in an increase in CD83+, CD86+ plasmacytoid DC2 cells with a concordant decrease in CD80+ monocytoid DC1 cells. These stimulate Th2 T-helper cells to secrete Th2 cytokines, which indirectly inhibit Th1-mediated alloreactivity.

Clinical efficacy of ECP in cGVHD NK cells and HLA-DR+ cells after 9 months of therapy.41,42 In two additional studies in adult patients, improvement A number of small studies have reported efficacy of ECP was reported in sclerodermatous skin manifestations, but to in GVHD (Table 2). Owsianowski et al39 first reported a lesser degree in visceral sites such as mucocutaneous dis- improvement in lichenified skin changes, joint contractures, ease and liver involvement.43 and sicca syndrome in one patient with chronic GVHD after Two recent studies reported results of ECP in 11 and 15 starting ECP, with normalization of the CD4/CD8 ratio and patients, respectively.44,45 All patients had failed first- and increase in NK cells from 8 to 20%. Rosetti et al40 sub- second-line therapies, including corticosteroids and CsA. In sequently reported improvement in skin, liver, and pulmon- one study ECP therapy was initiated late after the onset of ary GVHD in children, resulting in tapering or discontinu- cGVHD (median 510 days), while in the other it was ation of immunosuppressive. In this study, patients received initiated earlier (median 178 days). This difference may be ECP for 2 consecutive days every 3 weeks for 6 months, relevant when comparing the outcomes of ECP in these then monthly. Responders were noted to have a normaliz- two studies. In the group treated early, 12/15 patients had ation of CD4/CD8 ratios and decrease in numbers of CD56+ improvement in skin score, 11/11 in mucocutaneous dis-

Table 2 Studies of extracorporeal photopheresis in GVHD

Author No. patients Response Characteristics of responses

Owsianowski M et al, 1994 1 1 Case report, improvement in skin, joints, sicca syndrome Rossetti et al, 1996 9 4 Response in skin in 3/5, lung in 2/4, GI Miller et al, 1998 12 Response in skin, liver; no response in oral GVHD Child et al, 1999 11 9 Response in skin in 9/10, lung in 2/5, liver in 1/5 More improvement with twice monthly vs once monthly treatment Abhvankar et al, 1998 5 3 Response in skin and joints; steroids tapered in responders Bishop et al, 1999 33 Response in skin in 14/22; in liver in 4/4, in oral in 8/18, 4/12 in ocular, 2/7 in pulmonary, 2/7 in GI Greinix et al, 1998 15 12 Response in skin in 12/15, in liver in 7/10, oral in 11/11, joints in 4/4, ocular in 56

