Paroxysmal nocturnal haemoglobinuria (PNH) is a unique disorder characterised by the triad of intravascular haemolysis, thrombosis and bone marrow failure. In the early seventies it was shown that PNH is a clonal disease; and in the nineties the molecular basis of the PNH abnormality was elucidated. However, what makes a PNH clone expand is still not known. Here, we suggest that this is due to somatic cell selection, resulting from the presence in the patient of autoreactive T cells that target glycosylphosphatidylinositol (GPI) in the context of an MHC-like molecule on the surface of haemopoietic stem cells. PNH cells would escape damage precisely because they have lost most or all of their ability to produce GPI.
If we look back at our efforts to understand the nature of a disease condition, there are few examples with such a variegated history as paroxysmal nocturnal haemoglobinuria (PNH). In fact, whenever a new advance in PNH has answered a specific question, it has posed at the same time a new question. For instance, the early finding of haemoglobinuria clearly indicated intravascular haemolysis,1 but the cause was puzzling because a high-titre autoantibody was not found. Next it was shown that haemolysis was due to abnormal susceptibility to complement,2,3 but one wondered why this abnormality was limited to only a subset of all red cells. This led to a successful test of the notion that PNH was a clonal disorder due to a somatic mutation:4,5 but very soon this seemed to clash with the finding that not just one, but so many different surface proteins were deficient in PNH cells. This paradox was solved by the finding that all these proteins had a common non-protein moiety, a glycolipid which has since become known as the glucosylphosphatidylinositol (GPI) anchor, because it tethers each one of these proteins to the cell's surface by virtue of the fact that its fatty acid residues are embedded in the membrane's lipid bilayer6 (see Figure 1). Thus, deficiency of one of the enzymes required for the biosynthesis of GPI became a good candidate for explaining the PNH phenotype, but it was puzzling how a single mutation (ie on one allele only), could cause complete deficiency of an enzyme. This difficulty was solved when the biosynthetic gene PIG-A was cloned and mapped to the X chromosome, thus neatly explaining how one hit could cause complete loss of function of the gene product.7 Today, we can define PNH as an acquired clonal disorder of the haemopoietic stem cell (HSC). Somatic mutations in the X-linked PIG-A gene of one or more HSCs result in the deficiency of GPI-anchored proteins from the surface of blood cells. The mutated (PNH) HSC(s) are able to expand to such an extent that in a patient with PNH the PNH blood cells may comprise up to >90% of the total haemopoiesis. However, we still do not know what determines (1) the size of the PNH clone, (2) the severity of the patient's pancytopenia and (3) the increased propensity to venous thrombosis: ie the three most important features of this condition in terms of clinical course and clinical outcome.8 Here, we advance a specific hypothesis that may help to answer these questions.
The vast expansion of a PNH clone that is seen in a patient with PNH cannot take place without a growth advantage. We have previously suggested that this growth advantage, rather than being absolute (as it is in leukaemia), might be relative or conditional, ie dependent on a particular bone marrow environment.9 Specifically, an injury to the normal (GPI+) HSC might spare PNH (GPI−) HSC. While one might certainly conceive of physical, chemical or biological agents being able to discriminate between GPI+ and GPI− HSC, an obvious possibility is that the damaging agent is part of an auto-immune process (see Figure 2) especially, since this is a widely accepted pathogenetic mechanism in the closely related condition, idiopathic aplastic anaemia (IAA).10 Here, we explore this model, leading on to the notion that the target of this auto-immune process could be the GPI molecule itself on HSCs.
PIG-A mutations are necessary but not sufficient to cause PNH disease
First, we summarize the current evidence that, once a PIG-A mutation has taken place, there must be a mechanism for positive selection of the mutant cells, or negative selection against the non-mutated cells (dual pathogenesis of PNH). There are essentially four lines of evidence. (1) PIG-A mutations are found regularly in normal people.11 (2) Non-mutated hematopoietic cells are decreased in absolute terms in patients with PNH (as in IAA).12 (3) Patients with PNH not infrequently have more than one mutant clone; and sometimes a different PNH clone can be shown to be dominant in the same patient at different points in time.13 (4) In mice with targeted inactivation of the pig-a gene the PNH cell population does not increase in time, and often tends to disappear.14,15,16 This last finding does not support positive selection for mutant cells; by implication, it favours instead the notion that negative selection against non-mutant cells dominates the picture in human PNH patients.
