Autologous Stem Cell Transplantation in Autoimmune Diseases

Autologous Stem Cell Transplantation in Autoimmune Diseases Jakob Passwega and Alan Tyndallb Since 1996, approximately 1,000 patients have received an autologous hematopoietic stem cell transplant (HSCT) as treatment for a severe autoimmune disease (AD). The European Group for Blood and Marrow Transplantation (EBMT)/European League Against Rheumatism (EULAR) Autoimmune Disease Working Party have registered more than 800 patients and works in close collaboration with networks in the United States where several hundred more AD patients have been similarly transplanted. The majority of ADs were multiple sclerosis (MS), systemic sclerosis (SSc), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), juvenile idiopathic arthritis, and immune cytopenias. Many patients have experienced long-term disease-free remissions and immune reconstitution studies have shown in some cases that a “resetting” of autoimmunity is possible. The initially high treatment-related mortality (TRM) is reduced significantly in the later years, and the phase I/II experience is now being verified in several international prospective randomized clinical trials. In addition, the past several years have seen a growing interest in the role and potential therapeutic application of mesenchymal stem cells (MSC) in the immunomodulation of AD, as in the early experience with acute-graft-versus host disease (GvHD). Semin Hematol 44:278-285 © 2007 Elsevier Inc. All rights reserved.

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he rationale for hematopoietic stem cell transplantation (HSCT) is based on studies in experimental animal models of autoimmune disease (AD)1 and observations of remissions of autoimmune diseases in patients treated with HSCT for hematological malignancies.2 Various protocols have been employed depending on the underlying disease and individual experience of transplant centers, but most were based on autologous HSCT due to the reduced toxicity compared to allogeneic HSCT and its attendant graft-versus-host disease (GvHD). Although the principle therapeutic component of HSCT is the immunoablation, evidence suggests that other components of the procedure may modulate the safety and effectiveness of the procedure and, as such, HSCT may be more than just a means to dose-escalate immunosuppressive medication. A key difference with so-called “targeted therapies” is that HSCT nonspecifically affects a wide array of immunecompetent cells, which include B and T lymphocytes, thus creating space for a new immunological repertoire, generated aDivision

of Hematology, University Hospital Geneva, Geneva, Switzerland. of Rheumatology, Felix-Platter Spital, Basel, Switzerland. Supported in part by the Horton Foundation, Switzerland, and by the European League Against Rheumatism (EULAR). Address correspondence to Professor Alan Tyndall, Department of Rheumatology, University of Basel, Felix-Platter Spital, Burgfelderstrasse 101, Basel 4012, Switzerland. E-mail: [email protected] bDepartment

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from hematopoietic stem cells.3 Depending on the components of the immunoablative regimen, stem cells may or may not be targeted as well. Most regimens employed in autoimmune disease contained high doses of cyclophosphamide with or without antithymocyte globulin (ATG), which is nonmyeloablative because stem cells are resistant to cyclophosphamide. While HSCT quickly became an established treatment for many hemato-oncological conditions since it was first used to treat leukemia more than 30 years ago, its application in AD has long been hampered by concerns that HSCT might not be feasible or was too toxic in immunosuppressed patients with poor functional status and organ involvement from the underlying rheumatic disease. Despite some initial negative experiences, the feasibility of HSCT in AD has now been firmly established (Table 1). With respect to safety and efficacy, some trends have emerged from retrospective database analyses and prospective pilot studies, acknowledging the limitations inherent in such studies.4 More intense regimens were associated with higher treatment-related mortality but a slightly lower probability of relapse, although differences in regimens, patient entry criteria, and outcome parameters preclude more refined analyses. Importantly, safety of HSCT has improved as best illustrated by the dramatic decrease of transplant-related mortality (TRM) in patients with severe systemic sclerosis (SSc).