Bone Marrow Transplantation Extracorporeal photopheresis in chronic GVHD FM Foss et al 722 ease, 7/10 in hepatic enzymes, and 5/6 in ocular symptoms. Further, cGVHD is a more heterogeneous disease than In the group treated late after onset of cGVHD, response aGVHD, making interpretation of phase II clinical trial rate in the skin was still high (10/10 patients), but only results difficult. Acute GVHD is believed to be a Th1- 2/4 with oral and 1/5 with liver involvement demonstrated driven disease wherein inflammatory cytokines such as IL- response in these visceral sites. In this group, the best 2 and IFN␥ as well as the monokines IL1 and TNF␣, responses in both skin and visceral disease were observed contribute to tissue damage.47–49 In murine models, donor in patients who started ECP treatment less than 10 months CD4-enriched cells of Th2 phenotype have been shown to from the time of BMT. A dose intensity effect was demon- prevent aGVHD without affecting engraftment, suggesting strated in that improvement was greater over the first 4–6 that they play a role in downregulating the Th1 response50 months when patients were receiving ECP twice monthly. and, in other studies, administration of a Th1 inhibitor Clinical improvement in patients treated with ECP on (TAK-603) markedly reduced the mortality associated with these studies enabled tapering of immunosuppressive aGVHD.51 Chronic GVHD has been proposed by Ferrara agents and corticosteroids. The median time to discontinu- and colleagues52 to be a Th2-mediated disease, but recent ation of corticosteroids was 80 days, and the median dur- studies demonstrating upregulation of Th1 cytokines IFN␥ ation of response after discontinuation of ECP was 12 and IL-12 in peripheral blood mononuclear cells implicate, months, with 14% of patients experiencing a recurrence of in part, a Th1-driven mechanism similar to that seen in symptoms of cGVHD after discontinuation.45 Despite the acute alloreactivity.49,52,53 need for intravenous access for ECP therapy in this group To elucidate the immunomodulatory effects of ECP on of immunocompromised patients, there was no increase in putative effectors of cGVHD, we initiated a clinical trial to infection rate and no increase in reactivation of CMV evaluate lymphocyte and dendritic cell function in patients infection. receiving ECP for 2 consecutive days every 2 weeks.54 The median time from BMT in our population was 667 days (range, 101–600 days). Seven of 10 patients had a clinical ECP in acute GVHD response to ECP, with improvement in skin GVHD and joint contractures in patients with extensive scleroderma- The largest series of patients with aGVHD treated with tous skin disease. Visceral improvement in ocular involve- ECP was reported by Greinix et al,46 who noted a 60% ment was noted in 5/7, in oral GVHD in 5/8, and in liver response rate in 21 patients with steroid refractory grades in 2 of 3. Immunosuppressive therapy was decreased or II–IV aGVHD. Patients were treated for 2 consecutive days discontinued in seven of 10 patients. In contrast to reports at 1- to 2-week intervals until improvement and then every by Child et al,44 who noted a lower response rate in patients 2–4 weeks until maximal response. Complete responses who initiated ECP late in the course of cGHVD, four of were reported in all nine patients with grade II, 67% with five patients starting ECP therapy more than 10 months grade III, and 12% with grade IV aGVHD. Responses were after BMT responded. noted in the skin (60%) and liver (67%), but there were The detection of a clonal population of T cells in patients no responses in patients who had extensive involvement with scleroderma suggests that one explanation for the including skin, liver and gut. Corticosteroids were discon- efficacy of ECP in this setting is induction of an anti- tinued in the complete responders at a median of 53 days idiotype response against host tissue-reactive T cell clones. after initiation of ECP, and cyclosporin A was gradually When nine patients with cGVHD were analyzed for clonal tapered. populations of alloreactive T cells by spectratype analysis, In this study, six patients died, five of infection and one we detected oligoclonal or monoclonal populations in from complications of gut GVHD. Of the responders, four seven. We observed a clinical response in all patients in developed cGVHD and eight are alive without cGVHD. whom clonal populations were detected. No clear mono- The maximal response to ECP was observed after 2 months clonal peaks were seen in two patients who did not respond of therapy. In contrast to patients with cGVHD, patients to ECP. While the relevance of these monoclonal T cell treated early after stem cell infusion experienced throm- populations in cGVHD patients has not been defined, ECP bocytopenia, anemia and leukopenia associated with ECP may generate an anti-idiotype response in cGVHD similar therapy. There were no reported effects of ECP on estab- to its effect against Sezary leukemia cells and autoreactive lishment of the graft, but this remains an ongoing question clones in scleroderma. as this therapy becomes more widely implemented in the early peritransplant period. Importance of antigen presenting cells in cGVHD pathogenesis Immunomodulatory mechanisms of ECP in GVHD The most pronounced effect of ECP in our patients was a Unlike CTCL, there are few data describing the impact of modulation of dendritic cell populations. In all responding ECP on the underlying immunopathogenesis of cGVHD. patients, a 50% or greater decrease in circulating CD80+ While aGVHD is understood as a disease of acute alloreac- and CD123+ dendritic cell populations was noted with no tivity, the etiology of cGVHD is controversial and is marked change in CD28 expression on lymphocytes, sug- believed to be either an extension of aGVHD and/or a result gesting no effect of ECP to modulate class I major histoc- of dysfunctional immune reconstitution with generation of ompatibility complex-restricted T cell function. The autoantibodies and tissue auto-reactive T cell clones. decrease in antigen presenting cells paralleled a decrease

