The role of CD8+ T-cell clones in immune thrombocytopenia
Amna Malik, Anwar A Sayed, Panpan Han, Michelle M H Tan, Eleanor Watt, Adela Constantinescu-Bercu, Alexander T H Cocker, Ahmad Khoder, Rocel C Saputil, Emma Thorley, Ariam Teklemichael, Yunchuan Ding, Alice C J Hart, Haiyu Zhang, Wayne A Mitchell, Nesrina Imami, James T B Crawley, Isabelle I Salles-Crawley, James B Bussel, James L Zehnder, Stuart Adams, Bing M Zhang, Nichola Cooper, Amna Malik, Anwar A Sayed, Panpan Han, Michelle M H Tan, Eleanor Watt, Adela Constantinescu-Bercu, Alexander T H Cocker, Ahmad Khoder, Rocel C Saputil, Emma Thorley, Ariam Teklemichael, Yunchuan Ding, Alice C J Hart, Haiyu Zhang, Wayne A Mitchell, Nesrina Imami, James T B Crawley, Isabelle I Salles-Crawley, James B Bussel, James L Zehnder, Stuart Adams, Bing M Zhang, Nichola Cooper
Abstract
Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.
Conflict of interest statement
Conflict-of-interest disclosure: The authors declare no competing financial interests.
© 2023 by The American Society of Hematology.
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References
- Rodeghiero F, Stasi R, Gernsheimer T, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood. 2009;113(11):2386–2393.
- Cooper N, Ghanima W. Immune thrombocytopenia. N Engl J Med. 2019;381(10):945–955.
- Cooper N, Kruse A, Kruse C, et al. Immune thrombocytopenia (ITP) world impact survey (I-WISh): impact of ITP on health-related quality of life. Am J Hematol. 2021;96(2):199–207.
- Terrell DR, Neunert CE, Cooper N, et al. Immune thrombocytopenia (ITP): current limitations in patient management. Medicina (Mex) 2020;56(12):667.
- Provan D, Arnold DM, Bussel JB, et al. Updated international consensus report on the investigation and management of primary immune thrombocytopenia. Blood Adv. 2019;3(22):3780–3817.
- Neunert C, Terrell DR, Arnold DM, et al. American Society of Hematology 2019 guidelines for immune thrombocytopenia. Blood Adv. 2019;3(23):3829–3866.
- Harrington WJ, Minnich V, Hollingsworth JW, Moore CV. Demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura. J Lab Clin Med. 1951;38(1):1–10.
- Shulman NR, Marder VJ, Weinrach RS. Similarities between known antiplatelet antibodies and the factor responsible for thrombocytopenia in idiopathic purpura. Physiologic, serologic ans istopic studies. Ann N Y Acad Sci. 1965;124(2):499–542.
- van Leeuwen EF, van der Ven JT, Engelfriet CP, von dem Borne AE. Specificity of autoantibodies in autoimmune thrombocytopenia. Blood. 1982;59(1):23–26.
- Ghanima W, Khelif A, Waage A, et al. Rituximab as second-line treatment for adult immune thrombocytopenia (the RITP trial): a multicentre, randomised, double-blind, placebo-controlled trial. Lancet. 2015;385(9978):1653–1661.
- Mahévas M, Azzaoui I, Crickx E, et al. Efficacy, safety and immunological profile of combining rituximab with belimumab for adults with persistent or chronic immune thrombocytopenia: results from a prospective phase 2b trial. Haematologica. 2021;106(9):2449–2457.
- Bussel J, Arnold DM, Grossbard E, et al. Fostamatinib for the treatment of adult persistent and chronic immune thrombocytopenia: results of two phase 3, randomized, placebo-controlled trials. Am J Hematol. 2018;93(7):921–930.
- Kuter DJ, Efraim M, Mayer J, et al. Rilzabrutinib, an oral BTK inhibitor, in immune thrombocytopenia. N Engl J Med. 2022;386(15):1421–1431.
- Newland AC, Sánchez-González B, Rejtő L, et al. Phase 2 study of efgartigimod, a novel FcRn antagonist, in adult patients with primary immune thrombocytopenia. Am J Hematol. 2020;95(2):178–187.
- Vrbensky JR, Moore JE, Arnold DM, Smith JW, Kelton JG, Nazy I. The sensitivity and specificity of platelet autoantibody testing in immune thrombocytopenia: a systematic review and meta-analysis of a diagnostic test. J Thromb Haemost. 2019;17(5):787–794.
- Rogier T, Samson M, Mourey G, et al. Antiplatelet antibodies do not predict the response to intravenous immunoglobulins during immune thrombocytopenia. J Clin Med. 2020;9(6):1998.
- Chugh S, Darvish-Kazem S, Lim W, et al. Rituximab plus standard of care for treatment of primary immune thrombocytopenia: a systematic review and meta-analysis. Lancet Haematol. 2015;2(2):e75–e81.
- 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(10):4245–4254.
- Semple JW, Freedman J. Increased antiplatelet T helper lymphocyte reactivity in patients with autoimmune thrombocytopenia. Blood. 1991;78(10):2619–2625.
- Semple JW, Rebetz J, Maouia A, Kapur R. An update on the pathophysiology of immune thrombocytopenia. Curr Opin Hematol. 2020;27(6):423–429.
- Olsson B, Andersson PO, Jernås M, et al. T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nat Med. 2003;9(9):1123–1124.
- 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(6):1247–1253.
- Guo L, Yang L, Speck ER, et al. Allogeneic platelet transfusions prevent murine T-cell–mediated immune thrombocytopenia. Blood. 2014;123(3):422–427.
