CHAPTER 1 Basic sc*ience of ITP Editor: John W. Semple
1.1 Megakaryocyte differentiation and platelet produc*tion A.S. Weyrich
1.2 Autoimmune mechanisms and T regula*tory cell disturbances in ITP K. Yazdanbakhsh
1.3 Mouse models o*f ITP J.W. Semple
1.4 Peptide therapy for patients with* ITP S.J. Urbaniak * Basic science of ITP
1.1 Megakaryocyte differen*tiation and platelet production
Andrew S. Weyrich CHAPTER 1.1 • Megakaryocyte differentiation and platelet production
1. Introduction Platelets are anucleate cells that circulate in the bloodstream for approximately 10 days. The average adult must produce roughly 1 x 10 11 platelets per day to maintain normal platelet counts, a level of production that increases dramatically in a variety of clinical scenarios (1). In 1906, Wright provided the first evidence that megakaryocytes give rise to blood platelets. Since then, our understanding of the molecular basis of thrombopoiesis has progressed substantially and is arguably in a logarithmic growth phase. This chapter will review our current understanding of thrombopoiesis and highlight how the field is evolving. The history of megakaryocytes and platelets is fairly young. In 1841, Addison first described platelets and in 1882, Bizzozero named and identified platelets in the circulation and determined that they could induce clotting. In 1890, Howell named megakaryocytes, and 16 years later, Wright discovered that these were actually the precursors of platelets. Thus, the late 1880’s and early 1900’s were a period of prolific activity in the elucidation of megakaryocytes. Several discoveries were made about platelets and also about erythropoietin, which implied that a humoral substance also regulated platelet production though its exact nature was not yet known. In 1958, the term “thrombopoietin” (TPO) was coined, which means stimulator of “thrombopoiesis” or “platelet production”. In the early 1990’s, this substance was identified, and in 1994, several articles published in Science and Nature demonstrated that TPO can, in fact, increase platelet counts. Since that time, recombinant TPO and, more recently, TPO mimetics have been used clinically, as discussed in later chapters.
2. Megakaryocytes and thrombopoietin Platelets sprout from the cytoplasm of megakaryocytes (2, 3). Megakaryocytes are rare cells in the bone marrow; there is also literature to suggest that megakaryocytes can be found in peripheral blood and in the lungs. In vivo , it has been shown that bone marrow derived megakaryocytes produce platelets. Megakaryocytes originate from a stem cell progenitor in the bone marrow ( Figure 1 ) (4). In the presence of cytokines that include TPO, interleukins such as IL-3 and IL-6, and SDF (stromal-cell derived factor) the pluripotent haematopoietic stem cell (HPC) goes through endomitosis and DNA replication to produce a mature megakaryocyte. From the cytoplasm of the mature megakaryocyte sprouts numerous platelets. Though all above-stated cytokines are important, TPO has been found to be critical in this process (1). TPO signals via c-Mpl, which is expressed by megakaryocyte progenitors, megakaryocytes and their progeny. TPO is an acidic glycoprotein produced mainly
13 IMMUNE THROMBOCYTOPENIA Figure 1: Regulation of megakaryopoiesis
Megakaryopoiesis Cytokines and chemokines F ) 4 S T HSC R C E C
- l X M a C i / l G i
1 , - m
CFU-GEMM 2 F a 1 f D -
S
, , T 1 M 1 A CFU-EM -
C , ( 6
l -
p , 3 M
BFU-EM - / L O I P 4 T F CFU-Meg P
Mature megakaryocyte
Platelets
Modified from (4)
by the liver and the kidneys, and also by some stromal cells of the bone marrow. It stimulates thrombopoiesis by both enhancing proliferation of megakaryocyte progenitors and supporting differentiation of these progenitors into platelet producing cells. The discovery of TPO has led to the development of new drugs to treat thrombocytopenia (5) and the evolvement of megakaryocyte cultures that reconstitute platelet formation in vitro (2, 6). In conditions of thrombocytosis or thrombocytopenia, TPO levels are altered appropriately ( Figure 2 ) (1). In fact, platelets in the periphery help to adjust TPO levels. In this regard, when TPO levels are too high, the platelets take up TPO, which then leads to a reduction in megakaryocytes in the bone marrow and reduced platelet production. In conditions of thrombocytopenia, the bone marrow senses increased levels of TPO, as the platelets are not present in high enough concentrations to “quench” it. This leads to increased numbers of bone marrow megakaryocytes, raising the platelet count. Thus, this delicate balance is controlled both in the periphery and in the bone marrow, with TPO serving as a sensor.
3. Proplatelet formation The proplatelet theory, put forth by Joseph Italiano and his group (2, 7, 8), is
14 THE HANDBOOK FIRST EDITION CHAPTER 1.1 • Megakaryocyte differentiation and platelet production
Figure 2: Thrombopoietin regulates platelet production
Liver Kidney
TPO
Thrombocytosis Thrombocytopenia
Bone marrow Megakaryocyte
Reduced Enhanced thrombopoiesis thrombopoiesis
Modified from (1) currently well accepted. According to this theory, proplatelet formation is a prerequisite step used by megakaryocytes to package granules and organelles into platelets. Haematopoietic progenitors mature and eventually reach a stage where they begin to take on platelet and megakaryocyte phenotypic markers and have granules in their cytoplasm. In the final stages of maturation, they undergo endomitosis, where the DNA replicates but the cell does not divide. There is maturation of the cytoplasm and then microtubules start moving towards the periphery. In the final stages of this process, cytoplasmic extensions are generated into which organelles and granules are targeted into bulbs that become individual platelets. The extensions eventually reach into venous sinusoids and are released into the circulation and are clipped off, a process that releases individual platelets into the circulation. Proplatelets are characteristically rich in tubulin and have the appearance of beads linked by thin cytoplasmic bridges. Microtubules propel proplatelet formation and granules ride microtubular-rich shafts before entering nascent platelet buds. Microtubules also facilitate platelet release (3, 8). Our group has looked at this process by studying platelet formation in megakaryocytes generated from pluripotent HPC isolated from human cord blood (9). Human progenitors have a very small amount of cytoplasm, but are rich in ribosomes and translational machinery so they can support cell division and proliferation. When TPO is added to these cultured HPC, together with other cytokines, such as IL-3,
15 IMMUNE THROMBOCYTOPENIA the haematopoietic progenitor proliferates and begin to differentiate into a megakaryocytes that express platelet markers such as integrin IIb (Figure 3 ). Thus, one of the key things that TPO does is to increase the number of megakaryocyte progenitor cells present in the bone marrow. In human in vitro culture systems, progenitor cells differentiate into megakaryocytes over a period of twelve to thirteen days. During the final stages of maturation, the differentiated megakaryocytes have distinct organelles, granules and long proplatelet extensions sprout from their cytoplasm ( Figure 4 ) (6, 10). Thus, the process in humans is very similar to those originally described in murine model systems.
Figure 3: Haematopoietic CD34 + cells differentiate into megakaryocytes that a express integrin IIb
CD34 + cells Megakaryocyte Megakaryocytes precursors
This research was originally published in Blood. Foulks et al. PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids. Blood 2009;113:6699-6706. © the American Society of Hematology (9). It shows freshly isolated CD34 + cells (culture day 0), megakaryocyte precursors (culture day 7), or differentiated megakaryocytes (culture day 13) that are adherent to immobilised fibrinogen for 1 hour. The cells are stained for sialic acids (wheat germ agglutinin [WGA],
green) and integrin aIIb (red). Scale bar = 50 mm.
4. RNAs and translational machinery in platelets Recent evidence shows that platelets are not as simple as has been thought. Like all other cells they have ongoing protein synthesis and protein turnover to allow them to maintain equilibrium and homeostasis under normal conditions. Our group and others have shown, that during proplatelet production, in addition to the platelets receiving granules, cytoskeletal and tubular elements, messenger RNAs (mRNA), microRNAs, and translational machinery are also delivered to the platelets (Figure 4) (6, 10, 11). Our hypothesis has been that megakaryocytes transfer mRNA to anuclear platelets. In the case of megakaryocytes, it was thought for a long time that the mRNA got
16 THE HANDBOOK FIRST EDITION CHAPTER 1.1 • Megakaryocyte differentiation and platelet production
Figure 4: Haematopoietic CD34 + cells that differentiate into megakaryocytes develop proplatelet extensions
Day 8 Day 14 Transition phase Proplatelets Adherent 1 hr 6 S / A G W
This research was originally published in Blood. Weyrich et al. mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets. Blood 2007;109:1975-1983. © the American Society of Hematology (6). It shows that megakaryocytes transfer granules to proplatelets during thrombopoiesis. The megakaryocytes were cultured from CD34 + haematopoietic stem cells and stained with WGA, which localises to sialic acid-rich granules. The panel shows a megakaryocyte precursor (culture day 8) and differentiated megakaryocytes that are adherent to fibrinogen. Over time, fibrinogen-adherent megakaryocytes transition into proplatelets as described (6). WGA staining is identified by red fluorescence. S6K1 (green fluorescence), which controls translation, was also present in the cells as previously reported (6). Scale bars = 20 mm. translated into functional protein, and that protein was then packaged into platelets. But our group, as well as others, have now shown that in addition to packaging proteins into platelets, the megakaryocyte also delivers mRNA to platelets. We also surprisingly found that megakaryocytes transfer a subset of precursor mRNAs and requisite splicing machinery to platelets (10). Platelets receive approximately 4,000-6,000 mRNAs from parent megakaryocytes. These mRNAs are very translatable. Like mRNAs from nucleated cells, they have poly- A tails and 5’ caps (11). Figure 5 shows an example of mRNA expression in megakaryocytes and platelets (12). The top panel shows a human megakaryocyte that is giving off proplatelet shafts, and shows mRNA in the dark plaques in the cytoplasm, which also goes out into the individual platelets. mRNA is also detected in platelets freshly isolated from human blood that were left untreated or adhered to extracellular matrix (bottom panel). Circulating platelets are capable of translating mRNA into protein. Freshly-isolated human platelets readily take up labelled amino acids, which are incorporated into newly-synthesised proteins. In situ studies reveal that all the platelets in the population take up the label, indicating that protein synthesis is not restricted to young platelets (13). Synthetic events in platelets are blocked by puromycin, which is a translational inhibitor.
17 IMMUNE THROMBOCYTOPENIA Figure 5: Megakaryocytes transfer mRNA to platelets
TF Pre-mRNA TF Pre-mRNA A Megakaryocytes Proplatelets T R T R
o n
TF Pre-mRNA TF mRNA B Quiescent platelets Activated platelets T R T R
o n
This figure is taken from Schwertz et al., 2006. Originally published in J Exp Med doi:10.1084/jem.20061302 (12). Panel A shows in situ tissue factor (TF) precursor mRNA expression in the cytoplasm of megakaryocytes (top left) and in proplatelets that extend from megakaryocytes (top right). The bottom panels are controls where reverse transcriptase (RT) was omitted from the RT reaction (no RT). Panel B shows tissue factor precursor mRNA expression in quiescent platelets (top left) and mature tissue factor mRNA expression in platelets adherent to fibrinogen in the presence of thrombin for 1 h (top right). The bottom panels are controls where reverse transcriptase (RT) was omitted from the RT reaction (no RT). Interleukin- 1b precursor and mature mRNA has also been detected in situ in megakaryocytes and platelets (10).
18 THE HANDBOOK FIRST EDITION CHAPTER 1.1 • Megakaryocyte differentiation and platelet production
5. Ongoing platelet formation and differentiation When platelets are removed from the body and cultured, they use their protein synthetic machinery to develop extensions that resemble proplatelets that derive from the cytoplasm of megakaryocytes (13). This indicated to us that platelets could do the same thing as megakaryocytes, and further experiments indicated that was the case ( Figure 6 ). The left-hand panel shows freshly isolated platelets-small round cells that are all around two microns without any extensions or sprouts. Over six hours in culture, the platelets begin to take on long extensions. They vary in their
Figure 6: Freshly-isolated platelets extend projections with distinct cell bodies
A Baseline Cultured n i t c A
B Baseline Cultured
This research was originally published in Blood. Schwertz et al. Anucleate platelets generate progeny. Blood 2010;115:3801-3809. © the American Society of Hematology (13). In panel A, freshly-isolated human platelets were fixed immediately (baseline) or cultured for 6 hours in suspension. The green platelets are stained with phalloidin, which identifies polymerised actin. The bottom row displays corresponding transmission images. Scale bars = 5 mm. Panel B displays a lower magnification of freshly- isolated platelets that were stained for actin at baseline or after they were cultured for 6 hours. Scale bars = 10 mm.
