- Email: [email protected]
Thrombopoietin receptor agonists: a new immune modulatory strategy in immune thrombocytopenia?
Author’s Accepted Manuscript Thrombopoietin Receptor Agonists: A New Immune Modulatory Strategy in ITP? Alexandra Schifferli, Thomas Kühne
www.elsevier.com/locate/enganabound
PII: DOI: Reference:
S0037-1963(16)30022-1 http://dx.doi.org/10.1053/j.seminhematol.2016.04.010 YSHEM50866
To appear in: Seminars in Hematology Cite this article as: Alexandra Schifferli and Thomas Kühne, Thrombopoietin Receptor Agonists: A New Immune Modulatory Strategy in ITP?, Seminars in Hematology, http://dx.doi.org/10.1053/j.seminhematol.2016.04.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Thrombopoietin receptor agonists: A new immune modulatory strategy in ITP? Alexandra Schifferli, MD1, Thomas Kühne, MD1
1
Department of Oncology/Hematology, University Children’s Hospital Basel, Basel, Switzerland
Keywords: ITP; immune modulation; thrombopoietin receptor agonist; microparticles, TGF-β
Correspondence: Dr. Alexandra Schifferli, Department of Hematology/Oncology, University Children’s Hospital Basel, Spitalstrasse 33, 4031 Basel, Switzerland. E-mail: [email protected] Telephone: 0041 61 704 12 12 Fax: 0041 61 704 12 13
Conflict of interest statement: The authors declare no conflict of interest
2 Abstract In 2008, new drugs that mimic the effects of thrombopoietin became available for the treatment of primary immune thrombocytopenia, e.g., romiplostim and eltrombopag. These drugs activate the thrombopoietin receptor, stimulate the production of megakaryocytes, and increase the production of platelets. Important clinical observation has been gained, such as unexpected long-term remission after stopping thrombopoietin receptor agonists. The pathophysiology of this unforeseen cure is currently the subject of discussion and is investigated in clinical trials and laboratory research projects. Here we evaluate the different hypotheses on how thrombopoietin receptor agonists can affect the immune system, particularly the induction of tolerance, and by which mechanisms this may be achieved.
Introduction Thrombopoietin receptor agonists (TPO-RAs) have been designed to stimulate megakaryopoiesis. In 2008, the FDA approved two drugs, romiplostim and eltrombopag, for the treatment of primary immune thrombocytopenia (ITP). Both drugs exhibit high efficacy with acceptable adverse effects (1-3). The success story of TPO-RAs appears to be greater than expected. Data exist suggesting that stimulation of the receptor for thrombopoietin (TPO), the c-MPL receptor, may induce immunological mechanisms to restore immune tolerance in ITP (4, 5). ITP is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. The dysregulation of the immune system in ITP involves the innate and adaptive immune system. Multiple mechanisms underlie the development of autoimmunity, most of which involve
3 disturbances in regulatory circuits, cytokine synthesis, and signaling pathways. Patients with ITP harbor an imbalance in the T-cell subsets, with a shift toward Th1 and Th17 cells (6, 7). Moreover, it has been shown that there is a decrease in regulatory T and B cells (8, 9). These changes can be defined by the measurement of cytokines. Patients with ITP have a shift toward interleukin-2 (IL-2), interferongamma (IFN-γ), and IL-17 cytokines, all corresponding to a pro-inflammatory/immune response, as well as a drop in many cytokines, such as IL-10, transforming growth factor-β1 (TGF-β),IL-4, and IL-35. In this review article, we will discuss new insights into possible immunomodulatory functions of TPO-RAs.
Clinical background Recently, reports on long-term remission after discontinuation of TPO-RAs in adult patients with chronic ITP have been published. Several case reports, retrospective data, and a clinical trial revealed sustained platelet counts above 100 × 109/L after discontinuation of romiplostim in up to 30% of the patients (10-12). Červinek et al retrospectively analyzed 46 patients with relapsed and refractory ITP treated with TPO-RAs (10). In 11 of these cases, TPO-RA therapy (romiplostim, 7; eltrombopag, 4) could be successfully stopped, with a median follow-up of 33 months. The probability of remission appeared not to depend on previous treatment, splenectomy, or disease duration. In a case study of eight romiplostim trials, Bussel et al (12) examined patients with sustained remission after discontinuation of romiplostim; remission was identified in 27 patients. No clear predictors of remission were identified; however, a number of patients had ITP for <1 year and received
4 romiplostim for <1 year. This observation was supported by a prospective trial in patients with ITP lasting for <6 months; remission was observed in approximately 1/3 of adult patients who were treated with romiplostim with a median time (range) to onset of 27 (6–57) weeks (11).
