Vuong, L. | bioRxiv Science

bioRxiv2020

IRF8 Governs Tumor-Associated Macrophage Control of T Cell Exhaustion

Tumor progression is associated with overstimulation of cytotoxic T lymphocytes (CTLs), resulting in a dysfunctional state of exhaustion. How T cell exhaustion is elicited in the tumor remains poorly understood. Here we show that tumor-associated macrophages (TAMs) present cancer cell antigen and induce CTL exhaustion through a gene expression program dependent on the transcription factor interferon regulatory factor-8 (IRF8). In a transgenic model of murine breast cancer, CTL priming was supported by IRF8-dependent dendritic cells; yet, CTL exhaustion required TAM expression of IRF8, and its ablation suppressed tumor growth. An analysis of the highly immune-infiltrated human renal cell carcinoma tumors revealed abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. The IRF8 signature co-segregated with T cell exhaustion markers and was negatively associated with long-term patient survival. Thus, CTL exhaustion is promoted by TAMs via IRF8, and this crosstalk may be disrupted in TAM-targeted therapies.

immunology↗

bioRxiv2020

Fusion Pore Formation Observed During SNARE-Mediated Vesicle Fusion with Pore-Spanning Membranes

Planar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve docking and elementary steps of the fusion process with single large unilamellar vesicles (LUVs). However, in previous studies, we monitored only lipid mixing and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into LUVs containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex {Delta}N49 prepared on porous silicon substrates. By dual color spinning disc fluorescence microcopy with a time resolution of 20 ms, we could unambiguously distinguish between bursting vesicles and fusion pore formation. Owing to the aqueous compartment underneath the PSMs, vesicle bursting turned out to be an extremely rare event (< 0.01 %). From the time-resolved dual color fluorescence time traces, we were able to identify different fusion pathways including remaining three-dimensional postfusion structures with released content and flickering fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation follows the merger of the two lipid membranes by only about 40 ms. STATEMENT OF SIGNIFICANCEDespite great efforts to develop in vitro fusion assays to understand the process of neuronal fusion, there is still a huge demand to provide single vesicle fusion assays that simultaneously report on all intermediate states including three-dimensional postfusion structures and fusion pore formation including flickering pores without the underlying artifact of vesicle bursting. Here, we show that pore-spanning membranes (PSMs) are ideal candidates to fulfill these demands. Owing to their planarity and the second aqueous compartments, they are readily accessible by fluorescence microscopy and provide sufficient space so that vesicle bursting becomes negligible. Dual color fluorescence microscopy allows distinguishing between different fusion intermediates and fusion pathways such as "kiss and run" fusion as well as flickering fusion pores.

biophysics↗