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Table 1 EBMT/EULAR Autoimmune Disease Autologous HSCT Database Disease and Disease Category Neurological disorders Multiple sclerosis Myasthenia gravis Other Rheumatological disorders Systemic sclerosis Rheumatoid arthritis Juvenile idiopathic arthritis Systemic lupus erythematosus Dermatomyositis Mixed connective tissue disease Behcet’s disease Psoriatic arthritis Ankylosing spondylitis Sjõgren’s syndrome Other Vasculitides Wegener’s disease Cryoglobulinemia Takayasu’s disease Classical polyarteritis nodosa Microscopic polyangiitis Churg-Strauss syndrome Other Hematological immunocytopenias Immune thrombocytopenia Pure red cell aplasia Autoimmune hemolytic anemia Evans syndrome Pure white cell aplasia Other Gastrointestinal Enteropathy Inflammatory bowel disease Ulcerative colitis Other/unknown Total

No. 281 2 13 136 93 49 78 8 4 6 2 2 1 12 7 4 2 2 2 1 9 18 6 10 8 2 10 2 6 2 26 803

NOTE. Status as of March 2007 Courtesy of Professor D. Farge, EBMT, AD, WP.

6.7 years. Of the 178 evaluable transplanted patients, 163 received a peripheral blood stem cell transplant, mobilized with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) in 126 and followed by conditioning with the quadruple therapy BEAM (carmustine BCNU, etoposide, cytarabine Ara-C, and melphalan ) and ATG in 74 patients. BEAM alone was used in 30 patients. Purging, mostly by CD34 selection, was used in 97 patients and was not applied in 77 patients. The absolute TRM was 5.3%, with no deaths occurring in the last 3 years, probably due to more careful patient selection such as excluding patients with a very high Extended Disability Scoring System (EDSS) score or severe comorbidity such as active infection.

Systemic Sclerosis (scleroderma) Among the first 65 transplanted patients, 70% had an improvement of 25% or more in the skin score (measured by the modified Rodnan method), with a TRM of 12.5%.7 Several protocols were used, mostly either cyclophosphamidebased (4 g/m2 cyclophosphamide), mobilization and cyclophosphamide 200 mg/kg body weight conditioning, or radiation (8 Gy plus cyclophosphamide 120 mg/kg body weight). With further patient recruitment and longer term follow-up, the TRM of the EBMT-registered patients fell, considered to be related to more careful patient selection. Lung function tended to stabilize and some factors were identified as potentially hazardous for HSCT, for example, pulmonary hypertension greater than 50 mm Hg mean pulmonary arterial pressure, severe cardiac involvement, severe pulmonary fibrosis, and uncontrolled systemic hypertension. Long-term follow-up of this cohort showed an overall TRM of 8.5%, no further transplant-related deaths, and a trend toward durable remissions (Fig 1).8 For this subset of SSc, there is so far no proven disease-modifying therapy capable of controlling the disease. A multicenter US study of 19 SSc patients that administered a regimen of cyclophosphamide 120 mg/kg, total-body irradiation (TBI) 8 Gy, and equine ATG 90 mg/kg body weight, plus a CD34 selected graft product showed a sustained benefit in 12 patients at median follow-up of 14.7

Phase I/II Studies Multiple Sclerosis The first 85 patients registered in the EBMT database showed a 3-year progression-free survival of 78% in the patients with secondary progressive disease, and 66% in those with primary progressive disease. There were five deaths from treatment and two from progressive disease.5 Gadolinium-enhancing lesions, considered to be a surrogate marker of activity, were mostly eliminated, but functional deterioration may continue in advanced disease due to inflammation triggered progressive apoptosis and brain atrophy. A more recent analysis was performed on 183 registered cases, of which 99 were the secondary progressive form, 19 primary progressive, and 41 relapsing forms of the disease.6 Median age was 34 years and median disease duration was

Figure 1 The selected outcomes of sustained remission, remission then relapse, and treatment-related mortality are compared and contrasted among four different AD undergoing HSCT: SLE (n ⫽ 53), SSc (n ⫽ 57), MS (n ⫽ 85), and RA (n ⫽ 73).