Theoretical background Several theories on the induction of immune tolerance by TPO-RAs are proposed: -
Unknown and unexpected effects of the c-MPL receptor (e.g., c-MPL receptor on immune cells)
-
Unknown effects of TPO-RAs, which may not directly be related to the stimulation of the c-MPL receptor and involving the immune system (e.g., cross-reaction with activation or inhibition of other receptors and pathways of the immune system)
-
Immune modulation following increased platelet mass. Two mechanisms may be involved: 1. induction of immune tolerance by exposure to high-dose antigen or 2. innate platelet immune activity
At present, the latter theory appears to be the most plausible explanation. Platelets are more than just pro-coagulant fragments; indeed, accumulating evidence suggests an immunomodulatory role for platelets and platelet-derived micro-particles (PMP). Although they do not replicate or contain nuclei, platelets have multiple roles in host defense against infections and are involved in innate and adaptive immune response mechanisms (13-16). Thus, platelets seem to be part of the immune response. This theory is also consistent with the evolution of the hematological system. In many invertebrates and early vertebrates, one cell type (the hemocyte) carries out
5 hemostatic as well as host-defense functions. Platelets have the ability to bind to infectious agents, secrete many immunomodulatory cytokines and chemokines (alpha granules), and express receptors for various immune effector and regulatory functions, such as pattern recognition receptors (PRRs) (e.g., toll-like receptors), CD40L, and denaturated MHC class 1 molecules. Furthermore, platelets contain mRNA that originates from megakaryocytes and are capable of carrying out independent protein synthesis under different environmental stress, and they are able to release membrane micro-particles known to be involved in a variety of pathophysiological responses including immune responses, inflammation, angiogenesis, and tissue regeneration. In this respect, platelets are truly immunological cells.
Research background TPO-RA’s increase platelet production in the bone marrow and subsequently induce a rise in peripheral platelet count. This acute increase in platelet mass could induce tolerance mechanisms. The following five research topics support our theory that TPO-RAs could have an immunomodulatory function (figure 1).
1. Induction of immune tolerance by exposure to high-dose antigen T cell anergy is a key tolerance mechanism suppressing unwanted T-cell activation against self by rendering lymphocytes functionally inactive following antigen contact. Antigen (Ag) plays an important role in anergy induction, where high supra-optimal doses lead to the unresponsive phenotype (18-20). An antigen density of >100 peptide-MHC complexes/cell was found to be the transition point
6 between dominant activation and inhibition cascades, whereas higher antigen doses induced an anergic functional state (21). A rapid increase of platelet mass such as TPO-RA effects increase the antigen mass in patients with ITP, and so may inactivate T cells. However, it is unknown how much the Ag density rises with TPO-RAs and if this rise is sufficient to inactivate autoimmune T cells.
2. Platelet (micro-particles)-mediated immunoregulatory pathways Micro-particles, also known as ectosomes or micro-vesicles, are important mediators of intercellular communication. They are released by budding directly from the cell membrane surface either spontaneously or in response to various stimuli. Micro-particles contain cytosolic substances and phosphatidylserine in their membrane. Depending on their cellular origin, micro-particles have been associated with a broad spectrum of biological activities. In the last 10 years, it has been shown that platelet-derived-microparticles (PMP) have effects on the innate immune system, as well as on the induction of adaptive immunity with possibly tolerogenic activity (22). Increasing the mass of platelets, as caused by TPO-RAs, will necessarily increase the mass of circulating micro-particles. In a study using stored platelets, micro-particles appeared to show antiinflammatory and immunosuppressive properties (23). Indeed, the purified PMP reduced the release of TNF-α and IL-10 by activated macrophages. The effects on TNF-α lasted for 24 h, suggesting that micro-particles induced a modification of the differentiation of macrophages. PMP also induced the release of TGF-β from macrophages. In addition, the differentiation of monocytes to immature and mature dentritic cells (DC), as well as their activity, was modified by the presence of micro-particles. These data indicate that micro-particles shed by stored
7 platelets have intrinsic properties that downregulate macrophage and modify DC differentiation toward less reactive states. In another study (24), exposure of activated CD4+ T cells to PMP decreased their release of IFNγ, TNFα, and IL-6, and increased the production of TGF-β1. Concomitantly, micro-particles-exposed CD4+ T cells displayed increased frequencies of CD25high Foxp3+ regulatory T cells (Tregs). This was explained by induced differentiation of naïve T cells into Tregs, since micro-particles had no effect on pre-differentiated Tregs. These phenomena were dose-dependent and micro-particle-specific. PMP-mediated effects were partly dependent on platelet-derived TGF-β1.
