experimental basis of hematopoietic stem cell transplantation for treatment of autoimmune diseases...

8/7/2019 Experimental basis of hematopoietic stem cell transplantation for treatment of autoimmune diseases rev!

1/12

Experimental basis of hematopoietic stem cell transplantationfor treatment of autoimmune diseases

D. W. van BekkumCrucell B.V., Leiden, The Netherlands

Abstract: Experiments with animal models of au-toimmune disease provided the rational and stim-ulus for the current, clinical studies of autologousstem cell transplantation for the treatment of avariety of severe, refractory, autoimmune dis-eases. The discoveries that led to the recognition of the key role of hematopoietic stem cells and thesuccessful treatment of autoimmune diseases withbone marrow transplants are reviewed. The rele-vance of spontaneous and induced autoimmunedisease models for the development of clinical

treatment regimens is discussed. Most of the inves-tigations with autologous stem cell transplantationhave been performed with induced autoimmunedisorders: in rats with adjuvant arthritis and in ratsor mice with experimental, allergic encephalomy-elitis, the current model for multiple sclerosis. Themain aspects of this translational research were theconditioning regimens and the degree of T celldepletion of the graft as determinants of remissioninduction and the incidence of relapses. Theemerging recommendations are compared with theoutcome so far of the clinical studies. J. Leukoc. Biol. 72: 609620; 2002.

Key Words: transplants bone marrow arthritis encephalomy-elitis

INTRODUCTION

The pivotal role of the hematopoietic system in the develop-ment of autoimmune diseases (AID) was rst suggested morethan 30 years ago by Morton and Siegel [1] who described thedevelopment of antinuclear antibodies in normal mice after thetransplantation of bone marrow (BM) from NZB mice. NZB isan inbred strain of mice that spontaneously develop a syn-drome resembling systemic lupus erythematosus (SLE). Adop-tive transfer of the potential to develop AID as well as itsprevention with BM cells from resistant donors were subse-quently demonstrated to hold for several other AID in experi-mental animals.

The clinical literature contains sporadic case reports of thetransfer of AID and allergic disorders with donor BM to pa-tients treated for leukemia or aplastic anemia. These includethrombocytopenic purpura, thyroiditis, diabetes type I, celiacdisease, and myasthenia gravis [2]. After the discovery thatfull-blown AID of experimental animals can be cured by allo-

geneic BM transplantation (BMT), the records of patients whowere long-term survivors of treatment with allogeneic BMT for leukemia or aplastic anemia were searched for those withcoexisting AID at the time of transplantation. The searchrevealed 21 patients suffering from rheumatoid arthritis (RA),psoriasis, Crohns disease, ulcerative colitis, SLE, or insulin-dependent diabetes type 1 (IDD1), all of whom experienced acomplete remission of their autoimmune disorder. The trans-plant-associated risks of allogeneic BMT, however, precludedits use in AID. It was only after our unexpected nding thatautologous BMT could also cure experimental AID that trans-

plantation of BM or peripheral blood stem cells (PBSC) wereintroduced into the clinic as a treatment option for refractoryAID. Thus far, the results of these clinical studies, whichcomprise several hundreds of patients, have established ahighly predictive value of the disease models that were used.

RELEVANCE OF ANIMAL MODELS FORHUMAN AID

For appraisal of the predictive value of experiments with AIDmodels, an analysis of the causes and nature of the humandiseases is needed. Many if not all of the human AID are Tcell-initiated or -mediated. In the majority, the presence of autoantibodies in the serum of the affected subjects is nowconsidered an associated or epiphenomenon. The activation of T lymphocytes against self-antigens may be induced by therelease of an excessive amount of tissue-specic antigens towhich the organism has not become tolerant during develop-ment, as in the case of sympathetic ophthalmia. Although thetarget antigens have been identied for many of the humanAID, the inducing agents are unknown for most, despite ex-tensive epidemiological research. This failure suggests thatexposure to such antigens is ubiquitous and that affected

individuals must have an unusually high responsivenessapredisposition that is genetically determined. However, al-though numerous reports describe the linkage of certain major histocompatibility complex genes of the human leukocyte an-tigen (HLA) and the D/DR regions with specic AID, thelinkages are far from absolute [3]. Also, concordance inmonozygotic twins is limited: in multiple sclerosis (MS), 20

Correspondence: D. W. van Bekkum, Crucell B.V., P.O. Box 2048 2301 CA,Leiden, The Netherlands

Received February 5, 2002; revised May 1, 2002; accepted June 4, 2002.

Journal of Leukocyte Biology Volume 72, October 2002 609


2/12

30%; in Crohn s disease, 44%; and in RA, 11% [4], indicatingthat genetic and environmental factors are involved.

The animal models of AID are of two distinct categories: thespontaneous (or hereditary) and the induced forms. In the rstcategory, the disease develops spontaneously in a large pro-portion or in all of the individuals of a so-called autoimmunestrain of mice or rats. Well-known examples are the lupus-likesyndromes in several inbred mouse strains and diabetes inNOD mice and BB rats.

The induced AID require immunization with certain anti-gens to develop and do so only in selected inbred strains thesusceptible ones or responders and not in others the resis-tant or nonresponder strains. The best described models areadjuvant arthritis (AA) in rats and experimental allergic en-cephalitis (EAE), which can be induced in many species. Thelatter is considered the most appropriate animal model for MS.Susceptibility and resistance to inducible AID have beenshown to be genetically determined by cross-breeding experi-ments.

A continuous subject of debate is the question of what typeof disease model is expected to best predict ef cacy of newtherapeutic modalities in human AID. The induced modelsappear to be the favored candidates, as they share a dualethiology with their human counterparts. However, some of thespontaneous AID of animals have also been found to bestrongly inuenced if not dependent on environmental factors,such as microbes, food, and hormones. For instance, HLA-B27transgenic rats do not develop the characteristic colitis andarthritis when reared germ free [5]. In germ-free NZB mice, theincidence and severity of renal lesions were much lower than inconventional controls [6], but in MRL-lrp mice similar exper-iments did not provide any evidence for a role of infectiousagents in the development of their lupus-like disease [7].Exposure to a common rat virus, the Kilman rat virus, initiatesautoimmune IDD1 in some but not all rat strains that arenormally not susceptible to spontaneous diabetes [8, 9]. On theother hand, elimination of environmental viruses by Caesarianderivation of diabetes-prone BB rats increased the incidenceand rate of development of diabetes [10]. Other environmentalfactors that have been identi ed in diabetes-prone rodents arethe diet and hormones. A semi-puri ed diet prevents diabetesin BB rats [11], and a raised environmental temperature, whichdecreases food intake, reduced the incidence of diabetes inNOD mice [12]. Exposure to environmental stressors enhancedthe onset and increased the incidence of diabetes in BB rats[13], and sex hormones in uence the occurrence of insulinitisand diabetes, sialitis, and dacryoadenitis in NOD mice [14].

Interestingly, in several instances, the micro ora or hor-mones was also found to modify the susceptibility to inductionof AID. F344 rats are resistant to induction of adjuvant arthritisunder conventional conditions but are highly responsive in thegerm-free state [15]. This was not the case for collagen-inducedarthritis (CIA), to which F344 rats are equally resistant under both conditions. However, CIA was markedly enhanced ingerm-free (susceptible) DA rats as compared with conventionalones [16]. In this context, it should be reminded that graftversus host disease (GvHD), which has many pathologicalfeatures in common with SLE and autoimmune colitis, is

strongly inuenced by the composition of the micro ora inexperimental animals [17] and human patients [18].

The disease pattern of EAE is profoundly in uenced by theplasma level of corticosteroids. Nonresponsive PVG rats de-velop severe and fatal EAE when they are adrenalectomizedprior to induction. Adrenalectomy of the responder Lewis ratalters its usual pattern of acute remitting, nonrelapsing EAEinto nonremitting fatal disease [19]. The original reaction pat-tern is restored by replacement therapy with corticosteroids.Rats of the low-responder strain WAG have an incidence of 10% following immunization, which increases to 50% in adre-nalectomized animals [20].

Another factor identi ed as inuencing EAE development isthe presence of cyclophosphamide (CY)-sensitive suppressor cells. Abrogation of the resistance of acridine orange rats [21]and of BALB/c mice [22] to encephalitogenic immunizationswas obtained by pretreatment with low dose (20 mg/kg) CY.The incidence of subclinical EAE as de ned by histologicallesions in the central nervous system (CNS) of WAG ratsincreased from 37% to 86% by pretreatment with a similarlylow dose of CY, but the incidence of clinical EAE remainedunaltered at 10% [21].

In view of these data, the differences between the sponta-neous and the inducible animal models may well be gradualrather than fundamental. In both cases, there is a geneticdisposition that allows activation of antiself immunity; in thecase of the spontaneous diseases, activation is by relativelyweak, ubiquitous antigens; in the case of the inducible AID, bystrong, speci c antigens. Both mechanisms accommodate arole for infectious microorganisms acting as the initiating stim-ulus by providing antigens that resemble tissue targets (so-called mimicry) or as regulators of the immune reactivity,similar to adjuvants, hormones, and dietary factors. All thesedeterminants, genetic as well as environmental, have also been

implicated in the pathogenesis of human AID. The etiology of human AID is undisputedly multifactorial. In almost all auto-immune conditions, there is a familial tendency. Many AID areinduced by drugs: more than 70 different drugs have beenreported to induce SLE. Furthermore, many xenobiotics, e.g.,food supplements, heavy metals, and environmental toxins,have been linked to the development of SLE-like illnesses.Although in many patients with AID, the causative agentremains unknown, the low concordance in identical twinsseems to argue in favor of the induced disease models as beingmore realistic tools for translational research.

