Akhbariyoon, H. | Search bioRxiv

bioRxiv2026

Nanoscale 3D profiling of the T cell membrane reveals CD2 enrichment at microvilli tips, positioning adhesion near TCR zones in the immunological synapse

The T cell membrane features a specialized molecular and topological organization critical for signaling and immune function. During antigen detection, T cells utilize finger-like protrusions called microvilli to dynamically scan the antigen-presenting cell (APC) surface. Enriched with T cell receptors (TCRs) and key signaling molecules, microvilli serve as primary signaling hubs, yet their nanoscale architecture remains poorly defined. Upon antigen engagement, TCR activation drives the formation of the immunological synapse (IS), a highly organized membrane contact with the APC critical for T cell function. However, profiling IS architecture at the nanoscale remains technically challenging. Here, we introduce NanoMAP (Nanoscale Membrane Architecture Profiling), an expansion microscopy-based platform that resolves receptor organization on T cell microvilli and within the IS at 35-60 nm resolution. Using NanoMAP, we show that effector CD8+ T cells possess a 5.1-fold higher microvillar density than naive CD8+ T cells. Mapping the adhesion receptors CD2 and LFA-1 on the T cell surface reveals that CD2 exhibits a stronger preference for localizing to microvilli tips compared to TCR and LFA-1. Within microvilli, CD2 displays a strong spatial association with TCR clusters, contrasting with a markedly weaker TCR-LFA-1 association. At the IS, TCR and CD2 co-occupy close membrane contacts, while LFA-1 is excluded to more distal regions. Upon termination of activation, TCR clusters selectively disengage from microvilli, whereas CD2 and LFA-1 persist. These results suggest a coordinated, activation-dependent organization of adhesion receptors that drives microvillar adhesion during scanning and stabilizes IS membrane contacts. Together, NanoMAP establishes a powerful framework for dissecting nanoscale membrane architecture and spatial signaling in T cells.

immunology↗

bioRxiv2025

Identifying the therapeutic potential of Niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the Tumor Microenvironment

BackgroundTumor cells frequently develop immune resistance through interferon-{gamma} (IFN-{gamma})-induced PD-L1 expression, acquisition of cancer stem cell (CSC)-like features, and adaptation to hypoxia within the tumor microenvironment (TME). Although IFN-{gamma} activates both STAT1 and STAT3, how these pathways interact to regulate immune evasion under hypoxia remains unclear. MethodsUsing the MC38 murine colorectal cancer model and T cell-tumor spheroid co-culture assays, we examined how IFN-{gamma} signaling through STAT1 and STAT3 influences PD-L1 expression, CSC plasticity, and cytotoxic T cell function under normoxic and hypoxic conditions. Pharmacologic inhibitors and siRNA knockdown were used to dissect pathway function, and Niclosamide, an FDA-approved anthelmintic, was evaluated as a dual STAT1/STAT3 inhibitor. ResultsWe found that IFN-{gamma} primarily induced PD-L1 through STAT1 activation, while CSC plasticity was associated with STAT3 signaling. STAT1 and STAT3 displayed reciprocal regulation--blocking one enhanced activation of the other. Niclosamide effectively inhibited phosphorylation of both STAT1 and STAT3, which led to suppressed PD-L1 upregulation and reduced CSC enrichment. In addition, it also partially inhibited hypoxia-induced HIF-1 expression. In co-culture assays, Niclosamide improved T cell infiltration and reduced exhaustion under hypoxic conditions, resulting in improved T cell killing. ConclusionsOur findings identified Niclosamide as a potent dual STAT1/3 inhibitor capable of reversing IFN-{gamma} and hypoxia-driven immune evasion. Repurposing Niclosamide may represent a promising strategy to enhance the efficacy of immune checkpoint blockade in solid tumors. key messagesInterferon-{gamma} (IFN-{gamma}) enhances cytotoxic T cell function but also promotes tumor immune evasion by upregulating PD-L1 and inducing cancer stem cell- like properties. Our study identifies a reciprocal regulatory mechanism between STAT1 and STAT3 in IFN-{gamma}-treated tumor cells that shapes immune evasion outcomes. We demonstrate that Niclosamide, an FDA-approved anthelmintic, acts as a dual STAT1/STAT3 inhibitor, effectively suppressing PD-L1 induction, limiting cancer stemness, and reducing HIF-1 expression under hypoxia. Niclosamide also restores T cell infiltration and decreases exhaustion in a 3D tumor spheroid model. By repurposing Niclosamide, this work provides a feasible approach to enhance the efficacy of immune checkpoint blockade and guide future translational and clinical studies in immunotherapies against solid tumors.

immunology↗