3. Release of platelet TGF-β It is known that platelets contain the largest amount of transforming growth factorβ (TGFβ) in the body (25). TGF-β is believed to be important in the regulation of the immune system by Tregs and the differentiation of both Tregs and Th17 cells from CD4+ T cells (26, 27). An increased mass of platelets could increase the concentration of TGF-β and induce tolerance mechanisms.
4. Induction and improved immunosuppressive function of adaptive Tregs with TPO-RA Bao et al. studied immunologic profiles of patients with chronic ITP before and during treatment with TPO-RAs (28). Treg activity was improved in patients on treatment, and the improvement correlated with a reduction in IL-2-producing CD4+ T cells, consistent with a dampening of immune responses. There was a concomitant increase in total circulating TGF-β1 levels in patients receiving treatment, and the levels of TGF-β1 correlated with the degree of improvement in
8 platelet counts. This suggests that platelets in patients on TPO-RA treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-β1 because of high platelet counts and/or PMP turnover. This last hypothesis was supported by a cross-sectional study (28), showing increased TGF-β1 levels in addition to improved Treg activity in responders, but not in nonresponders. In an ITP mouse model, TPO treatment promoted peripheral induction of Tregs in conjunction with elevated circulating TGF-β levels and sustained platelet recovery (29). Again, it is hypothesized that a burst in platelet numbers correlates with increased TGF-β1 levels, which in turn increases the regulatory compartment.
5. Increased regulatory B cell (Bregs) function with TPO-RA In another study, Li et al. showed increased Breg (CD19 + CD24hiCD38hi) function in patients successfully treated with TPO-RAs (30). The frequency of Bregs improved as the platelet counts increased upon treatment. Because B-cell regulatory activity in vitro requires activation through CD40 engagement it is supposed that CD40L expressed on and in platelets, may directly contribute to improved Breg activity. However, these data are of limited value, since only five patients were studied, and the data must be confirmed by larger longitudinal studies.
9 Conclusions We believe that TPO-RAs exhibit tolerogenic activities because of increased platelet and probably because of micro-particle mass. PMP have been shown to exert in vitro immunosuppressive activities on phagocytes, modify the differentiation and maturation of DC toward less reactive states, and induce differentiation of CD4+ T cells toward functional Tregs, partly because of increased TGF-β1 levels. All these changes and probably the concomitant induction of T-cell anergy may represent the mechanism by which TPO-RAs enhance peripheral tolerance in ITP.