ADJUVANT ARTHRITIS IN BUFFALO (BUF)RATS

A large variety of antigens and immunization schedules wereknown to induce various forms of inammatory arthritis insusceptible, experimental animals. Among the antigens arecomplete Freund s adjuvant (CFA), collagens, and streptococ-cal cell-wall preparations. The most widely studied are theLewis rat and CFA, but in our hands, as in most other labo-ratories, immunization of Lewis rats with adjuvant induced atypically acute form of the disease of short duration, resultingin resistance to reinduction after remission. We then studied a

610 Journal of Leukocyte Biology Volume 72, October 2002 http://www.jleukbio.org


3/12

variety of mouse, rat, and guinea pig strains using adjuvant andcollagen as inducing agents and compared the clinical pictureas well as the histopathology of the lesions. Our conclusionfrom this extensive exploration was that AA in the BUF ratstrain provides the most resemblance to RA in humans. In theBUF rat, a single, intracutaneous inoculation of Mycobacteriumtuberculosis in CFA causes a chronic, progressive type of polyarthritis within 3 4 weeks in over 80% of the animals. Theinammation involves chie y the distal extremities, as achronic, proliferative synovitis with pannus formation, destruc-tion of cartilage and subchondral bone, vasculitis, pericapsular brosis, and extensive, reactive bone formation. Clincally,there is swelling and redness of the affected joints. During theacute and the progressive, chronic stage, the in amed jointsare reddish and painful. In some animals, the in ammationrecedes after 10 weeks at the earliest; in others, in ammationcontinues for as long as 30 weeks, by which time the experi-ments were usually terminated. Following extinction of theinammation, spontaneously or as a result of treatment, theosseous deformities persist. Spontaneous remissions occur inonly 12% of the animals. Reimmunization at 15 weeks after induction did not alter the clinical condition. Treatment withcyclosporin A of rats during the acute phase causes partialregression but the arthritis exacerbates and progresses againafter discontinuation of the drug.

EXPERIMENTAL AUTOIMMUNEENCEPHALOMYELITIS IN RODENTS

EAE is an inammatory demyelating disease of the CNS thatcan be induced in certain strains of rodents and in monkeys byimmunization with whole spinal cord or puri ed myelin pro-teins and CFA. This review is limited to EAE in rodents. In the

Lewis rat, symptoms last for 57 days, followed by completerecovery. Thereafter, the animals are resistant to reinduction.The neurological symptoms of acute EAE are probably due toa combination of reversible demyelination and edema causedby the inammation.

Relapsing EAE develops after a similar immunization inSJL/J mice, in Biozzi mice, in BUF rats, and in Lewis rats, butin the latter only if pretreated with low-dose cyclosporin A.Relapses are characterized by one or more new episodes of paralysis and paresis after the animals have recovered from therst attack. Most of the animals recover after each subsequentattack; some 10% of the BUF rats succumb during that period.The spontaneous relapses are accompanied by more wide-spread in ammation in the CNS and more pronounced demy-elination. After 40 50 days, relapses no longer develop spon-taneously, but can be reinduced by another immunization.Such induced relapses are likely due to activation of memory Tcells, as the latent period is 2 6 days less than in the case of primary immunization. It remains a matter of speculation howto interpret spontaneous and induced relapses as models for relapses that occur in MS patients. In all cases, the relapses areassociated with are-up of the in ammatory processes in theCNS at old sites or at new locations. As it is unknown whatcauses relapse in MS, notably if it represents a nonspeci cactivation or a re-exposure to the sensitizing antigen, it cannot

be decided whether induced relapses in the animals have anycounterpart in the clinic. Nevertheless, I and others have recordedthe incidence of induced relapses after BMT, reasoning that it willprovide information on the presence of residual memory cells.

In the Biozzi mouse, immunization with spinal cord tissueinduces a chronic form of EAE, which eventually leads to moreextensive demyelination than is observed in the remittingrelapsing EAE of rats. EAE in Biozzi mice is therefore a better model for studying the effects of therapeutic interventions onchronic, persistent demyelating disease, which is mainly due tothe scars from previous in ammations.

AID AS DISEASES OF THE HEMATOPOIETICSTEM CELLS

Having discussed the causes of AID, I will focus on theimmune system itself, which is instrumental in initiating andmaintaining the lesions of AID. Failure to abstain from or tolimit self-destruction is regarded as a defect of regulatoryimmune mechanisms. The question is whether this occurs atthe level of the lymphoid cell population; for example, adysregulation resulting from an abnormal generation toomuch or not enough of certain lymphocyte subpopulations(effector or suppressor cells). Examples are the multiform,autoimmune syndrome that develops in mice and Syrian ham-sters following neonatal thymectomy [23] and the AID that areinduced by treatment with cyclosporin A of lethally irradiatedrats rescued with syngeneic BM grafts [24].

An alternative hypothesis is based on the nding that thepotential for developing spontaneous AID can be transferred byBMT to lethally irradiated animals from a nonautoimmunestrain, and reversely, autoimmune-prone animals do not de-

velop the disease when grafted at an early age, before thedisease is manifest, with BM from a normal strain. This prop-erty was discovered in NZB mice, which spontaneously de-velop a lupus-like syndrome, by Morton and Siegel [1] whopostulated that some defect. . .may exist in the NZB mouse atthe level of the hemopoietic stem cell. Similar transfers weredemonstrated in several different spontaneous AID strains aslisted in Table 1 . It also led to a re-emphasis, most strongly byIkehara et al. [30], of AID being stem cell diseases. However,this conclusion is not fully justi ed as long as the transfershave not been accomplished with highly puri ed stem cells.For the time being, it seems appropriate to use the term stemcell-associated diseases.

Regarding the inducible disease models, studies have beenperformed with hematopoietic chimeras of resistant recipientsand susceptible donors and vice versa ( Table 2 ). After chi-merism had been established, these animals were immunizedwith the appropriate antigen. Similar to the ndings in thespontaneous models, responsiveness is generally dictated bythe BM donor genotype.

Among the notable exceptions is CIA in DBA/1 mice. Ar-thritis could not be induced in DBA/1 3 SWR chimeras,which might be a result of the C5 de ciency of the recipients.Surprisingly, the SWR 3 DBA/1 chimeras were susceptible.In this particular case, the inherent potential to react against

van Bekkum Stem cell transplants for autoimmune disease 611


4/12

collagen may be present in the so-called resistant donor strainbut masked by its C5 de ciency.

In the EAE group of experiments, those reported by Korn-

gold et al. [47] are contrasting with the other data. In their paper, they demonstrate that the responses of the chimeras aredictated by the recipient strain, similarly when the CNS anti-gen used for induction was of BALB/c or B10.S origin. Inanother publication of the same year, Lublin et al. [48] con-rmed these results with the SJL-derived antigen, but immu-nization with the B10.S antigen induced a high incidence of clinical EAE in B10.S 3 SJL and SJL 3 B10.S chimeras. Asatisfactory explanation has not emerged so far.

TREATMENT OF FULLY DEVELOPEDEXPERIMENTAL AID WITH ALLOGENEIC BMT

In view of the transfer data described above, it was logical toinvestigate if full-blown AID might be cured by BMT from

resistant animals. As this involves allogeneic donors, engraft-ment requires ablation of the lymphohemopoietic system of therecipient (to be designated as conditioning in analogy with the

treatment of leukemia). Successful treatment with allogeneicBMT was rst reported in 1985 by Ikehara et al. [53] inMLR/lpr and BXSB mice with overt lupus-like disease and inNOD mice with insulinitis [54]. Ikehara s group continued topublish similar therapeutic effects in other hereditary, autoim-mune conditions ( Table 3 ). Grafting of fetal thymus and fetalbone fragments in addition to T cell-depleted allogeneic BMimproved the results [57], which was attributed to facilitation of the engraftment of the BM stem cells. The problem withallogeneic BMT in treating SLE-type syndromes is that mea-sures have to be taken to prevent graft versus host reactions, asthese resemble some of the manifestations of SLE, notablydermatitis and the liver lesions.

Treatment of induced AID with allogeneic BM has beenequally successful as in the case of the spontaneous syndromes(Table 4 ). Complete lasting remissions and near absence of

TABLE 1. Transfer (T) and Prevention (P) of Spontaneous AID by Allogeneic BMT between Autoimmune and Normal Rodent Strains

Disease AI strain Non-AI strain T and/or P Ref.

SLE-like syndromes (mice) NZB, (NZB SJL/J)F1 (NZB C57BL)F1 BALB/c, SJL/J, C57BL T 1NZB BALB/c, B10.D2, C57BL, DBA/2 T/P a 2527MRL/lpr C3H nu/nu P 28MLR/lpr C57BL b T 29(NZW BXSB)F1 C3H, C57BL T 30BXSB CBA P 31

Polyarthritis (mice) NZB/KN C57BL P 32

Insulin-dependent diabetes mellitus NOD NON, (NOD NON)F1 T 33(mice) NOD (NOD B10)F1 T 34NOD C57BL/6B10.BR/cd T/P 35

(rats) BB BB diabetes-resistant subline P 36BB WF P 37

Motheaten syndrome (mice) me/me / T 38Skin brosis (mice) tsk / T 39Multisystem AID (rats) HLA-B27 transgenic Nontransgenic T/P 40

a Partly puri ed stem cells were transplanted by one group of investigators [25]. b T cell-depleted BM cells and bone as a source of stromal cells were grafted.