What is the target and what is the mechanism of negative selection?
We will make the specific assumption that an autoreactive population of cytotoxic T cells inhibits the growth or causes the demise of non-mutated HSC: ie we postulate that these cells have a differential effect on GPI+ and GPI− HSCs (escape model).9 An increased frequency of clonal T cell expansions in patients with PNH is in line with this concept.17 In addition, we have recently observed two patients with an association of T-LGL and PNH.18 According to this model, we consider four possible alternative targets and cognate mechanisms of selection.
The deficiency of GPI-anchored molecules from the surface of PNH cells helps in itself to protect them from T cell-mediated attack
Indeed, it is conceivable that a GPI-linked protein is essential for effective recognition or killing by a cytotoxic T cell. However, we and others have shown in internally controlled experiments that there is no significant difference in the ability of T cells or NK cells to kill PNH haemopoietic cells vs normal, isogenic haemopoietic cells.19
The target of autoaggressive T cells is an epitope belonging to the PIG-A protein itself
Because PIG-A is an intracellular protein it can become itself antigenic only if critical peptide(s) arising through endogenous processing pathways are then displayed to pathogenic cytotoxic T cells in the groove of an HLA class I molecule.20 Thus, non-PNH HSCs would process and present the putative PIG-A-derived peptide and become targets of T cells; in contrast, PNH HSCs would escape the T cell attack because, by failing to produce the PIG-A protein as a result of a PIG-A mutation they would not present the critical peptide. A strong argument against this possibility is the fact that PIG-A is ubiquitously expressed:7 therefore, the immune process would be expected to damage cells in a variety of tissues. Instead, PNH is strictly a disease of haemopoiesis. In addition, there are many missense mutations in PIG-A in PNH,21 and at least some of them would still produce the putative critical peptide(s). Therefore, it is unlikely that PIG-A is the target we are looking for.
The target of autoaggressive T cells is an epitope derived from a GPI-anchored protein
Although proteins destined to be GPI-linked fail to land on the surface of PNH cells because they do not find the anchor, they are as efficiently transcribed and translated in PNH cells as in non-PNH cells. This would result, in PNH cells, in the accumulation of unprocessed proteins which, in association with chaperones residing in the endoplasmic reticulum, are transferred to the cytoplasm where they are degraded.22 Thus, although it has been suggested that PNH cells may present a critical epitope less efficiently than GPI+ cells,23,24 there is no experimental evidence to support this notion. Therefore, peptides from GPI-linked proteins do not seem at the moment good candidate targets either.
GPI itself is the target of autoaggressive T cells
It is now well established that, just as HLA molecules present peptide antigens, the β2-microglobulin-associated CD1 molecules (CD1a, CD1b, CD1c, CD1d), expressed on professional antigen presenting cells (APC; for example, dendritic cells), present lipids or glycolipids to T cells.25 CD1a, b and c-restricted, lipid-specific immune responses have been implicated in the control of intracellular pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae.25 Similarly, CD1d-restricted, glycolipid-specific T cell responses may be involved in the pathogenesis of autoimmune disease,26 as well as in the immunity against intracellular pathogens27 and possibly against certain types of cancer.28 A finding potentially very pertinent to PNH is the that murine CD1d contains GPI in its presentation groove,29 and parasite GPI may be the target of protective T cell immunity in murine malaria.30
On the basis of these data, we favour the hypothesis that CD1d-restricted, GPI-specific T cells are central to the pathogenesis of PNH. Specifically, we envisage that some of the expanded T cell clones – which we have demonstrated in PNH patients – recognize GPI presented in the context of CD1d by HSCs, and thus attack these cells. By contrast, the PNH HSC, since they synthesise very little GPI, or none at all, will be able to escape the T cell attack. This model implies that pluripotential HSCs must express CD1d at some stage. This has not yet been tested.
