Epigenetic assessment of foxp3 to determine the different origins of human regulatory T cells (Tregs) during HIV-1 infection

Tao Shi¹, Omar Farnos¹, Alexis Yero-Díaz¹, Sharada Swaminathan¹, Cecile Tremblay², Jean-Pierre Routy^{3, 4}, Cecilia T. Costiniuk^{3, 4}, Mohammad-Ali Jenabian¹

1. Department of Biological Sciences, Université du Québec à Montréal (UQAM), Montreal, QC, Canada,
2.Centre de Recherche du CHUM, Université de Montréal, Montreal, QC, Canada,
3. Research Institute of the McGill University Health Centre, Montreal, QC, Canada,
4. Chronic Viral Illness Service, McGill University Health Centre, Montreal, QC, Canada

Background: Immunosuppressive Tregs originate either from the thymus, or in the peripheral blood during inflammation. Demethylation of foxp3 locus is required for stable FoxP3 expression and suppressive functions of Tregs. Conserved non-coding sequences (CNS) regulate foxp3, controlling either thymic (CNS2) or peripheral (CNS1) origin of Tregs, overall phenotype stability (CNS0/CNS3), or transcription initiation (CNS0/proximal promoter). HIV infection is characterized by increased Treg frequencies and functions which contribute in HIV-related immune dysfunction. However, the epigenetic control of Tregs during HIV infection is understudied.

Methods: We assessed the epigenetic status of foxp3 gene and the distribution of Treg subsets among 6 study groups: HIV-infected individuals in acute and chronic phases, ART-treated individuals, elite controllers, immunological controllers and uninfected individuals (n=7-10/group). Bisulfite conversion followed by nested-PCRs of each region was performed on genomic DNA of FACS-sorted Tregs. foxp3 methylation status was assessed by Next Generation Sequencing using Illumina MiSeq technology (≈25,000 sequences/sample). In parallel, flow cytometry was performed to evaluate markers associated with recent thymic emigrants (CD31) and thymic Tregs (Helios).

Results: Frequencies of total Tregs and Helios+ thymic Tregs increased during acute and chronic infection, which was reduced by ART. Moreover, elite controllers were able to maintain Treg frequency similar to healthy levels. No changes were observed in the demethylation of CNS0, enhancer and proximal promoter regions within groups, whereas methylation levels of CNS1 and CNS2 were increased in acutely infected individuals. Interestingly, elite controllers represent a similar CNS1 signature than uninfected individuals.