TABLE 2. Transfer of Susceptibility (S) or Resistance (R) to Induction of AID by Allogeneic BMT

AID AI strains Non-AI strains Transfer of S and/or R Ref.

AA(rats) Lewis F344 S/R 41(rats) BUF WAG S/P 42

Streptococcal cell-wall arthritis(rats) Lewis F344 S/R 41

Collagen-induced arthritis(rats) WAG BUF S 20(mice) DBA/1 SWR No transfer a 43

EAE(rats) (Lewis BN)F1 BN S/Rb 44(guinea pigs) strain 13, (2 13)F1 strain 2 S/R 45(rats) Lewis Le-R S/R 46(mice) SJL/J B10.S No transfer 47, 48(mice) SJL/J B10.S S/R 49(rats) BUF WAG S/R 50(rats) (WAG BUF)F1 WAG S/R 50(rats) BUF BN.1B S/R 51(mice) BUF, Lewis, SJL/J CB-17-scid/scid S 52

a The SWR mouse is C5-de cient, and C5 seems to be required for the development of CIA. b T cell-depleted bone marrow.



5/12

relapses were obtained in rats suffering from AA or EAE bytreatment in the acute phase of the disease. In AA, BMT is onlyeffective when there is active in ammation going on. Whentreatment is given in the chronic phase, disease progression ishalted, but osseous deformations of the joints are not repaired.BMT failed to induce remissions in mice with CIA; this mayalso have been a result of the presence of irreversible jointdamage.

EAE in BUF rats is a remitting, relapsing disease that doesnot develop into a chronic phase in contrast to EAE in Biozzimice. In the acute stage of EAE, i.e., at 20 days post-immu-nization, Biozzi mice responded well to allogeneic BMT. Allanimals went into complete remission, and only one mouse hada mild relapse with full recovery. Without treatment, 80%recover from the rst attack. Of those, 76% suffer one or morerelapses and eventually recover or enter the chronic phase.

The chronic disease is characterized by extensive demyeli-nation, axonal loss, and gliosis. The majority of the remittinganimals experience one or more relapses. Mice treated withallogeneic BMT when chronically ill at day 108 post-immuni-zation did not respond at all to allogeneic BMT. Apparently,

treatment with BMT effectively halts the in ammatory pro-cesses but does not lead to repair of scar tissue in the nervoustissue.

Overall, the results obtained with allogeneic BMT are im-pressive, considering that the animals were quite sick whentreated and had almost no relapses. These ndings stimulateda search of the clinical records of long-term survivors of allogeneic BMT for patients with coexisting AID at the time of grafting. Twenty-one patients with a follow-up of 7 21 yearswere discovered. All experienced complete remission of their various AID and only one patient relapsed. These cases havebeen reviewed by Marmont [61] and Nelson et al. [62].

Despite such strong evidence of its therapeutic potency,allogeneic BMT has not been intentionally applied to the

treatment of severe AID so far, except for refractory, idiopathic,aplastic anemia, where it is a long-established therapy, pri-marily intended to replace the lost BM. The highly successfultreatment of this life-threatening AID with allogeneic BMT isperhaps the most convincing argument for extending this mo-dality to other AID. However, the considerable risk of trans-plantation-associated mortality has precluded this.

The discovery that autologous BMT is equally effective asallogeneic BMT in inducing complete remissions in rats withAA an EAE cleared the way for clinical application, as autol-ogous BMT is less risky. Among more than 500 patientsregistered so far as having been treated with autologous stemcells for severe refractory AID, the overall transplant-associ-ated mortality was 9%, with signi cant variation between dis-eases [63].

AUTOLOGOUS BMT

The use of autologous BM was by no means a rational approachin view of the evidence that AID are hematopoietic stem

cell-associated diseases. Experiments using autologous graftswere initiated after the unexpected nding that arthritic ratsresponded just as well to treatment with syngeneic BM fromhealthy donors as the ones grafted with allogeneic marrow.Actually, the experiments with syngeneic BMT were includedbecause of the (mistaken) assumption that they would serve asnegative controls. Even more surprisingly, reimmunization of the cured rats, at 24 h or at 28 days following the engraftment,did not induce any relapses [58].

Curiosity made us investigate treatment with autologous BM.Once more, against all expectations, autologous BM was just asef cacious as syngeneic marrow from healthy animals and as

allogeneic marrow [64]. These results were con rmed withgrafts of real autologous BM, which was harvested from the

TABLE 3. Treatment of Fully Developed, Spontaneous AID with Allogeneic BMT

AI strain a Normal donor strain Effect Ref.

NOD BALB/c nu/nu Resolution of insulinitis 54B/W, BXSB BALB/c nu/nu Regression of glomerular damage 53, 55

Reduction in circulating immuneComplexes or complete cures

MLR/lpr BALB/c nu/nu Complete cures 53MRL/lpr C57BL Complete resolution of glomerulonephritis, arthritis, and correction of immunological

abnormalities; stroma cell transplants required56

old MRL/ C57BLb

Cure of pancreatitis and sialoadenitis, normalization of T and B cell functions 57a Conditioning with lethal dose of TBI. b T cell-depleted BM and fetal bone fragments and fetal thymus transplanted.

TABLE 4. Treatment of Fully Developed, Induced AID with Allogeneic BMT

Disease Responsive strain Resistant strain Effect Ref.

AAa (rats) BUF WAG Complete remission 58CIAa (mice) DBA/1 BALB/c No remission; complete prevention of progression 59EAE b (rats) BUF WAG and BN.1B Complete remission; few relapses 50, 51EAE a (mice) Biozzi CBA Treated in acute phase: Complete remission; few relapses 60

Treated in chronic phase: No effecta Conditioning with lethal total body irradiation. b Conditioning with lethal total body irradiation or CY and busulfan.



6/12

femur of arthritic recipients by a surgical procedure, followedby total body irradiation (TBI) and intravenous return of their own BM cells [64].

Subsequent studies with autologous stem cells were alwaysperformed with BM harvested from animals with exactly thesame stage and severity of the disease as the recipients. Themarrow obtained in this way was termed pseudoautologous atthat time. By this procedure, unnecessary suffering of the verysick animals from the surgical intervention needed for BMcollection is avoided. For each experiment, about 100 rats wereimmunized, and when the disease was fully developed, eachanimal was scored using a grading scale for the clinical symp-toms. The animals were distributed over the various experi-mental groups and the donor group of rats, assuring that theaverage score of all groups was similar. Animals without symp-toms (10 20%) were always excluded. As the composition andproperties of pseudoautologous and autologous BM are identi-cal, the term autologous is used for both throughout this review.

In contrast to the preclinical experiments with autologousBMT, those with syngeneic transplants have little practicalsignicance, as identical twin donors are rarely available.However, any difference in the results of treatment betweenautologous and syngeneic cells is of great interest, as it mayshed light on the signi cance of activated T cells and memorycells in the graft. As can be seen from Table 5 , few publisheddata are available on syngeneic and autologous BMT in thetreatment of spontaneous AID. An early publication of Mortonand Siegel [27] contains an experiment with syngeneic BMT inlethally irradiated 6-month-old (NZB DBA/2) F1 mice. Atthat time, 45% of the recipients were positive for antinuclear antibodies. This proportion did not decline after the treatment,in contrast to the near complete disappearance after allogeneicBMT. The study did not contain a description of the clinicalcondition of the mice. The second publication [40] concerns

the failure to cure HLA-B27 transgenic rats with syngeneicBMT, as contrasted with the successes with allogeneic BM inthe same model. Good and Ikehara [70] may have investigatedsyngeneic and autologous BMT in the course of their extensivestudies on the treatment of spontaneous, immune disorders, asthey stated, Our preclinical studies do not support autologous

or syngeneic BMT for treatment of mice, which may alreadyhave developed systemic autoimmune disease. However, de-tails of such experiments have not been published.

It is the opinion of this reviewer that there is room for a moreexhaustive con rmation of these negative results, as there areindications that some of the SLE-like syndromes in mice mayrespond favorably to high dose immunomyeloablation and au-tologous BMT.

Karussis et al. [71] treated MLR/lpr mice, aged 9 10 weeks,with high-dose CY or 9 gray (Gy) TBI followed by syngeneicBMT. These mice remained disease-free for at least 36 weeks,and untreated controls began to die at week 16. Unfortunately,it is not stated whether the recipients were already sick at thetime of transplantation nor were the age and disease status of the BM donors reported.

Loor et al. [72] reviewed several reports of complete andlasting remissions in mice with spontaneous lupus-like dis-eases after treatment with sublethal TBI. Furthermore, long-term treatment with relatively high dose CY (100 mg/kg weeklyfor 16 weeks) prolonged survival of sick MRL/lpr mice, causedreversal of adenopathy, and prevented the development of arthritis and glomerulonephritis [73]. In the latter study, thefollow-up was only 6 weeks after the last administration of thedrug, which is not long enough. Nevertheless, sublethal TBIand high dose CY cause massive destruction of the lymphaticand hematopoietic cells followed by endogenous repopulationfrom primitive precursors. That process closely resembles therepopulation following lethal TBI and autologous BMT.

