NK Cell Reconstitution in Paediatric Leukemic Patients after T-Cell-Depleted HLA-Haploidentical Haematopoietic Stem Cell Transplantation Followed by the Reinfusion of iCasp9-Modified Donor T Cells.

T-cell-depleted (TCD) human leukocyte antigen (HLA) haploidentical (haplo) hematopoietic stem cell transplantation (HSCT) (TCD-haplo-HSCT) has had a huge impact on the treatment of many haematological diseases. The adoptive transfer of a titrated number of T cells genetically modified with a gene suicide can improve immune reconstitution and represents an interesting strategy to enhance the success of haplo-HSCT. Natural killer (NK) cells are the first donor-derived lymphocyte population to reconstitute following transplantation, and play a pivotal role in mediating graft-versus-leukaemia (GvL). We recently described a CD56lowCD16low NK cell subset that mediates both cytotoxic activity and cytokine production. Given the multifunctional properties of this subset, we studied its functional recovery in a cohort of children given α/ßT-cell-depleted haplo-HSCT followed by the infusion of a titrated number of iCasp-9-modified T cells (iCasp-9 HSCT). The data obtained indicate that multifunctional CD56lowCD16low NK cell frequency is similar to that of healthy donors (HD) at all time points analysed, showing enrichment in the bone marrow (BM). Interestingly, with regard to functional acquisition, we identified two groups of patients, namely those whose NK cells did (responder) or did not (non responder) degranulate or produce cytokines. Moreover, in patients analysed for both functions, we observed that the acquisition of degranulation capacity was not associated with the ability to produce interferon-gamma (IFN-γ Intriguingly, we found a higher BM and peripheral blood (PB) frequency of iCas9 donor T cells only in patients characterized by the ability of CD56lowCD16low NK cells to degranulate. Collectively, these findings suggest that donor iCasp9-T lymphocytes do not have a significant influence on NK cell reconstitution, even if they may positively affect the acquisition of target-induced degranulation of CD56lowCD16low NK cells in the T-cell-depleted haplo-HSC transplanted patients.

Reconstitution of multifunctional CD56lowCD16low natural killer cell subset in children with acute leukemia given α/ß T cell-depleted HLA-haploidentical haematopoietic stem cell transplantation.

We recently described the CD56lowCD16low subset of Natural Killer (NK) cells that both mediate cytotoxic activity and produce IFNγ, being more abundant in bone marrow (BM) than in peripheral blood (PB) of pediatric normal subjects. Given the multifunctional properties of this subset, we examined its development and functional recovery in a cohort of children undergoing α/ß T-cell depleted HLA-haploidentical haematopoietic stem cell transplantation (HSCT). The results obtained indicate that CD56lowCD16low NK cells are present in both PB and BM already at one month post-HSCT, with an increased frequency in BM of graft recipients as compared with normal subjects. During the first 6 months after HSCT, no difference in CD56lowCD16low NK cells distribution between PB and BM was observed. In comparison to normal subjects, CD56lowCD16low NK cells from transplanted patients show lower expression levels of CD25 and CD127 and higher levels of CD122, and accordingly, produce higher amounts of IFNγ after stimulation with IL-12 plus IL-15. The recovery of NK-cell cytotoxicity after HSCT was strictly restricted to CD56lowCD16low NK cells, and their ability to degranulate against K562 target cells or autologous leukemic blasts was completely restored only one year after HSCT. Based on the phenotypic and functional ability of reconstituted CD56lowCD16low NK cells, we suggest that they play an important role in host defense against leukemia relapse and infections after HSCT, and represent an ideal candidate for approaches of adoptive immunotherapy.

Effector Functions of Natural Killer Cell Subsets in the Control of Hematological Malignancies.

Treatment of hematological malignant disorders has been improved over the last years, but high relapse rate mainly attributable to the presence of minimal residual disease still persists. Therefore, it is of great interest to explore novel therapeutic strategies to obtain long-term remission. Immune effector cells, and especially natural killer (NK) cells, play a crucial role in the control of hematological malignancies. In this regard, the efficiency of allogeneic stem cell transplantation clearly depends on the immune-mediated graft versus leukemia effect without the risk of inducing graft versus host disease. Alloreactive donor NK cells generated following hematopoietic stem cell transplantation ameliorate the outcome of leukemia patients; in addition, in vivo transfer of in vitro expanded NK cells represents a crucial tool for leukemia treatment. To improve NK cell effector functions against resistant leukemia cells, novel immunotherapeutic strategies are oriented to the identification, isolation, expansion, and administration of particular NK cell subsets endowed with multifunctional anti-tumor potential and tropism toward tumor sites. Moreover, the relationship between the emergence and persistence of distinct NK cell subsets during post-graft reconstitution and the maintenance of a remission state is still rather unclear.

Multifunctional human CD56 low CD16 low natural killer cells are the prominent subset in bone marrow of both healthy pediatric donors and leukemic patients.

We phenotypically and functionally characterized a distinct CD56(low) natural killer cell subset based on CD16 expression levels in bone marrow and peripheral blood of healthy children and pediatric patients with acute lymphoblastic leukemia. Our findings demonstrate for the first time that CD56(low)CD16(low) natural killer cells are more abundant in bone marrow than in peripheral blood and that their frequency is further increased in children with acute lymphoblastic leukemia. Bone marrow and peripheral blood CD56(low)CD16(low) natural killer cells compared with CD56(low)CD16(high) natural killer cells express lower levels of killer inhibitory receptors, higher levels of CD27, CD127, CD122, CD25, but undetectable levels of CD57, suggesting that they have a higher proliferative and differentiation potential. Moreover, CD56(low)CD16(low) natural killer cells display higher levels of CXCR4 and undetectable levels of CX3CR1 and can be consistently and rapidly mobilized in peripheral blood in response to CXCR4 antagonist. Unlike CD56(low)CD16(high), both bone marrow and peripheral blood CD56(low)CD16(low) natural killer cells release IFNγ following cytokine stimulation, and represent the major cytotoxic natural killer cell population against K562 or acute lymphoblastic leukemia target cells. All these data suggest that CD56(low)CD16(low) natural killer cells are multifunctional cells, and that the presence of hematologic malignancies affects their frequency and functional ability at both tumor site and in the periphery.