Donor-Derived NK Cell Infusion Following Haploidentical Hematopoietic Stem Cell Transplant for High-Risk and Relapsed/Refractory Pediatric Leukemia
Authors: Rossitti Emanuela , Pagano Gennaro , Cacace Fabiana , Caprioli Valeria , D'Amico Maria Rosaria , De Simone Giuseppina , Sabbatino Maria Simona , Toriello Mario , Maisto Giovanna , Penta de Vera d'aragona Roberta , Cianflone Alessandra , Mirabelli Peppino , Wierda William G. , Tambaro Francesco Paolo 
Published: December 2024

Background
Natural killer (NK) lymphocytes are cells of innate immunity, involved in early defense against tumor and infections. Their function and activity are regulated by a balance between activator and inhibitory signals through surface receptors, such as the inhibitory killer-cell immunoglobulin-like receptors (iKIRs), that allow NK cells to discriminate between healthy self and to mediate cytolytic activity against malignant or virally infected cells. In haploidentical hematopoietic stem cell transplantation (haplo-HSCT), NK cells play a key role in preventing early relapse and virus reactivation and exert an efficient graft-versus-leukemia (GvL) effect, without increasing graft-versus-host disease (GvHD) incidence.
After haplo-HSCT, donor NK cells derived from CD34+ stem cells are immature cells with decreased cytolytic activity (CD56bright). Progression toward more mature and potent CD56dim NK cells is characterized by acquisition of KIRs, CD16 and CD57 and is completed in 4-6 weeks. Infusion of mature donor NK cells could be useful in patients undergoing haplo-HSCT, ensuring control of disease and viral reactivations, before and during engraftment in the recipient.
Patients and Methods
We evaluated the outcome of 5 pediatric patients with high-risk leukemia who were planned to receive haplo-HSCT with donor NK cells infusion following one of two different conditioning platforms, Post-Transplant Cyclophosphamide (PTCy) and T-αβ/B cell-depleted. Four patients were male. Median age was 1.2 years (0.7-18). Three patients out of five had infant leukemia, 2 with a refractory disease and 1 relapsed after HSCT, and the remaining 2 patients had relapsed-refractory disease. All patients received a median of 3 lines of chemotherapy and 1 received prior allo-HSCT. Donor was one of the parents for all patients, selected based on genetic, immunological and clinical criteria. Four patients received T αβ/B cell-depleted haplo-HSCT; 1 patient received PTCy haplo-HSCT. Conditioning regimen consisted of Busulfan/Treosulfan + Fludarabine and Thiotepa backbone for 4 patients; 1 patient received TBI. The median CD34+, CD3+ TCRαβ+, CD3+ TCRγδ+ dose was 14.06x106/Kg (7.16-19.8), 0.3125x106/Kg (0.089-0.239) and 16.19 x 106/Kg (6.3-19.81), respectively. The TNC dose was 5.8x108/Kg for patient undergone PTCy aplo-HSCT. Patients who underwent T αβ/B cells depletion haplo-HSCT received rituximab and letermovir for EBV and CMV prophylaxis, respectively. NK counts were monitored on day +7, +14, +21, +28 after transplantation and on day +7, +14, +21, +28 after NK infusion. All patients, except one because of the onset of hepatic acute GvHD, received donor NK infusion at median of 35 days after transplantation (31-62), triggered by the decrease of NK count. Median number of NK cells infused was 2 x 107/kg (0.67-2.6).
Results
The median time to neutrophils and platelets engraftment was 14 (13-20) and 10 (8-14) days, respectively. None of the patients developed CMV or EBV reactivation while ADV reactivation was observed in three patients but only one required antiviral treatment. Three patients experienced Grade 1-2 acute GvHD and 1 patient experienced chronic GvHD. All patients are in complete molecular remission (OS and EFS 100%) with full donor chimerism at a median follow up of 25 months (13-32).
Conclusion
Donor NK cell infusion after haplo-HSCT could represent an effective option to improve transplant outcome and prevent virus reactivation and early relapse in pediatric patients with very high-risk or relapsed/refractory leukemia. Mild, steroid-responsive acute GvHD was observed and limited chronic GvHD. These limited but very promising preliminary results need to be confirmed in larger cohorts.