Much more exhaustive investigations were performed withautologous and syngeneic BMT in rodents suffering from in-duced AID. Excellent therapeutic effects were seen in allstudies except for the one with CIA mice. Failure of these miceto enter complete remission was also observed after allogeneicBMT as discussed before.

The studies performed by Karussis et al. [69, 74] on EAEinduced in SJL mice are of particular interest, as they alsotreated the mice before the appearance of clinical disease withhigh dose CY (300 mg/kg) and syngeneic BM. The inductionschedule consisted of two immunizations, 1 week apart. Thelatent interval between the rst immunization and appearance

TABLE 5. Treatment of Fully Developed, Spontaneous, and Induced AID with Syngeneic or Autologous BMT

AID (strain) BM origin (conditioning) Effect Ref.

Spontaneous AID

(NZB DBA/2)F1 (mice) Syngeneic (TBI) None 27HLA-B27 (rats) Syngeneic (TBI) None 40

Induced AIDEAMG (Lewis) Autologous Reduction of a-AChR titer 65(rats) (CY TBI) Elimination of memory responseAA (BUF) Syngeneic (TBI) Complete remission 58(rats) Autologous (TBI) Complete remission 64, 66

CIA (DBA/1) Syngeneic (TBI) No remission(mice) Prevention of progression 59

EAE (BUF) Syngeneic (TBI) Complete remission 67(rats) Autologous (TBI) Complete remission 68

EAE (SJL/J) (mice) Syngeneic (CY) Complete remission 69EAE (Biozzi) Syngeneic Complete remission 60

(mice) Acute phase



7/12

of symptoms was between 14 and 18 days. When treated at day6 after the rst immunization, the development of the diseasewas completely prevented, and the mice became resistant torechallenge with the encephalitogenic agent. However, treat-ment at day 9 after the rst immunization was not preventive;it only delayed the onset of paralysis by about 1 week. Theauthors interpretation is that shortly after the second inocula-tion, the population of autoreactive and memory lymphocytes isat a maximum and cannot be suf ciently reduced by theconditioning with CY. Yet this explanation is dif cult to rec-oncile with the nding that the same regimen was highlyeffective when applied 3 days after the appearance of clinicaldisease.

In addition to the results with treatment of spontaneous andinduced AID, as collected in Table 5, we should mention thetreatment of adoptively transferred EAE with syngeneic BM byBurt et al. [75]. This is a relapsing form of EAE that can beevoked in SJL/J mice with lymph node cells from sensitized,syngeneic donors. The cells are stimulated in vitro with thedisease-initiating peptide, proteolipid protein 139 151 prior totransfer. These mice were treated at the peak of the acute phaseof the disease at 14 days after transfer or at day 74 during thechronic phase with a lethal dose of TBI or with TBI andcyclophospamide followed by rescue with syngeneic BM.Treatment in the early phase caused a signi cant clinical andhistological improvement, but there was no effect of treatmentof the chronically ill animals. This is reminiscent of the lack of responses of chronically ill Biozzi mice to treatment withallogeneic BM and supports the rule that BMT cannot repair scar lesions.

EXPERIMENTAL DATA OF TRANSLATIONALSIGNIFICANCE

Most of the detailed information concerning conditioning andtransplantation regimens for autologous BMT was obtainedwith two models of induced AID, namely AA and EAE, in BUFrats. The two models have a lot in common regarding theresponses to treatment, but there are also considerable differ-ences. In trying to translate the results into the clinic, one canchoose to adhere strictly to each speci c model, e.g., use theresults of the AA model only for RA and SLE treatmentstrategies and the results obtained with EAE, only for MS. Of course, this approach suffers from the restrictions imposed bythe imperfections of each model. Another way to apply the

results is to select the conditions from each model that seem tobe most favorable to the outcome and translate these into theclinical protocols until shown otherwise.

Detailed results of autologous BMT in AA andEAE

After high dose TBI (9 10 Gy), autologous BMT causes remis-sions of both diseases in all animals. In AA, 70% are completeresponders, and 30% are partial responders. Spontaneous re-lapses or exacerbations are extremely rare; also relapses arehardly ever inducible ( Table 6 ). In this disease model, theoutcome seems to be dominated by the intensity of the condi-

tioning. Even the addition of large numbers of autologousspleen cells or lymphocytes from the lymph nodes or from theperipheral blood did not adversely in uence the responses nor did it evoke relapses [66]. So far, this cannot be explained butit seems to be in line with our failure to passively transfer AAin BUF rats with lymphoid cells.

Rats with EAE respond to autologous BMT with a rapidregression of the neurological symptoms, but one or morespontaneous relapses occur in 30% of the animals [68]. Whensyngeneic BM instead of autologous BM is used for rescue, thespontaneous relapse rate is the same, suggesting that theserelapses are initiated by autoreactive cells that have survivedthe conditioning. This is in accordance with our nding that T

cell depletion of autologous or syngeneic BM grafts, whichreduced the T cells to 0.1%, did not diminish the spontaneousrelapse rate. Rat BM contains 2 3% T lymphocytes. In theseexperiments, the number of T lymphocytes (5 105 ) that wasreturned with the unmanipulated BM was in the same range asthe estimated number of residual T lymphocytes (10 6 ), whichexplains the futility of T cell depletion in this situation. Thisdoes not imply that the number of T lymphocytes returned withthe stem cells is irrelevant. On the contrary, the addition of autologous spleen cells containing 3 107 T lymphocytes tothe BM graft, which raises the proportion of T cells to over 50%, causes the spontaneous relapse rate to rise to 93%. (For comparison, unmanipulated human BM grafts may contain20 30% of T cells and human PBSC, up to 50%.) Thesendings contrast sharply with those in arthritic animals re-ferred to above where addition of autologous T cells had noinuence whatsoever.

This illustrates the dilemma of the translator: is T celldepletion to be recommended for clinical transplants or onlyfor the treatment of patients with MS? As will be explainedlater, the decision in this case can be made on more pragmaticgrounds.

Interestingly, the spontaneous relapse incidence in EAE wasonly 5% following allogeneic BMT as compared with 30% after autologous and syngeneic BMT [50, 51]. The difference is

TABLE 6. Incidence of Relapses after Treatment of ExperimentalAID with BMT Following High-Dose Conditioning

Disease RemissionSpontaneous

relapsesInducedrelapses Ref.

AA rats a

Syngeneic 100% 0% 0% 58Autologous 100% 0% 6% 66Allogeneic 100% 0% Not done

EAE mice b

Syngeneic 100% 7% 25% 69EAE rats c

Syngeneic 100% 29% 44% 67Autologous 100% 30% 72% 68Allogeneic 100% 5% 11% 50, 51

EAMG ratsd

Autologous 100% d Does not apply d 11% d 65

Conditioning: a TBI 9 Gy; b CY; c TBI 10 Gy; d CY TBI 6 Gy. In thisdisease model, the criterion for remission is a decrease of the antiacetylcholinereceptor titer. The equivalent of relapse is a secondary antibody responsefollowing reimmunization.



8/12

ascribed to a graft-versus-autoimmunity reaction by whichresidual, autoreactive cells of the recipient are eliminated, aterm introduced by A. M. Marmont in reference to the well-known graft-versus-leukemia effect of allogeneic BM grafts.The analogy with the treatment of leukemia may be extendedfurther by proposing that the treatment of severe AID shouldalso aim at eradication of as many autoreactive cells as possi-ble by conditioning and in case of autologous BM, by ex vivopurging of the graft prior to reinfusion.

The conditioning regimen

In both models, the best results have been obtained with thestrongest lymphomyeloablative regimens, e.g., the highest tol-erated dose of 9 10 Gy of TBI [66, 68]. Partial body irradiationof the affected tissues only (the CNS in the case of EAE or thelegs and tail in the case of AA) or shielding of those parts whileirradiating the rest of the body resulted, at best, in a limited,temporary remission [58, 67]. Fractionated irradiation wasinvestigated in the AA model [66] and proved to be as effectiveas single dose TBI, provided the total dose was properlyadjusted upward. In AA and EAE, CY alone or busulfan (BU)

alone at highest tolerated doses was less effective than highdose TBI, and the combined regimen of CY and BU wasequally effective. The combination of a lower dose of TBI (4 Gyor 7 Gy) with a lower dose of CY (2 60 mg/kg) was also aseffective as the highest dose of TBI. In experimental autoim-mune myasthenia gravis (EAMG), conditioning with CY alonewas inadequate, and a moderate dose of TBI had to be addedto get complete responses [65]. Notable features of high doseCY as the sole conditioning agent in AA were not only thelower rate of complete remissions, but also the substantialincidence (36%) of spontaneous exacerbations. In contrast,among 155 AA animals treated with high-dose TBI or the

combination regimens, only one relapse occurred.So far, conditioning of more than 70 patients with severe RAhas consisted of high dose CY, either as the sole agent or combined with anti-thymocyte globulin (ATG). Although themajority of the patients went into complete or partial remission,approximately two-thirds relapsed, usually within 1 year [63].It seems fair to conclude that the equivocal results with exper-imental arthritis have predicted this outcome. The main reasonfor the rheumatologists not to adopt the combination of CY andmoderate dose TBI, which is clearly superior in AA and EAE,has been fear of higher toxicity and mortality. By drawing oncemore on the analogy with the treatment of leukemia, the relapserate can only be reduced by intensifying the conditioning. Thecost of the latter is more toxicity, unless target cell speci citycan be improved upon.

