The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Summary Switch-like activation of the Spindle Assembly Checkpoint (SAC) is critical for accurate chromosomes segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the “eSAC”. The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins, and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34CD117 ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34CD117 ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.

ObjectiveNK cells are understudied in the context of metabolic disease and obesity. The goal of this study was to define the effect of NK cell ablation on systemic inflammation and glucose homeostasis in murine obesity.MethodsA transgenic murine model was used to study the effect of NK cell ablation on systemic inflammation and glucose homeostasis in the context of diet-induced obesity using flow cytometry, QRTPCR, and glucose tolerance and insulin sensitivity testing.ResultsNK cell ablation achieved a 3-4 fold decrease in NK cells but had no effect on T-cell levels in adipose tissues and spleen. NK cell ablation was associated with decreased total macrophage infiltration in intra-abdominal adipose tissue, but macrophage infiltration in subcutaneous adipose tissue and spleen was unaffected. NK cell ablation was associated with modest improvement in insulin sensitivity but had no effect on tissue transcript levels of inflammatory cytokines.ConclusionNK cells play a role in promoting intra-abdominal adipose tissue macrophage infiltration and systemic insulin resistance in obesity.