Bone Marrow Transplantation Extracorporeal photopheresis in chronic GVHD FM Foss et al 723 in CD8+ cells suggesting an overall suppression of alloreac- cells in cGVHD may be related to their expression of FAS tivity. We further examined the effects of ECP on func- ligand, whose expression is limited to Th1 cells.57 tional antigen presentation and found that after ECP, there The effects of ECP on dendritic cell function and was a significant attenuation of dendritic cell function, as Th1/Th2 subsets will be further explored in a randomized measured by proliferation of allogeneic and autologous clinical trial of ECP vs conventional immunosuppressive lymphocytes in a mixed lymphocyte reaction. These results therapy in patients with cGVHD. This trial will enroll suggest not only a decrease in numbers of DC1 dendritic patients with steroid refractory or steroid-dependent cells, but also a direct effect of ECP on dendritic cell cGVHD with skin manifestations. Patients will be given function. immunosuppressive agents (FK506, CSA) with a total of Earlier reports demonstrated that UVA exposure in mice 25 ECP treatments over 12 weeks in the treatment arm or inhibits both the number and function of dendritic Langer- immunosuppressive therapy alone in the control arm. The hans cells in the skin, resulting in immune tolerance to allo- efficacy endpoint will be improvement in skin GVHD. geneic skin grafts.55 The importance of antigen presenting Because of its effects on lymphocyte populations and cells in the initiation of chronic GVHD was demonstrated dendritic cell function, ECP may play a role as a prophylac- in a murine allogeneic bone marrow transplantation model tic modality in the prevention of GVHD in high risk in which it was demonstrated that the initial targets for patients. A recent pilot study using ECP prior to low-dose CD8+ T cells in GVHD were restricted to proteins TBI and allogeneic stem cell infusion has reported a sig- expressed by residual host antigen presenting dendritic nificant attenuation of aGVHD with preserved graft-versus- cells.56 When host dendritic cells were eradicated by con- disease effect.58 Further studies of the immunomodulatory ditioning or replaced with donor dendritic cells, thus effects of ECP in the setting of both acute and chronic decreasing the interaction between host antigen-presenting GVHD, as well as in host conditioning prior to allogeneic cells and donor CD8+ cells, GVHD was attenuated. The stem cell transplantation are needed to further understand immunomodulatory effects of ECP on effectors of GVHD the mechanism of action and optimal utilization of this are summarized in Table 3. novel therapeutic intervention in transplantation strategies. A direct effect of ECP on dendritic cell number and/or function would lead to a decrease in alloantigen recog- + nition, processing, and thus stimulation of CD8 immune References effectors or a Th1 response. This inhibition might be syner- gized by concordant effects on T cells to induce a shift 1 Remberger M, Ringden O, Blau IW et al. No difference in from Th1 to Th2 cytokine profiles. graft-versus-host disease, relapse, and survival comparing per- Based on these results, we propose that the mechanism ipheral stem cells to bone marrow using unrelated donors. of ECP in cGVHD is to alter alloreactivity by affecting Blood 2001; 98: 1739–1745. both allo-targeted effector T cells, as well as the antigen- 2 Beatty PG, Hansen JA, Longton GM et al. Marrow transplan- presenting machinery. While this proposed mechanism is tation from HLA-matched unrelated donors for treatment of contrary to what has been observed in CTCL patients in hematologic malignancies. Transplantation 1991; 51: 443– whom monocytoid dendritic cells are activated by ECP and 447. 3 Storek J, Gooley T, Siadak M et al. Allogeneic peripheral Th1/Th2 ratios are shifted in favor of Th1, it may be that blood stem cell transplantation may be associated with a high the net effect of ECP is to restore immune homeostasis. In risk of chronic graft-versus-host disease. Blood 1997; 90: CTCL, the secretion of Th2 cytokines by leukemia cells 4705–4709. inhibits, in a paracrine fashion, a Th1-mediated anti-tumor 4 Gaziev D, Polchi P, Galimberti M et al. Graft-versus-host dis- response, and ECP induces a shift toward Th1 cyokine ease after bone marrow transplantation for thalassemia: an secretion by inducing apoptosis of tumor cells and thus, analysis of incidence and risk factors. Transplantation 1997; enhanced processing and tumor antigen recognition. In the 63: 854–860. context of the immune dysregulation of cGVHD,with ongo- 5 Lazarus HM, Rowe JM. New and experimental therapies for ing auto and alloreactivity, the net inhibitory effect of ECP treating graft-versus-host disease. Blood Rev 1995; 9: 117– on both activated T cells and antigen presenting cells favors 133. 6 Urbano-Ispizua A, Garcia-Conde J, Brunet S et al. High inci- attenuation of ongoing Th1-mediated events, thereby dence of chronic graft-versus-host disease after allogeneic per- removing the inhibition to Th2 cytokine secretion and ipheral blood progenitor cell transplantation. The Spanish restoring the Th1/Th2 balance. This selective effect on Th1 Group of Allo-PBPCT. Haematologica 1997; 82: 683–689. 7 Ratanatharathorn V, Ayash L, Lazarus HM et al. Chronic graft-versus-host disease: clinical manifestation and therapy. Table 3 Immunomodulatory effects in extracorporeal photopheresis Bone Marrow Transplant 2001; 28: 121–129. 8 Vogelsang GB. How I treat chronic graft-versus-host disease. Deletion of host reactive T cells in autoimmune disorders and Blood 2001; 97: 1196–1201. cGVHD Induce apoptosis in CD4+ leukemia cells in CTCL 9 Shulman HM, Sale GE, Lerner KG et al. Chronic cutaneous Induction of autoreactive CD8+ T cells in CTCL (vaccine effect) graft-versus-host disease in man. Am J Pathol 1978; 91: Increase expression of FAS-L by T cells 545–570. Decrease Th1, increase Th2 cytokines in cGVHD 10 Eppinger T, Ehninger G, Steinert M et al. 8-Methoxypsoralen Decrease T cell response to mitogens and ultraviolet A therapy for cutaneous manifestations of Increase TNF secretion by monocytes graft-versus-host disease. Transplantation 1990; 50: 807–811. Decrease dendritic cell function in MLR 11 Aubin F, Brion A, Deconinck E et al. Phototherapy in the treatment of cutaneous graft-versus-host disease. Our prelimi-

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