Biology subjects

Vannini, N.

Publications and source records attributed to Vannini, N..

5 recordsLinked to original sources

bioRxiv2026

Closed, Automated CAR-T Cell Manufacturing: from Research to Point of Care production.

Background: Point-of-care manufacturing of CAR-T cells could reduce cost and improve patient access, but few closed, automated systems support both flexible process development and GMP production. Methods and results: We describe LimCORE, a single-use consumable for cell incubation and processing, implemented in LimGROW (manual system) and LimONE (closed and automated system). In LimGROW, T cells expanded 246 +/-18 fold over 10 days at optimized seeding density, versus 89 +/-9 fold in a G-Rex control, with similar viability (>90%) and phenotype. Process parameters directly transferred to LimONE, where automated, closed buoyancy-based CD3+ selection achieved 90.3 +/-3.5% purity and 59.2 +/-17.0% recovery in under two hours. Using this workflow, we manufactured CD19 CAR-T cells in a fully closed, automated 7-day LimONE process requiring 155 minutes of operator intervention. LimONE manufactured CAR-T cells reached 42.6 +/-2.4 fold expansion (532 +/-29x10e6; cells at harvest), with 43.6 +/-6.0% transduction efficiency and viability similar to G-Rex controls. CD4+/CD8+ ratios, CD8+ differentiation subsets, and cytotoxicity were comparable between platforms, while LimONE-manufactured cells showed increased spare respiratory capacity and oxygen consumption. Across four manufacturing runs on the LimONE, no hardware or software failures occurred, and automated liquid transfers and volume concentration stayed within +/-5% accuracy. Conclusions: These data show that LimCORE supports T cell expansion, selection, and complete CD19 CAR-T manufacturing with performance similar to existing platforms while reducing operator time, across R&D and GMP scale closed-system formats.

bioengineering↗

bioRxiv2025

Computational Inference of Metabolic Programs: A Case Study Analyzing the Effect of BRCA1 Loss

Metabolic reprogramming is a hallmark of cancer, yet how oncogenic drivers shape tumor metabolism across disease progression remains incompletely understood. In this study, we present iMSEA (in silico Metabolic State and Enrichment Analysis), a computational framework that infers flux-based metabolic states from omics profiles. Applying iMSEA to isogenic BRCA1-mutant and BRCA1-wild-type ovarian cancer cells, we identified a shift toward glycolysis, nucleotide biosynthesis, and redox imbalance, coupled with impaired oxidative phosphorylation. These predictions were validated with metabolomics, Seahorse, and SCENITH assays, demonstrating the accuracy of our approach. Extending the analysis to homologous recombination deficient patient tumors at single-cell resolution, we found that BRCA1-deficient cancers display heightened metabolic activity and site-specific adaptations, including altered central carbon fluxes, mitochondrial function, nucleotide biosynthesis, and lipid metabolism. By linking transcriptional programs to functional metabolic states, iMSEA reveals hidden metabolic liabilities in BRCA1-deficient ovarian cancer and provides a broadly applicable strategy for dissecting metabolic heterogeneity and therapeutic vulnerabilities in cancer.

systems biology↗

bioRxiv2023

Transcriptional reprogramming by IL-2 variant generates metabolically active stem-like T cells

Interleukin-2 receptor (IL-2R)-mediated intracellular signaling is a key regulator of T-cell fate decisions. While the potent signals generated by IL-2 engagement execute effector differentiation, elevated metabolic activities and rapid cellular expansion, IL-15 binding induces a stemness/memory phenotype and a quiescent metabolic state. Here, we demonstrate that weak but sustained signaling generated by a non-IL-2R-binding variant of IL-2 (IL-2v) drive proliferation/metabolic and stemness transcriptional programs, thereby reprogramming CD8+ T cells into a hybrid metabolically active stem-like state. We further show that IL-2v-induced T cells are capable of superior engraftment, persistence, and tumor control when utilized in adoptive cell therapy. Taken together, our study highlights the ability to fine-tune cytokine engagement of cognate receptors in order to generate therapeutically relevant T-cell states and further reveals the metabolic plasticity of the T-cell memory program.

systems biology↗

bioRxiv2023

Induction of mitophagy reverts age-associated decline of the hematopoietic and immune systems

Aging compromises hematopoietic and immune system functions, making elderly individuals especially susceptible to hematopoietic failure, infections and tumor development and thus representing an important medical target for a broad range of diseases. During aging, hematopoietic stem cells (HSCs) lose their blood reconstitution capability and commit preferentially toward myeloid lineage (myeloid-bias). These processes are accompanied by an aberrant accumulation of mitochondria in HSCs. The administration of the mitophagy-inducer Urolithin-A re-establishes the correct mitochondrial homeostasis in HSCs and completely restores the blood reconstitution capability of "old" HSCs. Moreover, Urolithin-A supplemented food restores lymphoid compartments, boosts HSCs function and improves the immune response to viral infection in old mice. Altogether our results demonstrate that targeting mitophagy reverts aging phenotype in the hematopoietic and immune system.

immunology↗

bioRxiv2022

TGFβ limits Myc-dependent TCR-induced metabolic reprogramming in CD8+ T cells

T cell activation is dependent upon the integration of antigenic, costimulatory and cytokine-derived signals and the availability and acquisition of nutrients from the environment. Furthermore, T cell activation is accompanied by reprogramming of cellular metabolism to provide the energy and building blocks for proliferation, differentiation and effector function. Transforming growth factor {beta} (TGF{beta}) has pleiotropic effects on T cell populations, having both an essential role in the maintenance of immune tolerance but also context-dependent pro-inflammatory functions. We set out to define the mechanisms underpinning the suppressive effects of TGF{beta} on mouse CD8+ T cell activation. RNA-sequencing analysis of TCR-stimulated T cells determined that Myc-regulated genes were highly enriched within gene sets downregulated by TGF{beta}. Functional analysis demonstrated that TGF{beta} impeded TCR-induced upregulation of amino acid transporter expression, amino acid uptake and protein synthesis. Furthermore, TCR-induced upregulation of Myc-dependent glycolytic metabolism was substantially inhibited by TGF{beta} treatment with minimal effects on mitochondrial respiration or mTOR activation. Thus, our data suggest that inhibition of Myc-dependent metabolic reprogramming represents a major mechanism underpinning the suppressive effects of TGF{beta} on CD8+ T cell activation.

immunology↗