Regulatory T Cell Reconstitution Is Associated with Acute Graft-Versus-Host Disease in Pediatric and Young Adult Hematopoietic Cell Transplant Recipients

BACKGROUND Acute Graft-versus-Host Disease (aGvHD) is a potentially life-threatening complication following hematopoietic cell transplantation (HCT). Early CD4+ T-cell reconstitution (>50 cells/microliter) is associated with improved survival for moderate to severe (grade 2-4) aGvHD, but the underlying mechanisms remain unclear. CD4+ regulatory T cells (Tregs) play a key role in maintaining immune tolerance and regulating immune reactions. Here, we explored potential associations between Treg frequencies (Treg/CD4 ratio) and 90-day onset of aGvHD in pediatric and young adult HCT recipients. METHODS All patients transplanted between 06/2020 and 02/2023 underwent standard flow cytometric immunophenotyping for two years (from day +14), using CLIA-approved panels, including a Treg panel. Tregs were identified as CD3+CD4+CD25+CD127low and analyzed for activated (HLA-DR+), memory (CD45RO+), and naïve (CD45RO−HLA-DR−) differentiation. Only patients with Treg data before the earliest onset of aGvHD, or with Treg values before the mean onset of GvHD (for patients with no GvHD), were considered for analyses. The main outcome of interest was the relationship between Treg reconstitution and the development of aGvHD before 90 days. Other outcomes of interest (survival and relapse) were also assessed using uni- and multivariable (MV) models. RESULTS Out of 101 transplanted patients, 51 had early Treg data collected before the onset of 90-day GvHD and were included in the analysis (median age: 12, range: 1–24 years); 14 (27%) received cord blood, 10 (20%) T-cell deplete peripheral blood stem cells and 27 (53%) unmodified bone marrow. 16 (35%) patients developed aGvHD (grade 2-4). Patients prior to the onset of aGvHD exhibited a rapid decline in Treg frequencies around day 25-30 post-HCT (Fig. 1). This decrease was absent in patients without GvHD or those on treatment for aGvHD. Additionally, higher Treg frequencies (> 0.0316) at day 90 were associated with decreased odds of aGvHD, adjusting for age at HCT (P=0.016; Fig. 2). Treg differentiation trajectory suggested that memory and activated, but not naïve Treg frequencies decreased before GvHD onset (Fig. 3 A&B). While no difference in Treg frequency was observed between no GvHD and grade 2-4 GvHD cases, elevated naïve Tregs were seen in grade 2-4 GvHD (Fig. 3 C), suggesting an anergic Treg profile associated with aGvHD. With a small number of observed events, no impact on survival and relapse was found. CONCLUSION In summary, our findings indicate a decline in Treg frequencies preceding GvHD onset, with GvHD development shifting the Treg profile towards a naïve, inactive state. These observations highlight the protective role of Tregs against aGvHD, emphasizing the need for further exploration into the role and kinetics of Treg reconstitution in preventing aGvHD.