The most likely candidate target cells in overt AID areactivated T lymphocytes and memory T lymphocytes. Thecharacteristics of these cell types are still poorly de ned.

Yet, there are some indications for differences in targetspeci city between radiation and CY. In treating rats sufferingfrom EAMG, Pestronk et al. [65] showed CY to be less effectivethan TBI in eliminating immunological memory. In this case,the cells involved were most likely B memory cells, but Tmemory cells (in this case against M. tuberculosis) were alsoreported to be CY-resistant [76].

In addition, there is clinical evidence that CY as a singleagent is inadequate for ablation of memory T cells. In nonsen-sitized patients with aplastic anemia, the allograft failure rateis low after conditioning with high dose CY alone. However, inpatients sensitized by multiple blood transfusions, most allo-grafts are rejected, and more intensive conditioning with acombination of TBI and CY is required to prevent take failure.Presently, there is not enough information regarding the num-bers and drug sensitivity of memory lymphocytes in patientswith advanced AID and how the size of these cell populationsvaries between patients.

We did not study fractionated TBI in EAE for two reasons.First, fractionation does not produce different effects fromsingle dose TBI, provided the total dose is adjusted for thefractionation effect. Secondly, I did not anticipate the use of TBI in MS patients, as irradiation induced an acute exacerba-tion of the neurological symptoms in rats with EAE. Thisreaction recedes after 24 h, but was fatal in a small proportionof cases. It occurred even after a dose as low as 1.5 Gy.However, such adverse effects were not encountered so far inMS patients after treatment with high dose TBI and CY for conditioning [77].

ATG is used as part of the conditioning regimen in severalclinical protocols of autologous BMT for severe AID [78] and juvenile idiopathic arthritis [79]. ATG was shown to protectagainst allogeneic GvHD in mice and monkeys even whenadministered before the BM [80, 81]. If used for the condition-ing in AID patients, it is recommended that the last dose beinjected at least 24 h or less before the stem cell reinfusion. Itmay then act on residual lymphocytes in the recipient as wellas on lymphocytes that are introduced with the graft [81].

Unfortunately, we could not evaluate the merits of ATG andits optimal application in conditioning for experimental AID,as the ant-rat ATG preparations available to us cross- reacted

with hematopoietic stem cells. There remains an urgent needfor more research on speci c T lymphocytolytic agents to beapplied in conditioning.

Postulated mechamisms of autologous stem celltransplantation and clinical relevance

The satisfactory, therapeutic results obtained with high doselympomyeloablative conditioning regimens discussed aboveare easily understood if in ammatory AID are regarded asbeing initiated and maintained by activated T cells. Treatmentof SLE and various connective tissue AID with moderate dosesof cytoreductive drugs such as CY and methotrexate was al-ready known to be effective in many cases so that the improvedresults with a higher dose is no surprise. The currently usedconditioning for severe RA with 200 mg/kg CY has resulted inroughly 50% complete remissions and a high relapse rate.Apparently, this dose is not completely myeloablative, as it wasassociated with rapid hematological recovery when used with-out stem cell rescue for treatment of severe AID [82]. In thelatter study comprising 25 patients, the complete remissionrate was also 50%, which suggests that the short-term re-sponses at least are determined by the intensity of the condi-tioning and not by the cellular composition of the autografts.

Many clinical teams engaged in autologous BMT for AID sofar prefer the use of CY alone or combined with anti-lympho-



9/12

cyte globulin (ALG). TBI is avoided mainly because of itsseveral delayed side effects, e.g., the development of excesstumors, cataracts, and, in children, inhibition of skeletalgrowth. Yet, as pointed out in a recent review [83], the alky-lating drug CY is also carcinogenic [84, 85], as is prolongedimmunosuppressive treatment [86]. Prolonged conventionaltreatment with high dose corticosteroids is known to causegrowth inhibition and cataract, but a moderate dose of 4 GyTBI does not have these sequels.

The experience with animal models indicates that there isroom for improvement by intensifying the conditioning, but of course that would require hematological support with autolo-gous stem cells unless more speci c lymphocytolytic agentscould be introduced. One promising drug is udarabine. It wasrecently used successfully (120 mg/m 2 over 4 days) in place of TBI in combination with CY and ATG for conditioning sixtransfusion-dependent patients with severe aplastic anemia for grafting of allogeneic BM [87, 88]. There were no take failures,and all patients achieved full donor chimerism. In Cynomolgusmonkeys, udarabine (250 mg/m 2 over 5 days) induced T- andB-cell lymphopenia and prolonged the survival of allogeneicskin grafts in naive and presensitized animals [89]. The drugschedule used caused transient neutropenia as the only sideeffect. Treatment of patients with refractory, severe RA withpulsed udarabine induced a reduction of naive and memoryCD4 T cells [90]. High dose udarabine (300 mg/m 2 in twocourses of 5 days) was used in combination with ALG followedby autologous stem cells in a pilot study for treating patientswith various severe AID [91]. This regimen was not toxic, andthe immediate responses resembled those after treatment withCY plus ATG, but follow-up was not long enough for other conclusions. There is certainly a strong need for sorting out, inthe appropriate, established animal models, the advantagesthis drug has to offer and how it can be applied best.

The induction of remission and persistenttolerance

How are the excellent, curative results with autologous BM inexperimental animals and the encouraging experience withmany patients with various severe AID to be explained?

The most favored hypothesis is that the reconstitution of theimmune system from a few stem cells represents a recapitula-tion of ontogenesis, which entails the acquisition of self-toler-ance. Burt et al. [92] provided evidence for such a mechanism.They found persistence of T lymphocytes that react with frag-ments 68 82 of myelin basic protein (MBP) in the spinal cordof Lewis rats in spontaneous, clinical remission from acuteEAE but not in rats that had been treated with high dose TBIand syngeneic BM to induce remission. Karussis et al. [69]claimed induction of tolerance in EAE mice after treatmentwith a 30% lethal dose of CY and rescue with syngeneic Tcell-depleted BM [69]. The induction of EAE and rechallengewere with mouse spinal cord homogenate in CFA. They mea-sured the proliferation response of lymphocytes obtained fromlymph nodes to the antigens guinea pig MBP (GMBP) andtuberculin-puri ed protein derivative (PPD). The responses toGMBP were weak or negative before and after rechallenge inthe treated animals as well as in the controls. The stimulationindex with PPD increased from 8 before to 46 after rechallenge

in the controls and from 2.3 to 3.8 in the treated group. It isdoubtful if this can be regarded as evidence for tolerance. Thespontaneous relapse rate was low in the treated group (onerelapse in 15 mice) as compared with 21 relapses in 15controls. Two out of eight mice suffered a relapse after rechal-lenge as compared with nine out of nine controls.

Apart from our ignorance about the details of tolerance at thecellular level, there is a clear tendency from the data inanimals that suboptimal conditioning results in less completeresponses (as in AA), more spontaneous relapses (as in EAE),and more induced relapses (as in AA and in EAE). Further-more, following optimal conditioning, the induced relapse rateis higher with autologous or syngeneic stem cell grafts thanwith allogeneic transplants (Table 6).

The causes of the different responses of AA and EAE toBMT remain subject to speculation. One attractive hypothesisis that if the genes determining susceptibility are weaklyexpressed, the resulting state of tolerance may not be brokeneasily. This may be the case in AA, where disease could not bereinduced after complete remission was obtained with autolo-gous BMT. On the other hand, in EAE rats, relapses could beinduced in a high proportion of animals after autologous BMT.At the extreme end of the range is the HLA-B27 transgenic ratthat cannot even be brought into remission with syngeneic stemcells. These animals bear up to 150 copies of the B27 gene,which assumedly makes them respond to a large variety of environmental antigenic stimuli with autoimmune reactions,thereby precluding the development of autotolerance.

RECOMMENDATIONS

Intensive conditioning, such as can be achieved best with highdose TBI, is expected to give the best therapeutic results,

presumably because it kills off the most T lymphocytes.Clearly, high priority should be given to a search for agentswith a clinically effective, therapeutic window between lym-photoxicity and myelotoxicity and with suf cient penetrationinto inamed tissues such as as spinal cord, brain, and joints.Treatment with such agents would leave the stem cell popula-tion intact, but whether tolerance would also develop under these conditions remains to be seen.

It stands to reason that reinfusing any numbers of T lym-phocytes that would add substantially to the residual popula-tion should be avoided. A BM graft for an adult patient maycontain as many as 4 109 and a mobilized peripheral bloodcell graft, up to 2 1010 T lymphocytes as compared with anestimated 3 108 residual T lymphocytes. That estimate isbased on conditioning regimes equivalent to 9 Gy TBI (singledose), which causes roughly a 3 log kill of lymphocytes. Thetotal T cell population in an adult is taken as 3 1011 . In viewof the uncertainties of these estimates and in analogy with thepolicy of maximal purging of tumor cells in autologous BMgrafts used for the treatment of leukemia, it was recommendedto T cell-deplete the autograft as completely as current tech-niques allow. This strategy was supported by the relapse of allof the rst ve patients who received unmanipulated, autolo-gous BM or mobilized peripheral blood cells [93]. The maxi-mum number of reinfused T cells was recommended to be less



10/12

than 10 5 per kg [94], requiring between 3 and 4 log depletionfor BM and mobilized peripheral blood cells, respectively. Inpractice, 10 6 T cells/kg seems more realistic as a maximum;this number being well below the estimates of residual T cells.However, it is unknown which subpopulations of T lympho-cytes are crucially involved in the development of relapses andwhat their radio- and chemosensitivity are.

