Background: Graft-versus-host disease (GVHD) refractory to immunosuppressive therapy (IST) remains a major complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) in pediatric patients, often compounded by post-transplant viral infections. Adoptive regulatory T cell (Treg) therapy offers a strategy for GVHD control, while donor-derived memory T cells may aid in targeted immunological recovery. This study reports on the safety and outcomes of combined Treg and memory T cell therapy in this high-risk setting.

Methods: Thirteen pediatric patients with steroid-refractory late acute or chronic GVHD received haploidentical Treg infusions. Treg cells were generated by ex-vivo polyclonal expansion in 7 cases or by direct flow-sorting (CD4+CD127low) using GMP-grade Tyto sorter (Miltenyi Biotec) after CD25⁺ magnetic enrichment. The median dose of Tregs was 0.5*106 Tregs/kg (0.25 - 1*106/kg), the median frequency of Tregs (FoxP3+) was 94% (58 - 98%). Eight patients with active viral infections also received donor memory T cells. Memory T cells were generated by direct flow cytometry-based sorting for CD45RO+ cells from donor leukapheresis products using GMP-grade Tyto sorter (Miltenyi Biotec). All patients had received multiple lines of prior IST, including glucocorticosteroids, cyclosporine, and biologics (tocilizumab, infliximab, abatacept, etarnecept, vedolizumab). IST was discontinued completely before Treg infusion. Nine patients recieved lymphodepletion with cyclophosphamide at 400 mg/m2. Patients were assessed for safety, immune reconstitution, responses to GVHD and viral infections, and survival, with a median follow-up of 582 days.

Results: The Treg and Treg+Tmemory cell therapy was well tolerated with no severe infusion-related adverse events or new infectious complications. Eleven of 13 (84%) patients experienced either complete or partial response of GVHD manifestations. Seven of 8 (88%) patients with viral infections experienced control or resolution. Immune monitoring confirmed sustained peripheral presence of infused Treg and memory T cell populations, along with recovery of CD4 and CD8 effector/memory compartments and virus-specific T cells. In 6 patients reactivation of GVHD was recorded with good response to IST. At 1 year post-infusion, 10 patients were evaluable. Among them, 8 had no evidence of GVHD and were completely off IST.

Conclusions: Combined haploidentical Treg and memory T cell therapy, with memory T cells generated solely by flow cytometry-based sorting, is feasible and safe in pediatric patients with refractory GVHD and viral infections. This approach resulted in complete IST withdrawal and GVHD control in the majority of evaluable patients at 1 year. The promising outcomes in immune reconstitution and viral control support further clinical investigation of this integrative cellular strategy for high-risk pediatric transplant recipients.

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2025
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