- Vrbensky JR, Nazy I, Clare R, Larché M, Arnold DM. T cell–mediated autoimmunity in immune thrombocytopenia. Eur J Haematol. 2022;108(1):18–27.
- Zhao C, Li X, Zhang F, Wang L, Peng J, Hou M. Increased cytotoxic T-lymphocyte-mediated cytotoxicity predominant in patients with idiopathic thrombocytopenic purpura without platelet autoantibodies. Haematologica. 2008;93(9):1428–1430.
- Qiu J, Liu X, Li X, et al. CD8+ T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia. Sci Rep. 2016;6(1):27445.
- Zhang F, Chu X, Wang L, et al. Cell-mediated lysis of autologous platelets in chronic idiopathic thrombocytopenic purpura. Eur J Haematol. 2006;76(5):427–431.
- Han P, Yu T, Hou Y, et al. Low-dose decitabine inhibits cytotoxic t lymphocytes-mediated platelet destruction via modulating PD-1 methylation in immune thrombocytopenia. Front Immunol. 2021;12:630693.
- Bartram J, Mountjoy E, Brooks T, et al. Accurate sample assignment in a multiplexed, ultrasensitive, high-throughput sequencing assay for minimal residual disease. J Mol Diagn. 2016;18(4):494–506.
- Bolotin DA, Poslavsky S, Mitrophanov I, et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods. 2015;12(5):380–381.
- Constantinescu-Bercu A, Grassi L, Frontini M, Salles-Crawley II, Woollard K, Crawley JT. Activated αIIbβ3 on platelets mediates flow-dependent NETosis via SLC44A2. Elife. 2020;9
- Zamora C, Cantó E, Nieto JC, et al. Binding of platelets to lymphocytes: a potential anti-inflammatory therapy in rheumatoid arthritis. J Immunol. 2017;198(8):3099–3108.
- Polasky C, Wendt F, Pries R, Wollenberg B. Platelet induced functional alteration of CD4+ and CD8+ T cells in HNSCC. Int J Mol Sci. 2020;21(20):E7507.
- Martin MD, Badovinac VP. Defining memory CD8 T cell. Front Immunol. 2018;9:2692.
- Appay V, van Lier RAW, Sallusto F, Roederer M. Phenotype and function of human T lymphocyte subsets: consensus and issues. Cytometry. 2008;73(11):975–983.
- Klenerman P, Oxenius A. T cell responses to cytomegalovirus. Nat Rev Immunol. 2016;16(6):367–377.
- Macallan DC, Borghans JAM, Asquith B. Human T cell memory: a dynamic view. Vaccines. 2017;5(1):5.
- Tickotsky N, Sagiv T, Prilusky J, Shifrut E, Friedman N. McPAS-TCR: a manually curated catalogue of pathology-associated T cell receptor sequences. Bioinformatics. 2017;33(18):2924–2929.
- Bakry R, Sayed D, Galal H, Shaker S. Platelet function, activation and apoptosis during and after apheresis. Ther Apher Dial. 2010;14(5):457–464.
- Krailadsiri P, Seghatchian J, Williamson LM. Platelet storage lesion of WBC-reduced, pooled, buffy coat-derived platelet concentrates prepared in three in-process filter/storage bag combinations. Transfusion (Paris) 2001;41(2):243–250.
- Precopio ML, Betts MR, Parrino J, et al. Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8+ T cell responses. J Exp Med. 2007;204(6):1405–1416.
- Krug LM, Dao T, Brown AB, et al. WT1 peptide vaccinations induce CD4 and CD8 T cell immune responses in patients with mesothelioma and non-small cell lung cancer. Cancer Immunol Immunother. 2010;59(10):1467–1479.
- Zhao Y, Nguyen P, Ma J, et al. Preferential use of public TCR during autoimmune encephalomyelitis. J Immunol. 2016;196(12):4905–4914.
- Amoriello R, Chernigovskaya M, Greiff V, et al. TCR repertoire diversity in multiple sclerosis: high-dimensional bioinformatics analysis of sequences from brain, cerebrospinal fluid and peripheral blood. EBioMedicine. 2021;68
- Chapman LM, Aggrey AA, Field DJ, et al. Platelets present antigen in the context of MHC class I. J Immunol. 2012;189(2):916–923.
- Marcoux G, Laroche A, Hasse S, et al. Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules. Blood. 2021;138(25):2607–2620.
- Zufferey A, Speck ER, Machlus KR, et al. Mature murine megakaryocytes present antigen-MHC class I molecules to T cells and transfer them to platelets. Blood Adv. 2017;1(20):1773–1785.
- Pariser DN, Hilt ZT, Ture SK, et al. Lung megakaryocytes are immune modulatory cells. J Clin Invest. 2021;131(1)
- Guo L, Shen S, Rowley JW, et al. Platelet MHC class I mediates CD8+ T-cell suppression during sepsis. Blood. 2021;138(5):401–416.
- Aslam R, Speck ER, Kim M, Freedman J, Semple JW. Transfusion-related immunomodulation by platelets is dependent on their expression of MHC class I molecules and is independent of white cells. Transfusion (Paris) 2008;48(9):1778–1786.
- Gouttefangeas C, Diehl M, Keilholz W, Hörnlein RF, Stevanović S, Rammensee HG. Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro. Blood. 2000;95(10):3168–3175.
- Ghio M, Contini P, Mazzei C, et al. Soluble HLA class I, HLA class II, and Fas ligand in blood components: a possible key to explain the immunomodulatory effects of allogeneic blood transfusions. Blood. 1999;93(5):1770–1777.
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