Rescue with highly puri ed CD34 stem cells is likely tocause an extended period of severe immunosuppression withincreased risks of infections and lymphoproliferative malignan-cies. Several cases of fatal activated macrophage syndrome have occurred recently in children following treatment for juvenile chronic arthritis with highly puri ed autologous stemcells. It should be noted that the CD34 cell selection tech-niques that are currently used also effectively remove B cells,natural killer cells, and macrophages, which may be unneces-sary and possibly harmful. That has been the rationale for someclinical teams to change to purging methods that speci callydeplete T lymphocytes only.

Finally, there is the option of using allogeneic stem cells,which in the EAE rat minimizes relapses and also showed apresumed graft-versus-autoimmune effect. Considering thehigher risks of transplantation-associated mortality of alloge-neic BMT, its exploration should be postponed until it becomesclear from the ongoing studies with autologous stem cells,which patients might bene t from allogeneic grafts. The mostthreatening side effect of allogeneic BMT is graft-versus-hostdisease, which represents an antiself immune reaction par excellence. Therefore, one risks replacing one severe diseasewith another. The use of allogeneic stem cells should speci -cally be discouraged in the treatment of SLE, systemic sclero-sis, and related syndromes, as certain lesions induced by thegraft-versus-host reactions, especially the ones associated withchronic GvHD, will be very hard to differentiate from lesions

from a relapse of the original AID.

REFERENCES

1. Morton, J. I., Siegel, B. V. (1974) Transplantation of autoimmune poten-tial. I. Development of antinuclear antibodies in H-2 histocompatiblerecipients of bone marrow from New Zealand Black mice. Proc. Natl.Acad. Sci. USA 71, 2162 2165.

2. Marmont, A. M. (1994) Immune ablation followed by allogeneic or autol-ogous bone marrow transplantation: a new treatment for severe autoim-mune diseases? Stem Cells 12, 125 135.

3. Charon, D. (1990) Molecular basis for human leukocyte antigen class IIassociations. Adv. Immunol. 48, 107 159.

4. Silman, A. J., MacGregor, A. J., Thomson, W., Holligan, S., Carthy, D.,Farhan, A., Ollier, W. E. (1993) Twin concordance rates for rheumatoidarthritis: results from a nationwide study. Br. J. Rheumatol. 32, 903 907.

5. Taurog, J. D., Richardson, J. A., Croft, J. T., Simmons, W. A., Zhou, M.,Fernandez-Sueiro, J. L., Balish, E., Hammer, R. E. (1994) The germfreestate prevents development of gut and joint in ammatory disease inHLA-B27 transgenic rats. J. Exp. Med. 180, 2359 2364.

6. Unnik, K. (1975) Comparative study of NZB mice under germfree andcontrolled conditions. J. Rheumatol. 2, 36 44.

7. Maldonado, M. A., Kakkanaiah, V., MacDonald, G. C., Chen, F., Reap,E. A., Balish, E., Farkas, W. R., Jennette, J. C., Madaio, M. P., Kotzin,B. L., Cohen, P. L., Eisenberg, R. A. (1999) The role of environmentalantigens in the spontaneous development of autoimmunity in MRL-lpr mice. J. Immunol. 162, 6322 6330.

8. Thomas, V. A., Woda, B. A., Handler, E. S., Greiner, D. L., Mordes, J. P.,Rossini, A. A. (1991) Altered expression of diabetes in BB/Wor rats byexposure to viral pathogens. Diabetes 40, 255 258.

9. Ellerman, K. E., Richards, C. A., Guberski, D. L., Shek, W. R., Like, A. A.(1996) Kilham rat triggers T-cell-dependent autoimmune diabetes inmultiple strains of rat. Diabetes 45, 557 562.

10. Like, A. A., Guberski, D. L., Butler, L. (1991) In uence of environmentalviral agents on frequency and tempo of diabetes mellitus in BB/Wor rats.Diabetes 40, 259 262.

11. Wang, G. S., Gruber, H., Smyth, P., Pulido, O., Rosenberg, L., Duguid,W., Scott, F. W. (2000) Hydrolysed casein diet protects BB rats fromdeveloping diabetes by promoting islet neogenesis. J. Autoimmun. 15,407 416.

12. Williams, A. J., Krug, J., Lampeter, E. F., Mans eld, K., Beales, P. E.,Signore, A., Gale, E. A., Pozzilli, P. (1990) Raised temperature reducesthe incidence of diabetes in the NOD mouse. Diabetologia 33, 635 637.

13. Carter, W. R., Herrman, J., Stokes, K., Cox, D. J. (1987) Promotion of diabetes onset by stress in the BB rat. Diabetologia 30, 674 675.

14. Hunger, R. E., Carnaud, C., Vogt, I., Mueller, C. (1998) Male gonadalenvironment paradoxically promotes dacryoadenitis in nonobese diabeticmice. J. Clin. Investig. 101, 1300 1309.

15. Kohashi, O., Kuwata, J., Umehara, K., Uemura, F., Takahashi, T., Ozawa,A. (1979) Susceptibility to adjuvant-induced arthritis among germfree,specic- pathogen-free, and conventional rats. Infect. Immun. 26, 791 794.

16. Breban, M. A., Moreau, M. C., Fournier, C., Ducluzeau, R., Kahn, M. F.(1993) Inuence of the bacterial ora on collagen-induced arthritis insusceptible and resistant strains of rats. Clin. Exp. Rheumatol. 11, 61 64.

17. van Bekkum, D. W., Knaan, S. (1977) Role of bacterial micro ora indevelopment of intestinal lesions from graft-versus-host reaction. J. Natl.Cancer Inst. 58, 787 790.

18. Heidt, P. J., Vossen, J. M. (1992) Experimental and clinical gnotobiotics:

inuence of the microora on graft-versus-host disease after allogeneicbone marrow transplantation. J. Med. 23, 161 173.

19. Mason, D., MacPhee, I., Antoni, F. (1990) The role of the neuroendocrinesystem in determining genetic susceptibility to experimental allergicencephalomyelitis in the rat. Immunology 70, 1 5.

20. van Gelder, M., Kinwel-Bohre, E. P., Mulder, A. H., van Bekkum, D.(1996) Both bone marrow- and non-bone marrow-associated factors deter-mine susceptibility to experimental autoimmune encephalomyelitis of BUF and WAG rats. Cell. Immunol. 168, 39 48.

21. Mostarica-Stojkovic, M., Petrovic, M., Lukic, M. L. (1982) Resistance tothe induction of EAE in AO rats: its prevention by the pre-treatment withcyclophosphamide or low dose of irradiation. Clin. Exp. Immunol. 50,311 317.

22. Lando, Z., Teitelbaum, D., Arnon, R. (1980) Induction of experimentalallergic encephalomyelitis in genetically resistant strains of mice. Nature287, 551 552.

23. de Vries, M. J., van Putten, L. M., Balner, H., van Bekkum, D. W. (1964)Le sions sugge rant une re activite autoimmune chez des souris atteintes dela runt disease apres thymectomie neonatale. Rev. Fr. Etud. Clin. Biol.9, 381397.

24. Bos, G. M., Majoor, G. D., Van Breda Vriesman, P. J. (1989) Perturbationof T-cell differentiation in lethally irradiated rats reconstituted with syn-geneic bone marrow and treated with cyclosporin-A. Thymus 14, 155 161.

25. Sardina, E. E., Sugiura, K., Ikehara, S., Good, R. A. (1991) Transplanta-tion of wheat germ agglutinin-positive hematopoietic cells to prevent or induce systemic autoimmune disease. Proc. Natl. Acad. Sci. USA 88,3218 3222.

26. de Heer, D. H., Edgington, T. S. (1977) Evidence for a B lymphocytedefect underlying the anti-X anti-erythrocyte autoantibody response of NZB mice. J. Immunol. 118, 1858 1863.

27. Morton, J. I., Siegel, B. V. (1979) Transplantation of autoimmune poten-tial. IV. Reversal of the NZB autoimmune syndrome by bone marrowtransplantation. Transplantation 27, 133 134.

28. Ikehara, S., Inaba, M., Ishida, T., Ogata, H., Hisha, H., Yasumizu, R.,Oyaizu, N., Sugiura, K., Toki, J., Takao, F., Than, S., Kawamura, M.,Nishioka, N., Nagata, N., Good, R. A. (1991) Rationale for transplantationof both allogeneic bone marrow and stromal cells in the treatment of autoimmune diseases. In New Strategies in Bone Marrow Transplantation:UCLA Symposia on Molecular and Cellular Biology, New Series, vol. 137(R. E. Champlin, R. P. Gale, eds.), New York, NY, Wiley-Liss, 251.

29. Miyashima, S., Nagata, N., Nakagawa, T., Hosaka, N., Takeuchi, K.,Ogawa, R., Ikehara, S. (1996) Prevention of lpr-graft-versus-host diseaseand transfer of autoimmune diseases in normal C57BL/6 mice by trans-plantation of bone marrow cells plus bones (stromal cells) from MRL/lpr mice. J. Immunol. 156, 79 84.