References 1. Newland A. Romiplostim: a breakthrough treatment for the management of immune thrombocytopenic purpura. Eur J Haematol Suppl. 2009;(71):20-5. 2. Bussel JB, Cheng G, Saleh MN, et al. Eltrombopag for the treatment of chronic idiopathic thrombocytopenic purpura. N Engl J Med. 2007;357:2237-47. 3. Imbach P, Crowther M, Thrombopoietin-Receptor Agonists for Primary Immune Thrombocytopenia. NEJM. 2011, 365;734-41. 4. 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;116:4639-45. 5. Nishimoto T, Numajiri M, Nakazaki H, et al. Induction of immune tolerance to platelet antigen by short-term thrombopoietin treatment in a mouse model of immune thrombocytopenia. Int J Hematol. 2014;100:341-4. 6. Ogawara H, Handa H, Morita K, et al. High Th1/Th2 ratio in patients with chronic idiopatic thrombocytopenic purpura. Eur J Haematol. 2003;71:283-8. 7. Zhang J, Ma D, Zhu X, et al. Elevated profile of Th17, Th1 and Tc1 cells in patients with immune thrombocytopenic purpura. Haematologica. 2009;94:1326-9. 8. Sakakura M, Wada H, Tawara I, et al. Reduced Cd4+Cd25+ T cells in patients with idiopathic thrombocytopenic purpura. Thromb Red. 2007;120:187-93. 9. Li X, Zhong H, Bao W, et al. Defective regulatory B-cell compartment in patients with immune thrombocytopenia. Blood. 2012;120:3318-25. 10. Červinek L, Mayer J, Doubek M. Sustained remission of chronic immune thrombocytopenia after discontinuation of treatment with thrombopoietin-receptor agonists in adults. Int J Hematol. 2015;102:7-11. 11. Newland A, Godeau B, Priego V, et al. A Final Analysis of a Phase 2, Single-Arm Study of Platelet (Plt) Responses and Remission Rates in Patients with Immune Thrombocytopenia (ITP) Receiving Romiplostim. ASH 2014 (Abstract 2775). 12. Bussel JB, Wang X, Lopez A, Eisen M. Case study of remission in adults with
10 immune thrombocytopenia following cessation of treatment with the thrombopoietin mimetic romiplostim. Int J Hematol. 2015;102:7-11. 13. Semple JW, Italiano JE Jr, Freedman J. Platelets and the immune continuum. Nat Rev Immunol. 2011;11:264-74. 14. Varon D, Shai E. Platelets and their microparticles as key players in pathophysiological responses. J Thromb Haemost. 2015;13 Suppl 1:S40-6. 15. Kapur R, Zufferey A, Boilard E, Semple JW. Nouvelle cuisine: platelets served with inflammation. J Immunol. 2015;194:5579-87. .
16. Yeaman M. Platelets: at the nexus of antimicrobial defence. Nat Rev Microbiol. 2014;12(6):426-37. 17. Morrell C., Aggrey A., Chapman L. et al. Emerging roles for platelets as immune and inflammatory cells. Blood. 2014;123(18):2759-67. 18. Ferber I, Scho¨nrich G, Schenkel J, et al. Levels of peripheral T cell tolerance induced by different doses of tolerogen. Science. 1994;263:674-6. 19. Viola A, Lanzavecchia A. T cell activation determined by T cell receptor number and tunable thresholds. Science. 1996;273:104-6. 20. Taams L, van Eden W, Wauben MH. Dose-dependent induction of distinct anergic phenotypes: multiple levels of T cell anergy. J Immunol. 1999;162:1974-81. ͒ 21. Wolchinsky R, Hod-Marco M, Oved K, et al. Antigen-dependent integration of opposing proximal TCR-signaling cascades determines the functional fate of T lymphocytes. J Immunol. 2014;192:2109-19. 22. Sadallah S, Eken C, Schifferli JA. Ectosomes as immunomodulators. Semin Immunopathol. 2011;33:487-95. 23. Sadallah S, Eken C, Martin PJ, Schifferli JA. Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells. J Immunol. 2011;186:6543-52. 24. Sadallah S, Amicarella F, Eken C, Iezzi G, Schifferli JA. Ectosomes released by platelets induce differentiation of CD4+T cells into T regulatory cells. Thromb Haemost. 2014;112:1219-29. 25. Assoian RK, Komoriya A, Meyers CA, Miller DM, Sporn MB. Transforming growth factor-β in human platelets: identification of a major storage site, purification, and characterization. J Biol Chem 1983;258,7155-60. 26. Zheng SG. The Critical Role of TGF-beta1 in the Development of Induced Foxp3+ Regulatory T Cells. Int J Clin Exp Med. 2008;1:192-202. 27. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009;27:485-517. 28. 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;116:4639-45. 29. Nishimoto T, Numajiri M, Nakazaki H, et al. Induction of immune tolerance to platelet antigen by short-term thrombopoietin treatment in a mouse model of immune thrombocytopenia. Int J Hematol. 2014;100:341-4. 30. Li X, Zhong H, Bao W, et al. Defective regulatory B-cell compartment in patients with immune thrombocytopenia. Blood. 2012;120:3318-25.
