P146 Investigating the role of T regulatory cells in the amelioration of RA in pregnancy
Authors: Raine Charles , Robinson George , Jury Elizabeth C , Giles Ian 
Published: April 2021

Abstract
Background/Aims
Disease activity of rheumatoid arthritis (RA) improves in around 60% of patients during pregnancy, but the mechanisms underlying this phenomenon remain unclear. T regulatory cells (Tregs) expressing the transcription factor FOXP3 serve to suppress inflammation and are known to play an important role in RA pathogenesis. These cells are also essential for the regulation of fetomaternal tolerance in pregnancy and protect against adverse pregnancy outcomes such as pre-eclampsia. Treg expansion in the third trimester of pregnancy has been associated with disease amelioration of RA. Therefore, we examine the role of Tregs in RA pregnancy in greater detail, using an extended flow cytometry panel of functional markers to correlate numbers and function of these cells with accurate disease activity assessments using musculoskeletal ultrasound (MSK-US).
Methods
Pregnant RA patients (RA-P) were recruited from September 2018 to March 2020 with disease activity assessments by DAS28(3)CRP and MSK-US in the third trimester (T3) and post-partum (PP). Control groups of age-matched non-pregnant female RA patients (RA-NP) with equivalent assessments and healthy pregnant (HP) patients were also recruited. MSK-US was undertaken using a Logiq S8 with a standard 22-joint assessment; mean Power Doppler (PD) scores were calculated for each time point. Blood samples were taken with isolation of peripheral blood mononuclear cells (PBMCs) by Ficoll gradient centrifugation. PBMCs were stained with fluorochrome-conjugated antibodies for CD3, CD4, CD25, CD127, FOXP3, CD39, CD69, CTLA-4 and CD45RA. Data were acquired using a BD LSR2 flow cytometer with a gating strategy to identify Tregs. Analysis was carried out using FlowJo software; all statistical tests were performed in SPSS.
Results
Cross-sectional analysis was undertaken at T3 and PP of RA-P (n = 15) and HP (n = 18) and non-pregnant RA (n = 18). There was no significant difference in the proportion or absolute numbers of Tregs between groups, but there was a significant increase in the mean fluorescent intensity (MFI) of the activation marker CD69 in RA-P vs RA-NP (95% CI for difference 27.0-215, p = 0.01) and in HP vs RA-NP (95% CI 80.1-281, p &lt; 0.01). In T3 there were no significant correlations between disease activity scores and Treg numbers/activation markers, but in PP there were significant negative correlations between both DAS28(3)CRP and PD score and %FOXP3+Treg (R=-0.88, p &lt; 0.01; R=-0.76, p = 0.03).
Conclusion
We found a significant increase in activation of Tregs measured by CD69 in T3 of both pregnancy groups, implying that the pregnant state enhances the function of these cells and may thus lead to amelioration of RA. Although disease activity measured by DAS28(3)CRP and MSK-US was not correlated with Treg activation in T3, the strong negative correlation of these scores with %FOXP3+ Tregs in PP may link a decline in their suppressive function with PP flare. Further work is underway to clarify these findings.
Disclosure
C. Raine: Grants/research support; CR has received funding for research from UCB. G. Robinson: None. E.C. Jury: None. I. Giles: Grants/research support; IG has received an unrestricted educational grant, speaker's fees and travel fees from UCB.