30. Ikehara, S., Kawamura, M., Takao, F., Inaba, M., Yasumizu, R., Than, S.,Hisha, H., Sugiura, K., Koide, Y., Yoshida, T. O., et al. (1990) Organ-specic and systemic autoimmune diseases originate from defects inhematopoietic stem cells. Proc. Natl. Acad. Sci. USA 87, 8341 8344.



11/12

31. Himeno, K., Good, R. A. (1988) Marrow transplantation from tolerantdonors to treat and prevent autoimmune diseases in BXSB mice. Proc.Natl. Acad. Sci. USA 85, 2235 2239.

32. Nakagawa, T., Nagata, N., Hosaka, N., Ogawa, R., Nakamura, K., Ikehara,S. (1993) Prevention of autoimmune in ammatory polyarthritis in maleNew Zealand black/KN mice by transplantation of bone marrow cells plusbone (stromal cells). Arthritis Rheum. 36, 263 268.

33. Serreze, D. V., Leiter, E. H., Worthen, S. M., Shultz, L. D. (1988) NODmarrow stem cells adoptively transfer diabetes to resistant (NODNON)F1 mice. Diabetes 37, 252 255.

34. Wicker, L. S., Miller, B. J., Chai, A., Terada, M., Mullen, Y. (1988)Expression of genetically determined diabetes and insulitis in the non-obese diabetic (NOD) mouse at the level of bone marrow-derived cells.Transfer of diabetes and insulitis to nondiabetic (NOD B10) F1 micewith bone marrow cells from NOD mice. J. Exp. Med. 167, 1801 1810.

35. LaFace, D. M., Peck, A. B. (1989) Reciprocal allogeneic bone marrowtransplantation between NOD mice and diabetes-nonsusceptible miceassociated with transfer and prevention of autoimmune diabetes. Diabetes38, 894 901.

36. Naji, A., Silvers, W. K., Kimura, H., Anderson, A. O., Barker, C. F. (1983)Inuence of islet and bone marrow transplantation on the diabetes andimmunodeciency of BB rats. Metabolism 32, 62 68.

37. Scott, J., Engelhard, V. H., Benjamin, D. C. (1987) Bone marrow irradi-ation chimeras in the BB rat: evidence suggesting two defects leading todiabetes and lymphopoenia. Diabetologia 30, 774 781.

38. Kuntz, L., Montecino-Rodriguez, E., Jachez, B., Roman, D., Loor, F.(1991) Adoptive transfer of viable motheaten pathology in sublethallyirradiated beige recipient mice. Immunology 73, 356 362.

39. Walker, M. A., Harley, R. A., DeLustro, F. A., LeRoy, E. C. (1989)Adoptive transfer of tsk skin brosis to / recipients by tsk bonemarrow and spleen cells. Proc. Soc. Exp. Biol. Med. 192, 196 200.

40. Breban, M., Hammer, R. E., Richardson, J. A., Taurog, J. D. (1993)Transfer of the inammatory disease of HLA-B27 transgenic rats by bonemarrow engraftment. J. Exp. Med. 178, 1607 1616.

41. van Bruggen, M. C., van den Broek, M. F., van den Berg, W. B. (1991)Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344 3 Lewis and in Lewis 3 F344 bone marrow chimeras. Cell. Immunol. 136,278 290.

42. van Bekkum, D. W. (1997) Autologous stem cell therapy for treatment of autoimmune diseases. In Autologous Marrow and Blood Transplantation: Proceedings of the Eigth International Symposium on Autologous Bone Marrow Transplantaion, Arlington, Texas (K. A. Dicke, A. Keating, eds.),Charlottesville, VA, Carden Jennings, 645 662.

43. Fujita, M., Mishima, M., Iwabuchi, K., Katsume, C., Gotohda, T., Oga-sawara, K., Mizuno, Y., Good, R. A., Onoe, K. (1989) A study on type IIcollagen-induced arthritis in allogeneic bone marrow chimaeras. Immu-nology 66, 422 427.

44. Singer, D. E., Moore, M. J., Williams, R. M. (1981) EAE in rat bonemarrow chimeras: analysis of the cellular mechanism of BN resistance.J. Immunol. 126, 1553 1557.

45. Ben-Nun, A., Otmy, H., Cohen, I. R. (1981) Genetic control of autoim-mune encephalomyelitis and recognition of the critical nonapeptide moi-ety of myelin basic protein in guinea pigs are exerted through interactionof lymphocytes and macrophages. Eur. J. Immunol. 11, 311 316.

46. Pelfrey, C. M., Waxman, F. J., Whitacre, C. C. (1989) Genetic resistancein experimental autoimmune encephalomyelitis. I. Analysis of the mech-anism of LeR resistance using radiation chimeras. Cell. Immunol. 122,504 516.

47. Korngold, R., Feldman, A., Rorke, L. B., Lublin, F. D., Doherty, P. C.(1986) Acute experimental allergic encephalomyelitis in radiation bonemarrow chimeras between high and low susceptible strains of mice.

Immunogenetics 24, 309 315.48. Lublin, F. D., Knobler, R. L., Doherty, P. C., Korngold, R. (1986)Relapsing experimental allergic encephalomyelitis in radiation bone mar-row chimeras between high and low susceptible strains of mice. Clin. Exp.Immunol. 66, 491 496.

49. Binder, T. A., Greiner, D. L., Grunnet, M., Goldschneider, I. (1993)Relative susceptibility of SJL/J and B10.S mice to experimental allergicencephalomyelitis (EAE) is determined by the ability of prethymic cells inbone marrow to develop into EAE effector T cells. J. Neuroimmunol. 42,2332.

50. van Gelder, M., van Bekkum, D. W. (1995) Treatment of relapsingexperimental autoimmune encephalomyelitis in rats with allogeneic bonemarrow transplantation from a resistant strain. Bone Marrow Transplant.16, 343 351.

51. van Gelder, M., Mulder, A. H., van Bekkum, D. W. (1996) Treatment of relapsing experimental autoimmune encephalomyelitis with largely MHC-

matched allogeneic bone marrow transplantation. Transplantation 62,810 818.

52. Jones, R. E., Bourdette, D. N., Whitham, R. H., Offner, H., Vandenbark,A. A. (1993) Induction of experimental autoimmune encephalomyelitis insevere combined immunode cient mice reconstituted with allogeneic or xenogeneic hematopoietic cells. J. Immunol. 150, 4620 4629.

53. Ikehara, S., Good, R. A., Nakamura, T., Sekita, K., Inoue, S., Oo, M. M.,Muso, E., Ogawa, K., Hamashima, Y. (1985) Rationale for bone marrowtransplantation in the treatment of autoimmune diseases. Proc. Natl. Acad.Sci. USA 82, 2483 2487.

54. Ikehara, S., Ohtsuki, H., Good, R. A., Asamoto, H., Nakamura, T., Sekita,K., Muso, E., Tochino, Y., Ida, T., Kuzuya, H. (1985) Prevention of typeI diabetes in nonobese diabetic mice by allogenic bone marrow transplan-tation. Proc. Natl. Acad. Sci. USA 82, 7743 7747.

55. Ikehara, S., Yasumizu, R., Inaba, M., Izui, S., Hayakawa, K., Sekita, K.,Toki, J., Sugiura, K., Iwai, H., Nakamura, T., et al. (1989) Long-termobservations of autoimmune-prone mice treated for autoimmune diseaseby allogeneic bone marrow transplantation. Proc. Natl. Acad. Sci. USA 86,3306 3310.

56. Ishida, T., Inaba, M., Hisha, H., Sugiura, K., Adachi, Y., Nagata, N.,Ogawa, R., Good, R. A., Ikehara, S. (1994) Requirement of donor-derivedstromal cells in the bone marrow for successful allogeneic bone marrowtransplantation. Complete prevention of recurrence of autoimmune dis-eases in MRL/MP-Ipr/Ipr mice by transplantation of bone marrow plusbones (stromal cells) from the same donor. J. Immunol. 152, 3119 3127.

57. Hosaka, N., Nose, M., Kyogoku, M., Nagata, N., Miyashima, S., Good,R. A., Ikehara, S. (1996) Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/ mice. Proc. Natl. Acad.Sci. USA 93, 8558 8562.

58. van Bekkum, D. W., Bohre, E. P., Houben, P. F., Knaan-Shanzer, S.(1989) Regression of adjuvant-induced arthritis in rats following bonemarrow transplantation. Proc. Natl. Acad. Sci. USA 86, 10090 10094.

59. Kamiya, M., Sohen, S., Yamane, T., Tanaka, S. (1993) Effective treatmentof mice with type II collagen induced arthritis with lethal irradiation andbone marrow transplantation. J. Rheumatol. 20, 225 230.

60. Van Gelder, M. (1995) Bone Marrow Transplantation for Treatment of Experimental Autoimmune Encephalomyelitis in Rats. Prospects for Ther-apy of Severe Multiple Sclerosis, 1163, Rijks Universiteit Leiden, Leiden,Ph.D. thesis.

61. Marmont, A. M. (2001) Immunoablation followed or not by hematopoieticstem cells as an intense therapy for severe autoimmune diseases. Newperspectives, new problems. Haematologica 86, 337 345.

62. Nelson, J. L., Torrez, R., Louie, F. M., Choe, O. S., Storb, R., Sullivan,K. M. (1997) Pre-existing autoimmune disease in patients with long-termsurvival after allogeneic bone marrow transplantation. J. Rheumatol.Suppl. 48, 23 29.