This hypothesis might also help to explain the pathogenesis of venous thrombosis, one of the most dreaded complications of PNH, since it affects up to 40% of patients and it is a leading cause of mortality.8 For reasons which have been obscure hitherto, in PNH venous thrombosis has a special predilection for intra-abdominal and cerebral veins.8 Interestingly, CD1d, among other tissues, is also expressed in the muscle layer of abdominal blood vessels.31
Testing hypothesis 4
The crucial proof for this model must consist in demonstrating the presence in PNH patients of autoreactive T cells that recognize the GPI molecule. The identification of antigen-specific T cells in clinical samples has relied traditionally on stimulating their growth by having the appropriate antigenic peptide presented by appropriate APC. The functional read-out consists in cytotoxicity, proliferation assays or cytokine secretion assays. A recent powerful advance has been the introduction of HLA class I-peptide tetrameric complexes32,33 (Figure 3a). By using tetramer technology it is possible to identify, quantitate and sort specific CD8+ T cells with a sensitivity as high as 0.01%.34,35
We propose therefore that using CD1d/β2m/GPI tetramers would be the best way to experimentally test our hypothesis. In order to provide proof of principle that this approach is feasible, we have already constructed CD1d tetramers, with the glycolipid α-galactosylceramide (α-GalCer) in the presentation groove. The sensitivity of this technique has been validated36 (see Figure 3b). The next step would be to construct CD1d tetramers with GPI instead of α-GalCer, and use them to stain lymphocytes from PNH patients and control individuals. CD1d/GPI tetramer+ T cells can be then flow-sorted, and their specificity confirmed in proliferation, cytotoxicity or cytokine secretion assays in the presence of CD1d-expressing APC pulsed with GPI. Finally, the role of these cells in the pathogenesis of PNH could be proved directly by showing, in functional assays, that they are able to inhibit normal haemopoiesis but not PNH haemopoiesis.
At the time the escape model for the pathogenesis of PNH was first proposed, more than a decade ago,9 it was merely a concept based on the need to explain why the PNH clone was able to expand, and yet it did not have the features of leukaemia. The model has proved to have heuristic value, because we now know the molecular basis for the PNH abnormality, we know that PNH clones are present but do not expand in normal people,11 and we know that PNH cells tend to disappear rather than to expand in mouse model systems.14,15,16 Moreover, we have evidence that the conditional selective agent we had postulated may consist in T cell clones, because we have demonstrated such clones in a substantial proportion of PNH patients.17 CD1d tetramer technology may help to prove that these clones have the properties that we have hypothetically ascribed to them. Most important, if the hypothesis is correct, this may open up new possibilities for the treatment of PNH, and possibly of other conditions in which similar pathogenetic mechanisms are in operation.