19 IMMUNE THROMBOCYTOPENIA appearance - the majority of them look like dumbbells, but there are also long extensions as well, just as would be seen coming off a megakaryocyte. Individual platelets, which were packed inside surfactant bubbles, also developed extensions. This indicates that one platelet can actually change into two platelets. This suggested to us that the same type of process is going on in the small platelet as in a megakaryocyte, indicating that differentiation may continue in platelets as they go out into the circulation. If you take high microscopy and look at the new cell bodies, you find that each bulbar extension has granules and organelles inside ( Figure 7 ). We also found that platelets with these newly formed cell bodies express critical markers, including a b b integrin IIb 3, -tubulin coils that go through the cell membrane, P-selectin in the granules, and respiring mitochondria. These are all found in the platelets that have been cultured for six hours, at similar levels to those be expected in freshly isolated platelets. Furthermore, if the cells are activated after six hours of culture, a b they express integrin IIb 3 in its activated form on the surface the same levels as in freshly isolated platelets (13). Cleavage furrows are also very prominent,
Figure 7: Human platelets that develop new cell bodies express critical biomarkers
Cultured
-tubulin WGA αllb β3 P-selectin β IgG Mitotracker Thrombin added s t e l e t a l p
d e t a l o s i - y l h s e r F
This research was originally published in Blood. Schwertz et al. Anucleate platelets generate progeny. Blood 2010;115:3801-3809. © the American Society of Hematology (13). Freshly-isolated human platelets were cultured (6 hrs) alone or in the presence of thrombin (far right panels). From left to right in the top row, the red stain identifies integrin aIIb b3, P-selectin, b-tubulin, control IgG, respiring mitochondria (Mitotracker), or sialic acids (WGA). Corresponding transmission images are shown in the bottom row. Scale bars = 5 mm.
20 THE HANDBOOK FIRST EDITION CHAPTER 1.1 • Megakaryocyte differentiation and platelet production indicating that this process of extension may have a molecular trigger that allows platelets to split away from one another (13). Again, we think that this process mirrors what goes on in the megakaryocyte and probably continues to some extent in the circulation. The hypothesis that platelets continue to differentiate in the circulation was originally proposed by Benhke and Forer in 1998 (14). Our observations support this idea. This suggests that platelets continue to adjust their phenotype in the circulation, which could be absolutely critical in conditions of thrombocytopenia. These experiments were done in culture systems, but we have also shown that platelets with multiple cell bodies are present in freshly-isolated whole blood (13). The frequencies of these types of platelets, whose aetiology is unknown, often increases in thrombocytopenic situations.
6. Conclusion Much has been learnt about platelet formation using megakaryocyte cultures that reconstitute platelet formation in vitro . Culture-derived megakaryocytes produce cytoplasmic extensions referred to as proplatelets, which ultimately bud individual platelets. Proplatelet formation is a prerequisite step used by megakaryocytes to package granules and organelles, granular constituents, b-tubulin, and cytoskeletal elements into circulating platelets. Proplatelet formation is a dynamic process that unfolds over hours. In vivo studies confirmed that bone marrow megakaryocytes project proplatelets through venous sinusoids (15). Proplatelet-like structures are also commonly observed in whole blood, especially when platelets are being produced at increased rates. These elongated structures are, in part, derived from proplatelets that fragment from the megakaryocyte cell body. However, there is also recent evidence that discoid platelets generate new cell bodies (13). Progeny formation by circulating platelets mirrors proplatelet formation by megakaryocytes. In this regard, the generation of new cell bodies from individual platelets occur over hours, is mediated by b-tubulin, and granules are partitioned into new platelet buds. Understanding the molecular signals that control these events, both in the megakaryocyte and circulating platelet, will lead to new discoveries and potentially improved treatments for thrombocytopenia.
Acknowledgements Hansjörg Schwertz, Neal Tolley, Jesse Rowley, author’s laboratory. Joe Italiano and Jonathan Thon, Harvard Medical School. Sarah Köster, University of Göttingen. Walter Kahr, Hospital for Sick Children, Toronto.
21 IMMUNE THROMBOCYTOPENIA References 1. Kaushansky K. The molecular mechanisms that control thrombopoiesis. J Clin Invest 2005; 115: 3339–3347. 2. Italiano JE Jr, Lecine P, Shivdasani RA, Hartwig JH. Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes. J Cell Biol 1999; 147: 1299–1312. 3. Italiano JE Jr, Patel-Hett S, Hartwig JH. Mechanics of proplatelet elaboration. J Thromb Haemost 2007; 5 (Suppl 1): 18–23. 4. Pang L, Weiss MJ, Poncz M. Megakaryocyte biology and related disorders. J Clin Invest 2005; 115: 3332–3338. 5. Bussel JB, Provan D, Shamsi T, et al. Effect of eltrombopag on platelet counts and bleeding during treatment of chronic idiopathic thrombocytopenic purpura: A randomised, double-blind, placebo-controlled trial. Lancet 2009; 373: 641–648. 6. Weyrich AS, Denis MM, Schwertz H, et al. mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets. Blood 2007; 109: 1975–1983. 7. Italiano JE Jr, Hartwig JH. Megakaryocyte development and platelet formation. In: Platelets, 2nd ed. A. D. Michelson, ed. Elsevier, San Diego 23–44, 2007. 8. Italiano JE Jr, RA Shivdasani. Megakaryocytes and beyond: The birth of platelets. J Thromb Haemost 2003; 1: 1174–1182. 9. Foulks JM, Marathe GK, Michetti N, et al. PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids. Blood 113: 6699–6706, 2009. 10. Denis MM, Tolley ND, Bunting M, et al. Escaping the nuclear confines: Signal-dependent pre-mRNA splicing in anucleate platelets. Cell 2005; 122: 379–391. 11. Weyrich AS, Schwertz HL, Kraiss W, Zimmerman GA. Protein synthesis by platelets: Historical and new perspectives. J Thromb Haemost 2009; 7: 241–246. 12. Schwertz H, Tolley ND, Foulks JM, et al. Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenicity of human platelets. J Exp Med 203: 2433–2440, 2006. 13. Schwertz HS, Koster WH, Kahr N, et al. Anucleate platelets generate progeny. Blood 2010; 115: 3801–3809. 14. Behnke O, Forer A. From megakaryocytes to platelets: Platelet morphogenesis takes place in the bloodstream. Eur J Haematol Suppl 1998; 61: 3–23. 15. Junt T, Schulze H, Chen Z, et al. Dynamic visualization of thrombopoiesis within bone marrow. Science 2007; 317: 1767–1770. 11: 546-554.
Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/itphandbook2010/
1. Besides bone marrow, presence of megakaryocytes has been described in which of the following organs? a) Kidney ...... b) Spleen ......
22 THE HANDBOOK FIRST EDITION CHAPTER 1.1 • Megakaryocyte differentiation and platelet production
c) Lung ...... d) Heart ...... e) Pancreas ......
2. The following cytokines have been described to be involved in the development of megakaryocytes, except: a) TPO ...... b) IFN- g ...... c) IL-3, -6 ...... d) SDF-1 ...... e) PF4 ......
3. In regards to proplatelet production, which of the following stages have been described in both murine and human model systems? a) Shafts ...... b) Branch points ...... c) Tips ...... d) Swellings ...... e) All of the above ......
4. Which of the following is a purpose of proplatelet production in favour of platelets? a) Transfer granules ...... b) Transfer cytoskeletal/tubular elements ...... c) Deliver mRNAs ...... d) Deliver microRNAs ...... e) All of the above ......
5. Platelets with newly-formed cell bodies express the following biomarkers, except: a b a) IIb 3...... b) β-tubulin ...... c) P-selectin ...... d) Oct-4 ...... e) Respiring mitochondria ......
23 IMMUNE THROMBOCYTOPENIA * Basic science of ITP
1.2 Autoimmune mechanis*ms and T regulatory cell disturbances in ITP
Karina Yazdanbakhsh CHAPTER 1.2 • Autoimmune mechanisms and T regulatory cell disturbances in ITP
1. Introduction This chapter will focus on the importance of immune regulation and the tolerance mechanisms that are operating in humans and mice to prevent auto reactivity, with a particular focus on regulatory T cells, known as “Tregs”, which are seen as master regulators of immune responses. I will then review how some of these mechanisms may be disturbed in autoimmune diseases with a focus on ITP and discuss some of our new data showing that patients who are taking thrombopoietic agents may have improved Treg activity, and I will speculate why that might be and the implications of such an improvement on disease pathogenesis.
2. Immune regulation and immunological tolerance There are three situations where immune regulation is important. Firstly, upon exposure to an antigen, for example during an acute infection, when the immune system mounts a specific immune response, when the adaptive immune response is switched on and lymphocytes are activated. This activation can potentially go on forever, causing damage to the host in addition to fighting the infectious agent. Immune control mechanisms are necessary to limit excessive lymphocyte activation, and the collateral damage that can occur. In a healthy individual, when the infection is over, and the antigen is no longer present, the majority of the lymphocytes are deleted, or they become memory T and B cells. However, if the antigen persists, lymphocytes are in theory constantly stimulated unless immune regulation mechanisms are present to limit the ongoing lymphocyte activation and the associated collateral damage. The disadvantage to this situation is that these suppressive mechanisms may lead to tolerance to tumour antigens or chronic infections. Last, but not least, immune regulation is needed to prevent responsiveness against self-antigens. In a healthy person, there’s a balance between effector T cells and regulatory T cells. If this balance is tipped towards the effector T cells, hyper immune responses or autoimmune diseases can develop. If the balance is tipped towards the regulatory T cells, this can lead to infections and cancer because the effector T cells are not able to do their job and remove the antigen.
3. Tolerance Tolerance can be defined as unresponsiveness of the lymphocytes to an antigen. To avoid autoimmunity, a normal immune system has in place several mechanisms for maintaining tolerance to self-antigens. These include the elimination of self- reactive T and B lymphocytes during selection in the thymus and bone marrow,
25 IMMUNE THROMBOCYTOPENIA respectively, a process referred to as central tolerance. In addition, self-reactive lymphocytes that escape deletion and enter the peripheral circulation are prevented from becoming functionally active and causing autoimmune diseases by peripheral tolerance mechanisms that include deletion, anergy (inactivation) or ignorance due to weak self reactivity, poor accessibility, or lack of suitable presentation (1). In autoimmunity, these tolerance mechanisms break down and an immune response is generated against self and auto-antigens. By definition, this can be pathologic. Autoimmunity can be systemic, as in lupus, or organ-specific, as in ITP. The pathogenesis whether systemic or organ-specific is believed to be, for the most part, due to genetic susceptibility combined with various environmental triggers, such as infection.
3.1 Central tolerance What we mean by central tolerance is the mechanism by which immature lymphocytes that recognise antigens are deleted in the generative lymphoid organs, i.e. the bone marrow for the B cells and the thymus for the T cells. In the case of B cells in the bone marrow, apart from the deletion mechanism, there can be changes in B cell receptor editing, where the lymphocyte no longer recognises the self-antigen. Also, in the case of CD4 cells in the thymus, upon recognition of self-antigen, the immature lymphocytes can develop into regulatory T cells. These are the cells that go into the periphery, get activated, and suppress activated lymphocytes.
3.2 Peripheral tolerance Although central tolerance mechanisms ensure the removal/deletion of self-reactive immature lymphocytes, some do escape central tolerance mechanisms, and enter the periphery as mature lymphocytes. Here, the peripheral tolerance mechanisms come into play. In the normal T cell response, two signals are needed for T cell activation. One is through the T cell receptor recognising the antigens complexed to the MHC present on the antigen-presenting cell, which in most cases is a macrophage or a dendritic cell. A second signal is also needed, which is sent through the co-stimulatory receptor CD28 on the T cell and B7 on the antigen-presenting cell. If this co- stimulation is missing, the antigen will be ignored. As a result, the self-antigen is not recognised, and the T lymphocyte is rendered inactive or otherwise known as anergised. Another way the T cell induces anergy is by expressing “off” signals, for example CTLA-4. Hence, the T cell is not activated and becomes functionally unresponsive. A third mechanism, like the one existing in central tolerance, is that the T cell is
26 THE HANDBOOK FIRST EDITION CHAPTER 1.2 • Autoimmune mechanisms and T regulatory cell disturbances in ITP deleted due to expression of Fas-Fas ligands or pro-apoptotic genes. In addition, peripheral tolerance can occur by active suppression by a functionally distinct subpopulation of CD4 + T cells, called regulatory T cells (Tregs) (2).
4. Regulatory T cells (Tregs) These regulatory T cells are the so-called suppressive T cells that were described by Gershon in the 70’s and 80’s. They have been shown to suppress harmful, pathological responses against self and now we know that they can also suppress foreign antigens, either directly or indirectly by acting on helper CD4 + cells, CD8 + cells, antigen-presenting cells, B cells, and NK cells. That’s why we think of them as master regulators. Tregs represent 1–2% of the lymphocyte population in both humans and mice, and 5–10% of the CD4 + population. One of their characteristic markers is CD25, the high- affinity receptor for IL-2. The identification of CD25 enabled researchers in the field to isolate these cells and further characterise them. Tregs also express the transcription factor FOXP3. It has been shown that FOXP3 is essential for the maintenance of regulatory T cells. Indeed, patients with mutations in human FOXP3 gene develop the IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, which is associated with a high incidence of autoimmune diseases (2). Naturally occurring Tregs develop in the thymus during T cell maturation and recognise self-antigens. In addition, inducible Tregs can develop from peripheral mature CD4 + cells, following exposure to an antigen. There is some debate over whether these inducible Tregs are always generated following an immune response. At present there is no consensus on subtype-specific markers that can distinguish between these natural and inducible Tregs. It is believed that the signals for the generation and maintenance of both the inducible and naturally occurring Tregs in the thymus and in the periphery are basically similar. Thus far, these signals are not unique to the Tregs population because non-regulatory T cells appear to use the same factors. For example, TGF- b, a pleiotrophic cytokine, is one of the molecules that is important for the generation and maintenance of Tregs. IL-2, which is the growth factor for normal, non-regulatory CD4 + T cells, is now believed to be an important factor for the generation and maintenance of Tregs. FOXP3 is expressed transiently even on activated non-Tregs, but constitutive expression of it is considered is necessary for the generation of Tregs. Different mechanisms of Treg-mediated suppression have been described. Whether these different mechanisms operate in all Tregs, in subsets of Tregs, in specific disease situations, or in specific microenvironments, is not known. Much work was initially done to show that Tregs suppress CD4 + effector cells directly. Subsequently
27 IMMUNE THROMBOCYTOPENIA it was shown that they can also indirectly inhibit them, via antigen-presenting cells.The suppressor cytokines, IL-10, TGF- b and IL-35 can cause cell-cycle arrest in effector T cells. As mentioned, Tregs express CD25, the high affinity receptor for IL-2. A mechanism whereby Tregs can suppress CD4 + effector cells is by removing IL-2, which is as mentioned above an important growth factor for effector T cells. As a result of IL-2 removal, effector cells can die by apoptosis. Other mechanisms of Treg suppression include enzyme-mediated cytolysis, as well as expression of galectins, which cause cell-cycle arrest. Effector T cell functions by Tregs can also be inhibited indirectly via downmodulation of antigen presentation to T cells by various mechanisms.
