Treg Subtypes Alteration in Patients with Primary Immune Thrombocytopenia
Authors: Ji Lili , Zhan Yanxia , Li Feng , Cheng Luya , Zou Shanhua , Chen Pu , Cheng Yunfeng 
Published: October 2019

Abstract
Background: Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that Treg cells were numerically or functionally deficient1-3. It was shown that human FoxP3+CD4+ T cells were composed of three phenotypically and functionally distinct subpopulations: CD45RA+FoxP3lo resting Treg cells (rTreg cells, group I) and CD45RA-FoxP3hi activated Treg cells (aTreg cells, group II), and CD45RA-FoxP3lo non-suppressive Treg cells (group III) 4. Our current study was aimed to determine whether the subtypes alterated in ITP patients or not.
Methods: Ten healthy volunteers (normal contral, NC) and 15 newly diagnosed ITP patients (platelet below 30×109/L) donated 2mL peripheral blood to test the percentages of peripheral Treg cells subtypes by Flow Cytometry (FCM) before and after first-line glucocorticoid treatment. Among them, 10 NCs and 9 ITP patients donated additional 20mL peripheral before treatment for Treg cells subtypes functional study. CD4+CD25-Teffs and CD4+CD25+Tregs were purified. CD4+CD25-Teffs, labeled with CFSE, were cultured with Tregs for 5 days. Treg cells subtypes and their IL-10 fluorescence intensity were determined by FCM.
Results: After treatment, 9 patients got complete or partial remission (ITP R), while the other 6 patiests were evaluated as non-remission (ITP NR). Before treatment, group I rTreg cells in ITP R pre ((11.77±4.71)%, p=0.0048)or ITP NR pre ((10.30±4.29)%, ,p=0.0071) patients were both lower than NC (21.71±7.61)%. As to group II aTreg cells, the percentage in ITP R pre (4.04±2.09)% was higher than NC ((1.40±0.69)%, p=0.0008) or ITP NR pre ((2.17±0.78)%, p=0.0339), and there was no statistic difference between NC and ITP NR pre (p=0.0652). The non-suppressive group III Treg cells was higher in ITP R pre ((84.07±4.93)%, p=0.0185) and ITP NR pre ((84.25±7.92)%, p=0.0090), when compared to NC (75.82±7.83)%.
After treatment, the subtypes in ITP R (ITP R post) were group I (14.69±5.74)%, group II (4.18±2.67)%, and group III (81.01±5.66)%, none of them had statistic difference when compared with ITP R pre. In the term of ITP NR patients, the treatment also didn't change the subtypes alteration (group I (10.41±6.16)%, group II (2.69±2.09)%, and group III (86.71±5.25)% in ITP NR post).
In the co-culture study, ITP patients' Treg cells subtypes altered in the same pattern. That is decreased group I (NC (21.67±10.71)%, ITP (8.94±8.38), p=0.0222), increased group II (NC (7.94±3.49)%, ITP (13.89±7.13)%, p=0.0327), and increased group III (NC (59.9±14.37)%, ITP (77.17±9.31)%, p=0.0330). When we looked into the IL-10 producing capacity (represented by the mean fluorescence intensity, MFI) of each subtypes, the group II Tregs cells produced the most IL-10 in NC (group I (636.5±378.8), group II (3012.0±3165.3), and group III (834.3±1365.4), p=0.0463) as well as ITP (group I (385.5±416.9), group II (3934.6±3633.3), and group III (556.0±416.6), p=0.0007). However, there were no statistic difference between NC and ITP in terms of IL-10 MFI.
Conclusions: Tregs cells subtypes percentage altered when ITP occurred. The increased group II aTreg cells may forecast the better glucocorticoid treatment efficacy.

Disclosures
No relevant conflicts of interest to declare.