Strubing P . Paroxysmale hamoglobinurie Dtsch Med Wochenschr 1882 8: 1–8
Dacie JV, Israels MCG, Wilkinson JF . Paroxysmal nocturnal haemoglobinuria of the Marchiafava type Lancet 1939 i: 479–482
Ham TH, Dingle JH . Studies on destruction of red blood cells. II. Chronic hemolytic anemia with paroxysmal nocturnal hemoglobinuria: certain immunological aspects of the hemolytic mechanism with special reference to serum complement J Clin Invest 1938 18: 657–672
Lewis SM, Dacie JV . The aplastic anaemia–paroxysmal nocturnal haemoglobinuria syndrome Br J Haematol 1967 13: 236–251
Oni SB, Osunkoya BO, Luzzatto L . Paroxysmal nocturnal hemoglobinuria: evidence for monoclonal origin of abnormal red cells Blood 1970 36: 145–152
Englund PT . The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors Annu Rev Biochem 1993 62: 121–138
Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T . Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria Cell 1993 73: 703–711
Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV . Natural history of paroxysmal nocturnal hemoglobinuria N Engl J Med 1995 333: 1253–1258
Rotoli B, Luzzatto L . Paroxysmal nocturnal haemoglobinuria Baillières Clin Haematol 1989 2: 113–138
Young NS, Maciejewski J . The pathophysiology of acquired aplastic anemia N Engl J Med 1997 336: 1365–1372
Araten DJ, Nafa K, Pakdeesuwan K, Luzzatto L . Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals Proc Natl Acad Sci USA 1999 96: 5209–5214
Maciejewski JP, Sloand EM, Sato T, Anderson S, Young NS . Impaired hematopoiesis in paroxysmal nocturnal hemo-globinuria/aplastic anemia is not associated with a selective proliferative defect in the glycosylphosphatidylinositol-anchored protein-deficient clone Blood 1997 89: 1173–1181
Luzzatto L, Nafa K . Genetics of PNH In: Young NS, Moss J (eds) PNH and GPI-linked Proteins Academic Press: San Diego 2000 pp 21–48
Tremml G, Dominguez C, Rosti V, Zhang Z, Pandolfi PP, Keller P, Bessler M . Increased sensitivity to complement and a decreased red blood cell life span in mice mosaic for a nonfunctional Piga gene (see comments) Blood 1999 94: 2945–2954
Rosti V, Tremml G, Soares V, Pandolfi PP, Luzzatto L, Bessler M . Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion (see comments) J Clin Invest 1997 100: 1028–1036
Murakami Y, Kinoshita T, Maeda Y, Nakano T, Kosaka H, Takeda J . Different roles of glycosylphosphatidylinositol in various hematopoietic cells as revealed by a mouse model of paroxysmal nocturnal hemoglobinuria Blood 1999 94: 2963–2970
Karadimitris A, Manavalan JS, Thaler HT, Notaro R, Araten DJ, Nafa K, Roberts IA, Weksler ME, Luzzatto L . Abnormal T-cell repertoire is consistent with immune process underlying the pathogenesis of paroxysmal nocturnal hemoglobinuria Blood 2000 96: 2613–2620
Karadimitris A, Li K, Notaro R, Araten D, Nafa K, Thertulien R, Ladanyi M, Stevens A, Roberts I, Luzzatto L . A novel association of T-LGL leukemia and PNH supports the central role f a T-cell-mediated immune response in the pathogenesis of PNH Blood 2000 96: 995a
Karadimitris A, Notaro R, Koehne G, Roberts IA, Luzzatto L . PNH cells are as sensitive to T-cell-mediated lysis as their normal counterparts: implications for the pathogenesis of paroxysmal nocturnal haemoglobinuria Br J Haematol 2000 111: 1158–1163
Pamer E, Cresswell P . Mechanisms of MHC class I-restricted antigen processing Annu Rev Immunol 1998 16: 323–358
Nafa K, Mason PJ, Hillmen P, Luzzatto L, Bessler M . Mutations in the PIG-A gene causing paroxysmal nocturnal hemoglobinuria are mainly of the frameshift type Blood 1995 86: 4650–4655
Wang Y, Chen D, Androlewicz MJ . The role of endoplasmic reticulum-associated protein degradation in MHC class I antigen processing Immunol Rev 1999 172: 67–72
Dunn DE, Liu JM, Young NS . Bone marrow failure in PNH In: Young NS, Moss J (eds) PNH and GPI-linked Proteins Academic Press: San Diego 2000 pp 115–131
Young NS, Maciejewski JP . Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes ‘Why? Why? Why?’ J Clin Invest 2000 106: 637–641