5. Immune regulation in auto-immune diseases It is believed that autoimmunity is inherited as a polygenic trait. Some of these traits or susceptibility genes are associated with multiple diseases and some are disease-specific. It is thought that susceptibility genes, together with environmental triggers, such as infection, precipitate the autoimmune pathology ( Figure 1 ). There
Figure 1: Pathogenesis of autoimmunity
Susceptibility genes Environmental trigger (eg. NOD2, PTPN22, CD25) (e.g. infections, tissue injury)
Failure of self-tolerance
Persistence of functional Activation of self-reactive self-reactive lymphocytes lymphocytes
Immune responses against self tissues
Mutations in several genes have now been identified that predispose the individual to succumb to autoimmune diseases. As a result of these mutations, autoreactive lymphocytes escape tolerance mechanisms. An environmental trigger, such as an infection or tissue injury, can then lead to activation of these self-reacting lymphocytes, resulting in an immune response against self-tissues. Adapted from Abbas
28 THE HANDBOOK FIRST EDITION CHAPTER 1.2 • Autoimmune mechanisms and T regulatory cell disturbances in ITP is clearly an imbalance between immune activation and control and there are many reports suggesting that there are defects in the generation or function of Tregs, or that the effector T cells are resistant to suppression by Tregs. The ultimate therapeutic goal in autoimmunity should be to restore control of these abnormally directed responses. Genome-wide studies have identified several genes that seem to be mutated in different autoimmune diseases. NOD2, for example, is a pattern-recognition molecule. Mutations in this gene are associated with about 25% of cases in Crohn’s disease. The gene encodes a phosphatase that interacts with important adaptor molecules in T cell receptor signalling. Mutations in CD25 have been associated with several autoimmune diseases. Because CD25 is a marker for Tregs, mutations associated with it may cause a general defect in Tregs. In the case of ITP, there are mutations that seem to be associated with TNF- a. These susceptibility genes seem somehow to cause failure of self-tolerance mechanisms. As a result, there is persistence of functional self-reacting lymphocytes. An environmental trigger, such as an infection or tissue injury, can then lead to activation of these self-reacting lymphocytes, resulting in an immune response against self-tissues. We know, for example, that ITP may be induced when there are antibodies against some viral antigens that cross-react with platelets. That could be the result of activation of some self-reacting lymphocytes. In many cases, as a result of breakdown of tolerance mechanisms, a dominant T cell subset emerges that determines the disease. In the normal response to infection, for example, a T helper type (Th)1, subset dominance is induced against many organisms. However, a Th1 dominance is also associated with systemic and organ-specific autoimmune diseases, including ITP. Initially, it was believed that a naïve T cell could differentiate either into a Th1 or a Th2 subset, depending on the cytokine microenvironment. Now we know that naïve CD4 + T cells can also differentiate into Th17 cells, which are more involved with organ-specific autoimmunity, and as well as into inducible Tregs and possibly other Th types such as Th follicular cells. In addition, it used to be thought that these different Th subsets were stably and terminally differentiated, but interconversion amongst these Th subsets has been reported and functional plasticity depending on the cytokine environment and activation of signature transcription factors can occur. Is it then possible, for example in a patient with ITP, to create a microenvironment favourable to the induction of Tregs, as opposed to Th1 cells? The answer is that it may be possible, but more research is required to understand what factors are required to avoid plasticity such that once we have induced a specific subset, that it remains stable.
29 IMMUNE THROMBOCYTOPENIA 6. Tregs in patients with ITP We currently know that the major pathogenetic mechanisms in ITP are anti-platelet antibodies that cause Fc receptor-mediated destruction in the RES cells, cytotoxic T cells, in addition to impaired platelet production. The cellular immune responses of patients with ITP have the characteristic hallmarks of breakdown in their T cell tolerance mechanisms. These include: • increased platelet-induced CD4 + helper T cell (Th) proliferation and IL-2 secretion • an oligoclonal expansion of CD4 + cells • defects in the apoptotic pathways • higher frequency of activated CD4 + cells (3–8). The abnormal Th cells have been suggested to direct autoreactive B cells to secrete autoantibodies, and consistent with this view is the demonstration that peptides derived from platelet GPIIb-IIIa protein that are recognised by pathogenic CD4+ T can induce anti-GPIIb–IIIa IgG production in vitro (9, 10). In addition to helper T cell involvement in the pathogenesis of ITP, abnormal CD8 responses involving cytotoxic T cell-mediated lysis of autologous platelets in patients with active ITP have been reported (11). Such abnormal lymphocyte responses in T and B cell compartments as well as the reported cytokine imbalance in patients with ITP (12) suggest potential defects in regulatory immune networks. Indeed, recent studies in patients with ITP have shown reduced levels of Treg-specific transcription factor FOXP3 mRNA (13) and protein (14) in circulating mononuclear cells and abnormal Treg function in spleen biopsies (15) as well as bone marrow (16). To test the hypothesis that the pathogenesis of chronic ITP may be directly related to the levels or function of circulating Tregs, we first compared the frequency of Tregs in peripheral blood of patients with chronic ITP (n=17) and controls (n=26) (17). The patient cohort with one exception did not have an active disease at the time of blood sample collection. All patients were undergoing various treatments for ITP including steroids plus IVIG, Syk inhibitors, and TPO mimetic agents. We did not observe any differences in the frequency of CD4 +CD25 hi population, characteristic of the Treg phenotype between patients and controls. There was also no difference between patients and controls in the percentage of the CD25 hi CD4 which expressed FOXP3, another Treg marker, nor were there significant differences in the at levels of expression of this FOXP3 in these CD25 hi populations (17). We then looked at the function of these Tregs using a Treg suppressive assay available for in vitro assessment of their activity. To do that, we sorted PBMCs based on the expression of CD25 into a CD4 +CD25 hi population (Tregs) and a CD4 +CD25 - population. When CD4 +CD25 - cells are stimulated through their T cell receptors with
30 THE HANDBOOK FIRST EDITION CHAPTER 1.2 • Autoimmune mechanisms and T regulatory cell disturbances in ITP anti-CD3 and a second signal presented by allogeneic antigen-presenting cells, they proliferate. If regulatory T cells are stimulated in the same way in vitro , they do not proliferate; the cells are anergic. When CD4 +CD25 cells are co-cultured with the CD4 CD25 hi Tregs, the Tregs will suppress the proliferation of the effector cells, to a degree depending on the ratio of the two cell types. We found that patient Tregs have less inhibitory activity on the proliferation of autologous effector T cells relative to the controls, at all ratios of Tregs: effector cells tested and using two different concentrations of anti-CD3 ( Figure 2 ). To understand whether the reduced suppressive activity in the patients is due to
Figure 2: Reduced suppressive activity in patients with ITP
anti-CD3 (0.5 mg/mL) 100% - p=0.02 80% - p=0.006 Patient (n=12) n o i t
i 60% - b
i p=0.049
h Control (n=15) n 40% - I
% 20% - 0% - 1:0 1:1 1:4 1:16 Ratio 4 +25 + : 4 +25 -
anti-CD3 (0.1 mg/mL) 100% - p=0.004 80% - Patient (n=13) n o
i p=0.02 t
i 60% - b i
h p=0.006 Control (n=10) n 40% - I
% 20% - 0% - 1:0 1:1 1:4 1:16 Ratio 4 +25 + : 4 +25 -
Proliferation by CD4 +CD25 hi Treg was analyzed in a cohort of patients with ITP as well as normal healthy controls (n=9). Sorted populations of CD4 +CD25 – T cells were stimulated with plate-bound 0.5 mg/mL (upper graph) or 0.1 µg/mL (lower graph) anti-CD3 antibodies and allogeneic accessory cells, alone (1:0) or cocultured at varying ratios (1:1, 1:4 and 1:16) with autologous sorted CD4 +CD25 hi cells. Patient Tregs have lower ability to suppress proliferation of CD4 +CD25 – T cells. Modified from (17)
31 IMMUNE THROMBOCYTOPENIA defective Tregs or resistance of their effector cells, we performed co-mixing suppressive assays in which Tregs from patients were added to effector cells from healthy volunteers, and vice-versa . When Tregs from the patient were added to the effector cells from the healthy individuals, there was reduced suppressive activity whereas the Tregs from the healthy control were able to suppress the effector cells from the patient indicating that the defect is in the Tregs of the patients. This data indicates that the defect is in the Treg compartment and not due to effector cells resistant to suppression (17). Other labs have also obtained data indicating that there are defective Tregs in ITP, either in numbers (13–15) or suppressive activity (18). If Tregs can be induced or activated to suppress autoimmune responses, then they could potentially be used therapeutically. A recent report (19) has had success with the first part. They showed that antigen-specific Tregs can be generated in vitro to platelet-specific Tregs from patients with ITP.
7. Effects of treatment on Tregs in ITP Interestingly, chronic ITP patients treated with rituximab have shown restored numbers of Tregs as well as restored regulatory activity as determined by in vitro cell proliferation assays (18). Similarly, improvement in Treg frequency and activity have been reported following treatment with high dose dexamethasone in patients with ITP (14). In addition, in vitro as well as animal studies indicate a positive effect of IVIG on Treg function (20, 21). Altogether, the data are consistent with the immunomodulatory nature of such treatment modalities. Thrombopoietic agents have been effective treatment options for patients with ITP, and since platelets seem to express a number of proteins which are immunomodulatory, such as Toll-like receptors, CD40 ligands and TGF, our hypothesis was that as the platelet turnover increases, the Treg functional activity may improve in patients taking thrombopoietic agents. We looked at a pre-treatment or untreated group of 10 patients, consisting of 8 females and of 2 males, who had been without treatment at least two weeks prior to the study (22). Three of them were splenectomised, there was a wide range of ages with a median of 58.5, and platelet count 22 ± 3 x 10 9/L. We also took a separate, on treatment group, who had been receiving different thrombopoietic agents for at least one month. The different agents were Nplate, eltrombopag and a new investigational thrombopoietic agent called AKR-501. Only two of the patients were common in both groups, but there were similar numbers of splenectomies in this patient group, as well as a similar median age, and it contained eight females and one male. The platelet count in the treated group was 141 ± 33 x 10 9/L.
32 THE HANDBOOK FIRST EDITION CHAPTER 1.2 • Autoimmune mechanisms and T regulatory cell disturbances in ITP
There was no difference in Treg frequency as assessed by FOXP3 expression between the pre-treatment versus the on-treatment group, but we found a significant increase in their T regulatory suppressive activity ( Figure 3 ) (22). It was not quite as high as in the controls, but the activity was significantly improved in patients on treatment. We also observed a good negative correlation between Treg suppressive activity and the percentage expression of IL-2-expressing CD4 cells indicating that there is dampening of the immune response. We also found increased levels of TGF- b in the plasma of patients on treatment, compared to pre- treatment patients. TGF- b correlated with platelet count; as platelet levels improve, TGF- b levels increased. TGF- b is an important molecule for Treg survival and expansion, as well as Treg suppressive activity. We also observed higher plasma levels of CD40-ligand, a proinflammatory cytokine, in patients with chronic ITP, which decreased on treatment. Altogether, the data suggests that there is improved Treg activity in patients on treatment with TPO agents, indicating that these agents have immunomodulatory activity. The increased Treg activity nicely correlates with dampening of the immune responses as the frequency of the IL-2 secreting T cells goes down. The reason for
Figure 3: Treg activity improves in patients on treatment with thrombopoietic agents
100% + - 100 - 1:1 Treg: CD4 CD25 p=0.045 - 90 1:4 Treg: CD4 +CD25 - - 80 75% - n - 70 o - i s
s -60 e r
p 50% - -50 p u
S -40
% 25% - -30 -20 -10 0 - -0 Pre- On Controls treatment treatment (n=9) (n=10) (n=9)
Suppression of proliferation by CD4 +CD25 hi Treg was analyzed in patients before treatment (n=10) and in patient on treatment with thrombopoietic agents (n=9) as well as normal healthy controls (n=9). Suppression of proliferation of autologous CD4 +CD25 – T cells was measured as described in Figure 3. Mean inhibition was calculated as 1 – (cpm incorporated in the coculture)/cpm of responder cells alone) x 100. For comparison, the healthy volunteer controls suppressive activity at 1:1 ratio (70%±2%) and at 1:4 ratio (50%±5%) is also indicated. Adapted from (22)
33 IMMUNE THROMBOCYTOPENIA this improved activity is not clear, but may be due to the increased TGF- b levels. We think that might be a result of increased platelet turnover, but this is all speculation based on the correlation studies. We are currently working on understanding exactly how it is that these patients’ Tregs are improved. The proposed mechanism for pathogenesis of ITP is shown in Figure 4 . We think that there may be an inherent defect in the Treg compartment in patients with ITP, maybe due to the presence of susceptibility genes. Due to environmental factors, such as trigger by an infection, autoreactive T and B cells that have escaped tolerance are activated, destroying the patients’ own platelets. The autoreactive immune response is not dampened due to the impaired Treg compartment. The increase in autoreactivity is associated with an increase in inflammatory cytokines, which further reduces the Treg activity. And so the cycle continues, as in chronic ITP for example. Upon treatment with TPO-R agents (Figure 3) platelet counts increase and megakaryocyte survival improves. As a result Treg activity improves. This may happen directly through interaction with platelets or perhaps through TGF- b released due to increased platelet turnover. As Treg activity improves, there is dampening of the immune of the IL-2 expressing CD4 cells and presumably the autoimmune response ( Figure 5 ). This would further improve platelet and megakaryocyte survival.
