is autoimmunity in AA dysregulated or antineoplastic?

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AA, the high prevalence of one or several abnormal hematopoietic clones and their resistance to apoptosis in newly diagnosed AA patients suggest that these ...
The Hematology Journal (2002) 3, 169 ± 175 ã 2002 The European Hematology Association All rights reserved 1466 ± 4680/02 $25.00 www.nature.com/thj

HYPOTHESIS

Seeing the good and bad in aplastic anemia: is autoimmunity in AA dysregulated or antineoplastic? Catherine Nissen*,1 and JoÈrg Schubert2 1

Department of Hematology, University Hospital, CH-4031 Basel, Switzerland; 2Internal Medicine I, Saarland University Medical School, D-66421 Homburg, Germany

Aplastic anemia (AA) is considered to be an autoimmune disease directed against hematopoietic stem cells (HSC), though knowledge on the inciting autoantigen(s) is scant. According to the traditional concept of autoimmunity the target tissue in autoimmune disease is essentially normal, and misdirected self-attack is caused by disturbed self-recognition. Recently, this theory has been challenged by the hypothesis that autoimmunity against solid tissues is directed against intrinsically abnormal, transforming cells, i.e. autoimmune reactions are essentially antineoplastic, attempting to eliminate cells signalling `danger'. This theory might apply to AA as well. Observations such as the dysplastic traits typical of non-severe AA, the high prevalence of one or several abnormal hematopoietic clones and their resistance to apoptosis in newly diagnosed AA patients suggest that these cell populations do not develop secondarily, but expand primarily und could be the primary target of AA, normal hematopoietic stem cells being destroyed as innocent bystanders. If bone marrow hypoplasia/ aplasia indeed re¯ects an immune reaction incited by outgrowth of transformed cells, immunosuppressive treatment of AA would have to be reconsidered, since a two-edged sword. As a consequence, AA patients with a hyperreactive immune system may require more intense immunosuppressive therapy (IS), whereas patients with an anergic immune system may fare better with IS of lower intensity than the currently recommended standard. The Hematology Journal (2002) 3, 169 ± 175. doi:10.1038/sj.thj.6200179 Keywords:

aplastic anemia; autoimmunity; antineoplastic reactivity

State of the art Acquired aplastic anemia is generally accepted to be an autoimmune disease directed against hematopoietic stem cells, though the o€ending autoantigen is unknown. In contrast to congenital hematopoietic failure syndromes with de®ned genetic abnormalities1 no single genetic defect causing acquired AA is known to date. As in other autoimmune disorders, however, genetic predisposition is likely, as evidenced e.g. by overrepresentation of HL-A DR22 and of HL-A DR4, certain subtypes being associated with poor clinical outcome.3 Also, hypersensitivity to primary noxious agents in acquired AA appears to be genetically determined.4 The immune regulatory defects and/or stimuli inciting autoimmunity in AA are not clear, the fact that AA responds to immunosuppressive therapy *Correspondence: C Nissen, Department of Haematology, UniversitaÈtskliniken, Kantonsspital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland; E-mail: [email protected] Received 14 March 2002; accepted 24 April 2002

(IS) remains the most convincing evidence for autocytotoxicity against HSC. After IS, AA patients are left with a drastically reduced HSC pool. Many of them develop paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndrome (MDS), or acute myelogenous leukemia (AML).5 ± 7 Recovering hematopoiesis after IS is characterized by dysplastic features.8 ± 12 PNH clones are characterized genetically by somatic mutation(s) in the PIG-A (phosphatidylinositol-glycan-a) gene. Normals harbor minute ± below 0.003% ± populations of PIG-A-mutated blood cells.13 As a consequence of PIG-A mutations, the constituents of the anchor for GPI-linked proteins are not assembled intracellularly, the anchor is therefore not expressed and GPI-linked proteins are absent from the cell surface. That PIG-A-mutated cells are not destined to become malignant is evidenced by their presence in normals and their ability to coexist long-term with normal hematopoietic cells in mice14 and humans.15,16

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The PIG-A-mutated clones in AA are exceptional,17 as they have a tendency to expand beyond their apparently physiological size transiently or permanently, with or without causing clinical signs of PNH, ie intravascular hemolysis and thrombosis. The prevalence of expanded PNH clones in untreated AA is high.18 The pathogenetic link between AA and PNH is intricate, but too apparent to be accidental. According to the immune selection/immune escape theory proposed by Luzzatto's group,19 ± 21 autoimmunity in AA is directed against either the GPI-anchor or a GPI-linked protein, PNH cells being spared owing to nonexpression of the GPI anchor. Luzatto's idea implies the outgrowth of a dysregulated clone of immunocytes against GPI-linked proteins or their surface anchor. PNH clone(s) would thereby assume a growth advantage leading to their expansion in the aplastic marrow. The skewed T-cell repertoire in PNH patients is interpreted as presence of speci®c T cell clones recognizing the GPI-anchor itself,22 possibly presented by CD1d-molecules.23 Simultaneous occurrence of cytotoxic large granular lymphocyte leukemia (LGL) and PNH,24,25 according to the immune selection/escape theory, indicates that PNH clones expand whenever normal hematopoiesis is compromised by T-cellmediated attack.

