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2430 Active Immune Thrombocytopenia (ITP) Disease Is Characterised By a Reduced Treg:CD8 Effector T Cell Ratio Which Is Modulated By Thrombopoietin-Receptor Agonists (TPO-RA)

Program: Oral and Poster Abstracts
Session: 311. Disorders of Platelet Number or Function: Poster II
Hematology Disease Topics & Pathways:
Diseases, autoimmune disorders, Bleeding and clotting, Therapies, ITP, Biological Processes, Technology and Procedures, Immune Disorders, immune mechanism, flow cytometry
Sunday, December 2, 2018, 6:00 PM-8:00 PM
Hall GH (San Diego Convention Center)

Anwar A. Sayed, MBBS, MSc1,2*, Amna Malik, PhD1*, Ahmad Khoder, MD, PhD, MRCP1*, George Adams, MBChB1* and Nichola Cooper, MRCP3

1Centre for Haematology, Department of Medicine, Imperial College London, London, United Kingdom
2Department of Medical Microbiology and Immunology, College of Medicine, Taibah University, Madinah, Saudi Arabia
3Imperial College Healthcare NHS Trust, London, United Kingdom

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated low platelet count and a skewed proinflammatory Th1/Th17 profile. However, little is known about the involvement of CD8+ cytotoxic T cells in ITP pathophysiology and whether they are regulated by regulatory T cells (Treg). Immunosuppressive therapy has been the mainstay treatment in ITP. More recently, Thrombopoietin receptor agonists (TPO­-RA); Romiplostim (Romi) and Eltrombopag (EPAG), have been increasingly used to stimulate megakaryocytopoiesis to produce more platelets. TPO-RAs are reported to induce complete remission in up to 30% of cases, with limited understanding of their impact on the immune system. Here we describe changes in T cell subsets in patients with ITP: how these changes are affected by disease activity and how TPO-RA may induce remission through modulating the immune system.

Methods: Multi-color flow cytometric panels were designed to characterize peripheral blood T cell subsets, including CD8+ T cell and Treg subsets, phenotypically as well as functionally through intracellular cytokine expression. To determine whether CD8+ cells were platelet specific, an IFNγ ELISpot assay was performed using platelets from a healthy donor and PBMC from both HC and patients. Forty patients with ITP were included: 13 were on Romi, 11 on EPAG and 16 on no treatment at the time of analysis. Of these 40 patients, 15 patients had active disease (AD) (platelet-count less than 30 x 109/L) and 25 had stable disease (SD) (> 30 x 109/L). These patients were compared with 26 age and gender-­matched healthy controls (HC). Data were presented as median values; Mann Whitney U and Kruskal Wallis tests were used with Dunn’s multiple comparisons correction; a P value of < 0.05 was considered significant.

Results: CD4/CD8 T cell ratio was significantly lower in patients compared to HC [1.77 vs. 3.97; P value < 0.001]. CD45RA+CD62L- Terminally-differentiated CD8+ T cells were significantly higher in patients compared to HC [66.3% vs. 8.56%; P value < 0.001]. This finding was more prominent in AD patients than those with SD [66% vs. 44.4%; P value < 0.05]. This effector population is polyfunctional, expressing high levels of proinflammatory cytokines including TNFα, IFNγ and Granzyme B when compared to HC [P value < 0.05]. Additionally, this population lacks the exhaustion markers PD-1 and Tim-3. Furthermore, these cells were reactive to platelets showing higher IFNγ-producing cells when co-cultured with platelets.

CD3+CD4+CD25hiCD127lo Treg frequency did not differ between patients and HC [P value>0. 05]. Treg functionality was preserved in these patients; no important changes were observed in their capacity to express interlukin­2 intracellularly, nor in the cell surface receptor (CD25) [P value > 0. 05]. However, Tregs were significantly lower in AD patients with compared to SD patients [2.32% vs. 4.46%; P value < 0.05]. Treg function is interactive with other T cell subsets and depends on its relative abundance in relation to other subsets; The Treg/effector CD8+ T cell ratio was significantly lower in patients compared to HC [0.06 vs. 0.16; P value < 0.01] and was also significantly lower in AD compared to SD patients [0.03 vs. 0.09 ; P value < 0.05].

EPAG-treated patients had a significantly lower effector CD8+ T cells compared to Romi-treated patients [42.4% vs 76.8%; P value <0.01]. Although EPAG did not have a direct effect on the frequency of Treg, the Treg/effector CD8+ T cell ratio was significantly lower than that in patients on Romi [0.04 vs. 0.11; P value < 0.05] because of the CD8+.T cell difference. The Treg/effector CD8+ T cell ratio in EPAG-treated patients was comparable to the ratio of HC [0.11 vs. 0.16; P value > 0.05].

Conclusion: While Th1/Th2 cell ratio is often considered as driving ITP, these results demonstrate the involvement of cytokine secreting effector CD8+ T cells in the disease pathogenesis. The imbalance in immune tolerance is also highlighted in the form of a significant reduction in the Treg/effector CD8 T cell ratio. The differences seen in T cell subsets between EPAG- and Romi-­treated patients suggest the potential for an additional, differential immunomodulatory effect between the two agents which is currently being explored.

Acknowledgement: The authors wish to thank Prof James B Bussel for his insightful comments and support of this work.

Disclosures: Cooper: Novartis: Consultancy, Honoraria; Amgen: Honoraria.

*signifies non-member of ASH