Biology subjects

Aguilar, O. A.

Publications and source records attributed to Aguilar, O. A..

6 recordsLinked to original sources

bioRxiv2026

Integrative Genomic, Single-Cell, and Functional Profiling of the CD48-CD244 Axis and NK-Cell Dysfunction in Multiple Myeloma

Multiple myeloma (MM) orchestrates immune evasion by subverting natural killer (NK) cell function. CD48, one of the most abundant NK-ligands on MM cells, paradoxically enhances NK-cell activation yet is associated with high-risk cytogenetics and poor patient survival. We integrated multi-omics (bulk and single-cell RNA-seq, ATAC-seq), genome-wide CRISPR-KO/a screens, and machine learning to dissect CD48 regulation and function. In human MM and V{kappa}*MYC mice scRNA-seq datasets, NK cells exhibit stepwise increases in inflammatory and exhaustion signatures and loss of cytotoxic potential as disease progresses. In vitro co-culture assays show CD48 overexpression on MM enhances initial NK-cell cytotoxicity and cytokine secretion, whereas chronic exposure leads to ex vivo NK dysfunction. In vivo, CD48-overexpressing V{kappa}*MYC tumors progress more slowly and extend host survival, while NK-cell depletion accelerates disease. These findings support a context-dependent role for CD48, potentiating acute NK responses while coexisting with chronic NK exhaustion, and suggest strategies to modulate CD48 for therapeutic benefit.

cancer biology↗

bioRxiv2025

Site-specific genome engineering of primary human natural killer cells for programmable anti-tumor function

Natural killer (NK) cells are emerging as a promising platform for engineered adoptive cell therapies. However, gene editing in NK cells remains challenging, and more effective strategies are needed. Here, we established a robust, feeder-free, and modular workflow for genome engineering in primary human NK cells, combining CRISPR/Cas9 with AAV6-mediated transgene delivery. Efficient site-specific transgene integration was achieved at various loci and can be coupled with concurrent disruption of the target locus in a single editing step. Furthermore, transgene expression was tunable according to the integration site and promoter. We applied this strategy to target a chimeric antigen receptor (CAR) transgene to a panel of inhibitory NK receptor loci, establishing a synergistic approach to enhance anti-tumor activity and facilitate the reliable comparison of CAR variants without expression bias. We identified TIGIT as an ideal locus that supports strong CAR expression and anti-tumor function. This genome engineering framework, which leverages multiple, complementary and precisely controlled genetic edits, can support the rational design of future NK-cell therapies tailored to overcome cell-intrinsic limitations and tumor-specific barriers.

immunology↗

bioRxiv2025

Natural Killer Cell Receptor Signaling and Activation Depend on Cell Cycle Stages

Receptor signaling in Natural Killer (NK) cells leads to post-translational modification (e.g., phosphorylation) of sub-cellular signaling proteins within minutes of receptor stimulation that eventually give rise to diverse effector functions including cell proliferation. Recent single-cell mass cytometry (i.e., CyTOF) experiments in macrophages showed variations of abundances of phosphorylated signaling proteins across cell cycle states indicating a dependence of cell signaling kinetics on an order of magnitude slower kinetics (~ several hours) of cell cycle transitions. We investigated cell cycle dependence of NKG2D signaling kinetics in NK cells by CyTOF measurements performed on IL-2-treated NKG2D-stimulated primary human CD56dim NK cells. The CyTOF experiments revealed monotonic or semi-monotonic increases of the average protein abundances of the majority of signaling proteins such as pCrkL, pPLC{gamma}2, and pErk, and the degranulation marker protein CD107a with progressing cell cycle states at specific time points post-NKG2D stimulation; however, several proteins such as pVav1, pS6, and pAkt, and early activation marker protein CD69 also showed non-monotonic variations in the average abundances with progressing cell cycle states. We used minimal mathematical and computational models coupling signaling and cell cycle processes to show that non-monotonic variations in the signaling protein abundances with progressing cell cycle stages are likely to arise in situations where protein synthesis and degradation and signaling kinetics are actively regulated by cell cycle processes.

immunology↗

bioRxiv2024

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca2+ influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca++ influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca2+ flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70. Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

immunology↗

bioRxiv2024

PVRL2 Suppresses Anti-tumor Immunity Through PVRIG- and TIGIT-Independent Pathways

PVRL2 is believed to act as an immune checkpoint protein in cancer; however, most insight into PVRL2s role is inferred from studies on its known receptor PVRIG. Here, we directly study PVRL2. PVRL2 levels are high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer shows a dramatic reduction in tumor growth that is immune dependent. This effect can be even greater than seen with deletion of PD-L1. PVRL2 functions by suppressing CD8 T and NK cells in the tumor microenvironment. Unexpectedly, the effect of PVRL2 loss on tumor growth remains in the absence of PVRIG. In contrast, PVRIG loss shows no additive effect in the absence of PVRL2. TIGIT blockade combined with PVRL2 deletion results in the greatest reduction in tumor growth. This effect is not recapitulated by the combined deletion of PVRL2 with its paralog PVR, the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the anti-tumor immune response with functions beyond that of its known receptor PVRIG. Importantly, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.

immunology↗

bioRxiv2023

Clonal stochasticity in early NK cell response to mouse cytomegalovirus is generated by mature subsets of varying proliferative ability

Natural killer (NK) cells are classically defined as innate immune cells, but experiments show that mouse cytomegalovirus (MCMV) infection in C57BL/6 mice can cause NK cells to undergo antigen-specific proliferation and memory formation, similar to adaptive CD8+ T cells. One shared behavior between CD8+ T cells and NK cells is clonal expansion, where a single stimulated cell proliferates rapidly to form a diverse population of cells. For example, clones derived from single cells are most abundant during expansion when they are primarily CD27- for NK cells and CD62L- for T cells, phenotypes derived from precursor CD27+ and CD62L+ cells, respectively. Here we determined the mechanistic rules involving proliferation, cell death, and differentiation of endogenous and adoptively transferred NK cells in the expansion phase of the response to MCMV infection. We found that the interplay between cell proliferation and cell death of mature CD27- NK cells and a highly proliferative CD27-Ly6C- mature subtype and intrinsic stochastic fluctuations in these processes play key roles in regulating the heterogeneity and population of the NK cell subtypes. Furthermore, we estimate rates for maturation of endogenous NK cells in homeostasis and in MCMV infection and found that only NK cell growth rates, and not differentiation rates, are appreciably increased by MCMV. Taken together, these results quantify the differences between the kinetics of NK cell antigen-specific expansion from that of CD8+ T cells and unique mechanisms that give rise to the observed heterogeneity in NK cell clones generated from single NK cells in the expansion phase.

immunology↗