J. Passweg and A. Tyndall

280 months.9,10 In some cases, remodeling and regression of collagen deposition have been described post-transplant.10

Rheumatoid Arthritis A retrospective analysis of the first 78 registered patients showed significant improvement, with 67% achieving an American College of Rheumatology 50% response (ACR-50) at some time post-transplant.11 Most of the patients had failed a median of five (range, two to nine) conventional diseasemodifying antirheumatic drugs (DMARDs ) before the transplant. Some degree of relapse was seen in 73% of patients post-transplant (Fig 1), but in most cases it was relatively easy to control with drugs that had proven ineffective pretransplant.5 A multicenter trial in Australia failed to show any advantage of CD34 selection of the graft after non myeloablative conditioning with cyclophosphamide.12 Further HSCT studies in rheumatoid arthritis (RA) are not currently being planned, probably reflecting the plethora of less toxic alternative experimental options such as tumor necrosis factor-alpha (TNF-␣) blockade, anti-CD20 monoclonal antibody, and co-stimulation blockade. However, so far no such therapy has demonstrated long-term drug-free remission, as was seen in approximately 10% of RA patients receiving HSCT.

Systemic Lupus Erythematosus Of the 55 registrations in the EBMT/EULAR database, most had either renal and/or central nervous system (CNS) involvement, and 21 had failed conventional cyclophosphamide treatment. A peripheral stem cell source after mobilization with cyclophosphamide and G-CSF was used in the majority. Twenty-three patients received conditioning with cyclophosphamide and ATG, 11 received cyclophosphamide plus TBI, and in four patients, other regimens were employed. An unselected graft was used in 29, with CD34 selection in 19. In those 53 patients with sufficient data for analysis, 66% achieved an initial remission, defined as a Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) of ⱕ3 and steroid reduction to less than 10 mg/d (Fig 1).13,14 Of these, approximately one third experienced some degree of return of disease activity. Ongoing maintenance therapy may reduce this relapse and will be addressed in prospective future trials. Burt et al reported on 50 patients with severe systemic lupus erythematosus (SLE) who were mobilized in a transplant protocol.15,16 One died as a result of infection following mobilization and another died 3 months later of active CNS lupus, having not proceeded to transplant. Among the 48 transplanted patients, 50% achieved remission, with autoantibodies becoming undetectable in some cases. The highdose chemotherapy consisted of cyclophosphamide 200 mg/ kg, methylprednisolone 1 g, and equine ATG 90 mg/kg.

no early deaths and a response rate of 57%.17 The registry of the European Group for Blood and Marrow Transplantation (EBMT) holds data on 38 transplants, autologous for 27 and allogeneic for nine patients.18 The disease entities and regimens used were more heterogeneous. The conditions encountered were autoimmune hemolytic anemia, Evans’ syndrome, immune thrombocytopenia, pure red cell aplasia, pure white cell aplasia, and thrombotic thrombocytopenic purpura. Patients had long-standing disease, having failed multiple prior treatments. Among 26 evaluable patients mobilized for autologous HSCT, three died of treatment-related causes, one died of disease progression, seven were nonresponders, six had transient responses, and nine had sustained partial or complete remissions. Of the seven evaluable patients receiving allogeneic HSCT, one died of treatmentrelated complications, one with a transient response died of progressive disease, and five showed a sustained response. Autologous and allogeneic HSCT may result in a positive response in a significant proportion of patients with autoimmune cytopenia of long duration.

Other Autoimmune Diseases The numbers of cases with vasculitis, Behçets disease, relapsing polychondritis, dermatomyositis, and other ADs are too small to draw meaningful conclusions, with further phase I and II standardized protocol pilot studies proceeding. However, some satisfying sustained responses to often fatal diseases such as Behçet lung disease19 and necrotizing vasculitis20 have been reported. Phase II/III protocols are being developed for Crohn’s disease (Autologous Stem Cell Transplantation International Crohn’s [ASTIC] study)21 and chronic inflammatory demyelinating polyneuropathy (CIDP). In both diseases, some patients fail to respond to conventional anti–TNF-␣ blockade or intravenous pooled immunoglobulin treatment, respectively. A positive outcome following autologous HSCT has been reported in a series of 12 patients with Crohn’s disease (conditioning with cyclophosphamide and ATG)22 and in seven patients with celiac disease with aberrant T cells (conditioning with fludarabine and melphalan).23