with: TGF-β↓ IL-4↓ IL-2↑ IFN-γ↑ IL-17↑ IL-10↓
Th2 shift
platelets
Th1 shift/ Th17 ↑ /Tregs↓/Bregs ↓
immune dysregulation in ITP:
immune modulation/ induction of tolerance
TPO-RA’s
TGF-β
CD40L TGF-β
immune modulation/ induction of tolerance
-
-
macrophages activity ↓ (in vitro) modified dendritic cells differentiaton and maturation (in vitro) Tregs ↑ (in vitro, mouse model and in vivo) Breg ↑ (in vivo)
immune modulation:
- due to high antigen mass
T-cell anergy
www.elsevier.com/locate/enganabound
PII: DOI: Reference:
S0037-1963(16)30022-1 http://dx.doi.org/10.1053/j.seminhematol.2016.04.010 YSHEM50866
To appear in: Seminars in Hematology Cite this article as: Alexandra Schifferli and Thomas Kühne, Thrombopoietin Receptor Agonists: A New Immune Modulatory Strategy in ITP?, Seminars in Hematology, http://dx.doi.org/10.1053/j.seminhematol.2016.04.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Thrombopoietin receptor agonists: A new immune modulatory strategy in ITP? Alexandra Schifferli, MD1, Thomas Kühne, MD1
1
Department of Oncology/Hematology, University Children’s Hospital Basel, Basel, Switzerland
Keywords: ITP; immune modulation; thrombopoietin receptor agonist; microparticles, TGF-β
Correspondence: Dr. Alexandra Schifferli, Department of Hematology/Oncology, University Children’s Hospital Basel, Spitalstrasse 33, 4031 Basel, Switzerland. E-mail: [email protected] Telephone: 0041 61 704 12 12 Fax: 0041 61 704 12 13
Conflict of interest statement: The authors declare no conflict of interest
2 Abstract In 2008, new drugs that mimic the effects of thrombopoietin became available for the treatment of primary immune thrombocytopenia, e.g., romiplostim and eltrombopag. These drugs activate the thrombopoietin receptor, stimulate the production of megakaryocytes, and increase the production of platelets. Important clinical observation has been gained, such as unexpected long-term remission after stopping thrombopoietin receptor agonists. The pathophysiology of this unforeseen cure is currently the subject of discussion and is investigated in clinical trials and laboratory research projects. Here we evaluate the different hypotheses on how thrombopoietin receptor agonists can affect the immune system, particularly the induction of tolerance, and by which mechanisms this may be achieved.
Introduction Thrombopoietin receptor agonists (TPO-RAs) have been designed to stimulate megakaryopoiesis. In 2008, the FDA approved two drugs, romiplostim and eltrombopag, for the treatment of primary immune thrombocytopenia (ITP). Both drugs exhibit high efficacy with acceptable adverse effects (1-3). The success story of TPO-RAs appears to be greater than expected. Data exist suggesting that stimulation of the receptor for thrombopoietin (TPO), the c-MPL receptor, may induce immunological mechanisms to restore immune tolerance in ITP (4, 5). ITP is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. The dysregulation of the immune system in ITP involves the innate and adaptive immune system. Multiple mechanisms underlie the development of autoimmunity, most of which involve
3 disturbances in regulatory circuits, cytokine synthesis, and signaling pathways. Patients with ITP harbor an imbalance in the T-cell subsets, with a shift toward Th1 and Th17 cells (6, 7). Moreover, it has been shown that there is a decrease in regulatory T and B cells (8, 9). These changes can be defined by the measurement of cytokines. Patients with ITP have a shift toward interleukin-2 (IL-2), interferongamma (IFN-γ), and IL-17 cytokines, all corresponding to a pro-inflammatory/immune response, as well as a drop in many cytokines, such as IL-10, transforming growth factor-β1 (TGF-β),IL-4, and IL-35. In this review article, we will discuss new insights into possible immunomodulatory functions of TPO-RAs.
