Biology subjects

Castiglioni, A.

Publications and source records attributed to Castiglioni, A..

4 recordsLinked to original sources

bioRxiv2026

Inhibition of integrin αvβ8-mediated TGFβ activation and active-TGFβ blockade promote anti-tumor immunity through distinct biological mechanisms

Transforming Growth Factor {beta} (TGF{beta}) is a potent immunosuppressor and a primary driver of resistance to cancer immunotherapy. While preclinical models have long suggested that TGF{beta} inhibition could synergize with immune checkpoint inhibitors, these effects have proven difficult to replicate in clinical settings. The highly regulated TGF{beta} pathway can be inhibited through various mechanisms, including neutralizing activated ligands or inhibiting upstream activators, such as integrins. Recent structural data demonstrated that integrin v{beta}8 can enable TGF{beta}1/3 signaling without releasing the active cytokines from their Latency-Associated Peptides, suggesting that ligand-blocking antibodies may have limited access to their epitopes. Here, we show that integrin v{beta}8 blockade, while achieving anti-tumor responses similar to those of anti-TGF{beta} antibodies, does so through a distinct mechanism of action. Anti-v{beta}8 is 3 orders of magnitude more potent at inhibiting v{beta}8-mediated TGF{beta} activity than a commonly used antibody against the mature form of the cytokine. Whereas TGF{beta} ligand inhibition has little effect on TGF{beta} signaling in tumor-draining lymph nodes (tdLN) and requires IFN{gamma} for its anti-tumor effects, v{beta}8 blockade strongly inhibits TGF{beta} signaling in tdLN and, in combination with PD-L1 blockade, drives tumor control through an IFN{gamma} -independent mechanism that strictly requires T cell egress from tdLN. Combined v{beta}8 and anti-PD-L1 blockade enhances antigen presentation in dendritic cells (DCs) and, unlike TGF{beta} ligand blockade, improves the efficiency of DC-induced T cell activation in response to cross-presented antigen. These findings suggest that v{beta}8 blockade can disable an immunologically critical source of TGF{beta} signaling that is not addressed by antibodies targeting TGF{beta} ligands, suggesting a promising new approach to TGF{beta} pathway modulation. One Sentence SummaryUnlike ligand-neutralizing antibodies, v{beta}8 blockade suppresses TGF{beta} in tdLN and boosts DC-T cell activation, a differentiated immunotherapy strategy.

immunology↗

bioRxiv2026

A Co-culture Cell-Based Reporter Assay for Quantitative Measurement of Integrin αvβ8-Mediated Activation of Latent TGF-β1

Integrin v{beta}8 is a major activator of latent transforming growth factor-{beta} (TGF-{beta}) and an emerging therapeutic target in cancer and immune regulation. However, functional assays that directly measure v{beta}8-mediated activation of latent TGF-{beta} in a physiologically relevant context remain limited. Here, we report a co-culture cell-based reporter assay for quantitative measurement of v{beta}8-mediated activation of latent TGF-{beta}1. NIH/3T3 reporter cells were engineered to express a SMAD-responsive NanoLuc reporter, constitutive firefly luciferase for internal normalization, and cell-surface GARP-latent TGF-{beta}1. When co-cultured with v{beta}8-expressing LN-229 cells, reporter cells produced a robust signal that directly reflected localized latent TGF-{beta}1 activation. The assay demonstrated stable expression of the required biological components, reproducible signal-to-background performance, and sensitivity to benchmark v{beta}8-blocking antibodies. Inhibition studies showed potent dose-dependent blockade by an anti-v{beta}8 antibody. In contrast, pan-TGF-{beta} neutralizing antibody displayed markedly weaker apparent potency, suggesting that targeting localized v{beta}8-mediated activation is more effective than neutralizing released TGF-{beta} in this assay context. The assay also enabled screening and ranking of anti-v{beta}8 antibodies, identifying several high-potency clones, and detected v{beta}8-mediated activation of a non-cleavable latent TGF-{beta}1 mutant. This platform provides a sensitive, internally normalized, and scalable approach for mechanistic studies and therapeutic discovery targeting the v{beta}8-TGF-{beta} axis.

immunology↗

bioRxiv2026

Fibroblast TGF-β3 promotes tissue-residency and survival of CD8 T cells in barrier tissues and tumors

Fibroblasts are key organizers of tissue architecture and immune cell homeostasis, yet how they shape adaptive immune function within non-lymphoid tissues remains incompletely understood. CD8+ tissue-resident memory T cells (TRM) provide localized protection against pathogens and contribute to tumor control, but the microenvironmental signals that maintain their persistence and survival are poorly defined. Here, we identify fibroblast-derived TGF-{beta}3 as a conserved stromal niche factor that specifically sustains CD8+ TRM in both steady-state and disease settings. Across human single-cell cross-tissue atlases, CD8+ TRM preferentially correlated with fibroblast abundance in healthy barrier tissues and multiple tumor types, and TGFB3 emerged as a key fibroblast-enriched candidate mediator. In human and murine co-culture systems, fibroblast-derived TGF-{beta}3 promoted CD8 TRM-like differentiation in vitro. Using a novel genetic in vivo model, inducible fibroblast-specific deletion of Tgfb3 reduced CD8 TRM across barrier tissues at steady state and impaired antigen-specific CD8 TRM formation following viral infection. In tumor models, genetic loss or antibody mediated neutralization of TGF-{beta}3 impaired CD8 T cell residency and cytotoxicity, induced dysfunction via proteotoxic stress and apoptotic programs, and accelerated tumor growth. These findings provide mechanistic insight into the limited efficacy of pan-TGF-{beta} blockade in cancer therapy. Collectively, we describe a novel fibroblast-CD8 T cell axis mediated by TGF-{beta}3 that sustains residency and restrains proteotoxic stress in barrier tissues and tumors.

immunology↗

bioRxiv2023

A novel, RAS-independent role for NF1 in microtubular dynamics and damage repair dictates sensitivity to T-DM1 in HER2-positive breast cancer

Antibody-Drug Conjugates (ADC) have revolutionized the treatment of several tumors, and extensive research is being devoted to the identification of predictive biomarkers. These are particularly sought after in fields, like breast cancer, in which multiple ADCs with identical target but different payloads have been approved. NF1 is a tumor suppressor widely mutated across several cancers, best characterized as an inhibitor of RAS signaling. Additional functions have been proposed but not deeply investigated, due to its large size and complex domain structure. Whether somatic NF1 mutations can be used to guide clinical decisions is not known. Here, combining patient data, in vitro/in vivo models and protein biochemistry, we show that NF1 loss sensitizes cancer cells to T-DM1, the first approved ADC in breast cancer, through a novel, RAS-independent function on microtubular dynamics and repair. NF1 exhibits all biochemical properties of a bona fide Microtubule-Associated Protein (MAP) and specifically enhances intratubular repair, a recently discovered phenomenon whose regulation remains poorly characterized. NF1 loss results in mitotic defects and low-grade aneuploidy in cell lines and patients. Increased sensitivity to T-DM1 upon NF1 loss is confirmed in breast cancer patients analysed across institutions in Europe and USA. Our results define NF1 as a key regulatory factor for microtubular repair and the first ADC payload-associated predictive biomarker identified to date.

cancer biology↗