Promotion of Engraftment by Triad of Pre-HCT Donor Lymphocyte Infusion, Distant Thymoglobulin and Approaching Cyclophosphamide in TCR_αβ+-cell Depleted HCT for Patients with Thalassemia Major

Background: Hematopoietic stem transplant (HCT) is a time-proved cure approach for patients with thalassemia major (TM). However, achieving successful transplant and managing graft-versus-host disease (GVHD) still pose significant challenges. Although a low incidence of GVHD has been observed in T_αβ⁺-cell depleted HCT (TDH) from haploidentical donors, graft failure (GF) remains a considerable problem, especially in TM patients. To enhance engraftment in TM-TDH, we conducted this prospective single-center clinical study including Pre-HCT donor lymphocyte infusion, distant Thymoglobulin (Thy) and approaching Cyclophosphamide (Cy).

Method: A total of 205 patients diagnosed with TM received TDH at the BMT-center, Taixin hospital, between April 2021 and March 2024. The median age of the patients was 7 years (ranging from 2 to 19 years), with 131 males and 74 females. The median follow-up time was 578 days (ranging from 11 to 1185 days). The conditioning regimen consisted of Thy on day -21. 3-Gy total body irradiation on day -10, donor lymphocytes infusion (2*10⁸/kg) on day-11, and Cy at a dose of 50mg/kg on days -8, -7 and -2 and 25mg/kg on day -1. Additionally, blood-concentration adjusted busulfan was administered on days -6 to -4, fludarabine at a dose of 40mg/m² on days -6 to -2, and thiotepa at 10mg/kg on day -3. For GVHD prophylaxis, short-term (< 6 months) low-dose tacrolimus or sirolimus with or without mycophenolate mofetil was used. The mean infused mononuclear cells were 10.02 (ranging from 2.96 to 21.50) *10⁸/kg and CD34⁺ cells were 25.92 (14.25-61.12)*10⁶/kg , T_γδ⁺-cells were 31.17 (5.18-162.6)*10⁶/kg , and NK cells were 99.35 (11.05-309.41)*10⁶/kg, respectively. The targeted infusion of T_αβ+-cells was aimed at 1.5-2.0*10⁵/kg.

Results: Out of the 205 patients, 200 achieved successful engraftment with median engraftment times of 16 (9-48) days for ANC to reach 0.5x10⁹/L, 11 (1-23) days for HB to reach 80g/L, and 10 (5-22) days for platelets to reach 20x10⁹/L, respectively. One patient engrafted test was not unavailable because of early death (day+11). GF was observed in four patients, and three of them underwent salvage alternative donor second HCT in a month and all full donor engrafted. One patient received infusion of cryopreserved auto-peripheral blood stem cell and continues to live with TM., Ten patients died, unfortunately, five died of Covid-19-associated bronchiolitis obliterans, the remains died of fulminant myocarditis, cytomegalovirus pneumonia, adenovirus encephalitis, severe multiple infected pneumonia, and post-transplant Immune hemolysis, respectively.

With a median follow-up of 578 days, the overall survival (OS) and thalassemia-free survival (TFS) were 94.5% and 93.1%, respectively. GF and transplant-related mortality (TRM) were 2.4% and 5.5%, respectively (Fig.1). Acute GVHD of grade III-IV was observed in 5.50% of patients, while mild and moderate chronic GVHD occurred in 9.50% of patients (Fig.1). Veno-occlusive disease was little and mild.

Conclusion: The effect of ATG on engraftment is bi-directional and depends on its timing; Active ATG before day 0 favors engraftment or vice versa. Coming conditioning can kill the alloreactive cells activated by pre-HCT donor lymphocyte infusion. Cy on day-1 can more depleted revived host lymphocytes in the end of conditioning. The current study proved the above three approaches could promote engraftment. We will identify which approach is the most important in the future.

No relevant conflicts of interest to declare.