Clinical background Recently, reports on long-term remission after discontinuation of TPO-RAs in adult patients with chronic ITP have been published. Several case reports, retrospective data, and a clinical trial revealed sustained platelet counts above 100 × 109/L after discontinuation of romiplostim in up to 30% of the patients (10-12). Červinek et al retrospectively analyzed 46 patients with relapsed and refractory ITP treated with TPO-RAs (10). In 11 of these cases, TPO-RA therapy (romiplostim, 7; eltrombopag, 4) could be successfully stopped, with a median follow-up of 33 months. The probability of remission appeared not to depend on previous treatment, splenectomy, or disease duration. In a case study of eight romiplostim trials, Bussel et al (12) examined patients with sustained remission after discontinuation of romiplostim; remission was identified in 27 patients. No clear predictors of remission were identified; however, a number of patients had ITP for <1 year and received
4 romiplostim for <1 year. This observation was supported by a prospective trial in patients with ITP lasting for <6 months; remission was observed in approximately 1/3 of adult patients who were treated with romiplostim with a median time (range) to onset of 27 (6–57) weeks (11).
Theoretical background Several theories on the induction of immune tolerance by TPO-RAs are proposed: -
Unknown and unexpected effects of the c-MPL receptor (e.g., c-MPL receptor on immune cells)
-
Unknown effects of TPO-RAs, which may not directly be related to the stimulation of the c-MPL receptor and involving the immune system (e.g., cross-reaction with activation or inhibition of other receptors and pathways of the immune system)
-
Immune modulation following increased platelet mass. Two mechanisms may be involved: 1. induction of immune tolerance by exposure to high-dose antigen or 2. innate platelet immune activity
At present, the latter theory appears to be the most plausible explanation. Platelets are more than just pro-coagulant fragments; indeed, accumulating evidence suggests an immunomodulatory role for platelets and platelet-derived micro-particles (PMP). Although they do not replicate or contain nuclei, platelets have multiple roles in host defense against infections and are involved in innate and adaptive immune response mechanisms (13-16). Thus, platelets seem to be part of the immune response. This theory is also consistent with the evolution of the hematological system. In many invertebrates and early vertebrates, one cell type (the hemocyte) carries out
5 hemostatic as well as host-defense functions. Platelets have the ability to bind to infectious agents, secrete many immunomodulatory cytokines and chemokines (alpha granules), and express receptors for various immune effector and regulatory functions, such as pattern recognition receptors (PRRs) (e.g., toll-like receptors), CD40L, and denaturated MHC class 1 molecules. Furthermore, platelets contain mRNA that originates from megakaryocytes and are capable of carrying out independent protein synthesis under different environmental stress, and they are able to release membrane micro-particles known to be involved in a variety of pathophysiological responses including immune responses, inflammation, angiogenesis, and tissue regeneration. In this respect, platelets are truly immunological cells.
Research background TPO-RA’s increase platelet production in the bone marrow and subsequently induce a rise in peripheral platelet count. This acute increase in platelet mass could induce tolerance mechanisms. The following five research topics support our theory that TPO-RAs could have an immunomodulatory function (figure 1).
1. Induction of immune tolerance by exposure to high-dose antigen T cell anergy is a key tolerance mechanism suppressing unwanted T-cell activation against self by rendering lymphocytes functionally inactive following antigen contact. Antigen (Ag) plays an important role in anergy induction, where high supra-optimal doses lead to the unresponsive phenotype (18-20). An antigen density of >100 peptide-MHC complexes/cell was found to be the transition point
6 between dominant activation and inhibition cascades, whereas higher antigen doses induced an anergic functional state (21). A rapid increase of platelet mass such as TPO-RA effects increase the antigen mass in patients with ITP, and so may inactivate T cells. However, it is unknown how much the Ag density rises with TPO-RAs and if this rise is sufficient to inactivate autoimmune T cells.
