Engineered T and NK cell therapies have widely been used to treat hematologic malignancies and solid tumors, with promising clinical results. Current chimeric antigen receptor (CAR) T cell therapeutics are, however, associated with treatment-related adverse events such as cytokine release syndrome (CRS) and are prone to immunologic exhaustion. CAR-NK therapeutics, while not associated with CRS, have limited in vivo persistence. We now demonstrate that a population of NK-like TCRαβ + CD8 T cells (T KLR ), identified and expanded ex vivo through its expression of the activating receptor NKG2C, can be transduced to express a second-generation CD19 CAR (1928z). These CAR+ T KLR demonstrates superior tumor clearance and longer persistence compared to 1928z CAR + conventional CD8 T cells (T Conv ) and 1928z CAR+ NK cells in mouse xenograft models. Moreover, CAR-modified T KLR had decreased expression of PD-1, TIM-3, and LAG-3 and elicited significantly reduced CRS compared to CAR + CD8 T Conv in mouse xenograft models. Similarly, NKG2C + T KLR engineered with a TCR targeting the NY-ESO-1 antigen exhibited robust tumor control compared to TCR-engineered CD8 T Conv in a mouse xenograft model. These data establish NKG2C+ T KLR as a promising platform for cell engineering, and offer a safer, more durable alternative to conventional CAR-T and CAR-NK therapies.
Chronic myelomonocytic leukemia (CMML) is a hematological neoplasm characterized by expansion of malignant monocytes. However, as a stem cell neoplasm, the disease clone comprises most blood lineages, including granulocytes. To investigate their pathogenic contribution, we performed extensive phenotypic, transcriptomic and functional characterization of CMML granulocytes. These cells exhibited reduced granularity and impaired maturation, the latter of which is associated with poor prognosis. Functionally, CMML granulocytes displayed reduced phagocytic capacity and impaired NETosis, together with aberrant gene expression programs. Notably, RETN , encoding the chemokine resistin, was uniquely upregulated in CMML granulocytes. Accordingly, resistin levels were 10-fold higher in CMML plasma, correlating with inferior survival. Ex vivo exposure to resistin directly blocked monocyte maturation and macrophage differentiation, partly via TLR4 interaction, recapitulating the classical monocytosis repartitioning phenotype that characterizes CMML. Resistin-treated monocytes upregulated gene signatures related to immune suppression and myeloid-derived suppressor cell phenotype. Consistent with these findings, CMML patients displayed higher Treg proportions and Th2/Th1 ratios. Furthermore, we demonstrated that resistin directly skews the Th2/Th1 ratio via binding to monocytes. Thus, CMML granulocytes produce high levels of resistin, which contributes to defective monocyte maturation and induction of myeloid-derived suppressor cell features in monocytes, thereby fostering an immune-suppressive state in this disease.
Primary central nervous system (CNS) vasculitis is a rare and devastating inflammatory cerebrovascular disease for which pathogenic mechanisms remain poorly defined, largely due to limited access to human tissue and the absence of validated experimental models. Here, we present a mouse model of CNS-restricted vasculitis based on inducible, endothelial-biased neoantigen expression, termed brain endothelial ovalbumin (BrEndO). In BrEndO mice, tamoxifen-induced ovalbumin (OVA) expression in cerebral endothelial cells (ECs) elicited a controlled endogenous CD8 + T cell response at the blood–brain barrier that remained clinically restrained. In contrast, peripheral immune activation through infection with OVA-expressing Listeria monocytogenes or systemic PD-L1 immune checkpoint blockade converted this regulated response into severe, CD8 + T cell–driven CNS vasculitis with vascular injury and parenchymal infiltration. Transcriptomic profiling of inflamed cerebral microvessels and spatial transcriptomic analyses demonstrated shared inflammatory and immune activation signatures between BrEndO mice and human primary CNS vasculitis brain biopsies. These findings identify antigen-specific endothelial immune recognition as a regulated process whose breakdown precipitates CNS-restricted vasculitis and establish BrEndO as an experimentally tractable platform to investigate mechanisms and modifiers of immune-mediated cerebrovascular inflammation.
Filamin C (FLNC) is an essential cytoskeletal protein required for cardiomyocyte structural integrity and mechanotransduction. Truncating FLNC variants cause haploinsufficiency associated with dilated and arrhythmogenic cardiomyopathies, yet no therapies currently address the underlying defect of FLNC deficiency. Gene replacement is challenging because the approximately 8.2-kb FLNC coding sequence exceeds the packaging capacity of conventional adeno-associated virus (AAV) vectors. Here, we engineered a triple-AAV strategy optimized for full-length FLNC reconstitution in the heart. Screening in HEK293 cells identified GP41-1 and IMPDH as a compatible orthogonal pair for efficient full-length FLNC reconstitution. In tamoxifen-inducible, cardiomyocyte-specific Flnc knockout mice, systemic delivery of optimized MyoAAV2A vectors restored full-length FLNC expression and re-established sarcomeric localization following cardiomyocyte FLNC depletion. A total vector dose of 1 × 10¹³ genome copies/kg restored FLNC to approximately endogenous abundance, preserved systolic function and ventricular dimensions, limited cardiac enlargement, attenuated fibrotic remodeling and prevented premature death. FLNC replacement also attenuated pathological transcriptional remodeling and compensatory changes in cytoskeletal and junction-associated proteins. These findings demonstrate that systemic split-intein-mediated reconstitution of full-length FLNC can preserve cardiac structure and function in vivo, providing preclinical support for a mutation-independent therapeutic strategy for FLNC-associated cardiomyopathy.
