Optimization of NK cell-based immune therapy strategies against pediatric acute B cell precursor leukemia using a human-murine xenotransplantation model

Abstract

Killer-immunoglobulin-like (KIR)-receptors on natural killer (NK) cells and their corresponding HLA ligands play an important role in self-/non-self-discrimination of NK cells. In mismatched KIR-KIR ligand constellations the balance of inhibitory and activating signals is shifted towards target cell lysis. Previous studies indicate that these KIR-KIR ligand mismatch constellations significantly increase the outcome for patients suffering from adult acute myeloid leukemia whereas adult acute B cell lymphocytic leukemia (B-ALL) seems to be relative resistant to NK cell-mediated cytotoxicity. Expression of KIR molecules is regulated via promoter methylation rendering expression susceptible for DNA methyltransferase (Dnmt) inhibitors. Using a NOD-scid IL2Rgammanull (NSG) xenotransplantation model two modes for NK cell-based therapies in pediatric B cell precursor ALL (BCP-ALL) were explored: First, the adoptive transfer of mature cytokine-activated NK cells into leukemia-bearing mice and second graft-versus-leukemia effects (GvL) mediated by transplant–emerging NK cells. Adoptive transfer experiments show that pediatric BCP-ALL can be targeted by mature NK cells in vivo. Furthermore, this work provides substantial evidence that mismatched KIR-KIRL constellations are superior in this setting. Sorting experiments reveal that mismatched KIR+ NK cells display enhanced functionality compared to KIR- NK cells, which is reversed in matched constellations. Mechanistically, superior alloreactivity of KIR-KIRL mismatched compared to KIR-KIRL matched NK cells can be attributed to the KIR+ ‘alloreactive’ subset. Considering graft-emerging NK cells mainly immature KIR- NK-cells in stem cell transplanted NSG mice (“humanized NSG”) can be detected, thus ideally resembling human NK cell ontogeny of patients post hematopoietic stem cell transplantation (HSCT). Under cytokine support there are relevant GvL effects towards primary B cell precursor leukemia in huNSG mice providing evidence that also early graft-emerging immature NK cells might contribute to alloreactivity. The Dnmt-inhibitor 5-azacytidine (5-AzaC) surprisingly does not increase KIR expression on NK cells as previously described for mature NK cells in vitro. 5-AzaC distinctly enhances immature and mature NK cell subsets and along with this the anti-leukemic response. It can therefore be proposed that 5-AzaC treatment early after HSCT promotes differentiation of NK cells which can contribute to relevant GvL effects in vivo. As a result, future protocols might consider exploitation of NK cell-mediated immuneresponses for relapsing pediatric BCP-ALL patients.