2. Platelet (micro-particles)-mediated immunoregulatory pathways Micro-particles, also known as ectosomes or micro-vesicles, are important mediators of intercellular communication. They are released by budding directly from the cell membrane surface either spontaneously or in response to various stimuli. Micro-particles contain cytosolic substances and phosphatidylserine in their membrane. Depending on their cellular origin, micro-particles have been associated with a broad spectrum of biological activities. In the last 10 years, it has been shown that platelet-derived-microparticles (PMP) have effects on the innate immune system, as well as on the induction of adaptive immunity with possibly tolerogenic activity (22). Increasing the mass of platelets, as caused by TPO-RAs, will necessarily increase the mass of circulating micro-particles. In a study using stored platelets, micro-particles appeared to show antiinflammatory and immunosuppressive properties (23). Indeed, the purified PMP reduced the release of TNF-α and IL-10 by activated macrophages. The effects on TNF-α lasted for 24 h, suggesting that micro-particles induced a modification of the differentiation of macrophages. PMP also induced the release of TGF-β from macrophages. In addition, the differentiation of monocytes to immature and mature dentritic cells (DC), as well as their activity, was modified by the presence of micro-particles. These data indicate that micro-particles shed by stored
7 platelets have intrinsic properties that downregulate macrophage and modify DC differentiation toward less reactive states. In another study (24), exposure of activated CD4+ T cells to PMP decreased their release of IFNγ, TNFα, and IL-6, and increased the production of TGF-β1. Concomitantly, micro-particles-exposed CD4+ T cells displayed increased frequencies of CD25high Foxp3+ regulatory T cells (Tregs). This was explained by induced differentiation of naïve T cells into Tregs, since micro-particles had no effect on pre-differentiated Tregs. These phenomena were dose-dependent and micro-particle-specific. PMP-mediated effects were partly dependent on platelet-derived TGF-β1.
3. Release of platelet TGF-β It is known that platelets contain the largest amount of transforming growth factorβ (TGFβ) in the body (25). TGF-β is believed to be important in the regulation of the immune system by Tregs and the differentiation of both Tregs and Th17 cells from CD4+ T cells (26, 27). An increased mass of platelets could increase the concentration of TGF-β and induce tolerance mechanisms.
4. Induction and improved immunosuppressive function of adaptive Tregs with TPO-RA Bao et al. studied immunologic profiles of patients with chronic ITP before and during treatment with TPO-RAs (28). Treg activity was improved in patients on treatment, and the improvement correlated with a reduction in IL-2-producing CD4+ T cells, consistent with a dampening of immune responses. There was a concomitant increase in total circulating TGF-β1 levels in patients receiving treatment, and the levels of TGF-β1 correlated with the degree of improvement in
8 platelet counts. This suggests that platelets in patients on TPO-RA treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-β1 because of high platelet counts and/or PMP turnover. This last hypothesis was supported by a cross-sectional study (28), showing increased TGF-β1 levels in addition to improved Treg activity in responders, but not in nonresponders. In an ITP mouse model, TPO treatment promoted peripheral induction of Tregs in conjunction with elevated circulating TGF-β levels and sustained platelet recovery (29). Again, it is hypothesized that a burst in platelet numbers correlates with increased TGF-β1 levels, which in turn increases the regulatory compartment.
5. Increased regulatory B cell (Bregs) function with TPO-RA In another study, Li et al. showed increased Breg (CD19 + CD24hiCD38hi) function in patients successfully treated with TPO-RAs (30). The frequency of Bregs improved as the platelet counts increased upon treatment. Because B-cell regulatory activity in vitro requires activation through CD40 engagement it is supposed that CD40L expressed on and in platelets, may directly contribute to improved Breg activity. However, these data are of limited value, since only five patients were studied, and the data must be confirmed by larger longitudinal studies.
9 Conclusions We believe that TPO-RAs exhibit tolerogenic activities because of increased platelet and probably because of micro-particle mass. PMP have been shown to exert in vitro immunosuppressive activities on phagocytes, modify the differentiation and maturation of DC toward less reactive states, and induce differentiation of CD4+ T cells toward functional Tregs, partly because of increased TGF-β1 levels. All these changes and probably the concomitant induction of T-cell anergy may represent the mechanism by which TPO-RAs enhance peripheral tolerance in ITP.
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with: TGF-β↓ IL-4↓ IL-2↑ IFN-γ↑ IL-17↑ IL-10↓
Th2 shift
platelets
Th1 shift/ Th17 ↑ /Tregs↓/Bregs ↓
immune dysregulation in ITP:
immune modulation/ induction of tolerance
TPO-RA’s
TGF-β
CD40L TGF-β
immune modulation/ induction of tolerance
-
-
macrophages activity ↓ (in vitro) modified dendritic cells differentiaton and maturation (in vitro) Tregs ↑ (in vitro, mouse model and in vivo) Breg ↑ (in vivo)
immune modulation:
- due to high antigen mass
T-cell anergy