Prospective Phase III Studies Multiple Sclerosis The phase I/II data on multiple sclerosis (MS) were exploited in developing the Autologous Stem Cell Transplantation International Multiple Sclerosis (ASTIMS) trial, with patients being randomized to either transplant with BEAM, ATG, and no purging or mitoxantrone. In the retrospective analysis, no patient receiving this regimen died of treatment-related causes, and using the ASTIMS selection criteria, TRM was only 1.8%. Further information is available on the website www.astims.org. This study is still recruiting and no interim analysis has yet been performed.

Severe Refractory Immune Cytopenias A phase II study by the US National Institutes of Health (NIH) has presented data on autologous HSCT in 14 patients with immune thrombocytopenia or Evans’ syndrome, with

Systemic Sclerosis The prospective Autologous Stem Cell Transplantation International Scleroderma (ASTIS) trial selects patients who have

ASCT in autoimmune disease less than 4 years of diffuse skin involvement and evidence of progressive and organ- or life-threatening disease, or a skin score of greater than 20 after 2 years of disease and elevated acute-phase reactants. The primary end point on which the trial is powered is event-free survival at 2 years, events being arbitrarily but precisely defined to capture irreversible and severe end-organ failure or death. Exclusion criteria are based on the phase I and II data to avoid an unacceptably high TRM risk, together with a minimal chance of clinically significant improvement. The treatment arm is mobilization with cyclophosphamide 4 g/m2 and G-CSF, followed by cyclophosphamide 200 mg/kg body weight conditioning plus ATG and a CD34-selected graft. The control arm is monthly intravenous pulse cyclophosphamide 750 mg/m2 for 12 months. The ASTIS trial is ongoing, and further details are available on the website: www.astistrial.com. So far, 90 patients have been randomized and there has been one “probable” transplant-related death. A similar study, the Scleroderma Cyclophosphamide or Transplant (SCOT) trial is running in the United States under the auspices of the National Institute of Allergy and Infectious Diseases (NIAID)/NIH using similar entry criteria end points and a similar control arm but with a treatment arm that includes radiation.

Mechanistic Aspects The profound degree of immunosuppression attained with HSCT has provided unique insights in the dynamics of the reconstituting immune system in relationship with the disease course. Although interpretation is difficult in autologous settings because the sources of mature lymphocytes cannot be discerned (for example, reinfused v residual stem cells, or expanded lymphocytes), some patterns have emerged. Specific autoantibodies did not disappear after HSCT in most cases despite long-term remissions. This has been consistently observed for Scl-70 autoantibodies in scleroderma patients, indicating that these autoantibodies were produced by nondividing long-lived plasma cells. Titers of rheumatoid factors dropped in RA patients after HSCT but failed to normalize and returned to pretreatment levels before relapses, in keeping with data from RA patients treated with rituximab.24 In SLE patients, anti-nuclear antibody and anti-ds DNA antibodies disappeared in many patients after HSCT, and returned to detectable levels during relapse.25 HSCT has been shown not only to affect B-cell populations but also to profoundly perturb the T-cell compartment, as illustrated by the normalization of the dysregulated T-cell receptor (TCR) repertoires in MS26 and SLE patients. In RA patients, analyses of synovial tissue-infiltrating lymphocytes suggested that relapses originate from lesional T cells that were not eliminated by immunoablation.27 In juvenile idiopathic arthritis (JIA) the numbers of functionally active CD4⫹ CD25⫹ regulatory T cells increased after HSCT, proving that HSCT restores immunoregulatory mechanisms.28Clearly, future clinical studies need to be supported by experimental protocols that evaluate the mechanistic aspects of HSCT.

