Methylation in the Promoter Region and Expression of FOX-P3 Correlate with Graft Versus Host Disease, Relapse and Survival after Allogeneic Stem Cell Transplantation
Authors: Gonzalez Cintia Manzano , Balsalobre Pascual , Serrano David , Macias Gabriela Rodriguez , Pineda Alfonso Gomez- , Diez-Martin Jose L. , Buno Ismael 
Published: April 2020

Abstract
Introduction: Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes that controls the expression of certain genes. FOX-P3 is a member of the forkhead/winged-helix family of transcriptional regulators which expression is constitutive in regulatory T lymphocytes CD4/CD25 (Treg). Recent evidence suggests that human effector T cells express FOX-P3, albeit transiently and with significantly lower levels. Therefore, FOX-P3 mehtylation within the promoter region and gene expression could play a role in allogeneic stem cell transplantation (alloSCT).
Objective: To analyze the association between the mehtylation status and the levels of expression of FOX-P3 with the dynamics of chimerism and the development of complications after alloSCT.
Patients and methods: The degree of methylation was quantified in a 600 pb CpG island within the regulatory sequence of FOX-P3 by quantitative methylation-specific real time-PCR from 32 peripheral blood (PB) samples obtained within the first month post-alloSCT. Moreover, the expression level of FOX-P3 mRNA was analyzed by real-time quantitative PCR in 18 patients from which RNA samples were available. Results were analyzed using Fisher’s exact test due to the reduced sample size.
Results: A statistically significant association (Table 1) was observed between a higher degree of methylation in the FOX-P3 promoter and a lower incidence of acute graft versus host disease (GVHD) grades II-IV (35.3% vs a 88.9% in low methylation patients; p=0.014). Moreover, patients with higher degree of methylation showed significantly higher incidence of disease relapse (41.2% vs a 0% in low methylation patients; p=0.05). The observation of a high degree of methylation would reflect a lower amount of FOX-P3 expressing cells, both Treg and effector cells. Since Treg comprises a minority population, such observation would result from relative low levels of effector T cells, which would explain the lower graft-versus-host and graft-versus-leukemia effect observed in high mehtylation group. Surprisingly, when FOX-P3 mRNA levels were analyzed, an association with statistical significance between a higher methylation status and a higher expression of FOX-P3 (90% vs 55.8%; p=0.047) was observed. It has been shown that FOX-P3 is mainly produced by Treg while effector T cells produce very low amounts of FOX-P3 mRNA. In this sense, a higher amount of Treg cells produces high FOX-P3 mRNA levels and reduces the number of effector T cells, resulting in a higher methylation status in PB samples, in which most cells would have methylated FOX-P3. Low FOX-P3 expression would, in turn, indicate a lower amount of Treg to suppress the immune activation, resulting in higher amount of effector T cells and therefore would be associated with a lower methylation. Additionally, a significant association between higher FOX-P3 expression and lower incidence of death (7.7% vs 80%; p=0.007) was observed. Moreover, the main cause of death (3/4) in the group with lower expression of FOX-P3 was acute GVHD. The lower amount of effector T cells in PB samples with high FOX-P3 expression would protect from the development of GVHD.
Conclusions: Both FOX-P3 mRNA expression and promoter methylation are of prognostic value for the development of complications post-SCT. These determinations would favor an early establishment of the immunotherapeutic options for an improved management of transplanted patients.
Table 1. Association between FOX-P3 promoter methylation and mRNA expression with chimerism and complications post-SCT.
MC, mixed chimerism; PB, peripheral blood; Fail/Reject., graft failure/rejection. p, Fisher`s Test.
Methylation MC (PB) Fail/Reject. aGVHD (II-IV) cGVHD Relapse Exitus High 6/19 (31.6%) 4/19 (21.01%) 6/17 (35.3%) 10/13 (76.9%) 7/17 (41.2%) 5/19 (26.3%) Low 2/9 (22.2%) 0/9 (0%) 8/9 (88.9%) 3/8 (37.5%) 0/9 (0%) 4/9 (44.4%) p NS NS 0.014 NS 0.05 NS Expression MC (PB) Fail/Reject. aGVHD (II-IV) cGVHD Relapse Exitus High 4/13 (30.8%) 2/13 (15.4%) 6/12 (50%) 10/11 (90.9%) 3/12 (25%) 1/13 (7.7%) Low 0/5 (0%) 0/5 (0%) 4/5 (80%) 3/4 (75%) 1/5 (20%) 4/5 (80%) p NS NS NS NS NS 0,007