Su1266 In Vitro Generated Regulatory T Cells From Blood Suppress Mucosal Effector T Cells in Crohn's Disease Patients

Abstract

Background: Thymically derived CD4+CD25hiCD127loFOXP3+ regulatory T cells (Tregs) maintain immunological tolerance and prevent inappropriate activation of multiple immune cell types. Quantitative and qualitative Treg defects are seen in inflamed Crohn's (CD) mucosa, suggesting that insufficient Treg-mediated suppressor function contributes to chronic mucosal inflammation in CD. Consequently, cell-based therapy with autologous Tregs to restore mucosal Treg numbers and function is conceptually attractive. The recent use of in vitro expanded umbilical cord blood (UCB) Tregs to prevent GvHD following UCB transplantation confirmed the safety of this approach in humans. The optimal method of generating Tregs in vitro from CD patients to produce a homogenous, phenotypically stable yet functionally suppressive population on expansion is unknown, as is the effect of these cells on target organs: mesenteric lymph node (MLN) and lamina propria (LP) immune cells in inflamed CD gut. Aim: To define the optimal method of GMP-compatible Treg isolation and expansion from CD patients' blood with reference to homogeneity, phenotypic stability and suppressive ability of the cell product. Results: Tregs were expanded for 2028 days in X-VIVO15 with 5% human AB serum, IL-2 and Rapamycin. Tregs initially isolated using GMP-compatible MACS-based enrichment had unacceptably high CD8+ contamination (.20%) and high expression of IL-17 and IFNγ (n=5 independent experiments). CD4+CD25+CD127lo Tregs were then sorted from CD blood on the basis of CD45RA expression and expanded a median of 127-fold (IQR 58-531) to yield a .97% CD4+CD25hiCD127loFOXP3+ population (n=10). Expanded CD45RA+ Tregs expressed functionally relevant markers including CTLA-4, ICOS, CD27 and CD62L ( .97% each) and TGF-β but not IL-10. CD45RA+ Tregs expressed ,1% IL-17 by FACS and had greater phenotypic stability than expanded CD45RATregs, with less spontaneous (p,0.05) and induced (p,0.01) IL-17 production by ELISA. CD45RA+ Tregs highly suppressed autologous CD4+CD25proliferation at 96h (median=97.3% [96.6%-99.1%] at 1:1 ratio); and were more effective than CD45RATregs at suppressing CD4+CD25activation (p=0.003), assessed by CD154 expression at 7h. Importantly, using CD3+ T responders from inflamed Crohn's resections, CD45RA+ Tregs suppressed MLN CD3+ proliferation (88.4% [53.8-93.2%]), MLN CD154 expression (82.9% [60.1-87.6%]) and LP CD3+ CD154 expression (64.3% [64.377 . 9%] ) i n a do s e d ep enden t manne r . Conc lu s i ons : FACS so r t i n g o f a CD4+CD25hiCD127loCD45RA+ precursor population is required to produce a homogenous, phenotypically stable in vitro expanded cell product from CD patients. For the first time, we show that in vitro expanded Tregs suppress immune cell activation in relevant mucosal immune niches, strengthening the rationale for clinical trials of autologous in vitro expanded Tregs in CD.