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Mesenchymal Stem Cell Immunomodulation of Autoimmune Disease Mesenchymal stem cells (MSC) have attracted attention in the past years in the areas of tissue engineering, as vehicles for gene therapy, as support cells for hematopoietic stem cell engraftment, and as antiproliferative and immunomodulating cells. The latter properties are currently being investigated as potential treatment of AD,29 analogous to their use in acute GvHD.30 MSCs are multipotent cells capable of differentiating in vitro and in vivo to different MSC lineages, including adipose, bone, cartilage, and myelosupportive stroma.31-34 Although initially identified in bone marrow, MSCs were found later in muscle, adipose tissue, synovial membranes, and other connective tissues of human adults.35-38 MSCs are isolated from other bone marrow– derived cells by adherence to plastic and consecutive passaging after which they proliferate to spindle-shaped cells in confluent cultures. As MSCs are rare, cannot be mobilized in vivo, and lack unique cell surface markers, current data are based on studies performed on cells expanded in vitro. MSCs have been defined by using a combination of phenotypic markers and functional properties. Controversy still exists over the in vivo phenotype of MSCs: however, ex vivo– expanded MSCs do not express the hematopoietic markers CD14, CD34, CD45, and major histocompatibility complex (MHC) class II.34,39 In addition to their multipotentiality, they can be identified as cells that stain positive for CD73, CD90, and CD105, and by flow cytometry.33,34,39-41 In vitro, MSCs have vast proliferative potential, can clonally regenerate, and can give rise to differentiated progeny. Regardless of whether or not MSCs are true stem cells, clinical benefit from MSC may not require sustained engraftment of large numbers of cells. It is possible that a therapeutic benefit can be obtained by local production of growth factors and a provision of temporary antiproliferative and immunomodulatory properties. MSCs rapidly expand more than one billion-fold when cultured in vitro. They secrete cytokines important for hematopoiesis and have the capacity to maintain and expand lineage-specific colony-forming units from CD34⫹ marrow cells in long-term bone marrow culture.42-44 MSCs are not immunostimulatory in vitro. MSC were originally thought to be immune-privileged in that they neither induce lymphocyte proliferation when co-cultured with allogeneic lymphocytes nor are they targets for CD8⫹ cytotoxic lymphocytes or KIRligand mismatched natural killer cells.45-48 However, recent data suggest that in a non-immunosuppressed murine host, allogeneic MSCs will be eliminated49 and that allogeneic MSC may be eliminated by natural killer cells.50 In vitro results indicate that MSC possess immunosuppressive properties. Rodent, baboon, and human MSC suppress T- and B-cell lymphocyte proliferation in mixed lymphocyte cultures (MLC) or when induced by mitogens and antibodies in a dose-dependent fashion.45-48,51-55 The suppression is MHC-

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282 independent and, in human cell cultures, the magnitude of suppression is not reduced when the MSC are separated from the lymphocytes in transwells, indicating that cell– cell contact is not required.45,48,56 The mechanisms underlying the immunosuppressive effect remain to be clarified. Di Nicola et al proposed that two soluble factors, hepatocyte growth factor (HGF) and transforming growth factor-␤1 (TGF-␤1) were involved. The addition of anti-HGF and anti–TGF-␤1 partially restored the proliferation of CD2⫹ cells in the presence of MHC.48 These results could not be reproduced using unfractionated peripheral blood lymphocytes.57 Aggarwal and Pittenger suggested that MSC-produced prostaglandin E2 accounted for reduced lymphocyte proliferation.58 Another study suggests that indoleamine 2,3-deoxygenase-mediated tryptophane depletion by MSC can act as a T-cell inhibitory effector mechanism,59 as has been shown for dendritic cells.60 Indoleamine 2,3-deoxygenase, which is induced by gamma interferon, catalyzes the conversion from tryptophan to kynurenine and inhibits T-cell responses. However, in the hands of Tse et al, neither MSC production of interleukin-10, TGF-␤1, and prostaglandin E2 nor tryptophane depletion in the culture medium was responsible for the immunosuppressive effect.45 In addition, MSC produce bone morphometric protein 2, which may mediate immunosuppression via the generation of CD8⫹ regulatory T cells.61 These controversial data may be due to the use of MSC generated by different techniques, the use of different stimuli, culture conditions, doses, and kinetics, and by the different lymphocyte populations tested. Such differences may in turn affect cytokine and chemokine secretion, with seemingly contradictory results. In addition, species-specific differences, particularly between murine and human MSC, add to the confusion.52 An immunosuppressive effect of MSC in vivo was first suggested in a baboon model, where infusion of ex vivo– expanded donor or third-party MSC delayed the time to rejection of histoincompatible skin grafts.53 MSC also downregulate bleomycin-induced lung inflammation and fibrosis in murine models, if given early (but not late) after the induction.62 MSC adopt an epithelial-like morphology. Of note is the fact that the epithelial crosstalk with endothelium via integrin ␣v␤6 controls alveolar flooding.63 A similar effect was seen in a murine hepatic fibrosis model (carbon tetrachloride– induced) using a MSC line bearing the fetal liver kinase-1 (FLK1) marker.64 Tissue protective effects also were seen in a rat kidney model of ischemia/reperfusion injury in which syngeneic MSC but not fibroblasts were used.65 Autologous bone marrow– derived MSC have been shown to be potently antiproliferative to stimulated T cells from normal subjects and autoimmune (RA, SSc, Sjõgrens syndrome, SLE) patients,66 and in SSc patients these MSC were normal in respect to proliferation, clonogenicity, and differentiation (Larghero et al, personal communication, May 2007).

