Background: Natural Killer (NK) cells have intrinsic anticancer activity that can be redirected toward acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) engineering. Here, we study the functional consequences of CAR binding affinity and targeted epitope on CAR-NK cell activation, cytolytic synapse formation, and antitumor activity. Methods: We characterized NK-92 and primary NK cell populations expressing variant affinity AML-specific CARs containing single-chain variable fragments (scFvs, 26292 or 7G3) targeting two epitopes on CD123. 26292 affinity variants were discovered through directed evolution of an error-prone mutagenic library, while 7G3 affinity variants were previously reported. The resulting CAR-NK cell panel was studied with in vitro binding, activation, and cytotoxicity studies and in mouse xenograft models. Results: 26292 and 7G3 CARs of variable CD123 binding affinities were highly expressed in NK cells and conferred antigen-specific activation in vitro. High-resolution imaging demonstrated greater clustering of high-affinity 7G3 CAR-NK cells and consequent AML target cell death in a short-term time lapse. Low-affinity 7G3 CAR-NK cells exhibited enhanced antigen density discrimination with greater membrane-proximal signaling, cytokine production, and cytotoxicity. In longer-term assays, low-affinity 7G3 CAR-NK cells demonstrated more sustained killing of AML cells. In vivo testing highlighted greater expansion of low-affinity 7G3 CAR-NK cells in two xenograft models. Conclusions: Expression of 26292 and 7G3 CARs with a range of CD123 binding affinities in NK cells leads to antigen-specific activation and cytotoxicity against AML. Affinity-based differences in functional activation and antitumor activity are dependent on time course and are scFv/epitope specific.

The evolution of antiretroviral therapies (ART) has tremendously improved the life expectancy of people living with human immunodeficiency virus (HIV) (PLWH), which is currently similar to the general population. However, as PLWH are now living longer, they exhibit various comorbidities such as a higher risk of cardiovascular disease (CVD) and non-acquired immunodeficiency syndrome (AIDS)-defined malignancies. Clonal hematopoiesis (CH) is the acquisition of somatic mutations by the hematopoietic stem cells, rendering them survival and growth benefit, thus leading to their clonal dominance in the bone marrow. Recent epidemiologic studies have highlighted that PLWH have a higher prevalence of CH, which in turn is associated with increased CVD risk. Thus, a link between HIV infection and a higher risk for CVD might be explained through the induction of inflammatory signaling in the monocytes carrying CH mutations. Among the PLWH, CH is associated with an overall poorer control of HIV infection; an association that requires further mechanistic evaluation. Finally, CH is linked to an increased risk of progression to myeloid neoplasms including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), which are associated with particularly poor outcomes among patients with HIV infection. These bidirectional associations require further molecular-level understanding, highlighting the need for more preclinical and prospective clinical studies. This review summarizes the current literature on the association between CH and HIV infection.

Central nervous system (CNS) tumors are the most common solid malignancy in the pediatric population. These lesions are the result of the aberrant cell signaling step proteins, which normally regulate cell proliferation. Mitogen-activated protein kinase (MAPK) pathways and tyrosine kinase receptors are involved in tumorigenesis of low-grade gliomas. High-grade gliomas may carry similar mutations, but loss of epigenetic control is the dominant molecular event; it can occur either due to histone mutations or inappropriate binding or unbinding of DNA on histones. Therefore, despite the absence of genetic alteration in the classic oncogenes or tumor suppressor genes, uncontrolled transcription results in tumorigenesis. Isocitric dehydrogenase (IDH) mutations do not predominate compared to their adult counterpart. Embryonic tumors include medulloblastomas, which bear mutations of transcription-regulating pathways, such as wingless-related integration sites or sonic hedgehog pathways. They may also relate to high expression of Myc family genes. Atypical teratoid rhabdoid tumors harbor alterations of molecules that contribute to ATP hydrolysis of chromatin. Embryonic tumors with multilayered rosettes are associated with microRNA mutations and impaired translation. Ependymomas exhibit great variability. As far as supratentorial lesions are concerned, the major events are mutations either of NFkB or Hippo pathways. Posterior fossa tumors are further divided into two types with different prognoses. Type A group is associated with mutations of DNA damage repair molecules. Lastly, germ cell tumors are a heterogeneous group. Among them, germinomas manifest KIT receptor mutations, a subgroup of the tyrosine kinase receptor family.

Chimeric antigen receptor (CAR) T cells can effectively treat leukemias, but sustained antitumor responses can be hindered by a lack of CAR T-cell persistence. Cytotoxic effector T cells are short-lived, and establishment of CAR-T cells with memory to ensure immune surveillance is important. Memory T cells depend on cytokine support, with IL7 activation of the IL7 receptor (IL7R) being critical. However, IL7R surface expression is negatively regulated by exposure to IL7. We aimed to support CAR T-cell persistence by equipping CAR-T cells with a sustained IL7Rα signal. We engineered T cells to constitutively secrete IL7 or to express an anti–acute myeloid leukemia–targeted IL7Rα–chimeric cytokine receptor (CCR) and characterized the phenotype of these cell types. Canonical downstream signaling was activated in CCR-T cells with IL7R activation. When coexpressed with a cytotoxic CAR, functionality of both the CCR and CAR was maintained. We designed hybrid CAR–CCR and noted membrane proximity of the intracellular domains as vital for signaling. These data show cell-intrinsic cytokine support with canonical signaling, and functionality can be provided via expression of an IL7Rα domain whether independently expressed or incorporated into a cytotoxic CAR for use in anticancer therapy. Significance: To improve the phenotype of tumor-directed T-cell therapy, we show that provision of cell-intrinsic IL7R-mediated signaling is preferable to activation of cells with exogenous IL7. We engineer this signaling via independent receptor engineering and incorporation into a CAR and validate maintained antigen-specific cytotoxic activity.

Lay Summary: As we celebrate 50 years of newborn screening (NBS) for sickle cell disease (SCD), we must confront its hidden failure: the neglect of sickle cell trait (SCT). Identified in thousands of newborns annually, SCT status is often lost in a system with no clear owner for communicating its lifelong implications to families. This gap leads to missed opportunities for reproductive planning, widens health disparities, and undermines public trust. We present public health precedents that provide a pathway to integrating SCT counseling into routine care and urge stakeholders to move beyond identification to action and fulfill the original promise of NBS.