Figure 4: The proposed mechanism for pathogenesis of ITP
Environmental/infection Tregs
Autoreactivity Inflammatory cytokines
Platelets
ITP patients have an inherent defect in the Treg compartment. Following an environmental assault such as an infection, autoreactive T and B cells are activated but cannot be controlled by Tregs causing platelet destruction. The increase in autoreactivity is associated with an increase in inflammatory cytokines, further reducing the Treg activity
34 THE HANDBOOK FIRST EDITION CHAPTER 1.2 • Autoimmune mechanisms and T regulatory cell disturbances in ITP
Figure 5: Following treatment with TPO-R agents, platelet and megakaryocyte survival improves
Platelets and megakaryocytes survival
Inflammatory cytokines Treg T effector
Through either a direct interaction with platelets or indirectly through TGF- b released due to increased platelet turnover, Treg activity improves. As a result, there is dampening of autoreactivity, which further improves platelet and megakaryocyte survival
8. Conclusion A deficiency in generation and/or defective functions of Tregs may contribute to the loss of immunologic self-tolerance and pathogenesis in patients with ITP. In particular, failure to maintain immune suppression by Tregs may explain the reported platelet autoantigen-specific T cell proliferative responses and may contribute to suppressed megakaryocyte production in the bone marrow of patients with ITP. Our observations show that the Treg activity of chronic ITP patients on thrombopoietic agents is increased compared to patients before treatment and that the improved Treg activity correlates with decreased IL-2 production, suggesting that improved Treg activity is linked to dampening of immune responses. These results indicate that thrombopoietic agents possess immunomodulatory activity. In addition, we found that TGF- b1 levels in the platelet poor plasma strongly correlated with improved platelet counts and were increased in patients on treatment, while sCD40L, a proinflammatory cytokine was decreased. This raises the interesting possibility that platelets in patients on treatment may play a role in improving Treg function either directly through cell-cell interactions or indirectly by release of TGF- b1. In conclusion, the immune profiling studies indicate breakdown of immune tolerance mechanisms in patients with ITP. It is therefore likely that therapies that re-direct the immune system to a state of tolerance including the induction of functional regulatory T cells are likely to be the most effective for the treatment of ITP.
35 IMMUNE THROMBOCYTOPENIA Acknowledgements Weili Bao, Jin Yu, Susanne Heck and Wu He (Authors laboratory). James Bussel, Vivek Patel, Marissa Patel, Marissa Karpoff, Nayla Boulad, and Zeeshan Hafeez (Weill Cornell Medical College).
References 1. Walker LS, Abbas AK. Peripheral T cell tolerance. Nat Rev Immunol 2002; 2: 11–19. 2. Wing K, Sakaguchi S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol 2010; 11: 7–13. 3. Semple JW, Freedman J. Increased antiplatelet Y helper lymphocyte reactivity in patients with autoimmune thrombocytopenia. Blood 1991; 78: 2619–2625. 4. Ware RE, Howard TA. Phenotypic and clonal analysis of T lymphocytes in childhood immune thrombocytopenic purpura. Blood 1993; 82: 2137–2142. 5. Semple JW, Milev Y, Cosgrave D, et al. Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: Relationship to platelet phenotype and antiplatelet T-cell reactivity. Blood 1996; 87: 4245–4254. 6. Shimomura T, Fujimura K, Takafuta K, et al. Oligoclonal accumulation of T cells in peripheral blood from patients with idiopathic thrombocytopenic purpura. Br J Haematol 1996; 95: 732–737. 7. Shenoy S, Mohanakumar T, Chatila T, et al. Defective apoptosis in lymphocytes and the role of IL-2 in autoimmune hematologic cytopenias. Clin Immunol 2001; 99: 266–275. 8. Yoshimura C, Nomura S, Nagahama M, et al. Plasma-soluble Fas (APO-1, CD95) and soluble Fas ligand in immune thrombocytopenic purpura. Eur J Haematol 2000; 64: 219–224. 9. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive T cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura: Role in production of anti-platelet autoantibody. J Clin Invest 1998; 102: 1393–1402. 10. Kuwana M, Kaburaki J, Kitasato H, et al. Immunodominant epitopes on glycoprotein IIb- IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura. Blood 2001; 98: 130–139. 11. Olsson B, Andersson PO, Jernas M, et al. T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nat Med 2003; 9: 1123–1124. 12. Coopamah MD, Garvey MB, Freedman J, et al. Cellular immune mechanisms in autoimmune thrombocytopenic purpura: An update. Transfus Med Rev 2003; 17: 69–80. 13. Sakakura M, Wada H, Tawara I, et al. Reduced Cd4+Cd25+ T cells in patients with idiopathic thrombocytopenic purpura. Thromb Res 2007; 120: 187–193. 14. Ling Y, Cao X, Yu Z, Ruan C. Circulating dendritic cells subsets and CD4+Foxp3+ regulatory T cells in adult patients with chronic ITP before and after treatment with high-dose dexamethasome. Eur J Haematol 2007; 79: 310–316. 15. Liu B, Zhao H, Poon M-C, et al. Abnormality of CD4(+)CD25(+) regulatory T cells in idiopathic thrombocytopenic purpura. Eur J Haematol 2007; 78: 139–143.
36 THE HANDBOOK FIRST EDITION CHAPTER 1.2 • Autoimmune mechanisms and T regulatory cell disturbances in ITP
16. Olsson et al. Recruitment of T cells into bone marrow of ITP patients possibly due to elevated expression of VLA-4 and CX3CR1. Blood 2008; 112: 1078–1084. 17. Yu J, Heck S, Patel V, et al. Defective circulating CD25 regulatory T cells in patients with chronic immune thrombocytopenic purpura. Blood 2008; 112: 1325–1328. 18. Stasi R, Cooper N, Del Poeta G, et al. Analysis of regulatory T-cell changes in patients with idiopathic thrombocytopenic purpura receiving B cell-depleting therapy with rituximab. Blood 2008; 112: 1147–1150. 19. Zhang X-L, Peng J, Sun J-Z, et al. De novo induction of platelet-specific CD4+CD25+ regulatory T cells from CD4+CD25– cells in patients with idiopathic thrombocytopenic purpura. Blood 2009; 113: 2568–2577. 20. Kessel A, Ammuri H, Peri R, et al. Intravenous immunoglobulin therapy affects T regulatory cells by increasing their suppressive function. J Immunol 2007; 179: 5571–5575. 21. Ephrem A, Chamat S, Miquel C, et al. Expansion of CD4+CD25+ regulatory T cells by intravenous immunoglobulin: A critical factor in controlling experimental autoimmune encephalomyelitis. Blood 2008; 111: 715–722. 22. Bao W, Bussel JB, Heck S, et al. Improved regulatory T cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents. Blood 2010 Aug 5. [Epub ahead of print].
Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/itphandbook2010/
1. Regulatory T cells act directly or indirectly on the following cells: a) CD4 +...... b) CD8 +...... c) APCs ...... d) B cells ...... e) All of the above ......
2. Suppression of antigen presenting cells by T regulatory cells (Tregs) is mediated by the following, except: a) CTLA-4 ...... b) IL-10 ...... c) CD39 ...... d) Nrp1 ...... e) LAG3 ......
37 IMMUNE THROMBOCYTOPENIA 3. The following cytokines induce CD4 + differentiation or conversion, except: a) IFN- g ...... b) IL-12 ...... c) IL-4 ...... d) TNF ...... e) TGF- b ......
4. Which of the following mechanisms have been described to be involved in the immunopathology of ITP? a) Activated B cells ...... b) Activated T cell ...... c) T cell-mediated cytotoxicity ...... d) Cytokine imbalance ...... e) All of the above ......
5. Which of the following thrombopoietic agents has been shown to be effective in the treatment of ITP? a) Rituximab ...... b) IVIg ...... c) Eltrombopag ...... d) High dose dexamethasone ...... e) Damiplosim ......
38 THE HANDBOOK FIRST EDITION NOTES
39 IMMUNE THROMBOCYTOPENIA * Basic science of ITP
1.3 Mouse model*s of ITP John W. Semple CHAPTER 1.3 • Mouse models of ITP
1. Introduction ITP is an immune disorder characterised by petechiae, wet purpura, and, in some very rare cases, fatal intracranial haemorrhages. ITP used to be defined as a platelet count of less than 150 x 10 9 per litre, and was divided into acute and chronic forms: the acute form was a childhood disorder usually of abrupt onset and following an infectious illness, which often underwent spontaneous remission, whereas the chronic disorder lasted more than six months and was probably a true organ-specific autoimmune disease with presence of autoimmune antibodies and the presence of autoreactive T cells. In 2009, the nomenclature for ITP was changed. ITP is now called immune thrombocytopenia; the word purpura has been dropped. The platelet count for diagnosis now has now been reduced to less than 100 x 10 9 per litre, and four categories have been defined (1): • newly diagnosed ITP is within three months of diagnosis, • persistent ITP is between three and twelve months from diagnosis, • chronic ITP is now greater than twelve months in duration, and • the term severe ITP has been reserved for patients who exhibit bleeding diatheses.
2. Mechanisms of destruction in ITP For the last fifty to sixty years ITP has been considered an autoantibody-mediated disorder. Autoantibodies opsonise platelets, which are then very quickly destroyed by Fc receptor-mediated phagocytosis when they traverse the spleen. Harrington famously demonstrated this effect when he infused himself with plasma from a patient with ITP (2). There was a precipitous drop in his platelet counts and then they slowly rose again. He made similar observations in other subjects who volunteered to be infused with plasma. Further work showed that the autoantibodies were primarily directed against GpIIb/IIIa (CD41) and/or GpIbIX (CD42) (3). In patients who are infected with HIV and have ITP, the autoantibodies were found to react against a very specific portion of the IIIa molecule, the amino acids 49 to 66 (4). On binding to the platelets, they activate 12-lipoxygenase, which stimulates arachidonic acid metabolism and ultimately NADPH oxidase, with the production of superoxide and reactive oxygen species, so that the platelets undergo antibody-mediated autocytolysis. This seems to be unique to those patients who are infected with HIV, and will be discussed further in Chapter 2.1. In 2003, Wadenvik’s group showed that in some patients with ITP antibodies cannot be identified either in the plasma, or on the platelets, and they found that in these patients there are cytotoxic T cells that can bind to and destroy platelets
41 IMMUNE THROMBOCYTOPENIA independently of antibodies (5). The platelets die from apoptotic-like mechanisms. This has now been confirmed by others. ITP is a complex autoimmune disorder, at both the level of platelet destruction and immune T cell dysregulation. It appears that ITP is due to a deficiency of CD4 + T regulatory cells, as summarised in Chapter 1.2, which allow autoreactive lymphocytes to attack platelet antigens. In addition, platelets express on their surface a variety of pro and anti-inflammatory molecules, such as Toll-like receptors, that enable them to quickly bind to infectious agents and present them to the innate immune system. So it may very well be that platelets can dictate their own immunological function within the host by turning on macrophages when an infection is present.
3.The role of animal studies Several animal models have been used to study ITP ( Figures 1–3 ). At first glance, animal studies may not appear to offer valid information about human ITP. However, although laboratory animals differ from humans, out of the 23,000 genes that humans possess, all but about 300 are shared with the mouse. In fact, humans still have all their tail genes; they are just not expressed. There is a close relationship between most of the vertebrates and it is reasonable to assume they can act as good models for a variety of different immune disorders. If an anti-platelet antibody is given to mice, their platelet counts will fall after approximately a day and then slowly start to rise to normal levels as the antibodies are metabolised, just as Harrington (see ref above) showed in humans. Furthermore, practically everything that is known about the human immune system has come from
Figure 1: Mouse model of passive ITP
Injection
Anti-platelet antiserum (pre 1990) Immune OR thrombocytopenia (within 2-24hr) Anti-platelet monoclonal antibody (post 1990)
42 THE HANDBOOK FIRST EDITION CHAPTER 1.3 • Mouse models of ITP
Figure 2: Mouse model of secondary ITP
1. Drug +/- Anti-platelet antibody
Immune thrombocytopenia
2. Infection +/- Anti-platelet antibody
3. Nephritis SLE etc.
Figure 3: Mouse model of platelet-induced ITP
Immunise with Fetal immune Female WT platelets Mate with thrombocytopenia WT male
Platelet GPIIIa KO mouse
studying animals, such as mice. Both humans and laboratory animals have primary and secondary lymphoid structures, both have similar innate and adaptive immune systems, the complement genes are almost identical, both have Toll-like receptors and neutrophils, and all the types of lymphocytes and antigen-presenting cells are very similar. So one can probably extrapolate a fair amount of useful information from looking at animal models.