Animal Models of Autoimmunity Three reports of autoimmune animal model responses have been published recently. In the two murine models of exper-

imental allergic encephalomyelolithis (EAE), both clinical and histological improvement occurred. The responses were dependent on time of MSC treatment, the earlier the better, and were reversed with interleukin-2 treatment, indicating that anergy rather than apoptosis had occurred.67,68 However, in a murine model of arthritis, collagen-induced arthritis (CIA) was not improved by the addition of MSC and the in vitro immunosuppressive effects were reversed by the addition of TNF-␣. MSC were not found in the joints.69 However, a second murine arthritis model showed a positive outcome.70

MSC and Human Experience Ex vivo– expanded allogeneic MSC have been infused in several phase I studies.71-75 No adverse events during or after MSC infusion have been observed and no ectopic tissue formation has been noted. After infusion, MSC remain in the circulation for no more than an hour.74Although durable stromal cell chimerism has been difficult to establish, low levels of engrafted MSC have been detected in several tissues.75,72,76 It is possible that sufficient therapeutic benefit is obtained by local paracrine production of growth factors and the provision of temporary immunosuppression by MSC infusion. Infusion of haploidentical MSC to a patient with steroid resistant severe acute GvHD of the gut and liver promptly improved liver values and intestinal function.30 Upon discontinuation of cyclosporine, the patient’s acute GvHD recurred but was still responsive to a second MSC infusion. Lymphocytes from the patient, when investigated on multiple occasions after MSC infusion, continued to proliferate against lymphocytes derived from the haploidentical MSC donor in co-culture experiments. This suggests an immunosuppressive effect of MSC in vivo, rather than a development of tolerance. The EBMT is currently planning protocols for prevention and treatment of acute GvHD through the Stem Cell Subcommittee (W. Fibbe, K. Le Blanc, F. Frassoni, personal communication, July 2007). In conclusion, MSC appear capable of inducing antiproliferative and immunomodulatory effects in activated target cells and animal models of AD, while failing to either incite or be subject to immunological reactions across allogeneic barriers. The early results in human acute GvHD and apparently low toxicity justify further studies in AD.

Conclusion The past decade has seen the introduction of many agents, especially biologics, which have allowed a more successful control of AD manifestations. However the “Holy Grail” of tolerance induction has not yet been achieved. It could be that through harnessing the complex and multifaceted potential of cellular based therapies, especially HSCT, a “resetting” of autoaggressive immune reactions while maintaining protective immunity will be possible. In addition, the antiproliferative and immunomodulatory effect of MSC combined with their immunological privilege and seemingly low

ASCT in autoimmune disease toxicity may offer a new strategy for controlling and protecting vital organs from inflammatory, destructive autoimmune reactions.

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