63. Tyndall, A. (2001) Autologous hematopoietic stem cell transplantation for severe autoimmune disease with special reference to rheumatoid arthritis.J. Rheumatol. Suppl. 64, 5 7.

64. Knaan-Shanzer, S., Houben, P., Kinwel-Bohre, E. P., van Bekkum, D. W.(1991) Remission induction of adjuvant arthritis in rats by total bodyirradiation and autologous bone marrow transplantation. Bone MarrowTransplant. 8, 333 338.

65. Pestronk, A., Drachman, D. B., Teoh, R., Adams, R. N. (1983) Combinedshort-term immunotherapy for experimental autoimmune myasthenia gra-vis. Ann. Neurol. 14, 235 241.

66. van Bekkum, D. W. (2000) Conditioning regimens for the treatment of experimental arthritis with autologous bone marrow transplantation. BoneMarrow Transplant. 25, 357 364.

67. van Gelder, M., Kinwel-Bohre, E. P., van Bekkum, D. W. (1993) Treat-ment of experimental allergic encephalomyelitis in rats with total body

irradiation and syngeneic BMT. Bone Marrow Transplant. 11, 233 241.68. van Gelder, M., van Bekkum, D. W. (1996) Effective treatment of relaps-ing experimental autoimmune encephalomyelitis with pseudoautologousbone marrow transplantation. Bone Marrow Transplant. 18, 1029 1034.

69. Karussis, D. M., Slavin, S., Ben-Nun, A., Ovadia, H., Vourka-Karussis, U.,Lehmann, D., Mizrachi-Kol, R., Abramsky, O. (1992) Chronic-relapsingexperimental autoimmune encephalomyelitis (CR-EAE): treatment andinduction of tolerance, with high dose cyclophosphamide followed bysyngeneic bone marrow transplantation. J. Neuroimmunol. 39, 201 210.

70. Good, R. A., Ikehara, S. (1997) Preclinical investigations that subserveefforts to employ bone marrow transplantation for rheumatoid or autoim-mune diseases. J. Rheumatol. Suppl. 48, 5 12.

71. Karussis, D. M., Vourka-Karussis, U., Lehmann, D., Abramsky, O., Ben-Nun, A., Slavin, S. (1995) Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT). Clin. Exp.Immunol. 100, 111 117.



12/12

72. Loor, F., Jachez, B., Montecino-Rodriguez, E., Klein, A. S., Kuntz, L.,Pumio, F., Fonteneau, P., Illinger, D. (1988) Radiation therapy of spon-taneous autoimmunity: a review of mouse models. Int. J. Radiat. Biol.Relat. Stud. Phys. Chem. Med. 53, 119 136.

73. Smith, H. R., Chused, T. M., Steinberg, A. D. (1984) Cyclophosphamide-induced changes in the MRL-lpr/lpr mouse: effects upon cellular compo-sition, immune function, and disease. Clin. Immunol. Immunopathol. 30,51 61.

74. Karussis, D. M., Slavin, S., Lehmann, D., Mizrachi-Koll, R., Abramsky,O., Ben-Nun, A. (1992) Prevention of experimental autoimmune enceph-alomyelitis and induction of tolerance with acute immunosuppressionfollowed by syngeneic bone marrow transplantation. J. Immunol. 148,1693 1698.

75. Burt, R. K., Padilla, J., Begolka, W. S., Canto, M. C., Miller, S. D. (1998)Effect of disease stage on clinical outcome after syngeneic bone marrowtransplantation for relapsing experimental autoimmune encephalomyelitis.Blood 91, 2609 2616.

76. Orme, I. M. (1988) Characteristics and speci city of acquired immuno-logic memory to Mycobacterium tuberculosis infection. J. Immunol. 140,3589 3593.

77. Burt, R. K., Traynor, A. E., Pope, R., Schroeder, J., Cohen, B., Karlin,K. H., Lobeck, L., Goolsby, C., Rowlings, P., Davis, F. A., Stefoski, D.,Terry, C., Keever-Taylor, C., Rosen, S., Vesole, D., Fishman, M., Brush,M., Mujias, S., Villa, M., Burns, W. H. (1998) Treatment of autoimmunedisease by intense immunosuppressive conditioning and autologous he-matopoietic stem cell transplantation. Blood 92, 3505 3514.

78. Fassas, A., Anagnostopoulos, A., Kazis, A., Kapinas, K., Sakellari, I.,Kimiskidis, V., Tsompanakou, A. (1997) Peripheral blood stem cell trans-plantation in the treatment of progressive multiple sclerosis: rst results of

a pilot study. Bone Marrow Transplant. 20, 631 638.79. Wulffraat, N. M., Sanders, L. A., Kuis, W. (2000) Autologous hemopoieticstem-cell transplantation for children with refractory autoimmune disease.Curr. Rheumatol. Rep. 2, 316 323.

80. van Bekkum, D. W. (1970) Mitigation of acute secundary disease bytreatment of the recipient with antilymphocyte serum before grafting of hemopoietic cells. Exp. Hematol. 20, 3 4.

81. van Bekkum, D. W., Balner, H., Dicke, K. A., van den Berg, F. G.,Prinsen, G. H., Hollander, C. F. (1972) The effect of pretreatment of allogeneic bone marrow graft recipients with antilymphocytic serum on theacute graft-versus-host reaction in monkeys. Transplantation 13, 400 407.

82. Brodsky, R. A. (1999) Immunoabblative treatment of autoimmune diseaseswithout stem cell transplantation suppor. In Symposium on Autoimmune Disease, Immunoablation and Stem Cells: Towards Y2K , 28th AnnualMeeting ISEH, Monte Carlo.

83. van Bekkum, D. W. (1999) Effectiveness and risks of total body irradiationfor conditioning in the treatment of autoimmune disease with autologousbone marrow transplantation. Rheumatology (Oxford) 38, 757 761.

84. Zurcher, C., Varekamp, A. E., Solleveld, H. A., Durham, S. K., De Vries,A. J., Hagenbeek, A. (1987) Late effects of cyclophosphamide and totalbody irradiation as a conditioning regimen for bone marrow transplanta-tion in rats (a preliminary report). Int. J. Radiat. Biol. Relat. Stud. Phys.Chem. Med. 51, 1059 1068.

85. Radis, C. D., Kahl, L. E., Baker, G. L., Wasko, M. C., Cash, J. M.,Gallatin, A., Stolzer, B. L., Agarwal, A. K., Medsger Jr., T. A., Kwoh, C. K.(1995) Effects of cyclophosphamide on the development of malignancyand on long-term survival of patients with rheumatoid arthritis. A 20-year followup study. Arthritis Rheum. 38, 1120 1127.

86. Dantal, J., Hourmant, M., Cantarovich, D., Giral, M., Blancho, G., Dreno,B., Soulillou, J. P. (1998) Effect of long-term immunosuppression inkidney-graft recipients on cancer incidence: randomised comparison of two cyclosporin regimens. Lancet 351, 623 628.

87. Chan, K. W., Li, C. K., Worth, L. L., Chik, K. W., Jeha, S., Shing, M. K.,Yuen, P. M. (2001) A udarabine-based conditioning regimen for severeaplastic anemia. Bone Marrow Transplant. 27, 125 128.

88. Nishio, M., Nakao, S., Endo, T., Fujimoto, K., Takashima, H., Sakai, T.,Bacigalupo, A., Koike, T., Sawada, K. (2001) Successful non-myeloabla-tive stem cell transplantation for a heavily transfused woman with severeaplastic anemia complicated by heart failure. Bone Marrow Transplant.28, 783 785.

89. Goodman, E. R., Fiedor, P. S., Fein, S., Athan, E., Hardy, M. A. (1996)Fludarabine phosphate: a DNA synthesis inhibitor with potent immuno-suppressive activity and minimal clinical toxicity. Am. Surg. 62, 435 442.

90. Davis Jr., J. C., Fessler, B. J., Tassiulas, I. O., McInnes, I. B., Yarboro,C. H., Pillemer, S., Wilder, R., Fleisher, T. A., Klippel, J. H., Boumpas,D. T. (1998) High dose versus low dose udarabine in the treatment of patients with severe refractory rheumatoid arthritis. J. Rheumatol. 25,1694 1704.

91. Rabusin, M., Andolina, M., Maximova, N., Lepore, L., Parco, S., Tuveri,G., Jankovic, G. (2000) Immunoablation followed by autologous hemato-poietic stem cell infusion for the treatment of severe autoimmune disease.Haematologica 85, 81 85.

92. Burt, R. K., Burns, W., Ruvolo, P., Fischer, A., Shiao, C., Guimaraes, A.,Barrett, J., Hess, A. (1995) Syngeneic bone marrow transplantation elim-inates V beta 8.2 T lymphocytes from the spinal cord of Lewis rats withexperimental allergic encephalomyelitis. J. Neurosci. Res. 41, 526 531.

93. Euler, H. H., Marmont, A. M., Bacigalupo, A., Fastenrath, S., Dreger, P.,Hoffknecht, M., Zander, A. R., Schalke, B., Hahn, U., Haas, R., Schmitz,N. (1996) Early recurrence or persistence of autoimmune diseases after unmanipulated autologous stem cell transplantation. Blood 88, 3621 3625.

94. Tyndall, A., Grathwol, A. (1997) Concensus statement on blood andstemcell transplantation in autoimmune disease. Br. J. Rheumatol. 36,90 92.


experimental basis of hematopoietic stem cell transplantation for treatment of autoimmune diseases...

Documents