Functional Plasticity in CD4+ CD25+ CD127- Treg Population in Primary Immune Thrombocytopenia
Authors: Wu Boting , Zhan Yanxia , Li Feng , Cheng Luya , Zou Shanhua , Cheng Yunfeng 
Published: October 2019

Abstract
Background
As the most prevalent acquired bleeding disorder in adults, primary immune thrombocytopenia (ITP) and its underlying immune aberrations have been intensely investigated. Beyond the previously described Th1/Th2 imbalance, the role of Th17/Treg dysregulation has become the focus of attention. It was reported by our group that Tregs from untreated ITP patients demonstrated decreased IL-10 secretion and compromised control upon over-activated T effector cells (Li et al. 2015). The mechanism of Treg dysregulation under autoimmunity conditions is yet to be revealed. It has been recently argued by several research groups that the functional plasticity of Treg cell linage is dynamically regulated under inflammation via instability of Foxp3 expression, which means that Tregs can lose Foxp3 expression to gain effector T cell function in inflammatory milieu. The present study evaluated phenotypic features and gene expression traits of purified CD4+ T helper cells from ITP patients before and after glycocorticoids treatment, thus intending to investigate Treg functional plasticity among ITP patients.
Methods
CD4+ T helper cells were obtained via magnetic activated cell sorting from peripheral blood of 8 primary ITP patients before and after glycocorticoids treatment and 4 healthy volunteers. The phenotypic features were determined by FACS Canto II system with surface staining of CD25 and CD127, as well as cytoplasmic staining of IFN-γ and IL-17. Gene expression profiling was performed via QIAGEN Human T Helper Cell Differentiation PCR Array.
Results
The pre-treatment platelet count was (7±7)*109/L among 8 ITP patients (2 males and 6 females, median age 57.0 years), and the post-treatment platelet count was restored to (158±63)*109/L after high-dose dexamethasone regimen (40mg/d*4d). Gene expression profiling revealed that Foxp3 (-2.8 folds, p=0.001), TNF (-4.0 folds, p=0.003), and Stat1 (-2.3 folds, p=0.001) levels were significantly down-regulated among untreated ITP patients, while IL-17A (3.6 folds, p=0.05) was up-regulated with marginal statistical significance. The percentage of CD25+ CD127- population in CD4+ cells was similar among 3 groups (pre-treatment ITP: 3.1±0.6%, post-treatment ITP: 3.2±0.7%, health control: 3.4±0.8%). Among CD4+ CD25+ CD127- population, the percentage of IL-17+ cells was elevated in pre-treatment ITP patients (2.9±1.8% vs. 1.4±0.2%, p=0.17), and significantly decreased after high-dose dexamethasone regimen (1.5±1.1% vs. 2.9±1.8%, p=0.035), while the percentage of IFN-γ+ cells was similar among 3 groups (pre-treatment ITP: 18.5±12.6%, post-treatment ITP: 11.1±9.5%, health control: 18.5±3.8%).
Conclusions
Among primary ITP patients, Foxp3 expression was significantly decreased in their CD4+ T helper cells, which was inconsistent with the almost stable CD4+ CD25+ CD127- percentages determined by flow cytometry between ITP patients and healthy volunteers. Within the Treg population, we demonstrated elevated IL-17 expressing cells in pre-treatment ITP patients, which could be restored after high-dose dexamethasone regimen. These findings favored the argument of the functional plasticity of Treg cell linage during autoimmunity, and corresponded to Th17 dysregulation previously described in primary ITP.

Disclosures
No relevant conflicts of interest to declare.