4. Animal models of ITP There are two general types of autoimmune animal models, spontaneous models and induced models. The spontaneous models are usually serendipitously discovered within
43 IMMUNE THROMBOCYTOPENIA a breeding colony where a mutation is noticed phenotypically. These animals can then be bred to produce the observed trait. Examples include the famous animal models of the type I diabetes (NOD mouse) and the New Zealand Black mouse that shows symptoms of SLE. The other types of autoimmune models in animals are the induced models. These require some form of treatment of the animal to trigger the disease. Certain mouse strains, for example, when injected with spinal cord extract and adjuvant, will develop an experimental autoimmune encephalomyelitis, a disease that resembles MS. Primary chronic ITP is not unique to humans. The veterinary literature contains numerous clinical reports, clinical series, retrospective studies and reviews of diagnoses of ITP in a number of different domesticated animals, from dogs up to horses. However, no spontaneous model of ITP has been reported in laboratory animals. This difference may be due to the breeding schemes that we use. The domesticated animals, for example, are generally line bred, like purebred dogs, whereas laboratory animals are usually inbred. In contrast, many reports of induced models of ITP have been published in the literature, and have made valuable contributions to our understanding of pathogenic features of at least some of the different forms of ITP, and perhaps some of the therapies, such as IVIG. These types of animal model can be divided into five basic types. Passive ITP has been by far the most common model in the literature (used in 259 of 350 reported studies). There are also secondary models of ITP, of which there are three types: drug-induced, infectious, or related to other diseases. There are also models that can be termed platelet-induced ITP models.
5. Studies in animal models The types of questions that were asked even in the early nineteen fifties all the way up to present today are still relevant today. Some of these early questions studied are related to the following topics: • antibody-mediated platelet destruction • the effects of IVIG on this • the effects of thrombopoietin in immune thrombocytopenic mice • studies on megakaryocytopoiesis in thrombocytopenic animal models • how anti-platelet antiserum affects long-term bone marrow cultures • post-transfusion purpura in the dog • anti-platelet serum and its ability to inhibit atherosclerotic lesions • drug-induced thrombocytopenia • how thrombocytopenia influences bleeding and coagulation parameters.
44 THE HANDBOOK FIRST EDITION CHAPTER 1.3 • Mouse models of ITP
6. Passive ITP This is a relatively simple model where passive immunity is transferred to the animal by anti-platelet antiserum, or more recently by monoclonal antibodies (Figure 1). The antibodies are transferred to the animals, in this case the mouse, and a day later platelet counts show thrombocytopenia. Passive ITP models have been the mostly widely used models in animal studies of ITP. One of the first murine auto reactive monoclonal antibodies that was generated against platelets is called 6A6, and this antibody was used by the Ravetch group (6) to look at how this antibody causes thrombocytopenia and how IVIG can prevent this. There have been a number of later reports showing that in these passive immune models, IVIG is very effective at reducing the destruction caused by the antibodies (7–9). This provides a good model to try to understand the mechanisms of action of IVIg.
7. Secondary ITP Secondary ITP is ITP associated with another disease, a drug, or an infection. All of these types of secondary models have been studied in animals (Figure 2). Anti- platelet antibodies can be given in combination with various drugs to induce thrombocytopenia. Infections can modulate the thrombocytopenia generated by infusing anti-platelet antibodies. Certain animal models, such as the NZB animals which have a background of nephritis and SLE, develop ITP-like syndromes as they get older. These animals have been very useful for understanding how secondary ITP might arise. There have been a number of different animal models, over the last forty to fifty years, which have extensively looked at various types of drugs and how they can cause or modulate thrombocytopenia in animals. As early as the sixties, quinidine was used in an animal model and studied with regard to how it induced thrombocytopenia (10). Other models have studied hapten-carrier conjugates (11) and heparin-induced thrombocytopenia (12). Infectious agents have also been studied. A number of studies have looked at various bacteria and viruses, and have showed that viruses can enhance platelet destruction by anti-platelet antibodies (13). This has also been observed clinically in patients with ITP, who may have precipitous drops in their platelet counts triggered by infective episodes. It is probably a combination of the infectious agents with the anti-platelet antibody that causes an activation, for example, of the reticuloendothelial system and causes increased destruction (14). These types of mechanisms have been looked at in animal models. The oldest secondary ITP animal model is really the one that is associated with other
45 IMMUNE THROMBOCYTOPENIA diseases. The NZW x BXSB mouse, in fact, was originally a model of lupus, and these animals also suffered from coronary vascular disease. But if the males of this hybrid are allowed to develop to about three to four months of age, their platelet counts start to drop, and in fact they start to develop anti-platelet autoantibodies (15). These have been excellent models to try and dissect out how one disease can affect or initiate another immune disease.
8. Platelet induced ITP The passive and secondary animal models of ITP have been instrumental in understanding the pathophysiology of secondary ITP and the mechanisms of action of IVIG. However, they are really not ideal for studying the pathophysiology of chronic ITP, where both T cell and B cell autoimmune attack are focused on platelet-specific antigens. These are passive transfer models, so no role is played by T cells or B cells of the mouse itself and these models cannot therefore help in understanding how chronic ITP is initiated or perpetuated, and how T cells may help anti-platelet autoantibody production. A few years ago, our group developed a platelet specific animal model of ITP by utilizing knock-out mice. We found that if female mice knocked-out for the glycoprotein IIIa chain of the GPIIbIIIa complex on platelets were immunised against wild-type they became strongly immune against the wild type platelets (Figure 3). If these mice were then bred with a male wild type mouse, the offspring were thrombocytopenic, forming quite a good model of neonatal alloimmune thrombocytopenia purpura. The neonates were profoundly thrombocytopenic with bleeding diatheses but when the dams were treated with IVIG, there was a marked reduction in fetal morbidity and mortality (16). In the offspring of the immunised knock-out dams there were various degrees of bleeding, including cranial and abdominal bleeding, and many of the pups were stillborn because of massive haemorrhage. Our group has been studying this model for the past few years, trying to understand the mechanism of action of not only IVIg in this type of model, but also the immune response that occurs against the platelets in these fetal mice. We decided that we could try to develop a similar model that mimics severe ITP. We used the GPIIIa knock-out mice and immunised them against GPIIIa wild-type platelets ( Figure 4 ). Subsequently, we sacrificed the animals, removed their spleens and transferred their spleen cells into SCID mice. SCID mice have no T or B cells, so the spleen cells injected repopulate the SCID mouse immune system and we can understand how they might be able to influence the platelet counts in the individual transfers. We observed the phenotype of these mice and analysed platelet counts.
46 THE HANDBOOK FIRST EDITION CHAPTER 1.3 • Mouse models of ITP
Figure 4: Mouse model that mimics severe ITP
Wild type GPIIIa Production of high-titred (CD61)+BALB/C IgG anti-GPIIIa antibodies platelets transfused (x4) (>1:50,000) Syngeneic CD 61 KO mice
300 - 1:1,000 1:10,000 225 - r 1:50,000 e
b P
Immune CD61 KO m
u 150 - n
l l e C 75 -
Immune spleen cells 0- 10 0 10 1 10 2 10 3 10 4 Fluorescence
Transfer MACS deplete CD4 +, CD8 + or (5 x 10 4 - 1 x 10 5 cells) CD19 + lymphocytes
CB.17 SCID mice (2 Gy of radiation/asialo Gm1)
Observe phenotype and plateled counts
We also modified the spleen cells by depleting various lymphocyte populations in order to try and determine the role of the different lymphocytes. In control mice given spleen cells from a non-immune animal, or from an animal immunised against third party antigens, transient thrombocytopenia did occur at day seven after the transfer ( Figure 5 ), but this was due to the irradiation that we give the SCID mouse, and all the platelet counts in these animals recovered to normal levels by the second week after transfer. On the other hand, transfer of immune spleen cells showed completely different types of platelet count profiles ( Figure 6 ). Firstly, after transferring whole spleen cells that have not been depleted of lymphocytes, there was a continuous thrombocytopenia that develops and the counts did not recover. The mice remained thrombocytopenic and about 80% died from bleeding
47 IMMUNE THROMBOCYTOPENIA
Figure 5: Control
1500 - + Statistically significant / 9 L 1250 - ) 1000 - 750 - 500 - + 250 -
P (n=15) l a 0 -| | | | | | | t e
l 0 5 10 15 20 25 30 e t
c
o Days post transfer u n t
( x
Modified fr1 om (17) 0
by days 14-21. If the CD4 + helper T cells were removed from the spleen cells before transfer, the graph looked very similar to the control graph, indicating that the immune response generated in these GPIIbIIIa knock-out mice is critically dependent on T helper cells. In contrast, with removal of CD8 + T cells, which we called antibody-mediated thrombocytopenia because what is left is the B cells that were in the spleens transferred, the pattern was very similar to the non-depleted immune spleen cell transfers. There was profound thrombocytopenia and most of the mice, again, died after the second week after transfer. On the other hand, if the CD19 + B cells were removed from the spleens before transfer, which we called cell-mediated thrombocytopenia, all the mice became thrombocytopenic (Figure 6). Most of them did not die, but they remained thrombocytopenic. So we could not only observe antibody-mediated thrombocytopenia in these animals, but we could also see a cell- mediated form, and we confirmed that it was due to CD8 + T cells because if CD8 was depleted as well as CD19, the graphs were very similar to control (Figure 6). So this model demonstrated two different mechanisms of thrombocytopenia, mediated by the B cells and the T cells, just as Wadenvik’s group showed in human ITP patients (17). The mortality rate in these animals is shown in Figure 7 , and is very different from chronic ITP. Patients with chronic ITP do not necessarily have spontaneous bleeding problems, and fatal bleeding is extremely rare. In this animal model, however, the mice actively bled. In the non-depleted transfers, the mortality rate was about 80% by the third week, and most of the mice surviving were quite sick. When the helper
48 THE HANDBOOK FIRST EDITION CHAPTER 1.3 • Mouse models of ITP
Figure 6: Platelet counts in mice given immune spleen cells
A Non-depleted B CD4 + T cell depleted 1500 - (n=15) 1500 - (n=15) 1250 - 1250 -
1000 - 1000 -
750 - 750 - + 500 - + 500 - +
) CD8 & CD19 depleted L 250 - 250 - / 9 0
1 0 - 0 -
x 0510 15 20 25 30 0510 15 20 25 30 (
t n u o
c + + CD8 T cell depleted CD19 B cell depleted t
e +
l C (Antibody-mediated) D (CD8 T cell-mediated) e t
a (n=18) (n=14)
l 1500 - 1500 - P 1250 - 1250 - 1000 - 1000 - 750 - 750 - 500 - 500 - + 250 - + 250 - 0 - 0 - 0510 15 20 25 30 0510 15 20 25 30
+ Statistically significant Days post transfer
Modified from (17) cells were removed, the platelet counts were normal and there was no mortality associated with getting rid of helper cells. The antibody-mediated thrombocytopenia was associated with a high mortality. Also, the CD8 T cell-mediated thrombocytopenia had mortality, but not as great as the antibody-mediated. The mice exhibited a profound bleeding diathesis; by the second week of transfer: there was bleeding into the skull, the brain, and the abdomen. There were significant petechiae all over their bodies. The spleen was significantly enlarged because of the immune response taking place in these particular animals. We believe that, although this does not mimic the vast majority of chronic ITP, it does mimic the
49 IMMUNE THROMBOCYTOPENIA Figure 7: Mortality in mice given immune spleen cells
Non-depleted CD4 + T cell depleted A (n=15) B (n=15) 100 - 100 - 80 - 80 - 60 - 60 - 40 - 40 - 20 - 20 - 0 - 0 -
l 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 a v
i + +
v CD8 T cell depleted CD19 B cell depleted r + u (Antibody-mediated) (CD8 T cell-mediated) S C (n=18) D (n=14) % 100 - 100 - 80 - 80 - 60 - 60 - 40 - 40 - 20 - 20 - 0 - 0 - 0 5 10 15 20 25 30 0 5 10 15 20 25 30
Days post transfer
Modified from (17)
severe form, so should be useful in trying to understand why some patients with ITP bleed and why others do not. Looking at the bone marrow of these mice, we found megakaryocytes throughout the marrow, but in the ITP-transferred mice, the megakaryocytes were significantly abnormal as all the nuclei were condensed and they appeared to be undergoing apoptosis ( Figure 8 ). We are currently studying this further and have evidence to suggest that this apoptosis is induced by CD8 + T cells homing to the bone marrow and causing megakaryocyte destruction. Bone marrow production defects in ITP have been known since as early as 1946, when Dameshek showed that patients with ITP had abnormal non platelet-shedding megakaryocytes within the bone marrow. But antibodies can not only opsonise platelets in the periphery, they can actually traverse into the bone marrow and bind to megakaryocytes, destroying them or at least inhibiting their differentiation. We are looking at the hypothesis that the CD8 + T
50 THE HANDBOOK FIRST EDITION CHAPTER 1.3 • Mouse models of ITP
Figure 8: Apoptotic megakaryocytes within the bone marrow
WT ITP
WT = wild-type cells generated in this animal model can also move into the bone marrow and bind to the megakaryocytes, causing cytolysis and apoptosis. We also observed in the animal model that, like in human ITP, treatment with IVIg significantly reduced mortality, to about half ( Figure 9 ). Interestingly, it was possible to completely prevent mortality in the antibody-mediated form of the disease, but not if the disease was mediated by CD8 + T cells (17). This suggests a differential response between therapies depending on the main mechanism of destruction in each case of immune thrombocytopenia. Antibody-mediated thrombocytopenia is sensitive to IVIg therapy, whereas the cell-mediated form is resistant. This may be the reason why some patients fail therapy, because they have perhaps a different form of thrombocytopenia.
