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
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.
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