Functional recovery of NK cells after T-cell replete haploidentical HSCT: delayed licensing and poor anti-AML activity Open Access

• In the first months after PTCy-ATG haplo-HSCT, NK cells show immature phenotype, reduced cytotoxicity and loss of donor-derived licensing

• De novo KIR-mediated licensing is progressively reacquired post-transplant and is mostly influenced by the HLA-C genotype of the recipient

Haploidentical hematopoietic stem cell transplantation (h-HSCT) is increasingly used in patients lacking an HLA-matched donor. In this context, the combination of post-transplant cyclophosphamide (PTCy) and anti-thymocyte globulin (ATG) effectively prevents graft-versus-host disease, but its impact on the reconstitution of peripheral blood natural killer (NK) cell subsets remains insufficiently characterized. In this study, NK cell subsets were analyzed in depth in 56 adult recipients of unmanipulated h-HSCT with PTCy and ATG. Peripheral blood samples were collected at days +30, +60 and +100 post-transplant. NK cell immunophenotype and cytotoxic function were assessed using multiparameter flow cytometry with unsupervised clustering, and degranulation assays against lymphoid and myeloid targets. Data were compared with those from 200 healthy volunteers. In spite of early numerical reconstitution, NK cells exhibited an immature immunophenotype with a low expression of activation markers. Cytotoxic activity against lymphoid targets was preserved, but degranulation against acute myeloid leukemia (AML) cell-lines was significantly impaired across all NK subsets, including phenotypically mature NK cells. Cytomegalovirus reactivation was associated with an expansion of memory-like NK subsets, but did not enhance degranulation. Functional education via killer-cell immunoglobulin-like receptors was lost by day+30 and progressively reacquired from day +60 on, in a pattern primarily influenced by the HLA-C genotype of recipients. These results indicate that, following h-HSCT with PTCy and ATG, NK cell subsets recover in number but fail to achieve early functional competence, particularly against myeloid targets. Strategies aiming at restoring mature NK cell functions warrant prospective investigations, especially in high-risk myeloid malignancies.