9. Conclusion Animal models of immune thrombocytopenia have been with us since at least the 1950’s. The majority have been of the passive immune type, but others model secondary ITP either associated with drugs, infections or other diseases. These models have made significant contributions to our understanding of the pathogenesis of different forms of ITP, and also have really been instrumental in understanding how different therapies such as IVIG may rescue platelet counts. The newest models of platelet-specific protein knock-out animals are probably going to be important and allow for the elucidation of how platelet-reactive T cells mediate the pathophysiology of chronic severe ITP, at least.
51 IMMUNE THROMBOCYTOPENIA Figure 9: Effect of IVIg on mortality
A Non-depleted B CD8 + T cell depleted C CD19 + B cell depleted (Antibody-mediated) (CD8 + T cell-mediated) 100 - 100 - 100 -
80 - 80 - 80 - l a
v 60 - 60 - 60 - i v r u
S 40 - 40 - 40 -
% 20 - 20 - 20 -
0 - 0 - 0 - 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Days post transfer Albumin (n=8) IVIg 2g/kg (n=10) IVIg 2g/kg (n=10) IVIg 2g/kg (n=10) No treatment (15) No treatment (18) No treatment (14)
Modified from (17)
References 1. Rodeghiero F, Stasi R, Gernsheimer T, et al. Standardisation of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children. Blood 2009; 113: 2386–2393. 2. Harrington WJ, Minnich V, Hollingsworth JW, Moore CV. Demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura (1951). J Lab Clin Med 1990; 38: 1–10. 3. Hou M, Stockelberg D, Kutti J, Wadenvik H. Antibodies against GPIb/IX, GPIIb/IIIa, and other platelet antigens in chronic idiopathic thrombocytopenic purpura. Eur J Haematol 1995; 55: 307–314. 4. Nardi M, Feinmark SJ, Hu L, et al. Complement-independent Ab-induced peroxide lysis of platelets requires 12-lipoxygenase and a platelet NADPH oxidase pathway. J Clin Invest 2004; 113: 973–980. 5. Olsson B, Andersson PO, Jernas M, et al. T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nat Med 2003; 9: 1123–1124. 6. Samuelson A, Towers T, Ravetch JV. Anti-inflammatory activity of IvIg mediated through the inhibitory Fc receptor. Science 2001; 291: 484–486. 7. Teeling JL, Jansen-Hendriks L, Kuijpers TW, et al. Therapeutic efficacy of intravenous
52 THE HANDBOOK FIRST EDITION CHAPTER 1.3 • Mouse models of ITP
immunoglobulin preparations depends on the immunoglobulin G dimers: Studies in experimental immune thrombocytopenia. Blood 2001; 98: 1095–1099. 8. Crow AR, Song S, Semple JW, et al. IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity. Br J Haematol 2001; 115: 679–686. 9. Hansen RJ, Balthasar JP. Effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombocytopenia. Blood 2002; 100: 2087–2093. 10. Wakisaka G, Yasunaga K, Kuramoto A, et al. Experimental study of immune thrombocytopenia induced by quinidine. Arerugi 1964; 13: 686–689. 11. Kekomäki R, Kauppinen HL, Penttinen K, Myllylä G. Interactions of immune complexes and platelets in rabbits immunized with hapten-carrier conjugates. Acta Pathol Microbiol Scand [C]. 1977; 85: 207–214. 12. Reilly MP, Taylor SM, Hartman NK, et al. Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through FcgammaRIIA. Blood 2001; 98: 2442–2447. 13. Musaji A, Meite M, Detalle L, et al. Enhancement of autoantibody pathogenicity by viral infections in mouse models of anemia and thrombocytopenia. Autoimmun Rev 2005; 4: 247–252. 14. Semple JW, Aslam R, Kim M, et al. Platelet-bound lipopolysaccharide enhances Fc receptor-mediated phagocytosis of IgG opsonized platelets. Blood 2007; 109: 4803–4805. 15. Oyaizu N, Yasumizu R, Miyama-Inaba M, et al. (NZW x BXSB)F1 mouse. A new animal model of idiopathic thrombocytopenic purpura. J Exp Med 1988; 167: 2017–2022. 16. Ni H, Chen P, Spring CM, et al. A novel murine model of fetal and neonatal alloimmune thrombocytopenia: Response to intravenous IgG therapy. Blood 2006; 107: 2976–2983. 17. Chow L, Aslam R, Speck ER, et al. A murine model of severe immune thrombocytopenia is induced by antibody- and CD8+ T cell-mediated responses that are differentially sensitive to therapy. Blood 2010; 115: 1247–1253.
Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/itphandbook2010/
1. The following are components of the new nomenclature and diagnosis for immune thrombocytopenia (ITP), except: a) Newly diagnosed ITP ...... b) Acute ITP ...... c) Persistent ITP ...... d) Chronic ITP ...... e) Severe ITP ......
53 IMMUNE THROMBOCYTOPENIA 2. Which of the following immune components is/are shared by humans and mice? a) NK cells ...... b) Macrophages ...... c) T cells ...... d) Dendritic cells ...... e) All of the above ......
3. Which of the following animal models exist for immune thrombocytopenia? a) Acute ITP ...... b) Secondary ITP (diet induced) ...... c) Secondary ITP (infectious) ...... d) Secondary ITP (trauma induced) ...... e) Coagulation factors-induced ITP ......
4. Previous studies (experimental animal models) have described secondary ITP induced by the following drugs, except: a) Quinidine ...... b) Hapten-carrier conjugates ...... c) Coagulation factors ...... d) Heparin ...... e) Sulphonamide ......
5. Which of the following statements is/are true in regards to the passive and secondary animal models of ITP? a) They have helped in understanding the pathophysiology of acute ITP . . . b) They have helped in understanding the initiation or perpetuation of anti-platelet immunity ...... c) They have not helped in understanding the mechanism of action of IVIg ...... d) They are not ideal for studying chronic ITP pathophysiology ...... e) They are ideal for studying severe ITP pathophysiology ......
54 THE HANDBOOK FIRST EDITION NOTES
55 IMMUNE THROMBOCYTOPENIA * Basic science of ITP
1.4 Peptide therapy for p*atients with ITP
Stanislaw J. Urbaniak CHAPTER 1.4 • Peptide therapy for patients with ITP
1. Introduction This chapter will focus on the question of whether or not we can utilise our recent understandings of the basic immunology of ITP to develop targeted treatment that would be antigen-specific, addressing this from a clinical perspective. ITP is an autoimmune disorder in which autoantibodies are directed against an individual’s own platelets, leading to enhanced clearance of platelets and a consequent fall in platelet levels. ITP is a diverse syndrome (1). There are a large number of secondary causes, many associated with infections, disturbances of the immune system, haematopoietic diseases, and other causes, including drug- mediated platelet destruction. The majority of these secondary conditions, particularly in childhood, will recover spontaneously without any particular treatment, or by treating the underlying problem. The main problem is the large proportion of patients with so-called primary or idiopathic ITP, where the trigger is not known. Primary ITP is the commonest syndrome associated with severe bleeding, and about 75% are associated with IgG platelet-specific autoantibodies, most often directed against GPIIbIIIa or GPIbIX. Some antibody-negative cases are associated with platelet-specific cytotoxic T cells. Most ITP cases resolve satisfactorily with corticosteroids, with or without IVIg. In uncontrolled disease, splenectomy may be required, to remove a major site of platelet destruction and autoantibody production. In chronic cases, various immunosuppressive drugs are used in addition to corticosteroids. Unfortunately, most of these treatments are not specific for ITP and can have significant side effects. With better understanding of the regulation of the immune system, the nature of immune tolerance, and the mechanisms underlying human autoimmune disease, more rational treatments can be designed, including the possibility of antigen-specific immune regulation by peptide immunotherapy. As discussed in earlier chapters, the paradigm has been that there are autoantibodies against the platelet surface, which are then opsonised, and this causes the cells to be destroyed by the reticuloendothelial system. We now know that sometimes there is also decreased platelet production, which may respond well to new agents. More recently, evidence has accrued for the role of T cell-mediated toxicity. So there is now some rationale to explain how the conventional treatments are acting and also for some of the newer treatments that are currently being introduced. The basic problem with conventional treatments is that they are not specific. Corticosteroids have been used for a long time, as have immunosuppressive agents. They suppress the immune system, but they also suppress the abnormal immune response and can be associated with life-threatening and even lethal infections. It is therefore important to try and target treatment specifically towards the immune trigger that is responsible for the autoimmune response.
57 IMMUNE THROMBOCYTOPENIA 2. Tolerance A major feature of the immune system is “tolerance”, the ability not to react to certain antigens, especially “self” antigens, while still inducing protective immunity to foreign antigens. A number of different mechanisms for self-tolerance have been demonstrated in humans and animal models. These include: • the “censorship” of potentially autoreactive cells by killing them (central tolerance) or rendering them inherently unresponsive (peripheral tolerance) • lack of effective presentation of autoantigens • various forms of active immune regulation. Failure of these protective mechanisms can result in the emergence of clinically relevant autoimmune diseases, such as ITP, and specific treatments may be devised to correct or reverse these defects, such as peptide immunotherapy. The common feature of all the various presentations of ITP is loss of tolerance, or failure to delete or inactivate lymphocytes that recognise self-antigens, leading to antibody production and/or the presence of auto-aggressive T cells. Autoantibodies have been most widely studied, and are present in the majority of patients, either in the serum, or in platelet eluates. Most of the autoantibodies that are detected a b are directed against the integrin IIb 3 or GPIIbIIIa (CD61). This is not surprising since this is the fibrinogen receptor with the greatest number of copies, so it is the antigen that is expressed in largest amounts on platelets. These epitopes are external to the platelets, which is why they can be detected by the autoantibodies. More recently it has been shown that there are subsets of T lymphocytes that will attack the platelets directly, possibly by membrane-membrane contact or cytolytic enzyme processes. Thus the immune response includes both a B cell response and a T cell response which both require controlling. A central element in all that follows is the fact that T cells recognise short peptides derived from an antigen, when presented by antigen presenting cells APC ( Figure 1). The theory in thymic clonal tolerance is that cell peptides that have a high affinity for the cells result in deletion of the T cell by apoptosis and are removed. If T cells fail to recognise any of the peptides, which may be cryptic peptides, then there is no recognition and those cells die. The peptides that give trouble are those that are of low affinity, as these peptides can generate cells that survive in the circulation in low numbers against self. The other central tenet, in terms of activation of tolerance, is that two signals are needed in order to generate an innate immune response ( Figure 2 ). The key feature is costimulation, with or without a “danger” signal, such as the multi-array copies on bacteria that trigger the Toll receptor responses and cytokine responses. Under those circumstances there is triggering of the innate immune system, but when
58 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP
Figure 1: Thymic selection of T lymphocytes based on antigen recognition
T T
No recognition Apoptosis
T T T T
Deletion Survival (Apoptosis) APC APC
Low affinity High affinity
POSITIVE SELECTION NEGATIVE SELECTION
Figure 2: Anergy of T lymphocytes in absence of costimulation
T
T T T T CD28
B-7 Proliferation Unresponsive I n t e
r APC APC l e u k i n s Costimulation No costimulation “danger” signal Absence of “danger” signal
ACTIVATION ANERGY
59 IMMUNE THROMBOCYTOPENIA peptides are introduced in the absence of the “danger” signal, the T cells with the appropriate specificity become unresponsive, and when they are subsequently exposed to antigen, they do not respond, i.e. they are effectively tolerant. A third major element in relation to how one might approach immunoregulation is the recent emergence of the understanding of the balance between effector T cells and regulatory T cells, as discussed in Chapter 1.2. The natural Tregs are characterised by the expression of CD25 and FOXP3 and there are two major types of inducible Tregs recognised with their own cytokine specificities. The hypothesis is that in autoimmune disease, there is dysregulation between the effector cells that might induce the disease, and the regulatory cells controlling the immune response. With greater understanding, it may be possible to tip the balance towards regulation and away from disease.
3. Failure of tolerance There are essentially three elements in the failure of tolerance, which might be addressed in terms of peptide immunotherapy. These are: • failure to delete autoreactive T cells • presentation of “cryptic” autoantigens which escape regulation • loss of regulatory T cell numbers or function.
