Development of Quantitative Methylation-Specific PCR Assay for Assessment of Natural Tregs

Presentation Number: LB MON 81
Date of Presentation: April 3rd, 2017

Mohamed I. Husseiny Elsayed*1, Ahmed Akef Fahmy2, Weiting Du1, Angel Gu1, Anitha Rao1, Ding Wang1, Pablo Garcia1, Fouad R Kandeel1 and Kevin George Ferreri3
1Diabetes & Metabolic Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, 2National Research Center, Cairo, EGYPT, 3City of Hope, Duarte, CA


Tregs are long-lived cells that suppress immune responses in vivo in a dominant and antigen-specific manner. Therefore, therapeutic application of Tregs for transplant rejection and severe autoimmunity is an active area of investigation. A key advance for Treg therapy in humans is the finding that Tregs can be isolated and expanded in vitro while maintaining immunoregulatory function. Tregs characterized by CD4+CD25highCD127low with expression of FOXP3 which is critical for their development and function. Demethylation of the Treg-Specific Demethylation Region (TSDR) of FOXP3 has been suggested as a marker for natural Treg (nTreg) since all 15 CpGs in the TSDR are unmethylated in nTregs but fully methylated in all other cell types. Our aim was to develop an assay for routine assessment of the differential methylation pattern of the FOXP3 TSDR in patient-derived in vitro expanded nTregs. Human TSDR sequences from nTregs and pancreatic cells, representing the unmethylated and methylated sequences respectively, were amplified and cloned into vector plasmid for assay development and as assay standards. Droplet digital TaqMan probe-based qPCR (ddPCR) assays were developed using methylation-specific primers and probes which interrogate 4 CpGs to quantify both unmethylated and methylated sequences. The assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/µl to less than 1copies/µl (R=0.99) even in the presence of non-selective gDNA background. The assay was applied to quantify the unmethylation ratio of sorted CD4+CD25highCD127lowFOXP3+ human nTreg cells were expanded in vitro for 21 days in the presence of either Dynabeads or activators. There was a decrease in the unmethylation ratio of both groups of Tregs after expansion with almost the same ratio at days 10, 14, and 17 but the activator group showed a significant decrease in unmethylation at day 21 of expansion compared with Dynabeads. FACS analysis showed a decrease of the FOXP3+ Treg population at day 21 with activators compared with Dynabeads (57% to 72%, respectively). In addition the in vitro suppression activity of nTregs at day 21 was significantly decreased in nTregs expanded with activators compared with Dynabeads. Assessment of expanded human nTreg populations demonstrates that complete unmethylation of the TSDR is dependent on the growth conditions of the culture. Our findings suggest that the digital droplet MSP-based assay can be used for quantitative monitoring of nTreg expansion in vitro and this assay is very sensitive and highly specific to differentiate between nTregs and other cells. This assay will be useful for fast screening of nTreg preparations in our ongoing clinical protocol.


Nothing to Disclose: MIHE, AAF, WD, AG, AR, DW, PG, FRK, KGF