Enhanced engraftment and immune recovery in thalassemia via optimized conditioning for TCRαβ⁺-depleted haploidentical transplants

Abstract Background TCRαβ⁺/CD19⁺-depleted haplo-HSCT (TDH) reduces the risk of graft-versus-host disease (GVHD) in thalassemia major (TM) patients. However, high rate of graft failure (GF;15-30%) persist, potentially increasing infection risks. Contrary to reports associating TDH with increased infections, our center observed lower rates of severe infections(e.g., CMV pneumonia: 1.11%) following TDH compared to conventional HSCT, suggesting unique immune reconstitution (IR) advantages. To address the challenge of GF, we implemented a modified conditioning regimen incorporating pre-transplant ATG and PTCy on day -1. Concurrently, we investigated whether rapid early γδ T-cell and NK-cell reconstitution correlates with the observed reduction in severe infections. Methods We conducted a retrospective analysis of 239 TM patients (median age: 7 years; male:female = 149:90) who underwent TDH from June 2020 to September 2024. The patients were divided into two groups: TDH-A (n=77) received standard conditioning without day -1 Cy, while TDH-B (n=162) received added Cy (25 mg/kg) on day -1. Both groups shared the following conditioning regimen: Days -21 to -19: rATG (5 mg/kg); Day -11: donor lymphocyte infusion（DLI） (2×10⁸/kg); Days -8 and -7: cyclophosphamide (50 mg/kg/day); Days -6 to -4: busulfan (90-100 mg/m²/day; target CSS: 500-700; Days -6 to -2: fludarabine (40 mg/m²/day); Day -3: thiotepa (10 mg/kg); and Day -1: rituximab (100 mg/m²) for all patients. GVHD prophylaxis comprised tacrolimus/sirolimus (target level: 5-10 ng/mL), with mycophenolate mofetil added for high-risk patients. Result 1.The graft composited of 23.86×10⁶/kg (range 14.30-61.12) CD34 cells, with 11.90×10⁸/kg (6.15-21.5) mononuclear cell. The median time for hematopoietic reconstitution was as follows: neutrophils 16 days (range: 9-48), hemoglobin 11 days (1-23), platelets 10 days (5-22). Engraftment ：The GF rate was significantly lower in TDH-B (1.2% 2/162 vs. 9.1% 7/77 in TDH-A; P=0.004). Mechanism: Day -1 Cy eliminates DLI-activated alloreactive lymphocytes. 2.In acute GVHD: Grade II incidence was 5.63%, Grade III-IV incidence was 4.38%; in chronic GVHD, mild cases accounted for 11.88%, moderate cases 1.25%, with no severe cases reported. 3.GVHD risk is related with megadose CD34: CD34⁺ 25×10⁶/kg: Higher aGVHD risk (OR=3.1; 95% CI 1.5–6.4; P=0.004). CD34⁺ 30×10⁶/kg: Higher cGVHD risk (OR=2.8; 95% CI 1.2–6.5; P=0.040). 4.IR and infections: Infected patients showed markedly lower γδ-T, NK, and CD4⁺T cells vs. non-infected (12-month γδ-T: 275/μL vs. 369/μL; P0.01). Rapid γδ-T reconstitution (236/μL at 3 months) was critical for controlling severe infections. Conclusion 1.The innovative regimen (earlier ATG and day -1 Cy) reduced risk of GF and severe GVHD via depletion of activated lymphocytes. 2.Rapid γδ-T/NK-cell reconstitution is pivotal for controlling severe infections. 3.CD34⁺ dose ≤25×10⁶/kg reduces aGVHD risk without impairing engraftment. 4.Key finding: First evidence of a dose-time-response relationship between γδ-T-cell recovery speed and infection outcomes, offering novel immunomodulatory targets.