3.1 Failure to delete autoreactive T cells Central tolerance is always incomplete, because peripheral blood Th cells from healthy human donors can be induced to respond in vitro to recognise self-antigens. In ITP patients, lymphocytes are already activated in vivo and demonstrate an accelerated T cell response in vitro to autologous platelet-specific glycoproteins, and also autoantibody production in vitro . It is therefore the failure of mechanisms to control activation of autoreactive lymphocytes that drives disease, rather than deletion. It is considered that, in order for an active immune response to be generated, an antigen must not only be foreign, but present in the context of “danger” e.g. other signs of infection or tissue damage, which may explain the association of ITP with several infectious agents. There is good evidence that autoreactive T cells are present in the circulation in patients with ITP. Whole platelets were found to induce a helper Th1 immune response in ITP patients, but not in normals (2, 3).
3.2 Presentation of “cryptic” autoantigens which escape regulation Self-epitopes may be effectively hidden from Th cells, or “cryptic”, because they are inefficiently processed and presented by APC from the intact antigen. These epitopes may span the cut sites of APC processing enzymes and are subject to
60 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP
“destructive processing”, or they may bind MHC molecules too weakly to compete with other peptides for presentation. Inefficient presentation of particular epitopes will allow the respective Th cells to escape thymic deletion and remain in the periphery. Evidence to support this hypothesis has been obtained in human ITP where multiple T cell epitopes on the GPIIIa protein may be cryptic, are rarely recognised by Th cells of normal subjects, and fail to show predicted high affinity binding association with HLA-DR (4). Molecular mimicry to cryptic self-antigens may play a part in triggering some forms of autoimmunity.
3.3 Loss of regulatory T cell numbers or function Epitopes on antigens induce the expansion of multiple CD4 + Th cell clones and it is the balance of these clones and the cytokines they produce that determines the overall development and form of the immune response. Th cells can be classified as functional subsets, Th1 cells producing IFN- g, and Th2 cells producing IL-4. The balance between Th1/Th2 to some extent determines regulation of an immune response. Many models of autoimmune disease have a Th1 bias, which is also seen in ITP, and which is reversed on treatment. To counterbalance the effector Th subsets, at least two regulatory T cell (Treg) types have been identified. “Natural” Tregs, express the transcription factor FOXP3 and high levels of the surface activation marker CD25, differentiate in the thymus and have regulatory properties that depend on cell-cell contact. In contrast, “induced” Treg subsets develop from naïve cells in the periphery after antigen exposure, and inhibit effector responses by secretion of suppressive cytokines. Tr1 cells, secrete IL-10 and Th3 cells secrete TGF- b1. Patients with ITP have been shown to have several changes in T cell function which can be interpreted as loss of tolerance, e.g. reduction in number and function of CD25 +FOXP3 + Tregs, which can fluctuate with disease activity, and may reflect ongoing struggle between the effector and regulatory arms of the immune system, and which may be amenable to correction by specific peptide immunotherapy.
4. Studies using glycoprotein fragments The Japanese group led by Kuwana (3) have elegantly shown, using glycoprotein fragments, that there are epitopes recognised within these recombinant fragments in the circulation of patients with ITP ( Figure 3 ), i.e. there are particular stretches of the glycoproteins which are the targets for the autoimmune response. We ourselves have synthesised the entire sequence of the GPIIIa protein, in overlapping peptide sequences 15 amino acids long, in order to see if we could identity exactly which parts of the GPIIIa molecule were responsible for generating the autoimmune response.
61 IMMUNE THROMBOCYTOPENIA Figure 3: Th cells from ITP patients respond to epitopes on recombinant IIIa fragments
ITP2 ITP18 ITP21 n o i
t 6 - 4 - 4 - a r o ) p
r 4.5 - 3 - 3 - m o p c c n i ∆
3 - 2 - 2 - 3 e 0 n i 1
d I I I I i x I I I I I I 1.5 - I 1 - I 1 - I I ( I I I I b b b b I I I I m I I I I I I a a a a b b y a a a a I I a a 2 2 5 5 2 2 7 7 h a a t 1 1 2 4 2 4 6 6 5 5 0 - 0 0 - 0 0 - - 8 8 2 4 2 4 6 6 4 4 8 8 T 2 2 1 5 2 9 H S ------3 6 6 4 7 6 5 2 2 2 5 2 5 8 8 4 4 7 7 G G G 7 4 8 5 2 2 5 5 6 7 6 7 4 4 6 6 6 6 ------S S 9 9 2 5 2 5 1 1 2 2 2 2 T T 8 4 6 4 2 8 0 5 6 4 2 1 a 7 2 5 2 a I a a b a a I I I I I b b I I I I I I I I I
GPIIb-IIIa fragments
Modified from (3)
We confirmed that at the epitope-specific level, there are, in the autoimmune ITP patients, specific Th cells that recognise specific peptide epitopes (4). These are recall responses, which shows that there are antigen-specific Th cells that escape tolerance are present in the circulation ( Figure 4 ) and are ultimately responsible for triggering the effectors in their response. A feature of these epitopes is that they do have a low affinity for HLA class II. We identified seven particular epitopes that were present with greatest frequency ( Table 1 ). As one would predict for autoimmune epitopes, they have low affinity for MHC class 2 rather than high affinity, which is conventional in the immune response. Kuwana’s group (5) showed that lymphocytes from normal individuals do not respond to trypsin-digested GPIIb-IIIa, but cells from patients with ITP do ( Figure 5), suggesting the presence of cryptic autoantigens that escape regulation relevant to platelets and ITP. The hypothesis is that trypsin digestion has exposed epitopes that are not usually processed in normal individuals, but they are recognised in patients with ITP. Inspection of at the seven dominant peptides that we identified from our peptide scan confirmed that these epitopes can be cryptic, and Figure 6 shows the predicted locations of these particular sequences; one of them is actually transmembrane.
62 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP
Figure 4: Th cells from ITP patients respond to GPIIIa synthetic peptide sequences
1.5 -
1.0 -
0.5 -
) A
D 0 - S - / + 3
0 2 - 1
x
m 1 - p c (
n
o 0 - B i t a r o p r o
c 6 - n i
e n
i 4 - d i m y 2 - h T - H 3 0 - C
1.0 -
0.5 -
0 - D GPIIIa peptide stimulus
Modified from (4)
As discussed in Chapter 1.2, there is evidence that regulatory T cells are deficient in ITP, either in numbers or in function, and that they can recover when the patients are in remission. It appears these are general changes in regulatory CD25 + T cells, rather than in cells specific for any antigenic epitope. We found with our peptide scan method, that the responses to the pathogenic epitopes change over time, as
63 IMMUNE THROMBOCYTOPENIA Table 1 : GPIIIa peptide autoepitopes in ITP
Dominant Position on GPIIIa* HLA-DR Patients with AITP with PBMC peptide no. molecules bound response to peptide with high High-affinity DR No high-affinity affinity † expressed DR expressed 2(aa6-20) PSI domain None None AITP1, AITP3, AITP9, AITP10, AITP11, AITP18, AITP19, AITP20, AITP21, AITP22, AITP24 44(aa331-345) Spanning βA and DR04, DR07 AITP8, AITP24 AITP1, AITP3, hybrid domain AITP10, AITP18, AITP21 47(aa361-375) Hybrid domain None None AITP7, AITP9, AITP10, AITP11, AITP16, AITP19, AITP20, AITP21, AITP22, AITP23, AITP24, AITP26 53(aa421-435) Spanning hybrid and DR04, DR13 AITP11, AITP-12, AITP1, AITP8, PSI domain AITP18, AITP20 AITP9, AITP10, AITP22, AITP24 70(aa591-605) EGF-like domain None None AITP2, AITP7, AITP15, AITP20, AITP24 77(aa661-675) EGF-like domain DR04, DR15 AITP11, AITP16, AITP2, AITP8 AITP22 82(aa711-725) βTD domain DR01, DR08, #AITP1, AITP3, #AITP7, AITP12 (transmembrane/ DR11, DR13, DR15 AITP4, AITP5, cytoplasmic) AITP9, AITP10, AITP11, AITP16, AITP18, AITP19, AITP20, AITP21, AITP22, AITP24 *From structural analysis of b3 integrin. †Predicted using the Propred algorithm (http://www.im- tech.res.in/raghava/propred/). #Significant association between response to peptide 82 and expression of HLA-DR molecules to which peptide predicted to bind with high affinity (chi-square test = 10; p < 0.05). Epitopes are low affinity for MHC class II. AITP: auto-immune ITP. Modified from (4)
seen in other autoimmune diseases. This would be preliminary experimental evidence that there are platelet antigen-specific regulatory T cells in balance with effector cells in the circulation of ITP patients.
64 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP
Figure 5: Th cells from ITP patients respond to cryptic epitopes on denatured GPIIbIIIa n o
i P1 P3 P7 P5 t a r 20 - o p ) r o m c p n c
i
3 10 - e 0 n 1 i
d x i (
m 0 - y T T T T a a a a e e e e 9 9 9 9 5 5 5 1 1 1 5 1 h I I I I S S S S n n n n 5 5 5 5 7 7 7 4 4 4 7 4 I I I I t G G G G o o o o I I I - 2 2 2 2 5 5 5 8 8 8 5 8 - l l l l ------b a a a a H b b b I 8 8 8 8 4 4 4 6 6 6 4 6
3 I I I I 1 1 1 1 4 4 4 6 6 6 4 6 I I I P m m m m 2 2 2 5 5 5 2 5 P P P α α α α u u u u G
i i i i b b b b G G G α α α α α α α α
I I I I d d d d d b b b b b b b b I I I I d d d I I I I I I I I e e e e e I I I I I I I I e e e t M M M M t t t s s s s e e e e g i g g g i i i d d d d - - - - n i n n n i i i s s s s p p p p y r y y y r r r T T T T
Modified from (5)
5. Potential clinical applications of peptide therapy In autoimmune and allergic diseases, there are inappropriate immune responses which need to be modulated. The new information in the development of antigen-specific therapy in ITP, as in other autoimmune diseases, is identification of the “autopeptides”. As discussed, Th cells recognise short peptides processed from antigen and displayed bound to the MHC class II molecules of antigen presenting cells. Critical to recognition of the peptide by the T cell receptor (TCR) is a core epitope, typically of nine amino acids. Identification of peptide epitopes from well-defined autoantigens that trigger the specific immune response offers the possibility of two approaches: 1) re-establishing self-tolerance to the dominant Th epitopes on autoantigens, and 2) enhancing regulatory responses to distinct epitopes recognised by Treg cells. It has been shown from animal models of other autoimmune diseases that peptides containing dominant Th cell epitopes can prevent responses to the corresponding antigen when given in soluble form without adjuvant or if administered by the tolerogenic mucosal route. Importantly, induction to tolerance to only one dominant epitope, particularly if mediated by active immune regulation, can ablate responsiveness to the entire autoantigen from which it is derived and also to other, associated antigens by a process of bystander or linked suppression (6).
65 IMMUNE THROMBOCYTOPENIA Figure 6: GPIIIa peptide epitopes in ITP. Th-cell epitopes can be cryptic, and different from autoantibody binding sites
α β GPIIb-IIIa (Integrin IIb 3) CD61/CD41
βpropeller βA domain Dominant Position A peptide no. on GPIIIa M P44 2(aa6-20) PSI domain Hybrid domain P47 44(aa331-345) Spanning βA and P53 hybrid domain PSI domain P2 47(aa361-375) Hybrid domain Thigh domain 53(aa421-435) Spanning hybrid and PSI domain EGF repeats P70 70(aa591-605) EGF-like domain Calf P77 domains 77(aa661-675) EGF-like domain Cystatin-like domain 82(aa711-725) βTD domain (transmembrane/ cytoplasmic)
P82
Modified from (4)
To exploit a similar strategy in ITP, it is first necessary to identify suitable peptides. As described above, by screening a panel of short, overlapping peptides spanning the entire sequence of platelet GPIIIa for the ability to stimulate recall responses by peripheral blood Th cells from ITP patients, we were able to identify 7 immunodominant epitopes as candidates for further study. The strategy of “therapeutic vaccination” to reinstate tolerance with peptides is showing promise in a number of early human trials of immune-mediated disease, and the identification of a small number of dominant GPIIIa sequences that are recognised by autoreactive Th cells from most patients with ITP is the first step towards specific immunotherapy. Lessons can be learnt from the way that the natural immune system develops tolerance to infection ( Figure 7 ). In a normal situation where antigen is encountered in a pro-inflammatory environment, there is a risk of autoimmune reaction if the appropriate antigen-specific cells are present. However experiments over many
66 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP
Figure 7: Nature of antigen encounter determines the immune response
Hypersensitivity: Hyposensitisation: allergy and autoimmunity therapeutic tolerance Complex or insoluble antigen Soluble simple antigen y r
e (self or environmental) (peptide) h p
i Inflammatory cytokine Steady-state cytokine milieu r e
P milieu innate triggers No innate triggers
β s lL-12 lL-4 lL-10 TGF- c i t Activated a Steady-state h APC p APC m y L y r T 1 T 2 Treg
e H H h p i r e
P lgG lgE lgG4 lgA
Modified from (6) years, particularly in animal models, have shown that it is possible to present peptides from the same antigens in such a way that they do not trigger inflammatory cytokines or inadvertently trigger the innate immune system. In these situations, instead of triggering an immune response, the immune response is biased or deviated towards the production of regulatory T cells, which subsequently do not respond to the antigen. This is the key to the search for appropriate peptide immunotherapy. It has been shown from animal models of other autoimmune diseases that peptides containing dominant Th cell epitopes can prevent responses to the corresponding antigen when given in soluble form without adjuvant or if administered by a tolerogenic mucosal route. Importantly, induction to tolerance to only one dominant
67 IMMUNE THROMBOCYTOPENIA epitope, particularly if mediated by active immune regulation, can ablate responsiveness to the entire autoantigen from which it is derived and also to other, associated antigens by a process of bystander or linked suppression. There are now many examples of successful treatments based on this approach for inhibiting animal models of immune-mediated disease, including autoimmune haemolytic anaemia and responses to blood group antigens, and human trials are under way in patients with various allergies, rheumatoid arthritis, multiple sclerosis and type 1 diabetes (6).