Open questions and unexplained observations Which cells are the target of autoimmunity? According to the immune selection/immune escape theory the target of autoimmunity is a normal HSC expressing the GPI anchor, which is recognized as foreign by the immune system, anchor-negative PNH cells being spared. Whatever the mechanism may be by which T cells recognize the GPI anchor, we doubt that it is the target of autoimmunity in AA because: (i): An autoimmune attack against the GPI anchor would probably cause fatal ubiquitous tissue damage, since its expression is not restricted to HSC or (ii) PNH cells lacking the GPI-anchor, should be resistant to T cell attack. However, normal and GPI-de®cient cells are equally sensitive to the cytotoxic action of allogeneic T lymphocytes.26 As an alternative explanation it has been suggested that GPI-linked surface proteins on HSC such as ULBP's (Cytomegalovirus glycoprotein UL16 binding proteins)27 directly interfere with activating receptors (NKG2D) on NK cells.28 However, it is unknown to date if expression of these molecules is HSC speci®c, explaining why other tissues would be spared from such autoimmunity.

Subcellular localization of aplastic anemia-linked autoantigens In autoimmune disorders against solid tissues, peptides likely to elicit autoimmune reactions are `cryptic', i.e. intracellular, rather than surface-expressed, and preThe Hematology Journal

sented to the immune system upon desintegration of the cell.29,30 They induce T- rather than B-cell responses. In fact serum from most AA patients is stimulatory rather than inhibitory for HSC and thus probably does not contain antibodies against surface structures on HSC. In analogy to type I diabetes, multiple sclerosis and other autoimmune disorders the o€ending autoantigen in the AA/PNH-syndrome is likely to be intracellular rather than membrane-expressed. PNH cells contain the protein constituents of the GPI anchor in a non-assembled form but do not express them on the surface. It is therefore conceivable that such peptides play the role of `cryptic' antigen(s), which are sheltered from the immune system as long as the cells are intact. Accordingly, it has been suggested that PNH cells containing `hidden' GPI anchor protein fragments could be the primary target of autoimmunity.31,32 The three candidates of HSC-speci®c autoantigens described to date in AA are indeed intracellular proteins: heat shock protein,33 ribosomal nuclear protein S1234 and the ER-resided `kinectin'.35 Whether these putative autoantigens share properties with intracellular constituents of the non-expressed GPIanchor of PNH cells is not known. In a recent investigation CD4+ and CD8+ T cell clones showing Vb-restriction BV5 and BV13 respectively, have been isolated from patients with AA who express HLADRB1*15. These immortalized clones were cytotoxic against HSC in an HLA-restricted manner.36 Using these clones might be promising for the detection of further antigens recognised by the immune system.

Macrocytosis and monocytosis in aplastic anemia Untreated mild AA is indistinguishable from MDS,37,38 both have features of dysplasia, ie macrocytosis in the absence of vitamin de®ciency, increased HbF in the absence of a haemoglobinopathy and discrete morphological BM-abnormalities with or without chromosomal abnormalities. Macrocytosis is said to be a nonspeci®c sign of erythropoietic recovery, as typically seen eg during recovery from methotrexate toxicity. Spontaneous macrocytosis, however, is likely to re¯ect a defect at the DNA-level. During recovery after IS treatment of severe AA the same stigmata of dysplasia invariably appear and are particularly prominent during the early recovery phase. This suggests that a dysplastic population pre-exists, disappears when marrow failure becomes severe, and reappears upon recovery after IS. Macrocytosis has assumed a positive connotation because it heralds autologous marrow recovery after IS-treatment,39 despite the fact that it is an ackowledged sign of dysplasia. In the SAA/PNH/ MDS-syndrome macrocytosis could indicate that additional genetic abnormalities have occurred either in a PNH clone, which is known to be genetically unstable40 or that PIG-A mutation occurred in a previously transformed clone. Since in other cell systems previously mutated genetically unstable cells have a tendency to secondarily acquire PIG-A mutations,41 it is conceivable that the occurrence of a