Porcelli SA, Modlin RL . The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids Annu Rev Immunol 1999 17: 297–329
Wilson SB, Kent SC, Patton KT, Orban T, Jackson RA, Exley M, Porcelli S, Schatz DA, Atkinson MA, Balk SP, Strominger JL, Hafler DA . Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes Nature 1998 391: 177–181
Kumar H, Belperron A, Barthold SW, Bockenstedt LK . Cutting edge: CD1d deficiency impairs murine host defense against the spirochete, borrelia burgdorferi J Immunol 2000 165: 4797–4801
Toura I, Kawano T, Akutsu Y, Nakayama T, Ochiai T, Taniguchi M . Cutting edge: inhibition of experimental tumor metastasis by dendritic cells pulsed with alpha-galactosylceramide J Immunol 1999 163: 2387–2391
Joyce S, Woods AS, Yewdell JW, Bennink JR, De Silva AD, Boesteanu A, Balk SP, Cotter RJ, Brutkiewicz RR . Natural ligand of mouse CD1d1: cellular glycosylphosphatidylinositol Science 1998 279: 1541–1544
Schofield L, McConville MJ, Hansen D, Campbell AS, Fraser-Reid B, Grusby MJ, Tachado SD . CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells Science 1999 283: 225–229
Canchis PW, Bhan AK, Landau SB, Yang L, Balk SP, Blumberg RS . Tissue distribution of the non-polymorphic major histocompatibility complex class I-like molecule, CD1d Immunology 1993 80: 561–565
Ogg GS, McMichael AJ . HLA-peptide tetrameric complexes Curr Opin Immunol 1998 10: 393–396
Altman JD, Moss PAH, Goulder PJR, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM . Phenotypic analysis of antigen-specific T lymphocytes Science 1996 274: 94–96
Dunbar PR, Ogg GS, Chen J, Rust N, van der Bruggen P, Cerundolo V . Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood Curr Biol 1998 8: 413–416
He XS, Rehermann B, Lopez-Labrador FX, Boisvert J, Cheung R, Mumm J, Wedemeyer H, Berenguer M, Wright TL, Davis MM, Greenberg HB . Quantitative analysis of hepatitis C virus-specific CD8(+) T cells in peripheral blood and liver using peptide-MHC tetramers Proc Natl Acad Sci USA 1999 96: 5692–5697
Karadimitris A, Gadola S, Altamirano M, Brown D, Woolfson A, Klenerman P, Chen JL, Koezuka Y, Roberts IA, Price DA, Dusheiko G, Milstein C, Fersht A, Luzzatto L, Cerundolo V . From the cover: human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography Proc Natl Acad Sci USA 2001 98: 3294–3298
We warmly thank all the present and past members of the Human Molecular Genetics Laboratory in the Department of Human Genetics at MSKCC, New York; the ideas discussed in this paper have developed through the hard work of everyone in the PNH group. In particular, we thank David Araten, Monica Bessler, Khedoudja Nafa, Rosario Notaro and Vittorio Rosti. We also thank immensely Vincenzo Cerundolo and Stephan Gadola of Oxford, UK, for their role in the CD1 tetramer work. Finally, we thank Irene AG Roberts, London, for her enthusiastic support of our endeavours; and Bruno Rotoli of Napoli, Italy with whom for two decades we have shared efforts to shed light on the cellular pathogenesis of PNH. The authors’ research work has been supported by a grant from the National Institutes of Health (No. RO1 HL56778).
About this article
Cite this article
Karadimitris, A., Luzzatto, L. The cellular pathogenesis of paroxysmal nocturnal haemoglobinuria. Leukemia 15, 1148–1152 (2001). https://doi.org/10.1038/sj.leu.2402180
- paroxysmal nocturnal haemoglobinuria (PNH)
- stem cells
- clonal selection
- autoreactive T cells
- glycosylphosphatidylinositol (GPI)
This article is cited by
Long noncoding RNA FAM157C contributes to clonal proliferation in paroxysmal nocturnal hemoglobinuria
Annals of Hematology (2023)
A Pig-a conditional knock-out mice model mediated by Vav-iCre: stable GPI-deficient and mild hemolysis
Experimental Hematology & Oncology (2022)
Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study
Annals of Hematology (2019)
Nature Medicine (2006)
Telomere length dynamics in normal hematopoiesis and in disease states characterized by increased stem cell turnover