6. Approaches to inducing tolerance with peptides A number of approaches to inducing tolerance with peptides have been looked at experimentally. • Immunodominant peptide delivered by a tolerogenic route. One approach is to take the dominant peptides that trigger Th cells and deliver these particular peptides by a route that is biased to tolerisation, the mucosal route (see below). • Parenteral delivery of purified peptides. Another approach is to devise antigen- specific peptides, which are purer, soluble, without adjuvants and have been designed not to elicit any danger signals or second signals, and therefore bias the antigen-specific response to tolerance rather than inflammation. • Preferential stimulation of Tregs. A third approach, for which there is evidence in autoimmune haemolytic anaemia with the rhesus blood group is to identify epitopes that, instead of triggering Th cell responses, actually preferentially stimulate T regulatory cells and IL-10 induction. At present there is no data on this possibility in ITP. • Altered peptide ligands (APL). An approach of great scientific interest is taking the autoallergic peptides and modifying them, to enhance anergy. This is a theoretical approach, but in practice it has not actually worked yet. • TCR peptide therapy. A more recent concept is to reproduce the structure of the T cell receptor that recognises the autoantigenic peptide bound to the relevant MHC class II instead of reproducing the autoantigenic peptide. This is a novel concept; the major problem is that it would be highly MHC restricted, which is not always relevant for autoimmune diseases, which generally do not have any specific MHC class II restrictions.
7. The mucosal immune system and tolerance Most of the work on tolerisation using animal models that has moved on to the clinic has taken advantage of the features of the mucosal immune system. The mucosae are obviously, a barrier, against foreign antigens. As well as bacteria, we are also continuously exposed to food antigens in the gut and inhaled antigens
68 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP such as pollens in the nasal passages. The immune system has developed a way of dampening down unwanted responses to this foreign material. The common theme to these responses is that they involve regulatory T cells and inhibitory cytokines such as TGF- b or IL-10. Three mechanisms have been proposed to explain mucosal tolerance, which are not mutually exclusive: • ignorance of the antigen by the immune system (anergy) • deletion of T cells that respond to the inhaled or ingested antigen • generation of Tregs that suppress or down-regulate the response to the antigen. An interesting observation that was found through animal work is that there is a difference between what is called low dose tolerance and high dose tolerance. Low dose tolerance, in which repeated low doses of antigen are given, favours generation of regulatory T cells. On the other hand, giving repeated high doses of antigen tends to favour terminal deletion, or peripheral tolerance, by anergising the T cells. In practice, the majority of the development work has focused on low antigen doses.
8. Peptide epitopes for tolerisation There are many potential advantages to developing peptide immunotherapy rather than using immunosuppressive drugs. The therapy could be targeted against specific antigens that are the subject of the effector immune response. They are simple synthetic chemicals, so it should be easy to make them in a non-toxic format. Because they are short, synthetic peptides they will be cheap and easy to make, and easy to give as they do not need adjuvants to have an effect. However, there is always a concern in autoimmune diseases, that an auto aggressive response could be exacerbated, making the patient much worse by the treatment. There are nevertheless some features that help to identify suitable peptides. • They should be soluble in aqueous solution so they can be presented at a high concentration. This can be a challenge because some of the autoepitopes may be transmembrane, such as in multiple sclerosis. • Short peptide sequences without tertiary structure are an advantage, because if peptides fold in particular ways they can trigger a B cell and produce antibody response against the peptides, which occurs with some of the monoclonal antibody treatments. • They should be native protein sequences that nature has devised. If the sequences are modified (APL), they may behave unpredictably, and some clinical trials have actually been stopped as a result of unpredicted responses when using altered peptide ligands. • Ideally peptides would be active by the systemic route, subcutaneously is a
69 IMMUNE THROMBOCYTOPENIA favourite, or by the mucosal route. The two routes that have been studied most are oral administration of antigen or using the nasal mucosa. • Ideally, the peptides will not trigger the innate immune system and therefore not generate the “danger” signals that would make things worse rather than better. • A particular challenge is that these peptides are MHC-bound when presented to T cells, to make the product clinically useful. It is therefore an advantage to have peptides that are promiscuous in their MHC class II-type binding. An approach that has been taken by ourselves (4) and a number of others (6) is to select a limited number of epitopes that will cover a large number of MHC types, because each has their preferential binding; this is a solution to the problem of devising peptide therapy specific for every existing MHC class II type, which would be impossible. An advantage is that the natural peptides seem to be promiscuous anyway. • The ideal product would come from identifying those particular peptides that trigger regulatory T cells in an antigen-specific way. Despite many apparently successful animal models, a number of unforeseen problems have arisen in patients so that clinical studies have been limited. The problem with autoimmune disease, as opposed to alloimmune disease, or in transfusion or transplantation situations, is that it’s difficult to know which is the relevant antigen. Our understanding of the autoimmune response is still limited. Another challenge is that the immune system of the patient may be abnormal in the first place, and the information that you’re extracting from normal individuals may not actually apply to them. Last but not least, an animal model that works beautifully, may not work when applied in the clinic. Nevertheless, there are a number of peptide antigens which are now in the Phase I and Phase II clinical trials in the allergy field and the autoimmune field, including pollen allergy, bee venom allergy, rheumatoid arthritis and multiple sclerosis (6). Generally, the nature of tolerisation in these particular diseases is an increase in regulatory T cells and the production of increased levels of IL-10 and inhibitory cytokines. In the autoimmune field animal models have allowed the identification of relevant autoantigens, which are the targets for immunotherapy. In rheumatoid arthritis there is the bacterial heat shock protein dnaJP1, in multiple sclerosis the myelin basic protein, and in diabetes there is the heat shock protein 60 and some epitopes from insulin (6). These are all in Phase I studies and some are in Phase II. So far no serious side effects or mortality has been reported and we await the longer term studies to find out whether or not disease remission can be induced, which of course will take some time. So far no similar studies have been undertaken in ITP. The reason for that is that it has not previously been clear which antigens to target, although our data now indicates that there are seven peptides which are T
70 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP helper cell epitopes on GPIIIa that are recognised by most ITP patients ( Figure 8 ), and are low affinity MHC-promiscuous peptides. The next step is to look for a suitable animal model. In future, it may be possible to use the b-3 knock-out mice model
Figure 8: GPIIIa peptide autoepitopes in ITP
PPD - 86 - % AITP patients responding 85 - 84 - to GPIIIa peptides 83 - 82 - 81 - % Control donors responding 80 - 79 - to GPIIIa peptides 78 - 77 - 76 - % Individuals responding 75 - 74 - 73 - to control antigen PPD 72 - 71 - 70 - 69 - 68 - 67 - 66 - 65 - 64 - 63 - 62 - 61 - 60 - 59 - 58 - 57 - 56 - 55 - s 54 -
u 53 - l 52 - u 51 - 50 - m 49
i -
t 48 -
s 47 - Dominant Position 46 - e 45 -
d 44 - peptide no. on GPIIIa i 43 - t 42 - p 41 - e 40 - 2(aa6-20) PSI domain p 39 - 38 - a 37 - I 36 - I 35 I - 44(aa331-345) Spanning βA and 34 - P 33 - hybrid domain G 32 - 31 - 30 - 29 - 28 - 47(aa361-375) Hybrid domain 27 - 26 - 25 - 24 - 23 - 53(aa421-435) Spanning hybrid 22 - 21 - and PSI domain 20 - 19 - 18 - 17 - 16 - 70(aa591-605) EGF-like domain 15 - 14 - 13 - 12 - 77(aa661-675) EGF-like domain 11 - 10 - 9 - 8 - 7 - 82(aa711-725) βTD domain 6 - 5 - (transmembrane/ 4 - 3 - cytoplasmic) 2 - 1 - 100 80 60 40 20 0.0 20 40 60 80 100 % Individuals with T cell responding to GPIIIa peptides
Modified from (4)
71 IMMUNE THROMBOCYTOPENIA described in Chapter 1.3 to try to model tolerisation. If we can identify the most relevant Th epitopes then there would be two options ( Figure 9 ) for further studies: • to deliver them by the tolerogenic route, which would induce anergy, • to give them by repeated small doses, mimicking allergy immunotherapy it may be possible to induce a regulatory Th response. Thus there may be an antigen-specific way of switching off the entire autoimmune response.
Figure 9: Potential peptide immunotherapy
Immunodominant T cell epitopes Regulatory T cell epitopes (Tr1) ITP peptide A ITP peptide B
Mucosal route Tr Immunisation
Tolerance/Anergy Regulation: IL-10 help help Th1 B Th1 B
9. Conclusion The need for specific, effective, and safe treatment for patients with chronic ITP may be met by the development of peptide immunotherapy to re-induce Th tolerance to the platelet glycoproteins. Th cells recognise short peptides processed from antigen and displayed bound to the MHC class II molecules of antigen presenting cells. Critical to recognition of the peptide by the T cell receptor (TCR) is a core epitope, typically of nine amino acids. Identification of peptide epitopes from well-defined autoantigens that trigger the specific immune response offers the possibility of two approaches: 1. re-establishing self-tolerance to the dominant Th epitopes on autoantigens, and 2. enhancing regulatory responses to distinct epitopes recognised by Treg cells. It may be that we are now at the beginning of developing peptide-specific therapy for ITP.
72 THE HANDBOOK FIRST EDITION CHAPTER 1.4 • Peptide therapy for patients with ITP
Acknowledgements Prof Rob Barker, Professor of Immunology, University of Aberdeen, Hosea Sukati, PhD student, University of Aberdeen, and Dr Henry Watson, Consultant Haematologist, NHS Grampian, Aberdeen, who were my co-investigators for reference 4.
References 1. Cines DB, Bussel JB, Howard A, et al. The ITP syndrome: Pathogenic and clinical diversity. Blood 2009; 113: 6511–6521. 2. Semple JW, Freedman J. Increased antiplatelet T helper lymphocyte reactivity in patients with autoimmune thrombocytopenia. Blood 1991; 78: 2619–2625. 3. Kuwana M, Kaburaki J, Kitasato H, et al. Immunodominant epitopes on glycoprotein IIb- IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura. Blood 2001; 98: 130–139. 4. Sukati H, Watson HG, Urbaniak SJ, Barker RN. Mapping helper T-cell epitopes on platelet membrane glycoprotein IIIa in chronic autoimmune thrombocytopenic purpura. Blood 2007; 109: 4528–4538. 5. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive T cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura. Role in production of anti-platelet autoantibody. J Clin Invest 1998 102: 1393–1402. 6. Larche M, Wraith DC. Peptide-based therapeutic vaccines for allergic and autoimmune diseases. Nat Med 2005; 11: S69–76.
Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/itphandbook2010/
1. Which of the following mechanisms are involved in the development of ITP? a) Loss of self tolerance to platelet antigens ...... b) Antigenic cross reactivity: viral or bacterial infections ...... c) Neoantigens: altered antigen expression - drug induced ITP ...... d) Immune deficiency: association with other systemic autoimmune disorders ...... e) All of the above ......
2. The following agents belong to the second line treatment of ITP, except: a) IVIg ......
73 IMMUNE THROMBOCYTOPENIA b) Azathioprine ...... c) Cyclophosphamide ...... d) Rituximab ...... e) Romiplostim ......
3. The loss of self tolerance (failure to eliminate or deactivate self-reacting lymphocytes) results in the formation of autoantibodies. Which of the following statements is/are true in this regard? a) They are detected in about 15% of patients with primary ITP ...... b) The platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa) is the major autoantigen ...... c) Glycoproteins GPIb/IX, GPIa/IIa, and GPV are highly frequent ...... d) Epitopes are internal to the platelet surface ...... e) A subset of autoantibody have autoaggressive cytotoxic B cells against platelet ......
4. The following statements describe the role of the mucosal immune system in the development of tolerance, except: a) Prevents invasion of mucous membranes by dangerous pathogens ...... b) Prevents uptake of foreign antigens from ingested food, airborn matter and commensal micro-organisms ...... c) Prevents potentially harmful immune responses to these antigens if they enter the body ...... d) Induces tolerance by stimulating T cells in response to inhaled or ingested antigen ...... e) Induces generation of regulatory T cells that control and/or down modulate the inflammatory response against the antigen ......
5. Which of the following is/are ideal characteristics of peptide epitopes for tolerisation? a) Insoluble in aqueous solution (at high concentration) ...... b) Large linear sequences, avoiding tertiary structure ...... c) Inactive by systemic or mucosal administration ...... d) Trigger innate immune system (TLRs) ...... e) Appropriate MHC-binding characteristics ......
74 THE HANDBOOK FIRST EDITION