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PIG-A mutation unmasks or ¯ags another more harmful genetic abnormality. Examples of such additional genetic aberrations are upregulation of genes involved in cellular proliferation,42 dysregulation between proliferation and di€erentiation or other, yet illde®ned abnormalities.43 ± 45 Such genetic changes may be subtle and escape standard screening for transformation, yet conferring a growth advantage at the stem cell level. The selective outgrowth of human PNH cells when co-cultured with their normal counterparts in vitro46,47 or in vivo in the SCID mouse48 may indicate that PNH cells derived from patients have acquired such an additional abnormality. They would thus di€er from experimentally PIG-A mutated mouse cells which have no tendency to expand. Whether one or several of the above mentioned genetic changes underlie macrocytosis in AA, has not been tested. Relative or absolute monocytosis is often seen in non-transplanted AA patients,49 it is particularly pronounced in those who later develop monocytic leukemia. Interestingly, expression of EGR-1 in the mouse is associated with commitment of myelopoiesis towards the monocyte lineage, at the expense of selfrenewal of pluripotent hematopoietic stem cells.50,51 Thus, EGR-1 upregulation seen in PNH white cells42 may be one of the genetic alterations conferring a growth advantage explaining monocytosis in AA.

Apoptosis resistance of clonal cells in paroxysmal nocturnal hemoglobinuria

hypoplastic MDS could re¯ect the immune system's attempt to eliminate such transformed cells. This hypothesis is in agreement with the new concept of autoimmunity against solid tissues proposed by Reines et al.,63 who claims that gradual malignant transformation of the target cell ± rather than dysregulation of the immune system ± is the primary event in autoimmunity, transformed cell signaling `danger' to the immune system. He suggests that the cause of transformation is genetically induced premature aging. 59 ± 62

Figures are given to illustrate the hypothesis In Figure 1, a proposal for the relationship between AA, PNH and MDS is shown: Rather than being separate diseases, they are part of a syndrome, as previously proposed by Barrett et al.64 for the MDS/AA syndrome. The core or common denominator is a PIGA-mutated population which has undergone further mutation (PIG-A plus X). Patients with primary normo/hypercellular MDS or PNH are not part of the syndrome; however, all patients who develop hypoplasia/aplasia invariably belong to it, since their natural history includes PNH and/or MDS, whether AA was originally virus-, drug-, toxin-induced or idiopathic (unpublished results of the University Clinic Basel, Switzerland). One might argue that patients in whom AA develops from autoimmune unilineage disease, e.g.

Brodsky et al52 found that PIG-A mutated cells are apoptosis-resistant compared to non-mutated cells, Horikawa et al53 documented that granulocytes not only from PNH, but also from AA and MDS patients are relatively resistant to apoptosis. Ware et al54 and Bastisch et al55 showed that apoptosis resistance of PNH cells is not due to lack of GPI linked proteins, ie, that it is an additional characteristic of human PNH cells. Since resistance to apoptosis is an acknowledged sign of malignant transformation, the phenomenon in PNH cells may indicate that these cells have aquired genetic abnormalities in addition to the PIG-Amutation. What is the link between these apparently independent observations, and how could they help ®tting the AA/PNH-puzzle together?

Aplastic anemia is an antineoplastic reaction We propose that in the AA/PNH- syndrome praeleukemic transformation occurs before the onset of aplasia and is the cause of immune-mediated marrow failure, the immune attack being directed against transformed cells. Primary causes of transformation could be, eg, premature aging or chemically or virally induced genetic mutations within the genome of HSC. Telomere shortening, a common feature of AA56 could be the expression of such a defect. Upregulation of Fas, typical of AA57,58 and apoptosis, as observed in

Figure 1 Proposed pathogenic link between aplastic anemia (AA), myelodysplasia (MDS) and paroxysmal nocturnal hemoglobinuria (PNH). Although there is evident heterogeneity comparing these three diseases, our proposed pathogenic link could consist of a PIG-A mutation plus X (i.e. moleculular alterations within genes responsible for cellular growth and activation). The Hematology Journal

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from pure red cell aplasia (PRCA), have a di€erent pathophysiology with a better long-term prognosis. Clinical observations, however, show that these patients also have an increased risk of clonal evolution.65 The time when these patients develop pancytopenia from originally benign unilineage failure might indicate the turning point towards dysplasia. In Figure 2, we show a hypothetical sequence of events, depending on the patient's immune-competence. Figure 2A): A noxious agent, eg a virus, a toxic agent or a drug causes transient HSC damage and induces repair involving hyperproliferation during which the incidence of PIG-A mutations is increased. Since PNH clones are genetically unstable and disposed to further mutations,66 additional genetic changes providing a growth advantage are likely to occur. This would lead to the presentation of an aberrant internal image on MHC molecules and thus to T cell recognition and activation. In autoimmunity against solid organs `the clinical outcome of a struggle between incipient neoplasia and immunity will vary depending upon the degree of tumor-proneness and resistance of the individual'.63 In analogy, we propose that the immune reaction against transformed HSC can be of three basically di€erent types (Figure 2B):

Hyporeactive/anergic response In patients with a compromised immune system, the abnormal population expands and displaces normal HSC. PNH patients, though surviving long term with the abnormal clone have signs of immune incompetence: they are prone to infections and have low endogenous IL-2 production.31 Clinically, these patients present with PNH if the clone is stable and does not progress to leukemia,15 or with MDS/AML if the clone undergoes further transformation.

Hyperreactive response Normal cell (HSC) Cells with an isolated PIG-A mutation PIG-A mutated cells with abnormalities conferring a growth advantage Cells with additional cytogenetic abnormalities T lymphocytes Transformation route of HSC Cytotoxic activity Putative T cell activation Missing T cell activation

Figure 2 Sequence of events (see text): (A) HSCs with an isolated PIG-A-mutation occur in normal bone marrow. Exposure to toxic agents causes cellular damage and induces proliferative stress in HSC. PIG-A mutated cells or normal cells undergo mutations within genes involved in proliferation and di€erentiation leading to predysplasia. (B) Predysplastic cells alert the immune system, which reacts either with anergy, leading to expansion of abnormal clones, with a hyperreactive autoimmune reaction leading to severe aplastic aneamia or with a selective immune reaction leading to cure. The Hematology Journal

In patients predisposed to autoimmunity, the immune reaction against the abnormal population extends to normal HSCs by the mechanism of `epitope spreading'. This causes severe aplasia with neither normal nor abnormal cells detectable in the BM. The more vigorous the immune reaction, the more acute and serious is BM failure and thereby the chance of transformed clones to be decimated. This immune attack puts pressure on mutated cells which attempt to survive by the development of new, potentially resistant clones. Clinical experience has shown that young, previously healthy patients typically develop very severe hyperacute AA, whereas infants and elderly patients more often present with mild pancytopenia and signs of MDS. This di€erence could be due to age-related di€erences of immune competence. The rare AA patients who ultimately recover hematopoiesis with no residual hematological abnormalities show that suppression and possibly cure from predysplasia is possible, as evidenced by the natural history of

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many patients with the SAA/PNH-syndrome. The fact that clonal transformation can occur even decades after successful IS either means that the original premalignant clone is kept `dormant', or that new premalignant clones arise under long-term hematopoietic stress conditions imposed by gross reduction of the stem cell pool.32 67 ± 69

Selective response A speci®c immune reaction destroys the abnormal population, sparing normal HSC, leading to reconstitution of normal hematopoiesis. Clinically unrecognized expansion and subsequent immunological destruction of small potentially life-threatening cell populations is probably the most frequent event.

Possible consequences For IS treatment of AA a standard dose of immunosuppressive drugs is recommended. If, however, transformed clones are primary and the patient's immune system plays a pivotal role in their eradication, IS treatment of AA is a two-edged sword: suppression of autoimmunity, though inducing life-saving auto-

logous hematopoietic recovery, has the side e€ect of tolerization towards the preexisting abnormal cell populations. This may have more negative than positive e€ects if the transformation process has reached an advanced stage prior to treatment. Individual IS protocols tailored according to the patient's immune competence could be a consequence, those with a hyperimmune reaction requiring more intense, those with signs of immunological energy faring better with IS below the recommended standard. This would imply systematic analysis of immune parameters in patients undergoing IS for the SAA/ PNH/MDS syndrome.

Acknowledgements The critical and constructive scienti®c input from our colleagues, Aleksandra Filipowicz, Arnold Ganser, Alois Gratwohl, Anna Lyakisheva, Reinhold E Schmidt, Andre Tichelli and Andreas Tiede was of invaluable help. We are greatly indebted to Brandon Reines, Ghost Lab, Laboratory of Cellular and Molecular Immunology, NIAID, NIH, USA for his invaluable help in preparing the manuscript. This work was supported by the Swiss Science foundation, grants 32-36278.92, 32-45926.95 and 32-55694.98 and by the Deutsche Forschungs Gesellschaft grant 713/7-1.

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